0.05) (Table 3).\nOur data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).\n 3.5. Path Analysis Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).\nAdjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).\n 3.6. Temporal Validation We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).\nWe additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).\n\n3.1. Participants:\nOf 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.\n\n3.2. Sociodemographic Characteristics:\nTable 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.\nIn the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.\n\n3.3. Covariate Selection and Correlation Analysis:\nConsidering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).\nAs shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.\nEducation level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.\n\n3.4. Hierarchical Regression Analysis:\nOur data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).\n\n3.5. Path Analysis:\nAdjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).\n\n3.6. Temporal Validation:\nWe additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).\n\n4. Discussion:\nWe conducted a quantitative study to explore the potential mechanism of how stigma affects the likelihood of depression in asymptomatic carriers during the mid-stage of the COVID-19 outbreak in Shanghai. In the current study, depression was reported by 44.7% of participants. A high education level was a protective factor for depression. This study characterized the psychological status of COVID-19 asymptomatic carriers, underscoring the importance of psychiatric screening and early interventions in this subgroup. Moreover, we found a mediating role of entrapment and decadence on the relationship between social stigma and depression, which offers a new way to mitigate the high prevalence of depression among asymptomatic carriers in China.\nWe found that the prevalence of depression reported by asymptomatic carriers was comparable to the rate in COVID-19 patients in previous studies [7,43]. Since physicians prioritize the physical illnesses of hospital patients, it is not surprising that the mental health issues of this subpopulation did not receive attention. Asymptomatic carriers account for a large proportion of COVID-19 patients in China, which highlights the importance of raising awareness of psychiatric screening in infected patients. In addition, the final model showed that a higher education level was a protective factor for depression. Inconsistent findings were reported for the association between education level and psychological symptoms [7,9]. One explanation for our finding is that better-educated individuals tend to acquire the correct information and awareness about COVID-19, therefore being more likely to avoid some adverse psychological health outcomes.\nIn line with our hypothesis, entrapment and decadence mediate the relationship between stigma and depression in asymptomatic carriers during the epidemic. People who suffered from stigma were more likely to feel trapped and decadent, which, in turn, was associated with an increase depressive symptoms. Two conceptual frameworks described at the beginning of the article guided this mediation assumption complementarily. Moreover, we found that the indirect relationship between stigma and depression through entrapment was moderated by age group. This further supports the finding that stigma can affect depression via entrapment.\nMediation analysis is essentially a correlation analysis [44]. Based on accumulated theory and research, we hypothesized that entrapment mediates the relationship between stigma and depression. However, the existence of a correlation between stigma and depression may also be the result of reverse causality or the presence of confounding factors. We found a significant difference in the correlation coefficients between stigma and depression in the two age groups, which at least suggests that the correlation is not exactly the result of the two possible causes mentioned above (reverse causality and confounding factors) [44,45]. Otherwise, their correlation would not differ. Thus, the mediation effect exists.\nOur findings have three implications for the early detection of depression and effective mental health services for asymptomatic carriers. First and foremost, the timely assessment of stigmatizing stressors associated with COVID-19 must be carried out. Prior studies suggest that social stigma is not only a product of structural inequalities caused by bias and discrimination, but also the result of individual failures to cope adaptively with the stressors [15]. Ding K et al. also suggested that personal-level factors are more likely to be related to depression [46,47]. Therefore, the consideration of individuals’ stigmatizing stressors in early depression interventions is clearly warranted. This study focused on stigmatizing stressors related to job refusals and other overlooked situations. In future research, we believe there is a need to help infected individuals identify the well-defined type of stigma-related stress (e.g., being quarantined or treatment factors) they encounter to effectively help them cope with it successfully.\nSecond, response efforts to alleviate entrapment and decadence can be a crucial early intervention point for depression. Our main results shows that entrapment and decadence fully mediated the association between COVID-19-related stigma and depression. It appears that improving entrapment and decadence may effectively block the effects of stigma on depression. Specifically, an assessment of the psychological status of entrapment and decadence can be included in the early detection of depressive symptoms. Additionally, hospitals can implement remote mental health psychiatric counseling and screening programs through telemedicine or online mental health interventions [48,49]. Preventive strategies and early interventions are needed to adequately address early psychological abnormal states and avoid long-term mental health problems.\nThird, this research provides a basis for implementing individualized mental health intervention strategies. Our findings revealed that the indirect link between stigmatization and depression via entrapment is stronger in younger age groups, which implies we could give priority to younger infected individuals with respect to entrapment-reducing interventions. Meanwhile, we also need to find other influential factors of the mental state of entrapment and decadence for relatively high age groups in the future.\nThe current study involves several limitations. First, participants were recruited by non-probability sampling approaches, which may be subject to sampling bias. The non-response rate in this study was 18.4%, which may underestimate the prevalence of depression. Second, participants were recruited from one location, and we need to be cautious when applying the results to other areas. However, we did use a larger sample in our study, so the results may provide a worthy reference for the exploration of depressive mechanisms. Third, the cross-sectional study design limits our ability to establish causal mediation relationships. The mediating effect was developed and temporally validated based on the observational data. Future studies should trace changes in the psychological status of carriers at 376 later stages to further verify the causal mechanism. Fourth, other stigma-related stressors such as isolation factors or treatment factors were not studied. Future research could consider more stressors into account to understand the mechanism in depth.\n\n5. Conclusions:\nThis is one of the few studies to provide insights into the relationship between social stigma and depression among asymptomatic COVID-19 carriers in the post-epidemic era. Asymptomatic carriers were more likely to exhibit depression in the mid-stage of the pandemic. The current data showed the linkage between social stigma and depression through the mediating effects of entrapment and decadence. The routine assessment of stigma and screening for entrapment and decadence should be incorporated into early intervention strategies for depression among carriers to alleviate the psychological burden of the outbreak on society.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSince the advent of 2019 novel coronavirus (COVID-19), the coexistence between social stigma and depression symptoms (depression hereafter) in COVID-19 patients has been mentioned, but the mechanisms involved remains unclear. This study aimed to explore how the stigma affects depression during the mid-pandemic period.\n\nMethods:\nA cross-sectional survey using non-probability sampling was conducted among asymptomatic COVID-19 carriers in Shanghai, China (April 2022). An online questionnaire was used to obtain information on demographic characteristics and psychological traits. Logistic regression and path analysis were performed to analyze the depression risk factors and examine the mediation model, respectively.\n\nResults:\nA total of 1283 participants (59.6% men) were involved in this study, in which 44.7% of carriers reported having depression. Univariate analyses found that education level (OR 0.575; 95% CI 0.448-0.737) and doses of vaccine (OR 1.693; 95% CI 1.042-2.750), were significantly associated with depression among asymptomatic carriers. The association between social stigma and depression was fully mediated by their feelings of entrapment and decadence (indirect effect = 0.204, p < 0.001; direct effect = -0.059, p = 0.058). The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008).\n\nConclusions:\nMental health issues resulting from the COVID-19 pandemic are increasingly apparent in China and require urgent attention and responses. These findings provide new perspectives for the early prevention of depression in asymptomatic carriers."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nThe pandemic of 2019 novel coronavirus (COVID-19) has become a major global public health challenge [1,2]. It is self-evident that it not only caused the burden of physical illness but has also triggered a wide range of declines in mental well-being [3,4]. A meta-analysis suggested that the global prevalence of major depressive disorder increased the most among a series of negative psychology issues with the emergence of COVID-19. The prevalence of depression increased by 27.6% (25.1% to 30.3%), with a loss of disability-adjusted life-years of 4.94 million (33.6 to 68.7) [5]. Global levels of depression in the general population, health care providers and patients during the COVID-19 pandemic went up to 24.0% (21.0–27.1%) [6]. A meta-analysis revealed that psychological problems in infected patients were prominent, with a prevalence of 45% depression [7].\nAlthough risk factors for depression in vulnerable groups have been documented [8,9,10], current information regarding how the factors affect depression is lacking. Evidence suggested that COVID-19 survivors experienced higher stigma levels compared with healthy controls [11]. COVID-19-related stigma refers to a disagreeable or negative self-attitude that develops after infection or close contact with COVID-19 patients [12], including the negative deflection of social identity, with self-concept thus leading to a “spoiled identity” [12,13]. Additional evidence suggests a correlation between depression and social stigma. Liu et al. emphasized the importance of stigma in exacerbating the emotional impact of infected patients [9]. A prospective cohort study of COVID-19 survivors found that discrimination may partly account for the high incidence of depression after infection remission [14]. The identity-threat model of stigma raised by Brenda Major et al., which views stigma as a stressor, attempts to explain the effects of stigma on psychological well-being such as depression through coping strategies [15,16].\nThe economic consequences and psychosocial impacts of the COVID-19 pandemic are pervasive and profound [17,18,19]. Consequently, to avoid long-term mental health issues, there is an urgent need to clarify the mechanisms of the development of depression for COVID-19 patients [20,21]. The Social Rank Theory (SRT) proposes that depression evolves as a natural result of prolonged involuntary subordination [22]. Faced with an unfavorable situation (e.g., unachievable objectives, attempted change or challenge inhibited), individuals involuntarily yield adaptive responses through an automatic shutdown strategy [23]. If the situations persist and cannot be changed or escaped, the adaptive response will progressively change to maladjustment and eventually lead to depression [24,25]. Entrapment and decadence are precisely the key maladaptive defensive responses to this process. Entrapment is described as a common situation when there is a strong motivation to escape from an unsatisfying status, but that expectation cannot be met [22,26]. It could activate the Involuntary Defeat Strategy that, along with the feelings of decadence, forms a “depressogenic feedback loop” and ultimately contributes to the development of depression [27].\nTo our knowledge, little is known about the exact mechanisms on how stigma affects depression. Aiming to address this gap in understanding, we hypothesized and constructed a mediating model in which entrapment and decadence are mediators between social stigma and depression. The reasons are as follows: First, the identity-threat model of stigma posits that intrusive thinking and rumination about an event or issues occur when stigmatized individuals respond to stigma with involuntary engagement [15,28]. These two responses may have potential connection points with the assumed prerequisites of the SRT, that adverse situations are uncomfortable for individuals but cannot yet be accepted or escaped. That is, the identity-threat model of stigma implies that stigma affects entrapment and decadence. Second, entrapment and decadence are psychological signals prior to the onset of depression based on the SRT. Finally, a stigma-related stressor begins with external stimuli, while entrapment or decadence are inherently intrinsic psychological products, which satisfies the theoretical requirement of chronological order.\nThe primary objective of this study was to explore potential mediations between social stigma and depression via entrapment or decadence based on quantitative data from asymptomatic COVID-19 carriers in Shanghai, China. These findings may help us further understand the impact of the pandemic on COVID-19 patients’ mental health and inform subsequent intervention strategies and services for depression related to COVID-19.\n\n5. Conclusions:\nThis is one of the few studies to provide insights into the relationship between social stigma and depression among asymptomatic COVID-19 carriers in the post-epidemic era. Asymptomatic carriers were more likely to exhibit depression in the mid-stage of the pandemic. The current data showed the linkage between social stigma and depression through the mediating effects of entrapment and decadence. The routine assessment of stigma and screening for entrapment and decadence should be incorporated into early intervention strategies for depression among carriers to alleviate the psychological burden of the outbreak on society."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSince the advent of 2019 novel coronavirus (COVID-19), the coexistence between social stigma and depression symptoms (depression hereafter) in COVID-19 patients has been mentioned, but the mechanisms involved remains unclear. This study aimed to explore how the stigma affects depression during the mid-pandemic period.\n\nMethods:\nA cross-sectional survey using non-probability sampling was conducted among asymptomatic COVID-19 carriers in Shanghai, China (April 2022). An online questionnaire was used to obtain information on demographic characteristics and psychological traits. Logistic regression and path analysis were performed to analyze the depression risk factors and examine the mediation model, respectively.\n\nResults:\nA total of 1283 participants (59.6% men) were involved in this study, in which 44.7% of carriers reported having depression. Univariate analyses found that education level (OR 0.575; 95% CI 0.448-0.737) and doses of vaccine (OR 1.693; 95% CI 1.042-2.750), were significantly associated with depression among asymptomatic carriers. The association between social stigma and depression was fully mediated by their feelings of entrapment and decadence (indirect effect = 0.204, p < 0.001; direct effect = -0.059, p = 0.058). The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008).\n\nConclusions:\nMental health issues resulting from the COVID-19 pandemic are increasingly apparent in China and require urgent attention and responses. These findings provide new perspectives for the early prevention of depression in asymptomatic carriers."},"whole_article_text_length":{"kind":"number","value":14772,"string":"14,772"},"whole_article_abstract_length":{"kind":"number","value":296,"string":"296"},"other_sections_lengths":{"kind":"list like","value":[232,106,1992,30,424,308,220,517,238,250,88,338,57],"string":"[\n 232,\n 106,\n 1992,\n 30,\n 424,\n 308,\n 220,\n 517,\n 238,\n 250,\n 88,\n 338,\n 57\n]"},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["depression","stigma","entrapment","19","covid 19","covid","decadence","level","social","participants"],"string":"[\n \"depression\",\n \"stigma\",\n \"entrapment\",\n \"19\",\n \"covid 19\",\n \"covid\",\n \"decadence\",\n \"level\",\n \"social\",\n \"participants\"\n]"},"keybert_topics":{"kind":"list like","value":["incidence depression infection","psychosocial impacts covid","19 prevalence 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covid 19 | 19 | decadence | participants | entrapment decadence | social [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] depression | stigma | entrapment | covid | covid 19 | 19 | decadence | participants | entrapment decadence | social [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 | COVID-19 ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | Shanghai | China | April 2022 ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 1283 | 59.6% | 44.7% ||| 0.575 | 95% | CI | 0.448-0.737 | 1.693 | 95% | CI | 1.042 ||| 0.204, p < | 0.001 | 0.058 ||| 0.116 | 0.008 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | China ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 | COVID-19 ||| ||| COVID-19 | Shanghai | China | April 2022 ||| ||| ||| 1283 | 59.6% | 44.7% ||| 0.575 | 95% | CI | 0.448-0.737 | 1.693 | 95% | CI | 1.042 ||| 0.204, p < | 0.001 | 0.058 ||| 0.116 | 0.008 ||| COVID-19 | China ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 | COVID-19 ||| ||| COVID-19 | Shanghai | China | April 2022 ||| ||| ||| 1283 | 59.6% | 44.7% ||| 0.575 | 95% | CI | 0.448-0.737 | 1.693 | 95% | CI | 1.042 ||| 0.204, p < | 0.001 | 0.058 ||| 0.116 | 0.008 ||| COVID-19 | China ||| [SUMMARY]"}}},{"rowIdx":3353,"cells":{"title":{"kind":"string","value":"Trends in hospital discharges, management and in-hospital mortality from acute myocardial infarction in Switzerland between 1998 and 2008."},"pmid":{"kind":"string","value":"23530470"},"background_abstract":{"kind":"string","value":"Since the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Swiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Overall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","Databases, Factual","Female","Hospital Mortality","Humans","Male","Middle Aged","Myocardial Infarction","Patient Discharge","Switzerland","Treatment Outcome"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"Databases, Factual\",\n \"Female\",\n \"Hospital Mortality\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Myocardial Infarction\",\n \"Patient Discharge\",\n \"Switzerland\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3626665"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\nData from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\n Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.\nStatistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\nOverall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\n Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\nThe percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\n Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\nPercutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\n In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).\nAmong patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2)."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In Switzerland, a steep rise in hospital discharges and in revascularization\nprocedures for AMI occurred between 1998 and 2008."},"other_sections_titles":{"kind":"list like","value":["Background","Databases and available data","Statistical analysis","Characteristics of the patients","Intensive care unit","Revascularisation procedures","In-hospital mortality","Intensive care unit","Revascularisation procedures","In-hospital mortality","Study limitations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Databases and available data\",\n \"Statistical analysis\",\n \"Characteristics of the patients\",\n \"Intensive care unit\",\n \"Revascularisation procedures\",\n \"In-hospital mortality\",\n \"Intensive care unit\",\n \"Revascularisation procedures\",\n \"In-hospital mortality\",\n \"Study limitations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008.","Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.","Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.","Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.","The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).","Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).","Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).","The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.","Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.","An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.","This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].","The authors declare that they have no competing interests.","CI made part of the statistical analyses and wrote most of the article; PMV collected\ndata, made part of the statistical analysis and wrote part of the article; FP\nrevised the article for important intellectual content. PMV had full access to the\ndata and is the guarantor of the study. All authors read and approved the final\nmanuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\n"],"string":"[\n \"Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\\ntherapies and revascularization interventions. Indeed, management of AMI, namely\\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\\nthe findings from this study also apply to non participating centers, and whether\\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\\ndecided at the hospital or canton level, with little or no intervention from the\\nfederal bodies. The purpose of this study was to assess the trends in AMI management\\nand outcome for the whole country and for the main administrative regions, using\\ndata from the Swiss hospital discharge database for the period 1998–2008.\",\n \"Data from the hospital discharge database for years 1998 to 2008 was used. The\\ndatabase was provided by the Swiss federal office of statistics\\n(http://www.bfs.admin.ch) and covers 99% of public and private\\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\\ngender, age, length of stay, discharge status (main and secondary diagnoses,\\nvital status) and procedures. Four types of stays could be obtained:\\n“passing through”, patients who were admitted from another hospital\\nand transferred to another hospital; “inbound”, patients who were\\nadmitted from another hospital and managed on-site; “outbound”,\\npatients who were managed on-site and transferred to another hospital and\\n“in-house”, patients who were admitted and managed without being\\ntransferred to another hospital.\\nOverall length of stay was indicated in days and length of stay in an intensive\\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\\nfor seven-day mortality, patients who died after seven days were considered as\\nbeing alive.\\nMain and secondary diagnoses at discharge were coded using the International\\nClassification of Diseases, 10th revision (ICD10) of the World Health\\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\\nX = any number. The ICD10 coding does not distinguish between STEMI\\nand non-STEMI. Procedures were coded using the International Classification of\\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\\nprocedures were coded as binary (yes/no) variables.\\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\\nback to the initial event (and corresponding management) due to the\\nanonymization of the data.\\nPopulation statistics by age were also obtained from the Swiss federal office of\\nstatistics and used to calculate a discharge rate, expressed as the number of\\ndischarges per 100,000 adult (≥18 years) inhabitants.\",\n \"Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\\nCary, NC, USA). Results were expressed as average ± standard deviation and\\nas number of subjects and (percentage). Two sets of analyses were performed: the\\nmain analysis for trends in revascularization procedures included all hospital\\ndischarges, a supplementary analysis was carried out to limit the impact of\\ntransfers on significance levels and included only patients who were treated in\\none hospital (in-hospital).\\nTrends in the annual use of revascularisation procedures were assessed by\\nCochran-Armitage test for trend and further confirmed by multivariate logistic\\nregression adjusting for age, gender and type of transfer; when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Trends for\\nseven-day and overall mortality rates were assessed among inbound and in-house\\npatients only, as among “passing through” and outbound patients\\nmortality was zero. Multivariate models assessing trends for seven-day and\\noverall hospital mortality rates were adjusted for age, gender, ICU stay\\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Results were\\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\\nsignificance was assessed for p < 0.05.\",\n \"Overall, data from 102,729 hospital discharges from AMI were analyzed. The\\ncharacteristics of the patients discharged according to year are summarized in\\nTable 1. The percentage of women tended to\\ndecrease, while the mean age of patients at discharge increased by one year.\\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\\n221.3 in 2008 (Table 1).\\nCharacteristics of patients discharged from hospital with a main\\ndiagnosis of acute myocardial infarction, Switzerland, for period\\n1998–2008\\nResults are expressed as percentage or as\\nmean ± standard deviation. Trends assessed by\\nCochran-Armitage test or linear regression: p < 0.001\\nfor gender and age. § defined as the ratio between the number\\nof discharges and the adult (≥18 years) population,\\nexpressed per 100,000 inhabitants.\\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\\ndue to between-hospital transfers (inbound, outbound and passing through,\\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\\ntransfers in Mittelland (52%) and Ticino (68%).\\nNumber of patients discharged with a main diagnosis of acute\\nmyocardial infarction who were managed in a single hospital\\n(in-house), in Switzerland and by region, 1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were admitted from another hospital and managed\\non-site (inbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were managed on-site and transferred to another\\nhospital (outbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial admitted\\nfrom another hospital and transferred to another hospital (passing\\nthrough), in Switzerland and by region, 1998–2008.\",\n \"The percentage of hospitalizations with a stay in an Intensive Care Unit\\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\\ntrend < 0.001, Figure 5). This trend\\nwas further confirmed after multivariate adjustment for Switzerland. Differing\\ntrends were found according to the region considered: increase in Leman, Eastern\\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\\nin Mittelland (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results (Additional file\\n1: Figure S1).\\nTrends in intensive care unit (ICU) utilization for acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of hospital\\ndischarges with a diagnosis of acute myocardial infarction.\\nMultivariate analysis of the trends in revascularisation procedures\\nand in in-hospital mortality for patients discharged from hospital\\nwith a main diagnosis of acute myocardial infarction, Switzerland\\nand Swiss regions, for period 1998–2008\\nResults are expressed as multivariate adjusted Odds ratio and (95%\\nconfidence interval) for a one-year increase. ICU intensive\\ncare unit; CABG coronary artery bypass graft; DNC\\nmodel did not converge. Statistical analysis by multivariate\\nlogistic regression. For revascularization procedures, analysis was\\nconducted adjusting for age (continuous), gender and transfer type\\n(in-house, inbound, outbound and passing through) for each Swiss\\nregion; for Switzerland, a further adjustment was performed on\\nregion. For in-hospital mortality, analysis was conducted adjusting\\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\\nassistance (yes/no) and revascularization procedures (yes/no code:\\nbare stent, drug eluting stent and CABG) for each Swiss region; for\\nSwitzerland, a further adjustment was performed on region. §,\\npatients not transferred to another hospital (inboud or in-house);\\n§§, patients managed in a single hospital (in-house).\",\n \"Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\\n2008 (p for trend < 0.001, Figure 6).\\nThis trend was further confirmed after multivariate adjustment for Switzerland\\nand all regions considered (Table 2).\\nTrends in the use of drug-eluting and non-drug-eluting stents for\\nacute myocardial infarction in Switzerland, overall and by region,\\nfor the period 1998–2008. Results are expressed as\\npercentage of hospital discharges with a diagnosis of acute myocardial\\ninfarction.\\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\\nFigure 6). This trend was further confirmed after\\nmultivariate adjustment for Switzerland and all Swiss regions considered\\n(Table 2).\\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all regions concerned, although stronger for Central,\\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all Swiss regions, with the exception of Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\\nTrends in the use of coronary artery bypass graft (CABG) for acute\\nmyocardial infarction in Switzerland, overall and by region, for the\\nperiod 1998–2008. Results are expressed as percentage of\\nhospital discharges with a diagnosis of acute myocardial infarction.\\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\\nregions, with the exception of Ticino (Table 2).\\nFinally, no significant trends were found for thrombolysis, which increased from\\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\\ntrend = 0.09). This trend was further confirmed after multivariate\\nadjustment for Switzerland. Conversely, differing trends were found according to\\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\\ndecrease in Northwest and Ticino and no change in Mittelland and Central\\nSwitzerland (Table 2).\",\n \"Among patients not transferred to another hospital, seven-day in-hospital\\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\\ntrend < 0.01, Figure 8), while total\\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\\ntrend < 0.01, Figure 9). After\\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\\nchange in seven-day in-hospital mortality was found for Switzerland and all\\nSwiss regions (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results, with the exception\\nof an increase in mortality in Mittelland and Central Switzerland\\n(Table 2 and Additional file 4: Figure S4).\\nTrends in seven-day in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nTrends in overall in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nFor overall in-hospital death, no significant trend was found for Switzerland,\\nwhile differing trends were found for regions: decrease in Eastern; increase in\\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results, except that the decrease in\\nEastern Switzerland was no longer statistically significant (Table 2).\",\n \"The percentage of hospitalizations with ICU stay remained stable between 1998 and\\n2008 in Switzerland. This stabilization might be the consequence of improved\\nmanagement, requiring less ICU admissions before or after a revascularization\\nintervention. Another likely explanation is the increasing number of transfers,\\nthe patients being stabilized before being transferred to another hospital for\\nrevascularization. Still, considerable differences in the proportion of\\nhospitalizations with ICU and corresponding trends were found between regions,\\nsuggesting that other factors such as the number of ICU beds available or local\\noptions for AMI management might be at play. Unfortunately there is no available\\ninformation regarding the number of ICU beds in each region, so the precise\\nreasons for the regional differences in ICU admissions for AMI remain to be\\nfurther assessed.\",\n \"Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\\nregions were more responsive than others. For instance, in the Leman area DES\\nuse equaled but never exceeded bare stent use, while in South Switzerland\\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\\nsuch regional differences are more likely due to local preferences or to local\\nagreements regarding DES cost than to decisions based on scientific\\nevidence.\\nThe rate of CABG remained stable at 3% throughout the study period, a finding\\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\\nThis trend might partly be explained by the creation in 1999 of new medical\\ncenters with the capacity to carry out these interventions. Conversely, in\\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\\nlikely the result of the lack of required infrastructure. Overall, our results\\nshow that CABG rates evolved differently between Swiss regions, the most likely\\nexplanations being the existence of infrastructures and local preferences for\\ncertain types of revascularization procedures.\",\n \"An overall decrease in seven-day in-hospital mortality was found for Switzerland,\\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\\nsuggesting that most of the decrease in mortality is due to revascularization\\ninterventions and the improved quality of treatment according to the newest\\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\\ndays led to similar findings, although some regions presented a slight increase\\nin mortality (Additional file 5: Table S1). The rising\\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\\nmultivariate model as they are not reported systematically in the database.\\nSimilarly, it was not possible to assess whether the patients presented with a\\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\\nrevascularization interventions performed among patients discharged with a main\\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\\nassociated with in-hospital mortality from AMI in order to optimize patient\\nmanagement.\",\n \"This study has some limitations. Medicines are not included in the hospital\\ndischarge database, thus precluding the assessment of their trends. This absence\\nmight also explain the very low rates for thrombolysis, which are likely due to\\nthe absence of coding rather than a true absence of use. Furthermore, we lack\\ndata to assess whether the changes in thrombolytic therapy are related to\\nchanges in reporting levels or are actually real changes. The ICD10 coding does\\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\\nincrease in discharges from AMI during the study period might be related to the\\nchange in the definition of NSTEMI populations due to the generalization of the\\nuse of troponin measurements. Indeed, introduction of troponin measurements led\\nto the reclassification of many unstable angina patients into NSTEMI patients,\\nwith a 33% increase of the population of NSTEMIs from before to after the\\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\\nled to similar findings, with the exception that the increase in CABG was no\\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\\nbetween-hospital transfers and the anonymization of the data (which precluded\\nthe follow-up of the patients) complicated the interpretation of the trends.\\nStill, restricting the analysis to patients fully managed in a single hospital\\nled to similar results. It was not possible to assess “true” seven-\\nand 30-day mortality rates as no follow-up data was available for the patients\\ndischarged. The AMIS registry, which collects vital status data for AMI patients\\nhospitalized in the participating institutions, will be able to provide such\\ninformation [35]. Finally, there is no published information on the validity of\\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\\na value in agreement with the literature [36,37].\",\n \"The authors declare that they have no competing interests.\",\n \"CI made part of the statistical analyses and wrote most of the article; PMV collected\\ndata, made part of the statistical analysis and wrote part of the article; FP\\nrevised the article for important intellectual content. PMV had full access to the\\ndata and is the guarantor of the study. All authors read and approved the final\\nmanuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Databases and available data","Statistical analysis","Results","Characteristics of the patients","Intensive care unit","Revascularisation procedures","In-hospital mortality","Discussion","Intensive care unit","Revascularisation procedures","In-hospital mortality","Study limitations","Conclusion","Competing interests","Authors’ contributions","Pre-publication history","Supplementary Material"],"string":"[\n \"Background\",\n \"Methods\",\n \"Databases and available data\",\n \"Statistical analysis\",\n \"Results\",\n \"Characteristics of the patients\",\n \"Intensive care unit\",\n \"Revascularisation procedures\",\n \"In-hospital mortality\",\n \"Discussion\",\n \"Intensive care unit\",\n \"Revascularisation procedures\",\n \"In-hospital mortality\",\n \"Study limitations\",\n \"Conclusion\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008."," Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\nData from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\n Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.\nStatistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.","Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.","Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05."," Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\nOverall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\n Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\nThe percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\n Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\nPercutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\n In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).\nAmong patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).","Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.","The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).","Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).","Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).","Since the end of the MONICA study [12,13], few studies have assessed the management and outcome of AMI in\nSwitzerland. Our results show an increase in most revascularization procedures,\nnamely PCI, while CABG tended to stabilize or even to decrease in the most recent\nyears. Moreover, these trends strongly differ according to region. Overall, the\nchanges in revascularization procedure in Switzerland follow the ones observed in\nother countries, [6,7,14,15]. These findings are also in agreement with a previous study conducted on\n19,500 patients of the AMIS Plus registry [8], although the increase in PCI procedures observed [16] was considerably stronger than in ours. This difference is most likely\ndue to the overrepresentation of PCI procedures among the AMIS Plus registry\nhospitals as the Registry includes only selected voluntary teams.\nBetween 1998 and 2008, the number of hospital discharges for AMI increased over\ntwo-fold in Switzerland. This finding is in contradiction with some previous studies [17,18], but it should be noted that these studies showed declining rates of AMI\nrather than number of discharges. Part of this increase might be due to population\naging [2], although the observed increase in population age (from 66.5 years\nin 1998 to 67.6 years in 2008) would not lead to a doubling of the number of\nhospitalizations for AMI. Further, all analyses were adjusted for age. Another\npossibility would be a change in disease coding, but again this is unlikely as\nhospital discharge data has been shown to be reliable regarding the diagnosis of AMI [19] and as this study focused on a specific code. It is also possible that\nmore people are hospitalized for a second or even a third AMI (partly related to the\ndecrease in case fatality rate of the first AMI, leading to an increased incidence\nof 2nd or 3rd AMI). Another possibility is the change in\ncriteria defining AMI [20], which has been shown to increase the incidence of AMI [21]. The most likely explanation for the considerable increase in the number\nof hospital discharges for AMI in Switzerland is the steep rise in hospital\ntransfers, which accounted for almost half of hospital discharges for AMI in 2008.\nThe increase in hospital transfers might be due to the limited number of hospitals\ncapable of performing revascularization procedures, prompting smaller hospitals to\ntransfer their patients as recommended by European guidelines [22,23]. Still, considerable differences in the percentages of hospital\ndischarges due to transfers were noted: in 2008 the values ranged from 40% in Leman\nto 68% in Ticino. These differences might be due to local policies. As AMI\nmanagement is expensive [24], the increase in hospital discharges for AMI would add a considerable\npressure in Swiss health expenditures, which already increased from 9.6% in 1995 to\n11.4% of the Swiss GNP in 2009 [25].\n Intensive care unit The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\nThe percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\n Revascularisation procedures Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\nStent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\n In-hospital mortality An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\nAn overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\n Study limitations This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].\nThis study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].","The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.","Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.","An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.","This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].","In Switzerland, a steep rise in hospital discharges and in revascularization\nprocedures for AMI occurred between 1998 and 2008.","The authors declare that they have no competing interests.","CI made part of the statistical analyses and wrote most of the article; PMV collected\ndata, made part of the statistical analysis and wrote part of the article; FP\nrevised the article for important intellectual content. PMV had full access to the\ndata and is the guarantor of the study. All authors read and approved the final\nmanuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\n"," Trends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008, patients managed in a single hospital (in-house).\nResults are expressed as percentage of patients discharged with a\ndiagnosis of acute myocardial infarction.\nClick here for file\nTrends in the use of drug-eluting and non-drug-eluting stents for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008, patients managed in a single hospital\n(in-house). Results are expressed as percentage of patients discharged\nwith a diagnosis of acute myocardial infarction.\nClick here for file\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008, patients managed in a single hospital\n(in-house). Results are expressed as percentage of patients discharged\nwith a diagnosis of acute myocardial infarction.\nClick here for file\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008, patients managed in a single hospital (in-house).\nResults are expressed as percentage of patients discharged with a\ndiagnosis of acute myocardial infarction.\nClick here for file\nTrends in seven-day and overall in-hospital mortality, Switzerland and\nSwiss regions, for period 1998-2008\nClick here for file"],"string":"[\n \"Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\\ntherapies and revascularization interventions. Indeed, management of AMI, namely\\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\\nthe findings from this study also apply to non participating centers, and whether\\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\\ndecided at the hospital or canton level, with little or no intervention from the\\nfederal bodies. The purpose of this study was to assess the trends in AMI management\\nand outcome for the whole country and for the main administrative regions, using\\ndata from the Swiss hospital discharge database for the period 1998–2008.\",\n \" Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The\\ndatabase was provided by the Swiss federal office of statistics\\n(http://www.bfs.admin.ch) and covers 99% of public and private\\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\\ngender, age, length of stay, discharge status (main and secondary diagnoses,\\nvital status) and procedures. Four types of stays could be obtained:\\n“passing through”, patients who were admitted from another hospital\\nand transferred to another hospital; “inbound”, patients who were\\nadmitted from another hospital and managed on-site; “outbound”,\\npatients who were managed on-site and transferred to another hospital and\\n“in-house”, patients who were admitted and managed without being\\ntransferred to another hospital.\\nOverall length of stay was indicated in days and length of stay in an intensive\\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\\nfor seven-day mortality, patients who died after seven days were considered as\\nbeing alive.\\nMain and secondary diagnoses at discharge were coded using the International\\nClassification of Diseases, 10th revision (ICD10) of the World Health\\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\\nX = any number. The ICD10 coding does not distinguish between STEMI\\nand non-STEMI. Procedures were coded using the International Classification of\\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\\nprocedures were coded as binary (yes/no) variables.\\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\\nback to the initial event (and corresponding management) due to the\\nanonymization of the data.\\nPopulation statistics by age were also obtained from the Swiss federal office of\\nstatistics and used to calculate a discharge rate, expressed as the number of\\ndischarges per 100,000 adult (≥18 years) inhabitants.\\nData from the hospital discharge database for years 1998 to 2008 was used. The\\ndatabase was provided by the Swiss federal office of statistics\\n(http://www.bfs.admin.ch) and covers 99% of public and private\\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\\ngender, age, length of stay, discharge status (main and secondary diagnoses,\\nvital status) and procedures. Four types of stays could be obtained:\\n“passing through”, patients who were admitted from another hospital\\nand transferred to another hospital; “inbound”, patients who were\\nadmitted from another hospital and managed on-site; “outbound”,\\npatients who were managed on-site and transferred to another hospital and\\n“in-house”, patients who were admitted and managed without being\\ntransferred to another hospital.\\nOverall length of stay was indicated in days and length of stay in an intensive\\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\\nfor seven-day mortality, patients who died after seven days were considered as\\nbeing alive.\\nMain and secondary diagnoses at discharge were coded using the International\\nClassification of Diseases, 10th revision (ICD10) of the World Health\\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\\nX = any number. The ICD10 coding does not distinguish between STEMI\\nand non-STEMI. Procedures were coded using the International Classification of\\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\\nprocedures were coded as binary (yes/no) variables.\\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\\nback to the initial event (and corresponding management) due to the\\nanonymization of the data.\\nPopulation statistics by age were also obtained from the Swiss federal office of\\nstatistics and used to calculate a discharge rate, expressed as the number of\\ndischarges per 100,000 adult (≥18 years) inhabitants.\\n Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\\nCary, NC, USA). Results were expressed as average ± standard deviation and\\nas number of subjects and (percentage). Two sets of analyses were performed: the\\nmain analysis for trends in revascularization procedures included all hospital\\ndischarges, a supplementary analysis was carried out to limit the impact of\\ntransfers on significance levels and included only patients who were treated in\\none hospital (in-hospital).\\nTrends in the annual use of revascularisation procedures were assessed by\\nCochran-Armitage test for trend and further confirmed by multivariate logistic\\nregression adjusting for age, gender and type of transfer; when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Trends for\\nseven-day and overall mortality rates were assessed among inbound and in-house\\npatients only, as among “passing through” and outbound patients\\nmortality was zero. Multivariate models assessing trends for seven-day and\\noverall hospital mortality rates were adjusted for age, gender, ICU stay\\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Results were\\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\\nsignificance was assessed for p < 0.05.\\nStatistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\\nCary, NC, USA). Results were expressed as average ± standard deviation and\\nas number of subjects and (percentage). Two sets of analyses were performed: the\\nmain analysis for trends in revascularization procedures included all hospital\\ndischarges, a supplementary analysis was carried out to limit the impact of\\ntransfers on significance levels and included only patients who were treated in\\none hospital (in-hospital).\\nTrends in the annual use of revascularisation procedures were assessed by\\nCochran-Armitage test for trend and further confirmed by multivariate logistic\\nregression adjusting for age, gender and type of transfer; when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Trends for\\nseven-day and overall mortality rates were assessed among inbound and in-house\\npatients only, as among “passing through” and outbound patients\\nmortality was zero. Multivariate models assessing trends for seven-day and\\noverall hospital mortality rates were adjusted for age, gender, ICU stay\\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Results were\\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\\nsignificance was assessed for p < 0.05.\",\n \"Data from the hospital discharge database for years 1998 to 2008 was used. The\\ndatabase was provided by the Swiss federal office of statistics\\n(http://www.bfs.admin.ch) and covers 99% of public and private\\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\\ngender, age, length of stay, discharge status (main and secondary diagnoses,\\nvital status) and procedures. Four types of stays could be obtained:\\n“passing through”, patients who were admitted from another hospital\\nand transferred to another hospital; “inbound”, patients who were\\nadmitted from another hospital and managed on-site; “outbound”,\\npatients who were managed on-site and transferred to another hospital and\\n“in-house”, patients who were admitted and managed without being\\ntransferred to another hospital.\\nOverall length of stay was indicated in days and length of stay in an intensive\\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\\nfor seven-day mortality, patients who died after seven days were considered as\\nbeing alive.\\nMain and secondary diagnoses at discharge were coded using the International\\nClassification of Diseases, 10th revision (ICD10) of the World Health\\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\\nX = any number. The ICD10 coding does not distinguish between STEMI\\nand non-STEMI. Procedures were coded using the International Classification of\\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\\nprocedures were coded as binary (yes/no) variables.\\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\\nback to the initial event (and corresponding management) due to the\\nanonymization of the data.\\nPopulation statistics by age were also obtained from the Swiss federal office of\\nstatistics and used to calculate a discharge rate, expressed as the number of\\ndischarges per 100,000 adult (≥18 years) inhabitants.\",\n \"Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\\nCary, NC, USA). Results were expressed as average ± standard deviation and\\nas number of subjects and (percentage). Two sets of analyses were performed: the\\nmain analysis for trends in revascularization procedures included all hospital\\ndischarges, a supplementary analysis was carried out to limit the impact of\\ntransfers on significance levels and included only patients who were treated in\\none hospital (in-hospital).\\nTrends in the annual use of revascularisation procedures were assessed by\\nCochran-Armitage test for trend and further confirmed by multivariate logistic\\nregression adjusting for age, gender and type of transfer; when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Trends for\\nseven-day and overall mortality rates were assessed among inbound and in-house\\npatients only, as among “passing through” and outbound patients\\nmortality was zero. Multivariate models assessing trends for seven-day and\\noverall hospital mortality rates were adjusted for age, gender, ICU stay\\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\\nfor Switzerland, a further adjustment was performed on region. Results were\\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\\nsignificance was assessed for p < 0.05.\",\n \" Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The\\ncharacteristics of the patients discharged according to year are summarized in\\nTable 1. The percentage of women tended to\\ndecrease, while the mean age of patients at discharge increased by one year.\\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\\n221.3 in 2008 (Table 1).\\nCharacteristics of patients discharged from hospital with a main\\ndiagnosis of acute myocardial infarction, Switzerland, for period\\n1998–2008\\nResults are expressed as percentage or as\\nmean ± standard deviation. Trends assessed by\\nCochran-Armitage test or linear regression: p < 0.001\\nfor gender and age. § defined as the ratio between the number\\nof discharges and the adult (≥18 years) population,\\nexpressed per 100,000 inhabitants.\\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\\ndue to between-hospital transfers (inbound, outbound and passing through,\\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\\ntransfers in Mittelland (52%) and Ticino (68%).\\nNumber of patients discharged with a main diagnosis of acute\\nmyocardial infarction who were managed in a single hospital\\n(in-house), in Switzerland and by region, 1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were admitted from another hospital and managed\\non-site (inbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were managed on-site and transferred to another\\nhospital (outbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial admitted\\nfrom another hospital and transferred to another hospital (passing\\nthrough), in Switzerland and by region, 1998–2008.\\nOverall, data from 102,729 hospital discharges from AMI were analyzed. The\\ncharacteristics of the patients discharged according to year are summarized in\\nTable 1. The percentage of women tended to\\ndecrease, while the mean age of patients at discharge increased by one year.\\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\\n221.3 in 2008 (Table 1).\\nCharacteristics of patients discharged from hospital with a main\\ndiagnosis of acute myocardial infarction, Switzerland, for period\\n1998–2008\\nResults are expressed as percentage or as\\nmean ± standard deviation. Trends assessed by\\nCochran-Armitage test or linear regression: p < 0.001\\nfor gender and age. § defined as the ratio between the number\\nof discharges and the adult (≥18 years) population,\\nexpressed per 100,000 inhabitants.\\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\\ndue to between-hospital transfers (inbound, outbound and passing through,\\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\\ntransfers in Mittelland (52%) and Ticino (68%).\\nNumber of patients discharged with a main diagnosis of acute\\nmyocardial infarction who were managed in a single hospital\\n(in-house), in Switzerland and by region, 1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were admitted from another hospital and managed\\non-site (inbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were managed on-site and transferred to another\\nhospital (outbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial admitted\\nfrom another hospital and transferred to another hospital (passing\\nthrough), in Switzerland and by region, 1998–2008.\\n Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit\\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\\ntrend < 0.001, Figure 5). This trend\\nwas further confirmed after multivariate adjustment for Switzerland. Differing\\ntrends were found according to the region considered: increase in Leman, Eastern\\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\\nin Mittelland (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results (Additional file\\n1: Figure S1).\\nTrends in intensive care unit (ICU) utilization for acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of hospital\\ndischarges with a diagnosis of acute myocardial infarction.\\nMultivariate analysis of the trends in revascularisation procedures\\nand in in-hospital mortality for patients discharged from hospital\\nwith a main diagnosis of acute myocardial infarction, Switzerland\\nand Swiss regions, for period 1998–2008\\nResults are expressed as multivariate adjusted Odds ratio and (95%\\nconfidence interval) for a one-year increase. ICU intensive\\ncare unit; CABG coronary artery bypass graft; DNC\\nmodel did not converge. Statistical analysis by multivariate\\nlogistic regression. For revascularization procedures, analysis was\\nconducted adjusting for age (continuous), gender and transfer type\\n(in-house, inbound, outbound and passing through) for each Swiss\\nregion; for Switzerland, a further adjustment was performed on\\nregion. For in-hospital mortality, analysis was conducted adjusting\\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\\nassistance (yes/no) and revascularization procedures (yes/no code:\\nbare stent, drug eluting stent and CABG) for each Swiss region; for\\nSwitzerland, a further adjustment was performed on region. §,\\npatients not transferred to another hospital (inboud or in-house);\\n§§, patients managed in a single hospital (in-house).\\nThe percentage of hospitalizations with a stay in an Intensive Care Unit\\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\\ntrend < 0.001, Figure 5). This trend\\nwas further confirmed after multivariate adjustment for Switzerland. Differing\\ntrends were found according to the region considered: increase in Leman, Eastern\\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\\nin Mittelland (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results (Additional file\\n1: Figure S1).\\nTrends in intensive care unit (ICU) utilization for acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of hospital\\ndischarges with a diagnosis of acute myocardial infarction.\\nMultivariate analysis of the trends in revascularisation procedures\\nand in in-hospital mortality for patients discharged from hospital\\nwith a main diagnosis of acute myocardial infarction, Switzerland\\nand Swiss regions, for period 1998–2008\\nResults are expressed as multivariate adjusted Odds ratio and (95%\\nconfidence interval) for a one-year increase. ICU intensive\\ncare unit; CABG coronary artery bypass graft; DNC\\nmodel did not converge. Statistical analysis by multivariate\\nlogistic regression. For revascularization procedures, analysis was\\nconducted adjusting for age (continuous), gender and transfer type\\n(in-house, inbound, outbound and passing through) for each Swiss\\nregion; for Switzerland, a further adjustment was performed on\\nregion. For in-hospital mortality, analysis was conducted adjusting\\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\\nassistance (yes/no) and revascularization procedures (yes/no code:\\nbare stent, drug eluting stent and CABG) for each Swiss region; for\\nSwitzerland, a further adjustment was performed on region. §,\\npatients not transferred to another hospital (inboud or in-house);\\n§§, patients managed in a single hospital (in-house).\\n Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\\n2008 (p for trend < 0.001, Figure 6).\\nThis trend was further confirmed after multivariate adjustment for Switzerland\\nand all regions considered (Table 2).\\nTrends in the use of drug-eluting and non-drug-eluting stents for\\nacute myocardial infarction in Switzerland, overall and by region,\\nfor the period 1998–2008. Results are expressed as\\npercentage of hospital discharges with a diagnosis of acute myocardial\\ninfarction.\\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\\nFigure 6). This trend was further confirmed after\\nmultivariate adjustment for Switzerland and all Swiss regions considered\\n(Table 2).\\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all regions concerned, although stronger for Central,\\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all Swiss regions, with the exception of Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\\nTrends in the use of coronary artery bypass graft (CABG) for acute\\nmyocardial infarction in Switzerland, overall and by region, for the\\nperiod 1998–2008. Results are expressed as percentage of\\nhospital discharges with a diagnosis of acute myocardial infarction.\\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\\nregions, with the exception of Ticino (Table 2).\\nFinally, no significant trends were found for thrombolysis, which increased from\\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\\ntrend = 0.09). This trend was further confirmed after multivariate\\nadjustment for Switzerland. Conversely, differing trends were found according to\\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\\ndecrease in Northwest and Ticino and no change in Mittelland and Central\\nSwitzerland (Table 2).\\nPercutaneous revascularizations increased sevenfold, from 6.0% of all patients\\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\\n2008 (p for trend < 0.001, Figure 6).\\nThis trend was further confirmed after multivariate adjustment for Switzerland\\nand all regions considered (Table 2).\\nTrends in the use of drug-eluting and non-drug-eluting stents for\\nacute myocardial infarction in Switzerland, overall and by region,\\nfor the period 1998–2008. Results are expressed as\\npercentage of hospital discharges with a diagnosis of acute myocardial\\ninfarction.\\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\\nFigure 6). This trend was further confirmed after\\nmultivariate adjustment for Switzerland and all Swiss regions considered\\n(Table 2).\\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all regions concerned, although stronger for Central,\\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all Swiss regions, with the exception of Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\\nTrends in the use of coronary artery bypass graft (CABG) for acute\\nmyocardial infarction in Switzerland, overall and by region, for the\\nperiod 1998–2008. Results are expressed as percentage of\\nhospital discharges with a diagnosis of acute myocardial infarction.\\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\\nregions, with the exception of Ticino (Table 2).\\nFinally, no significant trends were found for thrombolysis, which increased from\\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\\ntrend = 0.09). This trend was further confirmed after multivariate\\nadjustment for Switzerland. Conversely, differing trends were found according to\\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\\ndecrease in Northwest and Ticino and no change in Mittelland and Central\\nSwitzerland (Table 2).\\n In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital\\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\\ntrend < 0.01, Figure 8), while total\\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\\ntrend < 0.01, Figure 9). After\\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\\nchange in seven-day in-hospital mortality was found for Switzerland and all\\nSwiss regions (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results, with the exception\\nof an increase in mortality in Mittelland and Central Switzerland\\n(Table 2 and Additional file 4: Figure S4).\\nTrends in seven-day in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nTrends in overall in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nFor overall in-hospital death, no significant trend was found for Switzerland,\\nwhile differing trends were found for regions: decrease in Eastern; increase in\\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results, except that the decrease in\\nEastern Switzerland was no longer statistically significant (Table 2).\\nAmong patients not transferred to another hospital, seven-day in-hospital\\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\\ntrend < 0.01, Figure 8), while total\\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\\ntrend < 0.01, Figure 9). After\\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\\nchange in seven-day in-hospital mortality was found for Switzerland and all\\nSwiss regions (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results, with the exception\\nof an increase in mortality in Mittelland and Central Switzerland\\n(Table 2 and Additional file 4: Figure S4).\\nTrends in seven-day in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nTrends in overall in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nFor overall in-hospital death, no significant trend was found for Switzerland,\\nwhile differing trends were found for regions: decrease in Eastern; increase in\\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results, except that the decrease in\\nEastern Switzerland was no longer statistically significant (Table 2).\",\n \"Overall, data from 102,729 hospital discharges from AMI were analyzed. The\\ncharacteristics of the patients discharged according to year are summarized in\\nTable 1. The percentage of women tended to\\ndecrease, while the mean age of patients at discharge increased by one year.\\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\\n221.3 in 2008 (Table 1).\\nCharacteristics of patients discharged from hospital with a main\\ndiagnosis of acute myocardial infarction, Switzerland, for period\\n1998–2008\\nResults are expressed as percentage or as\\nmean ± standard deviation. Trends assessed by\\nCochran-Armitage test or linear regression: p < 0.001\\nfor gender and age. § defined as the ratio between the number\\nof discharges and the adult (≥18 years) population,\\nexpressed per 100,000 inhabitants.\\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\\ndue to between-hospital transfers (inbound, outbound and passing through,\\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\\ntransfers in Mittelland (52%) and Ticino (68%).\\nNumber of patients discharged with a main diagnosis of acute\\nmyocardial infarction who were managed in a single hospital\\n(in-house), in Switzerland and by region, 1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were admitted from another hospital and managed\\non-site (inbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial\\ninfarction who were managed on-site and transferred to another\\nhospital (outbound), in Switzerland and by region,\\n1998–2008.\\nNumber of patients with a main diagnosis of acute myocardial admitted\\nfrom another hospital and transferred to another hospital (passing\\nthrough), in Switzerland and by region, 1998–2008.\",\n \"The percentage of hospitalizations with a stay in an Intensive Care Unit\\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\\ntrend < 0.001, Figure 5). This trend\\nwas further confirmed after multivariate adjustment for Switzerland. Differing\\ntrends were found according to the region considered: increase in Leman, Eastern\\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\\nin Mittelland (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results (Additional file\\n1: Figure S1).\\nTrends in intensive care unit (ICU) utilization for acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of hospital\\ndischarges with a diagnosis of acute myocardial infarction.\\nMultivariate analysis of the trends in revascularisation procedures\\nand in in-hospital mortality for patients discharged from hospital\\nwith a main diagnosis of acute myocardial infarction, Switzerland\\nand Swiss regions, for period 1998–2008\\nResults are expressed as multivariate adjusted Odds ratio and (95%\\nconfidence interval) for a one-year increase. ICU intensive\\ncare unit; CABG coronary artery bypass graft; DNC\\nmodel did not converge. Statistical analysis by multivariate\\nlogistic regression. For revascularization procedures, analysis was\\nconducted adjusting for age (continuous), gender and transfer type\\n(in-house, inbound, outbound and passing through) for each Swiss\\nregion; for Switzerland, a further adjustment was performed on\\nregion. For in-hospital mortality, analysis was conducted adjusting\\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\\nassistance (yes/no) and revascularization procedures (yes/no code:\\nbare stent, drug eluting stent and CABG) for each Swiss region; for\\nSwitzerland, a further adjustment was performed on region. §,\\npatients not transferred to another hospital (inboud or in-house);\\n§§, patients managed in a single hospital (in-house).\",\n \"Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\\n2008 (p for trend < 0.001, Figure 6).\\nThis trend was further confirmed after multivariate adjustment for Switzerland\\nand all regions considered (Table 2).\\nTrends in the use of drug-eluting and non-drug-eluting stents for\\nacute myocardial infarction in Switzerland, overall and by region,\\nfor the period 1998–2008. Results are expressed as\\npercentage of hospital discharges with a diagnosis of acute myocardial\\ninfarction.\\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\\nFigure 6). This trend was further confirmed after\\nmultivariate adjustment for Switzerland and all Swiss regions considered\\n(Table 2).\\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all regions concerned, although stronger for Central,\\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\\nfor Switzerland and all Swiss regions, with the exception of Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\\nTrends in the use of coronary artery bypass graft (CABG) for acute\\nmyocardial infarction in Switzerland, overall and by region, for the\\nperiod 1998–2008. Results are expressed as percentage of\\nhospital discharges with a diagnosis of acute myocardial infarction.\\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\\nregions, with the exception of Ticino (Table 2).\\nFinally, no significant trends were found for thrombolysis, which increased from\\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\\ntrend = 0.09). This trend was further confirmed after multivariate\\nadjustment for Switzerland. Conversely, differing trends were found according to\\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\\ndecrease in Northwest and Ticino and no change in Mittelland and Central\\nSwitzerland (Table 2).\",\n \"Among patients not transferred to another hospital, seven-day in-hospital\\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\\ntrend < 0.01, Figure 8), while total\\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\\ntrend < 0.01, Figure 9). After\\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\\nchange in seven-day in-hospital mortality was found for Switzerland and all\\nSwiss regions (Table 2). Restricting the analysis to\\npatients managed in a single hospital led to similar results, with the exception\\nof an increase in mortality in Mittelland and Central Switzerland\\n(Table 2 and Additional file 4: Figure S4).\\nTrends in seven-day in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nTrends in overall in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008. Results are expressed as percentage of patients\\ninbound or in-house discharged with a diagnosis of acute myocardial\\ninfarction.\\nFor overall in-hospital death, no significant trend was found for Switzerland,\\nwhile differing trends were found for regions: decrease in Eastern; increase in\\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\\n(Table 2). Restricting the analysis to patients\\nmanaged in a single hospital led to similar results, except that the decrease in\\nEastern Switzerland was no longer statistically significant (Table 2).\",\n \"Since the end of the MONICA study [12,13], few studies have assessed the management and outcome of AMI in\\nSwitzerland. Our results show an increase in most revascularization procedures,\\nnamely PCI, while CABG tended to stabilize or even to decrease in the most recent\\nyears. Moreover, these trends strongly differ according to region. Overall, the\\nchanges in revascularization procedure in Switzerland follow the ones observed in\\nother countries, [6,7,14,15]. These findings are also in agreement with a previous study conducted on\\n19,500 patients of the AMIS Plus registry [8], although the increase in PCI procedures observed [16] was considerably stronger than in ours. This difference is most likely\\ndue to the overrepresentation of PCI procedures among the AMIS Plus registry\\nhospitals as the Registry includes only selected voluntary teams.\\nBetween 1998 and 2008, the number of hospital discharges for AMI increased over\\ntwo-fold in Switzerland. This finding is in contradiction with some previous studies [17,18], but it should be noted that these studies showed declining rates of AMI\\nrather than number of discharges. Part of this increase might be due to population\\naging [2], although the observed increase in population age (from 66.5 years\\nin 1998 to 67.6 years in 2008) would not lead to a doubling of the number of\\nhospitalizations for AMI. Further, all analyses were adjusted for age. Another\\npossibility would be a change in disease coding, but again this is unlikely as\\nhospital discharge data has been shown to be reliable regarding the diagnosis of AMI [19] and as this study focused on a specific code. It is also possible that\\nmore people are hospitalized for a second or even a third AMI (partly related to the\\ndecrease in case fatality rate of the first AMI, leading to an increased incidence\\nof 2nd or 3rd AMI). Another possibility is the change in\\ncriteria defining AMI [20], which has been shown to increase the incidence of AMI [21]. The most likely explanation for the considerable increase in the number\\nof hospital discharges for AMI in Switzerland is the steep rise in hospital\\ntransfers, which accounted for almost half of hospital discharges for AMI in 2008.\\nThe increase in hospital transfers might be due to the limited number of hospitals\\ncapable of performing revascularization procedures, prompting smaller hospitals to\\ntransfer their patients as recommended by European guidelines [22,23]. Still, considerable differences in the percentages of hospital\\ndischarges due to transfers were noted: in 2008 the values ranged from 40% in Leman\\nto 68% in Ticino. These differences might be due to local policies. As AMI\\nmanagement is expensive [24], the increase in hospital discharges for AMI would add a considerable\\npressure in Swiss health expenditures, which already increased from 9.6% in 1995 to\\n11.4% of the Swiss GNP in 2009 [25].\\n Intensive care unit The percentage of hospitalizations with ICU stay remained stable between 1998 and\\n2008 in Switzerland. This stabilization might be the consequence of improved\\nmanagement, requiring less ICU admissions before or after a revascularization\\nintervention. Another likely explanation is the increasing number of transfers,\\nthe patients being stabilized before being transferred to another hospital for\\nrevascularization. Still, considerable differences in the proportion of\\nhospitalizations with ICU and corresponding trends were found between regions,\\nsuggesting that other factors such as the number of ICU beds available or local\\noptions for AMI management might be at play. Unfortunately there is no available\\ninformation regarding the number of ICU beds in each region, so the precise\\nreasons for the regional differences in ICU admissions for AMI remain to be\\nfurther assessed.\\nThe percentage of hospitalizations with ICU stay remained stable between 1998 and\\n2008 in Switzerland. This stabilization might be the consequence of improved\\nmanagement, requiring less ICU admissions before or after a revascularization\\nintervention. Another likely explanation is the increasing number of transfers,\\nthe patients being stabilized before being transferred to another hospital for\\nrevascularization. Still, considerable differences in the proportion of\\nhospitalizations with ICU and corresponding trends were found between regions,\\nsuggesting that other factors such as the number of ICU beds available or local\\noptions for AMI management might be at play. Unfortunately there is no available\\ninformation regarding the number of ICU beds in each region, so the precise\\nreasons for the regional differences in ICU admissions for AMI remain to be\\nfurther assessed.\\n Revascularisation procedures Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\\nregions were more responsive than others. For instance, in the Leman area DES\\nuse equaled but never exceeded bare stent use, while in South Switzerland\\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\\nsuch regional differences are more likely due to local preferences or to local\\nagreements regarding DES cost than to decisions based on scientific\\nevidence.\\nThe rate of CABG remained stable at 3% throughout the study period, a finding\\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\\nThis trend might partly be explained by the creation in 1999 of new medical\\ncenters with the capacity to carry out these interventions. Conversely, in\\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\\nlikely the result of the lack of required infrastructure. Overall, our results\\nshow that CABG rates evolved differently between Swiss regions, the most likely\\nexplanations being the existence of infrastructures and local preferences for\\ncertain types of revascularization procedures.\\nStent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\\nregions were more responsive than others. For instance, in the Leman area DES\\nuse equaled but never exceeded bare stent use, while in South Switzerland\\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\\nsuch regional differences are more likely due to local preferences or to local\\nagreements regarding DES cost than to decisions based on scientific\\nevidence.\\nThe rate of CABG remained stable at 3% throughout the study period, a finding\\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\\nThis trend might partly be explained by the creation in 1999 of new medical\\ncenters with the capacity to carry out these interventions. Conversely, in\\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\\nlikely the result of the lack of required infrastructure. Overall, our results\\nshow that CABG rates evolved differently between Swiss regions, the most likely\\nexplanations being the existence of infrastructures and local preferences for\\ncertain types of revascularization procedures.\\n In-hospital mortality An overall decrease in seven-day in-hospital mortality was found for Switzerland,\\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\\nsuggesting that most of the decrease in mortality is due to revascularization\\ninterventions and the improved quality of treatment according to the newest\\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\\ndays led to similar findings, although some regions presented a slight increase\\nin mortality (Additional file 5: Table S1). The rising\\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\\nmultivariate model as they are not reported systematically in the database.\\nSimilarly, it was not possible to assess whether the patients presented with a\\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\\nrevascularization interventions performed among patients discharged with a main\\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\\nassociated with in-hospital mortality from AMI in order to optimize patient\\nmanagement.\\nAn overall decrease in seven-day in-hospital mortality was found for Switzerland,\\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\\nsuggesting that most of the decrease in mortality is due to revascularization\\ninterventions and the improved quality of treatment according to the newest\\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\\ndays led to similar findings, although some regions presented a slight increase\\nin mortality (Additional file 5: Table S1). The rising\\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\\nmultivariate model as they are not reported systematically in the database.\\nSimilarly, it was not possible to assess whether the patients presented with a\\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\\nrevascularization interventions performed among patients discharged with a main\\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\\nassociated with in-hospital mortality from AMI in order to optimize patient\\nmanagement.\\n Study limitations This study has some limitations. Medicines are not included in the hospital\\ndischarge database, thus precluding the assessment of their trends. This absence\\nmight also explain the very low rates for thrombolysis, which are likely due to\\nthe absence of coding rather than a true absence of use. Furthermore, we lack\\ndata to assess whether the changes in thrombolytic therapy are related to\\nchanges in reporting levels or are actually real changes. The ICD10 coding does\\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\\nincrease in discharges from AMI during the study period might be related to the\\nchange in the definition of NSTEMI populations due to the generalization of the\\nuse of troponin measurements. Indeed, introduction of troponin measurements led\\nto the reclassification of many unstable angina patients into NSTEMI patients,\\nwith a 33% increase of the population of NSTEMIs from before to after the\\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\\nled to similar findings, with the exception that the increase in CABG was no\\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\\nbetween-hospital transfers and the anonymization of the data (which precluded\\nthe follow-up of the patients) complicated the interpretation of the trends.\\nStill, restricting the analysis to patients fully managed in a single hospital\\nled to similar results. It was not possible to assess “true” seven-\\nand 30-day mortality rates as no follow-up data was available for the patients\\ndischarged. The AMIS registry, which collects vital status data for AMI patients\\nhospitalized in the participating institutions, will be able to provide such\\ninformation [35]. Finally, there is no published information on the validity of\\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\\na value in agreement with the literature [36,37].\\nThis study has some limitations. Medicines are not included in the hospital\\ndischarge database, thus precluding the assessment of their trends. This absence\\nmight also explain the very low rates for thrombolysis, which are likely due to\\nthe absence of coding rather than a true absence of use. Furthermore, we lack\\ndata to assess whether the changes in thrombolytic therapy are related to\\nchanges in reporting levels or are actually real changes. The ICD10 coding does\\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\\nincrease in discharges from AMI during the study period might be related to the\\nchange in the definition of NSTEMI populations due to the generalization of the\\nuse of troponin measurements. Indeed, introduction of troponin measurements led\\nto the reclassification of many unstable angina patients into NSTEMI patients,\\nwith a 33% increase of the population of NSTEMIs from before to after the\\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\\nled to similar findings, with the exception that the increase in CABG was no\\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\\nbetween-hospital transfers and the anonymization of the data (which precluded\\nthe follow-up of the patients) complicated the interpretation of the trends.\\nStill, restricting the analysis to patients fully managed in a single hospital\\nled to similar results. It was not possible to assess “true” seven-\\nand 30-day mortality rates as no follow-up data was available for the patients\\ndischarged. The AMIS registry, which collects vital status data for AMI patients\\nhospitalized in the participating institutions, will be able to provide such\\ninformation [35]. Finally, there is no published information on the validity of\\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\\na value in agreement with the literature [36,37].\",\n \"The percentage of hospitalizations with ICU stay remained stable between 1998 and\\n2008 in Switzerland. This stabilization might be the consequence of improved\\nmanagement, requiring less ICU admissions before or after a revascularization\\nintervention. Another likely explanation is the increasing number of transfers,\\nthe patients being stabilized before being transferred to another hospital for\\nrevascularization. Still, considerable differences in the proportion of\\nhospitalizations with ICU and corresponding trends were found between regions,\\nsuggesting that other factors such as the number of ICU beds available or local\\noptions for AMI management might be at play. Unfortunately there is no available\\ninformation regarding the number of ICU beds in each region, so the precise\\nreasons for the regional differences in ICU admissions for AMI remain to be\\nfurther assessed.\",\n \"Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\\nregions were more responsive than others. For instance, in the Leman area DES\\nuse equaled but never exceeded bare stent use, while in South Switzerland\\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\\nsuch regional differences are more likely due to local preferences or to local\\nagreements regarding DES cost than to decisions based on scientific\\nevidence.\\nThe rate of CABG remained stable at 3% throughout the study period, a finding\\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\\nThis trend might partly be explained by the creation in 1999 of new medical\\ncenters with the capacity to carry out these interventions. Conversely, in\\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\\nlikely the result of the lack of required infrastructure. Overall, our results\\nshow that CABG rates evolved differently between Swiss regions, the most likely\\nexplanations being the existence of infrastructures and local preferences for\\ncertain types of revascularization procedures.\",\n \"An overall decrease in seven-day in-hospital mortality was found for Switzerland,\\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\\nsuggesting that most of the decrease in mortality is due to revascularization\\ninterventions and the improved quality of treatment according to the newest\\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\\ndays led to similar findings, although some regions presented a slight increase\\nin mortality (Additional file 5: Table S1). The rising\\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\\nmultivariate model as they are not reported systematically in the database.\\nSimilarly, it was not possible to assess whether the patients presented with a\\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\\nrevascularization interventions performed among patients discharged with a main\\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\\nassociated with in-hospital mortality from AMI in order to optimize patient\\nmanagement.\",\n \"This study has some limitations. Medicines are not included in the hospital\\ndischarge database, thus precluding the assessment of their trends. This absence\\nmight also explain the very low rates for thrombolysis, which are likely due to\\nthe absence of coding rather than a true absence of use. Furthermore, we lack\\ndata to assess whether the changes in thrombolytic therapy are related to\\nchanges in reporting levels or are actually real changes. The ICD10 coding does\\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\\nincrease in discharges from AMI during the study period might be related to the\\nchange in the definition of NSTEMI populations due to the generalization of the\\nuse of troponin measurements. Indeed, introduction of troponin measurements led\\nto the reclassification of many unstable angina patients into NSTEMI patients,\\nwith a 33% increase of the population of NSTEMIs from before to after the\\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\\nled to similar findings, with the exception that the increase in CABG was no\\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\\nbetween-hospital transfers and the anonymization of the data (which precluded\\nthe follow-up of the patients) complicated the interpretation of the trends.\\nStill, restricting the analysis to patients fully managed in a single hospital\\nled to similar results. It was not possible to assess “true” seven-\\nand 30-day mortality rates as no follow-up data was available for the patients\\ndischarged. The AMIS registry, which collects vital status data for AMI patients\\nhospitalized in the participating institutions, will be able to provide such\\ninformation [35]. Finally, there is no published information on the validity of\\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\\na value in agreement with the literature [36,37].\",\n \"In Switzerland, a steep rise in hospital discharges and in revascularization\\nprocedures for AMI occurred between 1998 and 2008.\",\n \"The authors declare that they have no competing interests.\",\n \"CI made part of the statistical analyses and wrote most of the article; PMV collected\\ndata, made part of the statistical analysis and wrote part of the article; FP\\nrevised the article for important intellectual content. PMV had full access to the\\ndata and is the guarantor of the study. All authors read and approved the final\\nmanuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\\n\",\n \" Trends in intensive care unit (ICU) utilization for acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008, patients managed in a single hospital (in-house).\\nResults are expressed as percentage of patients discharged with a\\ndiagnosis of acute myocardial infarction.\\nClick here for file\\nTrends in the use of drug-eluting and non-drug-eluting stents for acute\\nmyocardial infarction in Switzerland, overall and by region, for the\\nperiod 1998–2008, patients managed in a single hospital\\n(in-house). Results are expressed as percentage of patients discharged\\nwith a diagnosis of acute myocardial infarction.\\nClick here for file\\nTrends in the use of coronary artery bypass graft (CABG) for acute\\nmyocardial infarction in Switzerland, overall and by region, for the\\nperiod 1998–2008, patients managed in a single hospital\\n(in-house). Results are expressed as percentage of patients discharged\\nwith a diagnosis of acute myocardial infarction.\\nClick here for file\\nTrends in seven-day in-hospital mortality rates from acute myocardial\\ninfarction in Switzerland, overall and by region, for the period\\n1998–2008, patients managed in a single hospital (in-house).\\nResults are expressed as percentage of patients discharged with a\\ndiagnosis of acute myocardial infarction.\\nClick here for file\\nTrends in seven-day and overall in-hospital mortality, Switzerland and\\nSwiss regions, for period 1998-2008\\nClick here for file\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,"results",null,null,null,null,"discussion",null,null,null,null,"conclusions",null,null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null,\n null,\n \"conclusions\",\n null,\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["Acute myocardial infarction","Coronary revascularization","Epidemiology","Switzerland","Trends","Drug-eluting stent","Coronary artery bypass graft","Hospital discharge"],"string":"[\n \"Acute myocardial infarction\",\n \"Coronary revascularization\",\n \"Epidemiology\",\n \"Switzerland\",\n \"Trends\",\n \"Drug-eluting stent\",\n \"Coronary artery bypass graft\",\n \"Hospital discharge\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nCardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008.\n\nMethods:\n Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\nData from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\n Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.\nStatistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.\n\nDatabases and available data:\nData from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\n\nStatistical analysis:\nStatistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.\n\nResults:\n Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\nOverall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\n Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\nThe percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\n Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\nPercutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\n In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).\nAmong patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).\n\nCharacteristics of the patients:\nOverall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\n\nIntensive care unit:\nThe percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\n\nRevascularisation procedures:\nPercutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\n\nIn-hospital mortality:\nAmong patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).\n\nDiscussion:\nSince the end of the MONICA study [12,13], few studies have assessed the management and outcome of AMI in\nSwitzerland. Our results show an increase in most revascularization procedures,\nnamely PCI, while CABG tended to stabilize or even to decrease in the most recent\nyears. Moreover, these trends strongly differ according to region. Overall, the\nchanges in revascularization procedure in Switzerland follow the ones observed in\nother countries, [6,7,14,15]. These findings are also in agreement with a previous study conducted on\n19,500 patients of the AMIS Plus registry [8], although the increase in PCI procedures observed [16] was considerably stronger than in ours. This difference is most likely\ndue to the overrepresentation of PCI procedures among the AMIS Plus registry\nhospitals as the Registry includes only selected voluntary teams.\nBetween 1998 and 2008, the number of hospital discharges for AMI increased over\ntwo-fold in Switzerland. This finding is in contradiction with some previous studies [17,18], but it should be noted that these studies showed declining rates of AMI\nrather than number of discharges. Part of this increase might be due to population\naging [2], although the observed increase in population age (from 66.5 years\nin 1998 to 67.6 years in 2008) would not lead to a doubling of the number of\nhospitalizations for AMI. Further, all analyses were adjusted for age. Another\npossibility would be a change in disease coding, but again this is unlikely as\nhospital discharge data has been shown to be reliable regarding the diagnosis of AMI [19] and as this study focused on a specific code. It is also possible that\nmore people are hospitalized for a second or even a third AMI (partly related to the\ndecrease in case fatality rate of the first AMI, leading to an increased incidence\nof 2nd or 3rd AMI). Another possibility is the change in\ncriteria defining AMI [20], which has been shown to increase the incidence of AMI [21]. The most likely explanation for the considerable increase in the number\nof hospital discharges for AMI in Switzerland is the steep rise in hospital\ntransfers, which accounted for almost half of hospital discharges for AMI in 2008.\nThe increase in hospital transfers might be due to the limited number of hospitals\ncapable of performing revascularization procedures, prompting smaller hospitals to\ntransfer their patients as recommended by European guidelines [22,23]. Still, considerable differences in the percentages of hospital\ndischarges due to transfers were noted: in 2008 the values ranged from 40% in Leman\nto 68% in Ticino. These differences might be due to local policies. As AMI\nmanagement is expensive [24], the increase in hospital discharges for AMI would add a considerable\npressure in Swiss health expenditures, which already increased from 9.6% in 1995 to\n11.4% of the Swiss GNP in 2009 [25].\n Intensive care unit The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\nThe percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\n Revascularisation procedures Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\nStent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\n In-hospital mortality An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\nAn overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\n Study limitations This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].\nThis study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].\n\nIntensive care unit:\nThe percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\n\nRevascularisation procedures:\nStent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\n\nIn-hospital mortality:\nAn overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\n\nStudy limitations:\nThis study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].\n\nConclusion:\nIn Switzerland, a steep rise in hospital discharges and in revascularization\nprocedures for AMI occurred between 1998 and 2008.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nCI made part of the statistical analyses and wrote most of the article; PMV collected\ndata, made part of the statistical analysis and wrote part of the article; FP\nrevised the article for important intellectual content. PMV had full access to the\ndata and is the guarantor of the study. All authors read and approved the final\nmanuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\n\n\nSupplementary Material:\n Trends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008, patients managed in a single hospital (in-house).\nResults are expressed as percentage of patients discharged with a\ndiagnosis of acute myocardial infarction.\nClick here for file\nTrends in the use of drug-eluting and non-drug-eluting stents for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008, patients managed in a single hospital\n(in-house). Results are expressed as percentage of patients discharged\nwith a diagnosis of acute myocardial infarction.\nClick here for file\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008, patients managed in a single hospital\n(in-house). Results are expressed as percentage of patients discharged\nwith a diagnosis of acute myocardial infarction.\nClick here for file\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008, patients managed in a single hospital (in-house).\nResults are expressed as percentage of patients discharged with a\ndiagnosis of acute myocardial infarction.\nClick here for file\nTrends in seven-day and overall in-hospital mortality, Switzerland and\nSwiss regions, for period 1998-2008\nClick here for file\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSince the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap.\n\nMethods:\nSwiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed.\n\nResults:\nOverall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered.\n\nConclusions:\nIn Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates."},"background_conclusion_text":{"kind":"string","value":"Background:\nCardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008.\n\nConclusion:\nIn Switzerland, a steep rise in hospital discharges and in revascularization\nprocedures for AMI occurred between 1998 and 2008."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSince the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap.\n\nMethods:\nSwiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed.\n\nResults:\nOverall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered.\n\nConclusions:\nIn Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates."},"whole_article_text_length":{"kind":"number","value":12726,"string":"12,726"},"whole_article_abstract_length":{"kind":"number","value":365,"string":"365"},"other_sections_lengths":{"kind":"list like","value":[267,569,285,417,407,591,348,148,311,276,422,10,68,16],"string":"[\n 267,\n 569,\n 285,\n 417,\n 407,\n 591,\n 348,\n 148,\n 311,\n 276,\n 422,\n 10,\n 68,\n 16\n]"},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["hospital","switzerland","patients","2008","1998","trends","mortality","myocardial","acute","myocardial infarction"],"string":"[\n \"hospital\",\n \"switzerland\",\n \"patients\",\n \"2008\",\n \"1998\",\n \"trends\",\n \"mortality\",\n \"myocardial\",\n \"acute\",\n \"myocardial infarction\"\n]"},"keybert_topics":{"kind":"list like","value":["mortality switzerland swiss","myocardial infarction managed","hospital mortality switzerland","switzerland health policies","infarction switzerland overall"],"string":"[\n \"mortality switzerland swiss\",\n \"myocardial infarction managed\",\n \"hospital mortality switzerland\",\n \"switzerland health policies\",\n \"infarction switzerland overall\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] outcome | ami | study | decades | ami management outcome | management | plus | management outcome | amis plus | participating [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] procedures | hospital | icd10 | length | length stay | discharge | patients | stay | code | age [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | 2008 | switzerland | 1998 | trend | acute | myocardial | acute myocardial | patients | figure [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] rise hospital discharges | revascularization procedures ami | rise hospital discharges revascularization | ami occurred | ami occurred 1998 | ami occurred 1998 2008 | steep rise hospital discharges | discharges revascularization procedures ami | discharges revascularization procedures | discharges revascularization [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | patients | switzerland | 2008 | 1998 | ami | mortality | myocardial | trends | myocardial infarction [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | patients | switzerland | 2008 | 1998 | ami | mortality | myocardial | trends | myocardial infarction [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] the late nineties | AMI | Switzerland ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Swiss | years 1998 to 2008 ||| AMI | I21 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 102,729 | AMI ||| Intensive Care Unit | 38.0% | 1998 | 36.2% | 2008 | 0.001 ||| 6.0% | 39.9% | 0.001 ||| 1.3% | 16.6% | 0.001 ||| 2004 | 23.5% | 2008 | 0.001 ||| 1.0% to 3.0% | 0.001 ||| 0.2% | 1.7% | 0.001 ||| seven-day | 8.0% to 7.0% | 0.01 | 11.2% | 10.1% ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Switzerland | AMI | between 1998 and 2008 ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] the late nineties | AMI | Switzerland ||| ||| years 1998 to 2008 ||| AMI | I21 ||| ||| 102,729 | AMI ||| Intensive Care Unit | 38.0% | 1998 | 36.2% | 2008 | 0.001 ||| 6.0% | 39.9% | 0.001 ||| 1.3% | 16.6% | 0.001 ||| 2004 | 23.5% | 2008 | 0.001 ||| 1.0% to 3.0% | 0.001 ||| 0.2% | 1.7% | 0.001 ||| seven-day | 8.0% to 7.0% | 0.01 | 11.2% | 10.1% ||| ||| ||| Switzerland | AMI | between 1998 and 2008 ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] the late nineties | AMI | Switzerland ||| ||| years 1998 to 2008 ||| AMI | I21 ||| ||| 102,729 | AMI ||| Intensive Care Unit | 38.0% | 1998 | 36.2% | 2008 | 0.001 ||| 6.0% | 39.9% | 0.001 ||| 1.3% | 16.6% | 0.001 ||| 2004 | 23.5% | 2008 | 0.001 ||| 1.0% to 3.0% | 0.001 ||| 0.2% | 1.7% | 0.001 ||| seven-day | 8.0% to 7.0% | 0.01 | 11.2% | 10.1% ||| ||| ||| Switzerland | AMI | between 1998 and 2008 ||| [SUMMARY]"}}},{"rowIdx":3354,"cells":{"title":{"kind":"string","value":"Clinician preferences for computerised clinical decision support for medications in primary care: a focus group study."},"pmid":{"kind":"string","value":"31039120"},"background_abstract":{"kind":"string","value":"To improve user-centred design efforts and efficiency; there is a need to disseminate information on modern day clinician preferences for technologies such as computerised clinical decision support (CDS)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This study included focus groups and clinicians individually describing their ideal CDS. Three focus groups were conducted including prescribing clinicians from a variety of disciplines. Outcome measures included identification of favourable features and unintended consequences of CDS for chronic medication management in primary care. We transcribed recordings, performed thematic qualitative analysis and generated counts when possible."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"There were 21 participants who identified four categories of beneficial CDS features during the group discussion: non-interruptive alerts, clinically relevant and customisable support, presentation of pertinent clinical information and optimises workflow. Non-interruptive alerts were broadly defined as passive alerts that a user chooses to review, whereas interruptive were active or disruptive alerts that interrupted workflow and one is forced to review before completing a task. The CDS features identified in the individual descriptions were consistent with the focus group discussion, with the exception of non-interruptive alerts. In the individual descriptions, 12 clinicians preferred interruptive CDS compared with seven clinicians describing non-interruptive CDS."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Clinicians identified CDS for chronic medications beneficial when they are clinically relevant and customisable, present pertinent clinical information (eg, labs, vitals) and improve their workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. These features align with literature describing best practices in CDS design and emphasise those features clinicians prioritise, which should be considered when designing CDS for medication management in primary care. These findings highlight the disparity between the current state of CDS design and clinician-stated design features associated with beneficial CDS."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Decision Support Systems, Clinical","Female","Focus Groups","Grounded Theory","Humans","Male","Medical Order Entry Systems","Middle Aged","Practice Patterns, Physicians'","Primary Health Care"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Decision Support Systems, Clinical\",\n \"Female\",\n \"Focus Groups\",\n \"Grounded Theory\",\n \"Humans\",\n \"Male\",\n \"Medical Order Entry Systems\",\n \"Middle Aged\",\n \"Practice Patterns, Physicians'\",\n \"Primary Health Care\"\n]"},"pmcid":{"kind":"string","value":"7062316"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.\nClinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12\n\nUnfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.\nUnderstanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"The purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.\nEach focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.\nThe focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.\nFocus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.\nThe major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Twenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).\nClinician characteristics\n Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\n Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\nWhen asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\n Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\nWhen asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them."},"conclusions_title":{"kind":"string","value":"Open-ended discussion of beneficial features"},"conclusions_text":{"kind":"string","value":"When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them."},"other_sections_titles":{"kind":"list like","value":["What is already known?","What does this paper add?","Open-ended discussion of beneficial features"],"string":"[\n \"What is already known?\",\n \"What does this paper add?\",\n \"Open-ended discussion of beneficial features\"\n]"},"other_sections_texts":{"kind":"list like","value":["There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\nEnd user input in CDS design increases the likelihood of CDS to be successful.","Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.","Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’."],"string":"[\n \"There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\\nEnd user input in CDS design increases the likelihood of CDS to be successful.\",\n \"Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.\",\n \"Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null],"string":"[\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["What is already known?","What does this paper add?","Introduction","Methods","Results","Open-ended discussion of beneficial features","Discussion"],"string":"[\n \"What is already known?\",\n \"What does this paper add?\",\n \"Introduction\",\n \"Methods\",\n \"Results\",\n \"Open-ended discussion of beneficial features\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\nEnd user input in CDS design increases the likelihood of CDS to be successful.","Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.","Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.\nClinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12\n\nUnfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.\nUnderstanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.","The purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.\nEach focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.\nThe focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.\nFocus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.\nThe major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent.","Twenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).\nClinician characteristics\n Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\n Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\nWhen asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\n Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\nWhen asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.","Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.","This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care and the need to apply these principles to realise the benefits of CDS, which are pervasive in healthcare, yet do not currently produce consistently positive outcomes. We identified four main categories of CDS features for chronic medication management that clinicians find beneficial: clinically relevant and customisable, presentation of pertinent information and optimisation of workflow. These categories of features align with the best practices in CDS design and highlight aspects that clinicians perceive to be more or less beneficial, which should be prioritised when designing and implementing future CDS for chronic medications in primary care.\nInterestingly, focus group participants expressed strong dislike for interruptive CDS alerts during the open group discussion, which was the most common CDS suggested when individuals were asked to design the ideal CDS. This discrepancy may be due to effects of peer influence during the group discussion or realisation that interruptive alerts were helpful when designed well and that non-interruptive alerts are more likely to be overlooked or ignored. The discussion of whether the alerts should be interruptive or not aligns with literature supporting improved use and effectiveness of interruptive CDS16 22 34 35 and non-interruptive CDS being less likely to be viewed and therefore less likely to be effective.16 22 24 36 37 However, whether an alert is interruptive or not is only one feature of CDS and as the focus group conversation progressed, clinicians discussed other features of CDS that would improve usefulness of an interruptive alert, such as timing of the alert and the ability to customise the alert. These features may have led individual clinicians to favour interruptive alerts if the design incorporated these features. Although not explicitly discussed during the focus groups, the sentiment regarding passive versus active CDS highlights the importance of potentially reserving active CDS for high priority or high-risk patient care situations13 38–40 and carefully designing active CDS such that the other features are optimised to minimise clinician frustrations.\nAlthough there was no consensus in the timing of alerts, the discussion supports efforts to accommodate variations in individual clinician workflow and to personalise CDS to clinical workflows. However, to date, CDS have not been personalised to individual workflows, given the complexity required to account for the highly variable clinical workflows. Future research is needed to evaluate feasibility of such personalisation to workflows. For non-interruptive alerts, personalisation could include designing the CDS such that clinicians can access the information in the alert at a variety of different time points (eg, before entering the patient chart and/or on order entry and/or when signing a note), whereas such an approach with interruptive alerts would increase the frequency of interruptions and augment alert fatigue. Thus, for interruptive alerts, individualised personalisation of the alert timing by the clinician to meet their workflow needs would be the ideal state, but has not been realised yet. The ideal timing of passive or active CDS facilitates clinicians using the CDS without them noticing it, which can be challenging to accomplish in many situations, but nonetheless should be the goal in CDS design. Often personalisation is limited by technical constraints of the CDS software or EHR, which is continually evolving and may be less of a barrier with time and creative solutions.\nTo improve relevance and optimise their workflow, clinicians want CDS to be smarter, synthesising and presenting pertinent patient-specific information, with actionable recommendations that generates automated text in their clinical documentation and patient instructions. These findings are consistent with published recommendations on the ‘grand challenges’ that must be overcome for CDS to reach their potential and positively impact healthcare.39 Summarising pertinent information such as labs, vitals and allergies makes it easier for clinicians to quickly evaluate the appropriateness of a CDS recommendation and actionable links, such as ordering medications or labs, makes it easy to take action immediately. Further, the ability to populate clinical documentation and patient instructions with the plan would decrease duplicate work. Clinicians were specifically interested in auto-population of patient instructions to include titration schedules, timing of lab monitoring and pertinent adverse effects. Although not specifically mentioned in the focus groups, the clinical documentation could include routine monitoring that would assist in planning follow-up care. Some clinicians also expressed interest in presenting medication cost information with CDS recommendations. Although there are efforts to integrate medication costs into EHRs and CDS,41 there is currently no published literature describing the efficacy of such efforts. However, in other situations, such as ordering laboratory tests, clinicians did change ordering behaviour when informed of associated costs,42–45 suggesting the same may be true with medication ordering.\nAnother desired feature of the CDS was flexibility in response options, which is aligned with best practice principles in user-interface design. At times, clinicians felt forced to answer in a way not aligned with their intentions or the clinical scenario and wanted the option to better explain their actions in the form of free text that could be viewed later to trigger recall of their decisions. They also strongly wanted the option to permanently disable or temporarily dismiss CDS to alert at a more opportune time of their choosing. Clinicians who believed CDS would improve usefulness felt the ability to customise the CDS to the clinical scenario and individual workflow was very important. Principles of user-interface design includes ensuring clear and actionable response options,13 but flexibility in options is not well emphasised in the literature and can be critical to support the often nuanced clinical scenarios.\nClinicians had some concerns about unintended consequences, especially related to alert fatigue, mindlessly following CDS and inaccurate or incomplete information being presented by CDS. To avoid these problems, clinicians suggested implementing systematic processes for evaluation of CDS and inclusion of optional information and references. While inclusion of references in CDS are key to building trust, there are many additional strategies recommended to cultivate trust.13 Prioritising CDS recommendations according to specificity, urgency and relevance minimises obtrusiveness and increases trust.13 As CDS becomes increasingly pervasive in healthcare and we drive towards a Learning Health System, provision of a rationale with an assessment of the certainty or quality of the recommendation is important to engender trust.46 Further, as CDS are modified in response to clinician feedback, it is important to monitor for unintended consequences. For example, converting all CDS to be passive or giving clinicians the ability to permanently ‘snooze’ all CDS could result in missed opportunities to optimise evidence-based care for patients.\nThe findings of this study are limited by generalisability, given that participants represent clinicians at a single large academic medical centre using one integrated EHR platform for over 5 years and are exposed routinely to CDS. The results may be less representative of clinicians with less experience using an EHR and CDS, or using a different EHR platform, despite conversations focusing on broad concepts related to CDS that were not specific to an individual EHR. Further, participants represent a convenience sample based on ongoing professional relationships with the study investigators, thus introducing selection bias. However, a notable strength is that participants represent a variety of disciplines across outpatient practice settings and various expertise, with varying degrees of clinical practice responsibilities.\nClinicians characterised CDS for chronic medications as beneficial when it is clinically relevant and customisable, presents pertinent clinical information (eg, labs, vitals) and optimises workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. The design features align with literature describing best practices in CDS design and emphasise features that primary care providers prioritise when using CDS for chronic medication use. Despite awareness of these best practices in CDS design, many CDS are not designed with application of these best practices, which is thought to be one reason for the mixed outcomes of CDS to date.14 19 47 48 This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care. When designing CDS for chronic medications in primary care, developers should consider these user-centred design features and continually re-evaluate CDS design as technical capabilities of CDS and EHRs become more sophisticated."],"string":"[\n \"There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\\nEnd user input in CDS design increases the likelihood of CDS to be successful.\",\n \"Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.\",\n \"Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.\\nClinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12\\n\\nUnfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.\\nUnderstanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.\",\n \"The purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.\\nEach focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.\\nThe focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.\\nFocus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.\\nThe major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent.\",\n \"Twenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).\\nClinician characteristics\\n Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\\nClinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\\n Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\\nWhen asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\\n Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\\nWhen asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\",\n \"Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\",\n \"This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care and the need to apply these principles to realise the benefits of CDS, which are pervasive in healthcare, yet do not currently produce consistently positive outcomes. We identified four main categories of CDS features for chronic medication management that clinicians find beneficial: clinically relevant and customisable, presentation of pertinent information and optimisation of workflow. These categories of features align with the best practices in CDS design and highlight aspects that clinicians perceive to be more or less beneficial, which should be prioritised when designing and implementing future CDS for chronic medications in primary care.\\nInterestingly, focus group participants expressed strong dislike for interruptive CDS alerts during the open group discussion, which was the most common CDS suggested when individuals were asked to design the ideal CDS. This discrepancy may be due to effects of peer influence during the group discussion or realisation that interruptive alerts were helpful when designed well and that non-interruptive alerts are more likely to be overlooked or ignored. The discussion of whether the alerts should be interruptive or not aligns with literature supporting improved use and effectiveness of interruptive CDS16 22 34 35 and non-interruptive CDS being less likely to be viewed and therefore less likely to be effective.16 22 24 36 37 However, whether an alert is interruptive or not is only one feature of CDS and as the focus group conversation progressed, clinicians discussed other features of CDS that would improve usefulness of an interruptive alert, such as timing of the alert and the ability to customise the alert. These features may have led individual clinicians to favour interruptive alerts if the design incorporated these features. Although not explicitly discussed during the focus groups, the sentiment regarding passive versus active CDS highlights the importance of potentially reserving active CDS for high priority or high-risk patient care situations13 38–40 and carefully designing active CDS such that the other features are optimised to minimise clinician frustrations.\\nAlthough there was no consensus in the timing of alerts, the discussion supports efforts to accommodate variations in individual clinician workflow and to personalise CDS to clinical workflows. However, to date, CDS have not been personalised to individual workflows, given the complexity required to account for the highly variable clinical workflows. Future research is needed to evaluate feasibility of such personalisation to workflows. For non-interruptive alerts, personalisation could include designing the CDS such that clinicians can access the information in the alert at a variety of different time points (eg, before entering the patient chart and/or on order entry and/or when signing a note), whereas such an approach with interruptive alerts would increase the frequency of interruptions and augment alert fatigue. Thus, for interruptive alerts, individualised personalisation of the alert timing by the clinician to meet their workflow needs would be the ideal state, but has not been realised yet. The ideal timing of passive or active CDS facilitates clinicians using the CDS without them noticing it, which can be challenging to accomplish in many situations, but nonetheless should be the goal in CDS design. Often personalisation is limited by technical constraints of the CDS software or EHR, which is continually evolving and may be less of a barrier with time and creative solutions.\\nTo improve relevance and optimise their workflow, clinicians want CDS to be smarter, synthesising and presenting pertinent patient-specific information, with actionable recommendations that generates automated text in their clinical documentation and patient instructions. These findings are consistent with published recommendations on the ‘grand challenges’ that must be overcome for CDS to reach their potential and positively impact healthcare.39 Summarising pertinent information such as labs, vitals and allergies makes it easier for clinicians to quickly evaluate the appropriateness of a CDS recommendation and actionable links, such as ordering medications or labs, makes it easy to take action immediately. Further, the ability to populate clinical documentation and patient instructions with the plan would decrease duplicate work. Clinicians were specifically interested in auto-population of patient instructions to include titration schedules, timing of lab monitoring and pertinent adverse effects. Although not specifically mentioned in the focus groups, the clinical documentation could include routine monitoring that would assist in planning follow-up care. Some clinicians also expressed interest in presenting medication cost information with CDS recommendations. Although there are efforts to integrate medication costs into EHRs and CDS,41 there is currently no published literature describing the efficacy of such efforts. However, in other situations, such as ordering laboratory tests, clinicians did change ordering behaviour when informed of associated costs,42–45 suggesting the same may be true with medication ordering.\\nAnother desired feature of the CDS was flexibility in response options, which is aligned with best practice principles in user-interface design. At times, clinicians felt forced to answer in a way not aligned with their intentions or the clinical scenario and wanted the option to better explain their actions in the form of free text that could be viewed later to trigger recall of their decisions. They also strongly wanted the option to permanently disable or temporarily dismiss CDS to alert at a more opportune time of their choosing. Clinicians who believed CDS would improve usefulness felt the ability to customise the CDS to the clinical scenario and individual workflow was very important. Principles of user-interface design includes ensuring clear and actionable response options,13 but flexibility in options is not well emphasised in the literature and can be critical to support the often nuanced clinical scenarios.\\nClinicians had some concerns about unintended consequences, especially related to alert fatigue, mindlessly following CDS and inaccurate or incomplete information being presented by CDS. To avoid these problems, clinicians suggested implementing systematic processes for evaluation of CDS and inclusion of optional information and references. While inclusion of references in CDS are key to building trust, there are many additional strategies recommended to cultivate trust.13 Prioritising CDS recommendations according to specificity, urgency and relevance minimises obtrusiveness and increases trust.13 As CDS becomes increasingly pervasive in healthcare and we drive towards a Learning Health System, provision of a rationale with an assessment of the certainty or quality of the recommendation is important to engender trust.46 Further, as CDS are modified in response to clinician feedback, it is important to monitor for unintended consequences. For example, converting all CDS to be passive or giving clinicians the ability to permanently ‘snooze’ all CDS could result in missed opportunities to optimise evidence-based care for patients.\\nThe findings of this study are limited by generalisability, given that participants represent clinicians at a single large academic medical centre using one integrated EHR platform for over 5 years and are exposed routinely to CDS. The results may be less representative of clinicians with less experience using an EHR and CDS, or using a different EHR platform, despite conversations focusing on broad concepts related to CDS that were not specific to an individual EHR. Further, participants represent a convenience sample based on ongoing professional relationships with the study investigators, thus introducing selection bias. However, a notable strength is that participants represent a variety of disciplines across outpatient practice settings and various expertise, with varying degrees of clinical practice responsibilities.\\nClinicians characterised CDS for chronic medications as beneficial when it is clinically relevant and customisable, presents pertinent clinical information (eg, labs, vitals) and optimises workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. The design features align with literature describing best practices in CDS design and emphasise features that primary care providers prioritise when using CDS for chronic medication use. Despite awareness of these best practices in CDS design, many CDS are not designed with application of these best practices, which is thought to be one reason for the mixed outcomes of CDS to date.14 19 47 48 This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care. When designing CDS for chronic medications in primary care, developers should consider these user-centred design features and continually re-evaluate CDS design as technical capabilities of CDS and EHRs become more sophisticated.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,"intro","methods","results",null,"discussion"],"string":"[\n null,\n null,\n \"intro\",\n \"methods\",\n \"results\",\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["clinical decision support systems","electronic prescribing","primary health care"],"string":"[\n \"clinical decision support systems\",\n \"electronic prescribing\",\n \"primary health care\"\n]"},"whole_article_text":{"kind":"string","value":"What is already known?:\nThere are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\nEnd user input in CDS design increases the likelihood of CDS to be successful.\n\nWhat does this paper add?:\nClinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.\n\nIntroduction:\nComputerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.\nClinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12\n\nUnfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.\nUnderstanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.\n\nMethods:\nThe purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.\nEach focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.\nThe focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.\nFocus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.\nThe major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent.\n\nResults:\nTwenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).\nClinician characteristics\n Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\n Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\nWhen asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\n Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\nWhen asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\n\nOpen-ended discussion of beneficial features:\nClinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\n\nDiscussion:\nThis study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care and the need to apply these principles to realise the benefits of CDS, which are pervasive in healthcare, yet do not currently produce consistently positive outcomes. We identified four main categories of CDS features for chronic medication management that clinicians find beneficial: clinically relevant and customisable, presentation of pertinent information and optimisation of workflow. These categories of features align with the best practices in CDS design and highlight aspects that clinicians perceive to be more or less beneficial, which should be prioritised when designing and implementing future CDS for chronic medications in primary care.\nInterestingly, focus group participants expressed strong dislike for interruptive CDS alerts during the open group discussion, which was the most common CDS suggested when individuals were asked to design the ideal CDS. This discrepancy may be due to effects of peer influence during the group discussion or realisation that interruptive alerts were helpful when designed well and that non-interruptive alerts are more likely to be overlooked or ignored. The discussion of whether the alerts should be interruptive or not aligns with literature supporting improved use and effectiveness of interruptive CDS16 22 34 35 and non-interruptive CDS being less likely to be viewed and therefore less likely to be effective.16 22 24 36 37 However, whether an alert is interruptive or not is only one feature of CDS and as the focus group conversation progressed, clinicians discussed other features of CDS that would improve usefulness of an interruptive alert, such as timing of the alert and the ability to customise the alert. These features may have led individual clinicians to favour interruptive alerts if the design incorporated these features. Although not explicitly discussed during the focus groups, the sentiment regarding passive versus active CDS highlights the importance of potentially reserving active CDS for high priority or high-risk patient care situations13 38–40 and carefully designing active CDS such that the other features are optimised to minimise clinician frustrations.\nAlthough there was no consensus in the timing of alerts, the discussion supports efforts to accommodate variations in individual clinician workflow and to personalise CDS to clinical workflows. However, to date, CDS have not been personalised to individual workflows, given the complexity required to account for the highly variable clinical workflows. Future research is needed to evaluate feasibility of such personalisation to workflows. For non-interruptive alerts, personalisation could include designing the CDS such that clinicians can access the information in the alert at a variety of different time points (eg, before entering the patient chart and/or on order entry and/or when signing a note), whereas such an approach with interruptive alerts would increase the frequency of interruptions and augment alert fatigue. Thus, for interruptive alerts, individualised personalisation of the alert timing by the clinician to meet their workflow needs would be the ideal state, but has not been realised yet. The ideal timing of passive or active CDS facilitates clinicians using the CDS without them noticing it, which can be challenging to accomplish in many situations, but nonetheless should be the goal in CDS design. Often personalisation is limited by technical constraints of the CDS software or EHR, which is continually evolving and may be less of a barrier with time and creative solutions.\nTo improve relevance and optimise their workflow, clinicians want CDS to be smarter, synthesising and presenting pertinent patient-specific information, with actionable recommendations that generates automated text in their clinical documentation and patient instructions. These findings are consistent with published recommendations on the ‘grand challenges’ that must be overcome for CDS to reach their potential and positively impact healthcare.39 Summarising pertinent information such as labs, vitals and allergies makes it easier for clinicians to quickly evaluate the appropriateness of a CDS recommendation and actionable links, such as ordering medications or labs, makes it easy to take action immediately. Further, the ability to populate clinical documentation and patient instructions with the plan would decrease duplicate work. Clinicians were specifically interested in auto-population of patient instructions to include titration schedules, timing of lab monitoring and pertinent adverse effects. Although not specifically mentioned in the focus groups, the clinical documentation could include routine monitoring that would assist in planning follow-up care. Some clinicians also expressed interest in presenting medication cost information with CDS recommendations. Although there are efforts to integrate medication costs into EHRs and CDS,41 there is currently no published literature describing the efficacy of such efforts. However, in other situations, such as ordering laboratory tests, clinicians did change ordering behaviour when informed of associated costs,42–45 suggesting the same may be true with medication ordering.\nAnother desired feature of the CDS was flexibility in response options, which is aligned with best practice principles in user-interface design. At times, clinicians felt forced to answer in a way not aligned with their intentions or the clinical scenario and wanted the option to better explain their actions in the form of free text that could be viewed later to trigger recall of their decisions. They also strongly wanted the option to permanently disable or temporarily dismiss CDS to alert at a more opportune time of their choosing. Clinicians who believed CDS would improve usefulness felt the ability to customise the CDS to the clinical scenario and individual workflow was very important. Principles of user-interface design includes ensuring clear and actionable response options,13 but flexibility in options is not well emphasised in the literature and can be critical to support the often nuanced clinical scenarios.\nClinicians had some concerns about unintended consequences, especially related to alert fatigue, mindlessly following CDS and inaccurate or incomplete information being presented by CDS. To avoid these problems, clinicians suggested implementing systematic processes for evaluation of CDS and inclusion of optional information and references. While inclusion of references in CDS are key to building trust, there are many additional strategies recommended to cultivate trust.13 Prioritising CDS recommendations according to specificity, urgency and relevance minimises obtrusiveness and increases trust.13 As CDS becomes increasingly pervasive in healthcare and we drive towards a Learning Health System, provision of a rationale with an assessment of the certainty or quality of the recommendation is important to engender trust.46 Further, as CDS are modified in response to clinician feedback, it is important to monitor for unintended consequences. For example, converting all CDS to be passive or giving clinicians the ability to permanently ‘snooze’ all CDS could result in missed opportunities to optimise evidence-based care for patients.\nThe findings of this study are limited by generalisability, given that participants represent clinicians at a single large academic medical centre using one integrated EHR platform for over 5 years and are exposed routinely to CDS. The results may be less representative of clinicians with less experience using an EHR and CDS, or using a different EHR platform, despite conversations focusing on broad concepts related to CDS that were not specific to an individual EHR. Further, participants represent a convenience sample based on ongoing professional relationships with the study investigators, thus introducing selection bias. However, a notable strength is that participants represent a variety of disciplines across outpatient practice settings and various expertise, with varying degrees of clinical practice responsibilities.\nClinicians characterised CDS for chronic medications as beneficial when it is clinically relevant and customisable, presents pertinent clinical information (eg, labs, vitals) and optimises workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. The design features align with literature describing best practices in CDS design and emphasise features that primary care providers prioritise when using CDS for chronic medication use. Despite awareness of these best practices in CDS design, many CDS are not designed with application of these best practices, which is thought to be one reason for the mixed outcomes of CDS to date.14 19 47 48 This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care. When designing CDS for chronic medications in primary care, developers should consider these user-centred design features and continually re-evaluate CDS design as technical capabilities of CDS and EHRs become more sophisticated.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nTo improve user-centred design efforts and efficiency; there is a need to disseminate information on modern day clinician preferences for technologies such as computerised clinical decision support (CDS).\n\nMethods:\nThis study included focus groups and clinicians individually describing their ideal CDS. Three focus groups were conducted including prescribing clinicians from a variety of disciplines. Outcome measures included identification of favourable features and unintended consequences of CDS for chronic medication management in primary care. We transcribed recordings, performed thematic qualitative analysis and generated counts when possible.\n\nResults:\nThere were 21 participants who identified four categories of beneficial CDS features during the group discussion: non-interruptive alerts, clinically relevant and customisable support, presentation of pertinent clinical information and optimises workflow. Non-interruptive alerts were broadly defined as passive alerts that a user chooses to review, whereas interruptive were active or disruptive alerts that interrupted workflow and one is forced to review before completing a task. The CDS features identified in the individual descriptions were consistent with the focus group discussion, with the exception of non-interruptive alerts. In the individual descriptions, 12 clinicians preferred interruptive CDS compared with seven clinicians describing non-interruptive CDS.\n\nConclusions:\nClinicians identified CDS for chronic medications beneficial when they are clinically relevant and customisable, present pertinent clinical information (eg, labs, vitals) and improve their workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. These features align with literature describing best practices in CDS design and emphasise those features clinicians prioritise, which should be considered when designing CDS for medication management in primary care. These findings highlight the disparity between the current state of CDS design and clinician-stated design features associated with beneficial CDS."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nComputerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.\nClinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12\n\nUnfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.\nUnderstanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.\n\nOpen-ended discussion of beneficial features:\nWhen asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nTo improve user-centred design efforts and efficiency; there is a need to disseminate information on modern day clinician preferences for technologies such as computerised clinical decision support (CDS).\n\nMethods:\nThis study included focus groups and clinicians individually describing their ideal CDS. Three focus groups were conducted including prescribing clinicians from a variety of disciplines. Outcome measures included identification of favourable features and unintended consequences of CDS for chronic medication management in primary care. We transcribed recordings, performed thematic qualitative analysis and generated counts when possible.\n\nResults:\nThere were 21 participants who identified four categories of beneficial CDS features during the group discussion: non-interruptive alerts, clinically relevant and customisable support, presentation of pertinent clinical information and optimises workflow. Non-interruptive alerts were broadly defined as passive alerts that a user chooses to review, whereas interruptive were active or disruptive alerts that interrupted workflow and one is forced to review before completing a task. The CDS features identified in the individual descriptions were consistent with the focus group discussion, with the exception of non-interruptive alerts. In the individual descriptions, 12 clinicians preferred interruptive CDS compared with seven clinicians describing non-interruptive CDS.\n\nConclusions:\nClinicians identified CDS for chronic medications beneficial when they are clinically relevant and customisable, present pertinent clinical information (eg, labs, vitals) and improve their workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. These features align with literature describing best practices in CDS design and emphasise those features clinicians prioritise, which should be considered when designing CDS for medication management in primary care. These findings highlight the disparity between the current state of CDS design and clinician-stated design features associated with beneficial CDS."},"whole_article_text_length":{"kind":"number","value":14639,"string":"14,639"},"whole_article_abstract_length":{"kind":"number","value":341,"string":"341"},"other_sections_lengths":{"kind":"list like","value":[37,60,3538],"string":"[\n 37,\n 60,\n 3538\n]"},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["cds","clinicians","patient","alerts","interruptive","clinician","alert","information","stated","interruptive alerts"],"string":"[\n \"cds\",\n \"clinicians\",\n \"patient\",\n \"alerts\",\n \"interruptive\",\n \"clinician\",\n \"alert\",\n \"information\",\n \"stated\",\n \"interruptive alerts\"\n]"},"keybert_topics":{"kind":"list like","value":["cds facilitates clinicians","practices clinical decision","designing cds clinicians","computerised clinical decision","clinical decision support"],"string":"[\n \"cds facilitates clinicians\",\n \"practices clinical decision\",\n \"designing cds clinicians\",\n \"computerised clinical decision\",\n \"clinical decision support\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] clinical decision support systems | electronic prescribing | primary health care [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] clinical decision support systems | electronic prescribing | primary health care [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] clinical decision support systems | electronic prescribing | primary health care [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] clinical decision support systems | electronic prescribing | primary health care [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] clinical decision support systems | electronic prescribing | primary health care [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] clinical decision support systems | electronic prescribing | primary health care [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Decision Support Systems, Clinical | Female | Focus Groups | Grounded Theory | Humans | Male | Medical Order Entry Systems | Middle Aged | Practice Patterns, Physicians' | Primary Health Care [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Decision Support Systems, Clinical | Female | Focus Groups | Grounded Theory | Humans | Male | Medical Order Entry Systems | Middle Aged | Practice Patterns, Physicians' | Primary Health Care [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Decision Support Systems, Clinical | Female | Focus Groups | Grounded Theory | Humans | Male | Medical Order Entry Systems | Middle Aged | Practice Patterns, Physicians' | Primary Health Care [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Decision Support Systems, Clinical | Female | Focus Groups | Grounded Theory | Humans | Male | Medical Order Entry Systems | Middle Aged | Practice Patterns, Physicians' | Primary Health Care [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Decision Support Systems, Clinical | Female | Focus Groups | Grounded Theory | Humans | Male | Medical Order Entry Systems | Middle Aged | Practice Patterns, Physicians' | Primary Health Care [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Decision Support Systems, Clinical | Female | Focus Groups | Grounded Theory | Humans | Male | Medical Order Entry Systems | Middle Aged | Practice Patterns, Physicians' | Primary Health Care [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cds facilitates clinicians | practices clinical decision | designing cds clinicians | computerised clinical decision | clinical decision support [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cds facilitates clinicians | practices clinical decision | designing cds clinicians | computerised clinical decision | clinical decision support [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cds facilitates clinicians | practices clinical decision | designing cds clinicians | computerised clinical decision | clinical decision support [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cds facilitates clinicians | practices clinical decision | designing cds clinicians | computerised clinical decision | clinical decision support [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cds facilitates clinicians | practices clinical decision | designing cds clinicians | computerised clinical decision | clinical decision support [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cds facilitates clinicians | practices clinical decision | designing cds clinicians | computerised clinical decision | clinical decision support [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | patient | alerts | interruptive | clinician | alert | information | stated | interruptive alerts [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | patient | alerts | interruptive | clinician | alert | information | stated | interruptive alerts [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | patient | alerts | interruptive | clinician | alert | information | stated | interruptive alerts [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | patient | alerts | interruptive | clinician | alert | information | stated | interruptive alerts [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | patient | alerts | interruptive | clinician | alert | information | stated | interruptive alerts [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | patient | alerts | interruptive | clinician | alert | information | stated | interruptive alerts [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | design | end | user | cds design | end user | adoption | prototyping | medicine | preferences [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] focus | investigator | focus groups | groups | group | coding | themes | reviewed | investigator ket | ket [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] clinicians | cds | alerts | patient | interruptive | alert | stated | clinician | information | visit [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] clinicians | cds | alerts | patient | interruptive | alert | stated | clinician | visit | interruptive alerts [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | interruptive | alerts | design | patient | alert | clinician | information | cds design [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cds | clinicians | interruptive | alerts | design | patient | alert | clinician | information | cds design [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] CDS [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] clinicians | CDS ||| Three | clinicians ||| CDS ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 21 | four | CDS ||| ||| CDS ||| 12 | clinicians | CDS | seven | clinicians | CDS [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Clinicians | CDS ||| clinicians ||| CDS | clinicians | CDS ||| CDS | CDS [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CDS ||| clinicians | CDS ||| Three | clinicians ||| CDS ||| ||| 21 | four | CDS ||| ||| CDS ||| 12 | clinicians | CDS | seven | clinicians | CDS ||| Clinicians | CDS ||| clinicians ||| CDS | clinicians | CDS ||| CDS | CDS [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] CDS ||| clinicians | CDS ||| Three | clinicians ||| CDS ||| ||| 21 | four | CDS ||| ||| CDS ||| 12 | clinicians | CDS | seven | clinicians | CDS ||| Clinicians | CDS ||| clinicians ||| CDS | clinicians | CDS ||| CDS | CDS [SUMMARY]"}}},{"rowIdx":3355,"cells":{"title":{"kind":"string","value":"Rab11 regulates E-cadherin expression and induces cell transformation in colorectal carcinoma."},"pmid":{"kind":"string","value":"25117932"},"background_abstract":{"kind":"string","value":"In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Aged, 80 and over","Cadherins","Cell Line, Tumor","Cell Transformation, Neoplastic","Colorectal Neoplasms","Epithelial-Mesenchymal Transition","Female","Gene Expression Regulation, Neoplastic","HT29 Cells","Humans","Male","Middle Aged","rab GTP-Binding Proteins"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Cadherins\",\n \"Cell Line, Tumor\",\n \"Cell Transformation, Neoplastic\",\n \"Colorectal Neoplasms\",\n \"Epithelial-Mesenchymal Transition\",\n \"Female\",\n \"Gene Expression Regulation, Neoplastic\",\n \"HT29 Cells\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"rab GTP-Binding Proteins\"\n]"},"pmcid":{"kind":"string","value":"4137074"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\nThe study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\n Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\nHT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\n Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\nThe tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\n Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\nTissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\n Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\nHT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\n Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\nCells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\n Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\nResults are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\nThis study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\nTo examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\nIn order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\nHT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future."},"other_sections_titles":{"kind":"list like","value":["Background","Patients and ethics statements","Cell culture and transfection","Immunohistochemistry","Western blots","Trans-well cell migration assay","Immunofluorescence microscopy","Statistics","Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues","Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells","Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration","Rab11 induced colon cell transformation"],"string":"[\n \"Background\",\n \"Patients and ethics statements\",\n \"Cell culture and transfection\",\n \"Immunohistochemistry\",\n \"Western blots\",\n \"Trans-well cell migration assay\",\n \"Immunofluorescence microscopy\",\n \"Statistics\",\n \"Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues\",\n \"Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells\",\n \"Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration\",\n \"Rab11 induced colon cell transformation\"\n]"},"other_sections_texts":{"kind":"list like","value":["Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.","The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.","HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).","The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.","Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.","HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.","Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.","Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.","This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.","To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.","In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.","HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view."],"string":"[\n \"Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.\",\n \"The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\",\n \"HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\",\n \"The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\",\n \"Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\",\n \"HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\",\n \"Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\",\n \"Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\",\n \"This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\n\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\\n\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\n\\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\",\n \"To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\\n\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\",\n \"In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\\n\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\",\n \"HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\\n\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Patients and ethics statements","Cell culture and transfection","Immunohistochemistry","Western blots","Trans-well cell migration assay","Immunofluorescence microscopy","Statistics","Results","Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues","Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells","Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration","Rab11 induced colon cell transformation","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Patients and ethics statements\",\n \"Cell culture and transfection\",\n \"Immunohistochemistry\",\n \"Western blots\",\n \"Trans-well cell migration assay\",\n \"Immunofluorescence microscopy\",\n \"Statistics\",\n \"Results\",\n \"Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues\",\n \"Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells\",\n \"Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration\",\n \"Rab11 induced colon cell transformation\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study."," Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\nThe study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\n Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\nHT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\n Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\nThe tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\n Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\nTissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\n Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\nHT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\n Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\nCells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\n Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\nResults are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.","The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.","HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).","The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.","Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.","HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.","Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.","Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed."," Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\nThis study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\nTo examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\nIn order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\nHT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.","This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.","To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.","In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.","HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.","E-cadherin, which functions as an adhesion junction, has been demonstrated to be an important marker in cancer biology, as disassembly of E-cadherin is required for epithelial cell transformation and migration. E-cadherin is bound with most of the β-catenin located at the cytosolic side and also interacts with the actin cytoskeleton. β-catenin is the key component of the Wnt pathway that can be stimulated for cell growth and proliferation [10]. E-cadherin has also been shown to regulate initial cell-cell contacts formation and can further recruit exocyst components to the contacts for the formation of cell surface polarity [24]. Recently, the dark side of E-cadherin has been revealed gradually in pathological study of different cancers. The increased expression of E-cadherin was found to be associated with the formation of epithelium tumors. E-cadherin may play an important role in “collective cell migration” [25] and provide the “anchorage-independent” property of cancer cells [9]. In our study, significant over-expression of E-cadherin was found in colorectal cancer tissues. These findings are consistent with the previous study of Truant et al. [5], who demonstrated that the expression ratio of E-cadherin in reference to the normal adjacent tissue was increased in 57% of primary tumors. In contrast, only 30% of specimens were decreased in terms of the expression of E-cadherin in colorectal carcinoma. Regarding liver metastasis, a significantly higher expression of E-cadherin was found at stage I ~ II than at stage III ~ IV [6]. Therefore, the authors of the study suggested that the role of high expression of E-cadherin in colorectal cancer cells may be protective against widespread metastasis.\nAs Rab11, which functions as a recycling endosome, has been reported to play a role in regulating E-cadherin turnover in vitro, dysregulation may be associated with cancer formation. Our data subsequently demonstrated that the expression of Rab11 was also increased in colorectal cancer tissues. Co-overexpression of E-cadherin and Rab11 was found in 65.5% (74/113) of colorectal carcinomas, which suggests that dysregulation of both molecules might be associated with the occurrence and progression of colorectal carcinoma. The correlations of expressions of E-cadherin and Rab11 with stages of colorectal carcinoma were not statistically significant. However, the number of E-cadherin and Rab11 co-expressing cases was decreased in advanced stage cancers (81.8% in stage I, but 50% in stage IV). As we know, pathological progression from early adenomatous proliferation through adenomatous polyp, high grade dysplasia and ultimately, invasive colorectal carcinoma to metastasis occurs as a continuum. This progression coincides with the accumulation of multiple genetic alterations during neoplastic progression as originally described by Fearon and Volgelstein [26]. The regulatory alteration of E-cadherin and Rab11 may vary dynamically in the multistep model of progression of colorectal carcinoma in different stages.\nE-cadherin may participate in tumor progression through its associated cellular mechanisms. In epithelial cells, β-catenin acts as a linker between transmembrane E-cadherin and cytosolic actin fibers and forms a junctional adhesion complex. β-catenin is either stable connected to E-cadherin or translocated into the nucleus and binds to Lef/Tcf transcription factor upon stimulation for cell proliferation. Free cytosol β-catenin is degraded through ubiquitin-mediated degradation. Therefore, dynamic regulation of membrane E-cadherin and Rab11 may be necessary for cell proliferation and tumor growth. Rab11 was suggested to play a role in E-cadherin recycling and enhance membrane E-cadherin dynamics, which may be involved in cell signaling for cancer cell growth.\nThe in vitro experiments used GFP-Rab11 plasmid overexpressed in HT-29 cells induced cell transformation and migration. Intense staining of E-cadherin and Rab11 were also observed in infiltrated tumor nest cells. Actin dynamics are required for cell motility, and it has also been demonstrated that Rab11 interacts with RTK down-stream target Rac1 and controls moesin activity to regulate the endocytic cycle and actin cytoskeleton in cell migration and collective cell migration [27–29]. Rac1 has been shown to regulate actin nucleation via neural WASP (N-WASP) and the down-stream Arp2/3 complex [30]. A recent study has also shown that Rac1 activation is involved in twist1-induced cell transformation and migration [31]. Taken together, Rab11 plays a role not just in E-cadherin turnover but also improves the cytoskeleton reorganization for cell migration.\nDiscovering markers for cancer progression is important; however, the formation of cancer is multi-stepped, and specific protein expressions are restricted and responsible for different steps of EMT. Cell migration is an integrated process that requires continuous, coordinated formation and disassembly of adhesions. These processes are complex and require regulated interaction of numerous transcription factors such as snail/slug or twist, and activation of specific signaling pathways [32–35]. In this study, the expression of Rab11 was shown to be associated with cancer formation, and the expression of Rab11 also induced cell transformation, which is associated with cell motility. Moreover, Rab 11 could upregulate the expression of E-cadherin. Thus, Rab11 may be a useful maker together with E-cadherin for the diagnosis of colorectal cancer progression. However, the roles of other transcription factors in this mechanism have not been elucidated and require further investigation.\nIn the report of Anastasiadis et al. [9], several possible mechanisms were suggested to explain the positive role of E-cadherin in tumor progression. First, E-cadherin may cross-talk with EGFR signaling. Second, increased E-cadherin expression may up-regulate the expression of anti-apoptotic proteins, such as Bcl-2 and Bcl-xL. Third, E-cadherin may trigger the activation of the PI3K/AKT pathway through p85. In addition, extracellular N-terminal E-cadherin ectodomain “shedding” could be a role in tumor-promoting activities [9]. Our results showing that Rab11 up-regulated E-cadherin to induce the transformation of cancer cells might be another mechanism of alteration of neoplastic progression by E-cadherin.","Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future."],"string":"[\n \"Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.\",\n \" Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\\nThe study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\\n Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\\nHT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\\n Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\\nThe tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\\n Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\\nTissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\\n Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\\nHT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\\n Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\\nCells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\\n Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\\nResults are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\",\n \"The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\",\n \"HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\",\n \"The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\",\n \"Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\",\n \"HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\",\n \"Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\",\n \"Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\",\n \" Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\n\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\\n\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\n\\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\\nThis study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\n\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\\n\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\n\\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\\n Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\\n\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\\nTo examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\\n\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\\n Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\\n\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\\nIn order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\\n\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\\n Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\\n\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\\nHT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\\n\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\",\n \"This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\n\\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\\n\\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\\n\\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\",\n \"To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\\n\\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\",\n \"In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\\n\\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\",\n \"HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\\n\\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\",\n \"E-cadherin, which functions as an adhesion junction, has been demonstrated to be an important marker in cancer biology, as disassembly of E-cadherin is required for epithelial cell transformation and migration. E-cadherin is bound with most of the β-catenin located at the cytosolic side and also interacts with the actin cytoskeleton. β-catenin is the key component of the Wnt pathway that can be stimulated for cell growth and proliferation [10]. E-cadherin has also been shown to regulate initial cell-cell contacts formation and can further recruit exocyst components to the contacts for the formation of cell surface polarity [24]. Recently, the dark side of E-cadherin has been revealed gradually in pathological study of different cancers. The increased expression of E-cadherin was found to be associated with the formation of epithelium tumors. E-cadherin may play an important role in “collective cell migration” [25] and provide the “anchorage-independent” property of cancer cells [9]. In our study, significant over-expression of E-cadherin was found in colorectal cancer tissues. These findings are consistent with the previous study of Truant et al. [5], who demonstrated that the expression ratio of E-cadherin in reference to the normal adjacent tissue was increased in 57% of primary tumors. In contrast, only 30% of specimens were decreased in terms of the expression of E-cadherin in colorectal carcinoma. Regarding liver metastasis, a significantly higher expression of E-cadherin was found at stage I ~ II than at stage III ~ IV [6]. Therefore, the authors of the study suggested that the role of high expression of E-cadherin in colorectal cancer cells may be protective against widespread metastasis.\\nAs Rab11, which functions as a recycling endosome, has been reported to play a role in regulating E-cadherin turnover in vitro, dysregulation may be associated with cancer formation. Our data subsequently demonstrated that the expression of Rab11 was also increased in colorectal cancer tissues. Co-overexpression of E-cadherin and Rab11 was found in 65.5% (74/113) of colorectal carcinomas, which suggests that dysregulation of both molecules might be associated with the occurrence and progression of colorectal carcinoma. The correlations of expressions of E-cadherin and Rab11 with stages of colorectal carcinoma were not statistically significant. However, the number of E-cadherin and Rab11 co-expressing cases was decreased in advanced stage cancers (81.8% in stage I, but 50% in stage IV). As we know, pathological progression from early adenomatous proliferation through adenomatous polyp, high grade dysplasia and ultimately, invasive colorectal carcinoma to metastasis occurs as a continuum. This progression coincides with the accumulation of multiple genetic alterations during neoplastic progression as originally described by Fearon and Volgelstein [26]. The regulatory alteration of E-cadherin and Rab11 may vary dynamically in the multistep model of progression of colorectal carcinoma in different stages.\\nE-cadherin may participate in tumor progression through its associated cellular mechanisms. In epithelial cells, β-catenin acts as a linker between transmembrane E-cadherin and cytosolic actin fibers and forms a junctional adhesion complex. β-catenin is either stable connected to E-cadherin or translocated into the nucleus and binds to Lef/Tcf transcription factor upon stimulation for cell proliferation. Free cytosol β-catenin is degraded through ubiquitin-mediated degradation. Therefore, dynamic regulation of membrane E-cadherin and Rab11 may be necessary for cell proliferation and tumor growth. Rab11 was suggested to play a role in E-cadherin recycling and enhance membrane E-cadherin dynamics, which may be involved in cell signaling for cancer cell growth.\\nThe in vitro experiments used GFP-Rab11 plasmid overexpressed in HT-29 cells induced cell transformation and migration. Intense staining of E-cadherin and Rab11 were also observed in infiltrated tumor nest cells. Actin dynamics are required for cell motility, and it has also been demonstrated that Rab11 interacts with RTK down-stream target Rac1 and controls moesin activity to regulate the endocytic cycle and actin cytoskeleton in cell migration and collective cell migration [27–29]. Rac1 has been shown to regulate actin nucleation via neural WASP (N-WASP) and the down-stream Arp2/3 complex [30]. A recent study has also shown that Rac1 activation is involved in twist1-induced cell transformation and migration [31]. Taken together, Rab11 plays a role not just in E-cadherin turnover but also improves the cytoskeleton reorganization for cell migration.\\nDiscovering markers for cancer progression is important; however, the formation of cancer is multi-stepped, and specific protein expressions are restricted and responsible for different steps of EMT. Cell migration is an integrated process that requires continuous, coordinated formation and disassembly of adhesions. These processes are complex and require regulated interaction of numerous transcription factors such as snail/slug or twist, and activation of specific signaling pathways [32–35]. In this study, the expression of Rab11 was shown to be associated with cancer formation, and the expression of Rab11 also induced cell transformation, which is associated with cell motility. Moreover, Rab 11 could upregulate the expression of E-cadherin. Thus, Rab11 may be a useful maker together with E-cadherin for the diagnosis of colorectal cancer progression. However, the roles of other transcription factors in this mechanism have not been elucidated and require further investigation.\\nIn the report of Anastasiadis et al. [9], several possible mechanisms were suggested to explain the positive role of E-cadherin in tumor progression. First, E-cadherin may cross-talk with EGFR signaling. Second, increased E-cadherin expression may up-regulate the expression of anti-apoptotic proteins, such as Bcl-2 and Bcl-xL. Third, E-cadherin may trigger the activation of the PI3K/AKT pathway through p85. In addition, extracellular N-terminal E-cadherin ectodomain “shedding” could be a role in tumor-promoting activities [9]. Our results showing that Rab11 up-regulated E-cadherin to induce the transformation of cancer cells might be another mechanism of alteration of neoplastic progression by E-cadherin.\",\n \"Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,"results",null,null,null,null,"discussion","conclusions"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Rab11","E-cadherin","Colorectal carcinoma","Vesicle recycling","Epithelial mesenchymal transition"],"string":"[\n \"Rab11\",\n \"E-cadherin\",\n \"Colorectal carcinoma\",\n \"Vesicle recycling\",\n \"Epithelial mesenchymal transition\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nMost tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.\n\nMethods:\n Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\nThe study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\n Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\nHT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\n Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\nThe tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\n Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\nTissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\n Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\nHT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\n Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\nCells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\n Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\nResults are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\n\nPatients and ethics statements:\nThe study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\n\nCell culture and transfection:\nHT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\n\nImmunohistochemistry:\nThe tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\n\nWestern blots:\nTissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\n\nTrans-well cell migration assay:\nHT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\n\nImmunofluorescence microscopy:\nCells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\n\nStatistics:\nResults are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\n\nResults:\n Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\nThis study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\nTo examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\nIn order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\nHT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nElevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues:\nThis study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nProportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells:\nTo examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration:\nIn order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 induced colon cell transformation:\nHT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nDiscussion:\nE-cadherin, which functions as an adhesion junction, has been demonstrated to be an important marker in cancer biology, as disassembly of E-cadherin is required for epithelial cell transformation and migration. E-cadherin is bound with most of the β-catenin located at the cytosolic side and also interacts with the actin cytoskeleton. β-catenin is the key component of the Wnt pathway that can be stimulated for cell growth and proliferation [10]. E-cadherin has also been shown to regulate initial cell-cell contacts formation and can further recruit exocyst components to the contacts for the formation of cell surface polarity [24]. Recently, the dark side of E-cadherin has been revealed gradually in pathological study of different cancers. The increased expression of E-cadherin was found to be associated with the formation of epithelium tumors. E-cadherin may play an important role in “collective cell migration” [25] and provide the “anchorage-independent” property of cancer cells [9]. In our study, significant over-expression of E-cadherin was found in colorectal cancer tissues. These findings are consistent with the previous study of Truant et al. [5], who demonstrated that the expression ratio of E-cadherin in reference to the normal adjacent tissue was increased in 57% of primary tumors. In contrast, only 30% of specimens were decreased in terms of the expression of E-cadherin in colorectal carcinoma. Regarding liver metastasis, a significantly higher expression of E-cadherin was found at stage I ~ II than at stage III ~ IV [6]. Therefore, the authors of the study suggested that the role of high expression of E-cadherin in colorectal cancer cells may be protective against widespread metastasis.\nAs Rab11, which functions as a recycling endosome, has been reported to play a role in regulating E-cadherin turnover in vitro, dysregulation may be associated with cancer formation. Our data subsequently demonstrated that the expression of Rab11 was also increased in colorectal cancer tissues. Co-overexpression of E-cadherin and Rab11 was found in 65.5% (74/113) of colorectal carcinomas, which suggests that dysregulation of both molecules might be associated with the occurrence and progression of colorectal carcinoma. The correlations of expressions of E-cadherin and Rab11 with stages of colorectal carcinoma were not statistically significant. However, the number of E-cadherin and Rab11 co-expressing cases was decreased in advanced stage cancers (81.8% in stage I, but 50% in stage IV). As we know, pathological progression from early adenomatous proliferation through adenomatous polyp, high grade dysplasia and ultimately, invasive colorectal carcinoma to metastasis occurs as a continuum. This progression coincides with the accumulation of multiple genetic alterations during neoplastic progression as originally described by Fearon and Volgelstein [26]. The regulatory alteration of E-cadherin and Rab11 may vary dynamically in the multistep model of progression of colorectal carcinoma in different stages.\nE-cadherin may participate in tumor progression through its associated cellular mechanisms. In epithelial cells, β-catenin acts as a linker between transmembrane E-cadherin and cytosolic actin fibers and forms a junctional adhesion complex. β-catenin is either stable connected to E-cadherin or translocated into the nucleus and binds to Lef/Tcf transcription factor upon stimulation for cell proliferation. Free cytosol β-catenin is degraded through ubiquitin-mediated degradation. Therefore, dynamic regulation of membrane E-cadherin and Rab11 may be necessary for cell proliferation and tumor growth. Rab11 was suggested to play a role in E-cadherin recycling and enhance membrane E-cadherin dynamics, which may be involved in cell signaling for cancer cell growth.\nThe in vitro experiments used GFP-Rab11 plasmid overexpressed in HT-29 cells induced cell transformation and migration. Intense staining of E-cadherin and Rab11 were also observed in infiltrated tumor nest cells. Actin dynamics are required for cell motility, and it has also been demonstrated that Rab11 interacts with RTK down-stream target Rac1 and controls moesin activity to regulate the endocytic cycle and actin cytoskeleton in cell migration and collective cell migration [27–29]. Rac1 has been shown to regulate actin nucleation via neural WASP (N-WASP) and the down-stream Arp2/3 complex [30]. A recent study has also shown that Rac1 activation is involved in twist1-induced cell transformation and migration [31]. Taken together, Rab11 plays a role not just in E-cadherin turnover but also improves the cytoskeleton reorganization for cell migration.\nDiscovering markers for cancer progression is important; however, the formation of cancer is multi-stepped, and specific protein expressions are restricted and responsible for different steps of EMT. Cell migration is an integrated process that requires continuous, coordinated formation and disassembly of adhesions. These processes are complex and require regulated interaction of numerous transcription factors such as snail/slug or twist, and activation of specific signaling pathways [32–35]. In this study, the expression of Rab11 was shown to be associated with cancer formation, and the expression of Rab11 also induced cell transformation, which is associated with cell motility. Moreover, Rab 11 could upregulate the expression of E-cadherin. Thus, Rab11 may be a useful maker together with E-cadherin for the diagnosis of colorectal cancer progression. However, the roles of other transcription factors in this mechanism have not been elucidated and require further investigation.\nIn the report of Anastasiadis et al. [9], several possible mechanisms were suggested to explain the positive role of E-cadherin in tumor progression. First, E-cadherin may cross-talk with EGFR signaling. Second, increased E-cadherin expression may up-regulate the expression of anti-apoptotic proteins, such as Bcl-2 and Bcl-xL. Third, E-cadherin may trigger the activation of the PI3K/AKT pathway through p85. In addition, extracellular N-terminal E-cadherin ectodomain “shedding” could be a role in tumor-promoting activities [9]. Our results showing that Rab11 up-regulated E-cadherin to induce the transformation of cancer cells might be another mechanism of alteration of neoplastic progression by E-cadherin.\n\nConclusions:\nOur data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIn the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation.\n\nMethods:\nFor tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy.\n\nResults:\nIn immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration.\n\nConclusions:\nThis study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment."},"background_conclusion_text":{"kind":"string","value":"Background:\nMost tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.\n\nConclusions:\nOur data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIn the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation.\n\nMethods:\nFor tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy.\n\nResults:\nIn immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration.\n\nConclusions:\nThis study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment."},"whole_article_text_length":{"kind":"number","value":15522,"string":"15,522"},"whole_article_abstract_length":{"kind":"number","value":329,"string":"329"},"other_sections_lengths":{"kind":"list like","value":[713,251,167,409,159,125,209,59,1536,354,870,306],"string":"[\n 713,\n 251,\n 167,\n 409,\n 159,\n 125,\n 209,\n 59,\n 1536,\n 354,\n 870,\n 306\n]"},"num_sections":{"kind":"number","value":16,"string":"16"},"most_frequent_words":{"kind":"list like","value":["rab11","cadherin","cell","cells","tumor","cadherin rab11","expression","colon","patients","tissue"],"string":"[\n \"rab11\",\n \"cadherin\",\n \"cell\",\n \"cells\",\n \"tumor\",\n \"cadherin rab11\",\n \"expression\",\n \"colon\",\n \"patients\",\n \"tissue\"\n]"},"keybert_topics":{"kind":"list like","value":["cadherin tumor progression","cadherin trafficking epithelial","role cadherin carcinogenesis","epithelium tumors cadherin","metastasis cadherin protein"],"string":"[\n \"cadherin tumor progression\",\n \"cadherin trafficking epithelial\",\n \"role cadherin carcinogenesis\",\n \"epithelium tumors cadherin\",\n \"metastasis cadherin protein\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | cadherin | progression | cell | epithelial | tumor progression | role | recycling | metastasis | regulate [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | usa | cells | min | transfection | mm | dab | reagent | incubated | sections [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] rab11 | cadherin | tumor | cadherin rab11 | cells | cell | tissue | expression | figure | expressions [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] colorectal carcinoma | colorectal | carcinoma | promoted colon cancer cell | regulation dual | targeting progression | regulated cadherin expression promoted | targeting progression colorectal | targeting progression colorectal carcinoma | overexpression cadherin rab11 detected [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin | rab11 | cells | cell | tumor | patients | cadherin rab11 | expression | colon | gfp [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cadherin | rab11 | cells | cell | tumor | patients | cadherin rab11 | expression | colon | gfp [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] EMT ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 113 ||| GFP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 1.41 ± | 0.06 | 1.08 ± | 0.06 ||| 0.05 ||| 0.51 ± | 0.05 | 0.18 | 0.02 | 0.05 ||| 74 | 66.5% ||| GFP | HT-29 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] EMT ||| ||| ||| 113 ||| GFP ||| ||| 1.41 ± | 0.06 | 1.08 ± | 0.06 | 0.05 ||| 0.51 ± | 0.05 | 0.18 | 0.02 | 0.05 ||| 74 | 66.5% ||| GFP | HT-29 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] EMT ||| ||| ||| 113 ||| GFP ||| ||| 1.41 ± | 0.06 | 1.08 ± | 0.06 | 0.05 ||| 0.51 ± | 0.05 | 0.18 | 0.02 | 0.05 ||| 74 | 66.5% ||| GFP | HT-29 ||| ||| [SUMMARY]"}}},{"rowIdx":3356,"cells":{"title":{"kind":"string","value":"Reexamining treatment of high-grade T1 bladder cancer according to depth of lamina propria invasion: a prospective trial of 200 patients."},"pmid":{"kind":"string","value":"25535728"},"background_abstract":{"kind":"string","value":"Management of high-grade T1 (HGT1) bladder cancer represents a major challenge. We studied a treatment strategy according to substaging by depth of lamina propria invasion."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In this prospective observational cohort study, patients received initial transurethral resection (TUR), mitomycin-C, and BCG. Subjects with shallower lamina propria invasion (HGT1a) were followed without further surgery, whereas subjects with HGT1b received a second TUR. Association of clinical and histological features with outcomes (primary: progression; secondary: recurrence and cancer-specific survival) was assessed using Cox regression."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Median age was 71 years; 89.5% were males, with 89 (44.5%) cases T1a and 111 (55.5%) T1b. At median follow-up of 71 months, disease progression was observed in 31 (15.5%) and in univariate analysis, substaging, carcinoma in situ, tumour size, and tumour pattern predicted progression. On multivariate analysis only substaging, associated carcinoma in situ, and tumour size remained significant for progression."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In HGT1 bladder cancer, the strategy of performing a second TUR only in T1b cases results in a global low progression rate of 15.5%. Tumours deeply invading the lamina propria (HGT1b) showed a three-fold increase in risk of progression. Substaging should be routinely evaluated, with HGT1b cases being thoroughly evaluated for cystectomy. Inclusion in the TNM system should also be carefully considered."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Aged, 80 and over","Carcinoma in Situ","Carcinoma, Squamous Cell","Cohort Studies","Cystectomy","Female","Humans","Male","Middle Aged","Mucous Membrane","Neoplasm Grading","Neoplasm Invasiveness","Neoplasm Recurrence, Local","Reoperation","Urinary Bladder Neoplasms","Urinary Tract"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Carcinoma in Situ\",\n \"Carcinoma, Squamous Cell\",\n \"Cohort Studies\",\n \"Cystectomy\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Mucous Membrane\",\n \"Neoplasm Grading\",\n \"Neoplasm Invasiveness\",\n \"Neoplasm Recurrence, Local\",\n \"Reoperation\",\n \"Urinary Bladder Neoplasms\",\n \"Urinary Tract\"\n]"},"pmcid":{"kind":"string","value":"4453654"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.\nRecurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.\nThe association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.\nProgression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.\nThe low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.\nFinally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"With this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy."},"other_sections_titles":{"kind":"list like","value":[],"string":"[]"},"other_sections_texts":{"kind":"list like","value":[],"string":"[]"},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]"},"all_sections_titles":{"kind":"list like","value":["Materials and methods","Results","Discussion","Conclusions"],"string":"[\n \"Materials and methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Trial design and inclusion of this prospective observational cohort study began in April 2005. Since then, patients diagnosed at initial TUR with HGT1 BC were offered entry into this protocol. Transurethral resection BT included complete resection of all visible tumours, with a separate base biopsy. Cold cup biopsies to detect CIS were taken, depending on surgeon preference, in a standardised manner or according to EAU Guidelines (abnormal urothelium, non-papillary tumour appearance, or positive cytology) (Babjuk et al, 2013). All patients received a postoperative dose of intravesical mitomycin-C.\nTumours were graded according to the 2004 WHO system (2006) after pathological assessment of the total specimen. A two-tier system (Cottrell et al, 2007) was used to assess the depth of LP invasion: T1a when tumour involved the subepithelial connective tissue superficial to muscularis mucosae (MM); T1b when tumour was found at the level of or beyond MM. When MM was not identifiable, thick-walled blood vessels deep in LP served as a landmark, or alternatively location of MM in tumour-free tissue of the same TUR was used for reference. Only initial HGT1 tumours with a visible, clearly identifiable and disease-free muscularis propria were included in this study. Patients with LP invasion (T1) but without muscularis propria in the specimen represent ∼10% of all T1 in our centre. These cases, identified as Tx, did not fulfill the requirements to enter this protocol and, following guidelines, directly underwent a repeat resection.\nThis protocol was approved by the Hospital Ethics Committee and registered at clinicaltrials.gov (NCT02113501), and last evaluation of patients was performed in February 2014. Enrolled subjects received six weekly instillations of 2–8 × 108 UFC BCG-Tice (OncoTICE, Schering-Plough Canada Inc., Kirkland, QC, Canada) and, at 3 months after initial TUR, underwent either a second TUR post-BCG (T1b) or a cystoscopy plus cytology (T1a). Follow-up included BCG maintenance; the strategy is summarised in Figure 1 and described in detail in our previous report (Orsola et al, 2010). Baseline characteristics were compared between the T1a and T1b groups using Fisher's exact test.\nThe primary objective was the assessment of progression, recurrence, and CSS. Progression was defined as occurrence of an invasive tumour at post-BCG TUR, the development of muscle invasive or more advanced stage carcinoma, distant metastasis, or death from BC. Time to progression was defined as time from diagnosis of BC to the date when disease progression was observed, or censored on the last known date alive without progression. Recurrence was defined as the first evidence of any NMIBC detected on follow-up. Time to recurrence was defined as time from diagnosis of BC to the date when disease recurrence was observed, or censored at the last known date alive without disease recurrence, or at date of progression. Cancer-specific survival was defined as death from the same cancer or related causes.\nThe secondary objective was the assessment of prognostic factors. Eleven clinical and pathological variables were prospectively documented to evaluate their capacity to predict events. Kaplan–Meier analysis was used to summarise median time to recurrence and time to progression. Association of time to recurrence, time to progression, and CSS with the variables of interest was assessed using Cox regression models in univariate and multivariable models, reporting hazard ratios (HRs) and 95% CI.","Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.\nRecurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.\nThe association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.\nProgression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.\nThe low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.\nFinally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest.","In HGT1 BC, the proposed strategy of deferring a second TUR until after induction of BCG and doing so only in T1b cases, results in a global low progression rate of 15.5%. Cases in which tumours invaded the MM (T1b) had a three-fold increased risk of progression compared with those without MM invasion (T1a), even though the latter group was treated more conservatively. Our report goes on to confirm the role of CIS, tumour size, and tumour pattern as prognostic factors for worse outcomes in this group of high-risk non-muscle invasive BC.\nTo the best of our knowledge, this is the first report addressing the role of substaging HGT1 BC prospectively to guide therapeutic management. This novel strategy, together with the relatively large number of cases of initial HGT1, the long follow-up and the consistent treatment approach are the main strengths of our series. In addition, the evaluation of depth of tumour invasion was performed prospectively after TUR as part of the routine pathological report, whereas most reports on HGT1 are retrospective and observational. We cannot rule out bias due to patient selection since cases with recurrent tumours and those with no muscularis propria identified were not included; our series might also include some G2 cases (WHO 1974 classification) under the broader definition of HG BC. On the other hand, tumours that could have been understaged (early progressors or positive second TUR) were not excluded from the analysis in the understanding that this represents a more generalisable real-world practice, but might also be seen as a bias. Finally, our results are limited by the fact that the study was performed in a non-randomised manner. This limitation is widespread in the high-risk BC literature as most reports have retrospective observational designs, with few randomised studies (Martin-Doyle et al, 2014). Given the public nature of our health-care system, we chose to differentially allocate patients to treatment, using a more aggressive approach for T1b tumours, aiming to optimise resources.\nThe role of substaging has been well established throughout the past 20 years, with over 2500 published cases substaged according to invasion of the MM (Rouprêt et al, 2013). The relevance of this factor has been recently confirmed in the largest meta-analysis ever conducted on prognostic factors in HGT1 (Martin-Doyle et al, 2014). Because the evaluation of the depth of invasion is subject to interobserver and intraobserver variability, this parameter has been criticised for the last two decades, but there is growing evidence on the reproducibility of this evaluation. In a study specifically addressing this issue, Cottrell et al (2007) elegantly showed a high concordance rate among general pathologists after guidance based only in a presentation of the staging systems as described by the SIGN guideline. In their report, the median K concordance rate was 0.623, which is higher than that attained for Gleason score among pathologists (Bottke et al, 2013). In a more recent report, Rouprêt et al (2013) showed a reclassification rate for substaging of only 16% after central review of 587 HGT1 BC tumour sets from a multicentre data set. Other methods to substage HGT1 have been described using either millimetric evaluation of the depth of invasion (Cheng et al, 1999; Chang et al, 2012), measuring the size of the infiltrative tumour area as ⩽ or > 1 high-power field (Cheng et al, 1999; Bertz et al, 2011) or describing the extent of invasion (microinvasive or extensive) (van der Aa et al, 2005; van Rhijn et al, 2012). These alternative systems, however, have all been studied retrospectively in single data sets of patients and have not yet been externally validated (Martin-Doyle et al, 2014). A head-to-head comparison among these approaches in a large data set unfortunately is unavailable to date. Nonetheless, the fact that substaging remained a robust prognostic factor in our series (three-fold increase risk of progression in the adjusted multivariable model, and five-fold increase in the univariate model), even though patients with HGT1a underwent a more conservative approach demonstrates the magnitude of negative impact associated with depth of invasion.\nWe report a 15.5% progression rate, which is lower than the recently published pooled results of ∼20% progression rate for this high-risk group of tumours (van den Bosch and Alfred Witjes, 2011; Gontero et al, 2014; Martin-Doyle et al, 2014). Progression rates for high-risk BC have typically been considered to be over 30% and up to 50%, but more current data support that approximately one in five cases progress and will typically do so within 48 months of initial diagnosis (van den Bosch and Alfred Witjes, 2011). Our approach of proceeding with the second TUR after the 6-week induction of BCG (Orsola et al, 2010) was also used by Denzinger et al (2007) with an overall progression rate of 41% in 132 patients. We contend that the better outcome in our series might be attributable to a complete, deep, and radical initial TUR together with the exclusion of cases without clear muscularis propria in the TURBT specimen. It is possible the use of BCG (induction and maintenance) and the second TUR have also contributed an added therapeutic benefit. The combined influence of all these factors might explain the low rate of understaging we found in the second TUR (9.9%) as well as the low rate of positive findings at 3 months (Orsola et al, 2010), but the limitations mentioned previously should also be taken into account. Mitomycin-C might have only played a minor role given that no impact on progression has been demonstrated with this agent in all randomised published trials. Also the proposed approach in delaying the second TUR makes it impossible to accurately differentiate between true understaging and early invasive recurrence.\nOur study goes on to corroborate previous observations that CIS is a strong prognostic factor with a two-fold increase in risk of progression (Sylvester et al, 2006). In our report, the impact was even stronger with a 0.23 HR favouring lack of CIS. Interestingly, we observed a high rate of detection in those patients who, according to surgeon preference, underwent multiple random bladder biopsies. This adds to the debate of whether routine bladder biopsies, which we have previously defended (Orsola et al, 2010), should be standardised at the time of initial BC TUR (Millan-Rodriguez et al, 2000). The usefulness of random bladder biopsies in the broad population of NMIBC is controversial (Kiemeney et al, 1994), but our findings suggest that a high positive yield is obtained in HGT1 cases. Alternatively, the diagnosis of CIS is based on its detection in either peritumoral areas of the specimen or if suspicious areas are biopsied. This approach, currently recommended by guidelines (American Urological Association, 2007; Babjuk et al, 2013), is highly limited by interpretation and likely leads to underdetection of true CIS, even though fluorescence-guided TURBT might improve identification of suspicious areas of CIS (Witjes et al, 2014). Finally, the findings of our study suggest that larger tumours and those with a solid pattern are associated with a worse outcome in HGT1, are consistent with the relevance of these parameters in the broader NMIBC population. Combining these factors we observed that patients with T1b tumours that were larger than 3 cm and associated with CIS were 4.8 times more likely to progress than the rest.\nWe failed to demonstrate a worse prognosis for LVI and for females in any of the outcomes. The incidence of LVI was very low (under 5%) and, because its effects have been noted to correlate with depth of LP invasion, it is unclear whether LVI independently predicts outcome, or whether its detrimental impact is already captured by pathologic information on substaging (Martin-Doyle et al, 2014). Regarding gender, large population-based studies may be required to confirm a worse prognosis of females that has been suggested previously (Palou et al, 2012).\nIn summary, our report proposes a novel approach for HGT1 BC based on the selection of patients who will most benefit from a second TUR, specifically those with deep invasion of the MM (T1b). The main clinical implication of separately identifying almost half of HGT1 cases with T1a substaging as having a low (4%) progression rate is that they can be spared a second endoscopic procedure with no harm. More accurate risk stratification incorporating depth of invasion, associated CIS, and tumour size would enable improved resource allocation for reTUR. It would also help identify optimal candidates for prompt cystectomy, namely T1b tumours larger than 3 cm and with associated CIS. If the one in five HGT1 patients who will eventually develop a muscle invasive BC could be identified after diagnostic TUR, then cystectomy could be offered more selectively. Also, overtreatment and the morbidity and anxiety of undergoing additional procedures could be spared in patients with more indolent prognosis. Our results further support the notion that the ‘rule of thirds for HGT1' (i.e., one-third will never recur, one-third will require deferred cystectomy, and one-third will die of disease) is currently outdated.\nGiven the low impact of available scoring systems and biological markers (van Rhijn et al, 2012), and in view of this data, depth of invasion might be considered as the strongest prognostic factor for progression in HGT1. In addition, recent evidence suggests that histopathological classification into pTa, pT1a, and pT1b can be translated into a molecular signature, reflecting progressive dysregulation to more invasive stages (Descotes et al, 2013). Based on the prognostic impact of T1 substaging in the present and previous studies, we believe there is an argument for evolution of the current TNM classification. Together with tumour size and the detection of CIS an updated classification would help to identify the patients most suitable for cystectomy. Randomised control trials to evaluate the positive impact of this subclassification of tumours would also be beneficial.","With this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy."],"string":"[\n \"Trial design and inclusion of this prospective observational cohort study began in April 2005. Since then, patients diagnosed at initial TUR with HGT1 BC were offered entry into this protocol. Transurethral resection BT included complete resection of all visible tumours, with a separate base biopsy. Cold cup biopsies to detect CIS were taken, depending on surgeon preference, in a standardised manner or according to EAU Guidelines (abnormal urothelium, non-papillary tumour appearance, or positive cytology) (Babjuk et al, 2013). All patients received a postoperative dose of intravesical mitomycin-C.\\nTumours were graded according to the 2004 WHO system (2006) after pathological assessment of the total specimen. A two-tier system (Cottrell et al, 2007) was used to assess the depth of LP invasion: T1a when tumour involved the subepithelial connective tissue superficial to muscularis mucosae (MM); T1b when tumour was found at the level of or beyond MM. When MM was not identifiable, thick-walled blood vessels deep in LP served as a landmark, or alternatively location of MM in tumour-free tissue of the same TUR was used for reference. Only initial HGT1 tumours with a visible, clearly identifiable and disease-free muscularis propria were included in this study. Patients with LP invasion (T1) but without muscularis propria in the specimen represent ∼10% of all T1 in our centre. These cases, identified as Tx, did not fulfill the requirements to enter this protocol and, following guidelines, directly underwent a repeat resection.\\nThis protocol was approved by the Hospital Ethics Committee and registered at clinicaltrials.gov (NCT02113501), and last evaluation of patients was performed in February 2014. Enrolled subjects received six weekly instillations of 2–8 × 108 UFC BCG-Tice (OncoTICE, Schering-Plough Canada Inc., Kirkland, QC, Canada) and, at 3 months after initial TUR, underwent either a second TUR post-BCG (T1b) or a cystoscopy plus cytology (T1a). Follow-up included BCG maintenance; the strategy is summarised in Figure 1 and described in detail in our previous report (Orsola et al, 2010). Baseline characteristics were compared between the T1a and T1b groups using Fisher's exact test.\\nThe primary objective was the assessment of progression, recurrence, and CSS. Progression was defined as occurrence of an invasive tumour at post-BCG TUR, the development of muscle invasive or more advanced stage carcinoma, distant metastasis, or death from BC. Time to progression was defined as time from diagnosis of BC to the date when disease progression was observed, or censored on the last known date alive without progression. Recurrence was defined as the first evidence of any NMIBC detected on follow-up. Time to recurrence was defined as time from diagnosis of BC to the date when disease recurrence was observed, or censored at the last known date alive without disease recurrence, or at date of progression. Cancer-specific survival was defined as death from the same cancer or related causes.\\nThe secondary objective was the assessment of prognostic factors. Eleven clinical and pathological variables were prospectively documented to evaluate their capacity to predict events. Kaplan–Meier analysis was used to summarise median time to recurrence and time to progression. Association of time to recurrence, time to progression, and CSS with the variables of interest was assessed using Cox regression models in univariate and multivariable models, reporting hazard ratios (HRs) and 95% CI.\",\n \"Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.\\nRecurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.\\nThe association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.\\nProgression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.\\nThe low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.\\nFinally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest.\",\n \"In HGT1 BC, the proposed strategy of deferring a second TUR until after induction of BCG and doing so only in T1b cases, results in a global low progression rate of 15.5%. Cases in which tumours invaded the MM (T1b) had a three-fold increased risk of progression compared with those without MM invasion (T1a), even though the latter group was treated more conservatively. Our report goes on to confirm the role of CIS, tumour size, and tumour pattern as prognostic factors for worse outcomes in this group of high-risk non-muscle invasive BC.\\nTo the best of our knowledge, this is the first report addressing the role of substaging HGT1 BC prospectively to guide therapeutic management. This novel strategy, together with the relatively large number of cases of initial HGT1, the long follow-up and the consistent treatment approach are the main strengths of our series. In addition, the evaluation of depth of tumour invasion was performed prospectively after TUR as part of the routine pathological report, whereas most reports on HGT1 are retrospective and observational. We cannot rule out bias due to patient selection since cases with recurrent tumours and those with no muscularis propria identified were not included; our series might also include some G2 cases (WHO 1974 classification) under the broader definition of HG BC. On the other hand, tumours that could have been understaged (early progressors or positive second TUR) were not excluded from the analysis in the understanding that this represents a more generalisable real-world practice, but might also be seen as a bias. Finally, our results are limited by the fact that the study was performed in a non-randomised manner. This limitation is widespread in the high-risk BC literature as most reports have retrospective observational designs, with few randomised studies (Martin-Doyle et al, 2014). Given the public nature of our health-care system, we chose to differentially allocate patients to treatment, using a more aggressive approach for T1b tumours, aiming to optimise resources.\\nThe role of substaging has been well established throughout the past 20 years, with over 2500 published cases substaged according to invasion of the MM (Rouprêt et al, 2013). The relevance of this factor has been recently confirmed in the largest meta-analysis ever conducted on prognostic factors in HGT1 (Martin-Doyle et al, 2014). Because the evaluation of the depth of invasion is subject to interobserver and intraobserver variability, this parameter has been criticised for the last two decades, but there is growing evidence on the reproducibility of this evaluation. In a study specifically addressing this issue, Cottrell et al (2007) elegantly showed a high concordance rate among general pathologists after guidance based only in a presentation of the staging systems as described by the SIGN guideline. In their report, the median K concordance rate was 0.623, which is higher than that attained for Gleason score among pathologists (Bottke et al, 2013). In a more recent report, Rouprêt et al (2013) showed a reclassification rate for substaging of only 16% after central review of 587 HGT1 BC tumour sets from a multicentre data set. Other methods to substage HGT1 have been described using either millimetric evaluation of the depth of invasion (Cheng et al, 1999; Chang et al, 2012), measuring the size of the infiltrative tumour area as ⩽ or > 1 high-power field (Cheng et al, 1999; Bertz et al, 2011) or describing the extent of invasion (microinvasive or extensive) (van der Aa et al, 2005; van Rhijn et al, 2012). These alternative systems, however, have all been studied retrospectively in single data sets of patients and have not yet been externally validated (Martin-Doyle et al, 2014). A head-to-head comparison among these approaches in a large data set unfortunately is unavailable to date. Nonetheless, the fact that substaging remained a robust prognostic factor in our series (three-fold increase risk of progression in the adjusted multivariable model, and five-fold increase in the univariate model), even though patients with HGT1a underwent a more conservative approach demonstrates the magnitude of negative impact associated with depth of invasion.\\nWe report a 15.5% progression rate, which is lower than the recently published pooled results of ∼20% progression rate for this high-risk group of tumours (van den Bosch and Alfred Witjes, 2011; Gontero et al, 2014; Martin-Doyle et al, 2014). Progression rates for high-risk BC have typically been considered to be over 30% and up to 50%, but more current data support that approximately one in five cases progress and will typically do so within 48 months of initial diagnosis (van den Bosch and Alfred Witjes, 2011). Our approach of proceeding with the second TUR after the 6-week induction of BCG (Orsola et al, 2010) was also used by Denzinger et al (2007) with an overall progression rate of 41% in 132 patients. We contend that the better outcome in our series might be attributable to a complete, deep, and radical initial TUR together with the exclusion of cases without clear muscularis propria in the TURBT specimen. It is possible the use of BCG (induction and maintenance) and the second TUR have also contributed an added therapeutic benefit. The combined influence of all these factors might explain the low rate of understaging we found in the second TUR (9.9%) as well as the low rate of positive findings at 3 months (Orsola et al, 2010), but the limitations mentioned previously should also be taken into account. Mitomycin-C might have only played a minor role given that no impact on progression has been demonstrated with this agent in all randomised published trials. Also the proposed approach in delaying the second TUR makes it impossible to accurately differentiate between true understaging and early invasive recurrence.\\nOur study goes on to corroborate previous observations that CIS is a strong prognostic factor with a two-fold increase in risk of progression (Sylvester et al, 2006). In our report, the impact was even stronger with a 0.23 HR favouring lack of CIS. Interestingly, we observed a high rate of detection in those patients who, according to surgeon preference, underwent multiple random bladder biopsies. This adds to the debate of whether routine bladder biopsies, which we have previously defended (Orsola et al, 2010), should be standardised at the time of initial BC TUR (Millan-Rodriguez et al, 2000). The usefulness of random bladder biopsies in the broad population of NMIBC is controversial (Kiemeney et al, 1994), but our findings suggest that a high positive yield is obtained in HGT1 cases. Alternatively, the diagnosis of CIS is based on its detection in either peritumoral areas of the specimen or if suspicious areas are biopsied. This approach, currently recommended by guidelines (American Urological Association, 2007; Babjuk et al, 2013), is highly limited by interpretation and likely leads to underdetection of true CIS, even though fluorescence-guided TURBT might improve identification of suspicious areas of CIS (Witjes et al, 2014). Finally, the findings of our study suggest that larger tumours and those with a solid pattern are associated with a worse outcome in HGT1, are consistent with the relevance of these parameters in the broader NMIBC population. Combining these factors we observed that patients with T1b tumours that were larger than 3 cm and associated with CIS were 4.8 times more likely to progress than the rest.\\nWe failed to demonstrate a worse prognosis for LVI and for females in any of the outcomes. The incidence of LVI was very low (under 5%) and, because its effects have been noted to correlate with depth of LP invasion, it is unclear whether LVI independently predicts outcome, or whether its detrimental impact is already captured by pathologic information on substaging (Martin-Doyle et al, 2014). Regarding gender, large population-based studies may be required to confirm a worse prognosis of females that has been suggested previously (Palou et al, 2012).\\nIn summary, our report proposes a novel approach for HGT1 BC based on the selection of patients who will most benefit from a second TUR, specifically those with deep invasion of the MM (T1b). The main clinical implication of separately identifying almost half of HGT1 cases with T1a substaging as having a low (4%) progression rate is that they can be spared a second endoscopic procedure with no harm. More accurate risk stratification incorporating depth of invasion, associated CIS, and tumour size would enable improved resource allocation for reTUR. It would also help identify optimal candidates for prompt cystectomy, namely T1b tumours larger than 3 cm and with associated CIS. If the one in five HGT1 patients who will eventually develop a muscle invasive BC could be identified after diagnostic TUR, then cystectomy could be offered more selectively. Also, overtreatment and the morbidity and anxiety of undergoing additional procedures could be spared in patients with more indolent prognosis. Our results further support the notion that the ‘rule of thirds for HGT1' (i.e., one-third will never recur, one-third will require deferred cystectomy, and one-third will die of disease) is currently outdated.\\nGiven the low impact of available scoring systems and biological markers (van Rhijn et al, 2012), and in view of this data, depth of invasion might be considered as the strongest prognostic factor for progression in HGT1. In addition, recent evidence suggests that histopathological classification into pTa, pT1a, and pT1b can be translated into a molecular signature, reflecting progressive dysregulation to more invasive stages (Descotes et al, 2013). Based on the prognostic impact of T1 substaging in the present and previous studies, we believe there is an argument for evolution of the current TNM classification. Together with tumour size and the detection of CIS an updated classification would help to identify the patients most suitable for cystectomy. Randomised control trials to evaluate the positive impact of this subclassification of tumours would also be beneficial.\",\n \"With this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["materials|methods","results","discussion","conclusions"],"string":"[\n \"materials|methods\",\n \"results\",\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["high risk bladder cancer","HGT1","substaging","reTUR","prognosis"],"string":"[\n \"high risk bladder cancer\",\n \"HGT1\",\n \"substaging\",\n \"reTUR\",\n \"prognosis\"\n]"},"whole_article_text":{"kind":"string","value":"Materials and methods:\nTrial design and inclusion of this prospective observational cohort study began in April 2005. Since then, patients diagnosed at initial TUR with HGT1 BC were offered entry into this protocol. Transurethral resection BT included complete resection of all visible tumours, with a separate base biopsy. Cold cup biopsies to detect CIS were taken, depending on surgeon preference, in a standardised manner or according to EAU Guidelines (abnormal urothelium, non-papillary tumour appearance, or positive cytology) (Babjuk et al, 2013). All patients received a postoperative dose of intravesical mitomycin-C.\nTumours were graded according to the 2004 WHO system (2006) after pathological assessment of the total specimen. A two-tier system (Cottrell et al, 2007) was used to assess the depth of LP invasion: T1a when tumour involved the subepithelial connective tissue superficial to muscularis mucosae (MM); T1b when tumour was found at the level of or beyond MM. When MM was not identifiable, thick-walled blood vessels deep in LP served as a landmark, or alternatively location of MM in tumour-free tissue of the same TUR was used for reference. Only initial HGT1 tumours with a visible, clearly identifiable and disease-free muscularis propria were included in this study. Patients with LP invasion (T1) but without muscularis propria in the specimen represent ∼10% of all T1 in our centre. These cases, identified as Tx, did not fulfill the requirements to enter this protocol and, following guidelines, directly underwent a repeat resection.\nThis protocol was approved by the Hospital Ethics Committee and registered at clinicaltrials.gov (NCT02113501), and last evaluation of patients was performed in February 2014. Enrolled subjects received six weekly instillations of 2–8 × 108 UFC BCG-Tice (OncoTICE, Schering-Plough Canada Inc., Kirkland, QC, Canada) and, at 3 months after initial TUR, underwent either a second TUR post-BCG (T1b) or a cystoscopy plus cytology (T1a). Follow-up included BCG maintenance; the strategy is summarised in Figure 1 and described in detail in our previous report (Orsola et al, 2010). Baseline characteristics were compared between the T1a and T1b groups using Fisher's exact test.\nThe primary objective was the assessment of progression, recurrence, and CSS. Progression was defined as occurrence of an invasive tumour at post-BCG TUR, the development of muscle invasive or more advanced stage carcinoma, distant metastasis, or death from BC. Time to progression was defined as time from diagnosis of BC to the date when disease progression was observed, or censored on the last known date alive without progression. Recurrence was defined as the first evidence of any NMIBC detected on follow-up. Time to recurrence was defined as time from diagnosis of BC to the date when disease recurrence was observed, or censored at the last known date alive without disease recurrence, or at date of progression. Cancer-specific survival was defined as death from the same cancer or related causes.\nThe secondary objective was the assessment of prognostic factors. Eleven clinical and pathological variables were prospectively documented to evaluate their capacity to predict events. Kaplan–Meier analysis was used to summarise median time to recurrence and time to progression. Association of time to recurrence, time to progression, and CSS with the variables of interest was assessed using Cox regression models in univariate and multivariable models, reporting hazard ratios (HRs) and 95% CI.\n\nResults:\nTwo hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.\nRecurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.\nThe association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.\nProgression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.\nThe low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.\nFinally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest.\n\nDiscussion:\nIn HGT1 BC, the proposed strategy of deferring a second TUR until after induction of BCG and doing so only in T1b cases, results in a global low progression rate of 15.5%. Cases in which tumours invaded the MM (T1b) had a three-fold increased risk of progression compared with those without MM invasion (T1a), even though the latter group was treated more conservatively. Our report goes on to confirm the role of CIS, tumour size, and tumour pattern as prognostic factors for worse outcomes in this group of high-risk non-muscle invasive BC.\nTo the best of our knowledge, this is the first report addressing the role of substaging HGT1 BC prospectively to guide therapeutic management. This novel strategy, together with the relatively large number of cases of initial HGT1, the long follow-up and the consistent treatment approach are the main strengths of our series. In addition, the evaluation of depth of tumour invasion was performed prospectively after TUR as part of the routine pathological report, whereas most reports on HGT1 are retrospective and observational. We cannot rule out bias due to patient selection since cases with recurrent tumours and those with no muscularis propria identified were not included; our series might also include some G2 cases (WHO 1974 classification) under the broader definition of HG BC. On the other hand, tumours that could have been understaged (early progressors or positive second TUR) were not excluded from the analysis in the understanding that this represents a more generalisable real-world practice, but might also be seen as a bias. Finally, our results are limited by the fact that the study was performed in a non-randomised manner. This limitation is widespread in the high-risk BC literature as most reports have retrospective observational designs, with few randomised studies (Martin-Doyle et al, 2014). Given the public nature of our health-care system, we chose to differentially allocate patients to treatment, using a more aggressive approach for T1b tumours, aiming to optimise resources.\nThe role of substaging has been well established throughout the past 20 years, with over 2500 published cases substaged according to invasion of the MM (Rouprêt et al, 2013). The relevance of this factor has been recently confirmed in the largest meta-analysis ever conducted on prognostic factors in HGT1 (Martin-Doyle et al, 2014). Because the evaluation of the depth of invasion is subject to interobserver and intraobserver variability, this parameter has been criticised for the last two decades, but there is growing evidence on the reproducibility of this evaluation. In a study specifically addressing this issue, Cottrell et al (2007) elegantly showed a high concordance rate among general pathologists after guidance based only in a presentation of the staging systems as described by the SIGN guideline. In their report, the median K concordance rate was 0.623, which is higher than that attained for Gleason score among pathologists (Bottke et al, 2013). In a more recent report, Rouprêt et al (2013) showed a reclassification rate for substaging of only 16% after central review of 587 HGT1 BC tumour sets from a multicentre data set. Other methods to substage HGT1 have been described using either millimetric evaluation of the depth of invasion (Cheng et al, 1999; Chang et al, 2012), measuring the size of the infiltrative tumour area as ⩽ or > 1 high-power field (Cheng et al, 1999; Bertz et al, 2011) or describing the extent of invasion (microinvasive or extensive) (van der Aa et al, 2005; van Rhijn et al, 2012). These alternative systems, however, have all been studied retrospectively in single data sets of patients and have not yet been externally validated (Martin-Doyle et al, 2014). A head-to-head comparison among these approaches in a large data set unfortunately is unavailable to date. Nonetheless, the fact that substaging remained a robust prognostic factor in our series (three-fold increase risk of progression in the adjusted multivariable model, and five-fold increase in the univariate model), even though patients with HGT1a underwent a more conservative approach demonstrates the magnitude of negative impact associated with depth of invasion.\nWe report a 15.5% progression rate, which is lower than the recently published pooled results of ∼20% progression rate for this high-risk group of tumours (van den Bosch and Alfred Witjes, 2011; Gontero et al, 2014; Martin-Doyle et al, 2014). Progression rates for high-risk BC have typically been considered to be over 30% and up to 50%, but more current data support that approximately one in five cases progress and will typically do so within 48 months of initial diagnosis (van den Bosch and Alfred Witjes, 2011). Our approach of proceeding with the second TUR after the 6-week induction of BCG (Orsola et al, 2010) was also used by Denzinger et al (2007) with an overall progression rate of 41% in 132 patients. We contend that the better outcome in our series might be attributable to a complete, deep, and radical initial TUR together with the exclusion of cases without clear muscularis propria in the TURBT specimen. It is possible the use of BCG (induction and maintenance) and the second TUR have also contributed an added therapeutic benefit. The combined influence of all these factors might explain the low rate of understaging we found in the second TUR (9.9%) as well as the low rate of positive findings at 3 months (Orsola et al, 2010), but the limitations mentioned previously should also be taken into account. Mitomycin-C might have only played a minor role given that no impact on progression has been demonstrated with this agent in all randomised published trials. Also the proposed approach in delaying the second TUR makes it impossible to accurately differentiate between true understaging and early invasive recurrence.\nOur study goes on to corroborate previous observations that CIS is a strong prognostic factor with a two-fold increase in risk of progression (Sylvester et al, 2006). In our report, the impact was even stronger with a 0.23 HR favouring lack of CIS. Interestingly, we observed a high rate of detection in those patients who, according to surgeon preference, underwent multiple random bladder biopsies. This adds to the debate of whether routine bladder biopsies, which we have previously defended (Orsola et al, 2010), should be standardised at the time of initial BC TUR (Millan-Rodriguez et al, 2000). The usefulness of random bladder biopsies in the broad population of NMIBC is controversial (Kiemeney et al, 1994), but our findings suggest that a high positive yield is obtained in HGT1 cases. Alternatively, the diagnosis of CIS is based on its detection in either peritumoral areas of the specimen or if suspicious areas are biopsied. This approach, currently recommended by guidelines (American Urological Association, 2007; Babjuk et al, 2013), is highly limited by interpretation and likely leads to underdetection of true CIS, even though fluorescence-guided TURBT might improve identification of suspicious areas of CIS (Witjes et al, 2014). Finally, the findings of our study suggest that larger tumours and those with a solid pattern are associated with a worse outcome in HGT1, are consistent with the relevance of these parameters in the broader NMIBC population. Combining these factors we observed that patients with T1b tumours that were larger than 3 cm and associated with CIS were 4.8 times more likely to progress than the rest.\nWe failed to demonstrate a worse prognosis for LVI and for females in any of the outcomes. The incidence of LVI was very low (under 5%) and, because its effects have been noted to correlate with depth of LP invasion, it is unclear whether LVI independently predicts outcome, or whether its detrimental impact is already captured by pathologic information on substaging (Martin-Doyle et al, 2014). Regarding gender, large population-based studies may be required to confirm a worse prognosis of females that has been suggested previously (Palou et al, 2012).\nIn summary, our report proposes a novel approach for HGT1 BC based on the selection of patients who will most benefit from a second TUR, specifically those with deep invasion of the MM (T1b). The main clinical implication of separately identifying almost half of HGT1 cases with T1a substaging as having a low (4%) progression rate is that they can be spared a second endoscopic procedure with no harm. More accurate risk stratification incorporating depth of invasion, associated CIS, and tumour size would enable improved resource allocation for reTUR. It would also help identify optimal candidates for prompt cystectomy, namely T1b tumours larger than 3 cm and with associated CIS. If the one in five HGT1 patients who will eventually develop a muscle invasive BC could be identified after diagnostic TUR, then cystectomy could be offered more selectively. Also, overtreatment and the morbidity and anxiety of undergoing additional procedures could be spared in patients with more indolent prognosis. Our results further support the notion that the ‘rule of thirds for HGT1' (i.e., one-third will never recur, one-third will require deferred cystectomy, and one-third will die of disease) is currently outdated.\nGiven the low impact of available scoring systems and biological markers (van Rhijn et al, 2012), and in view of this data, depth of invasion might be considered as the strongest prognostic factor for progression in HGT1. In addition, recent evidence suggests that histopathological classification into pTa, pT1a, and pT1b can be translated into a molecular signature, reflecting progressive dysregulation to more invasive stages (Descotes et al, 2013). Based on the prognostic impact of T1 substaging in the present and previous studies, we believe there is an argument for evolution of the current TNM classification. Together with tumour size and the detection of CIS an updated classification would help to identify the patients most suitable for cystectomy. Randomised control trials to evaluate the positive impact of this subclassification of tumours would also be beneficial.\n\nConclusions:\nWith this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nManagement of high-grade T1 (HGT1) bladder cancer represents a major challenge. We studied a treatment strategy according to substaging by depth of lamina propria invasion.\n\nMethods:\nIn this prospective observational cohort study, patients received initial transurethral resection (TUR), mitomycin-C, and BCG. Subjects with shallower lamina propria invasion (HGT1a) were followed without further surgery, whereas subjects with HGT1b received a second TUR. Association of clinical and histological features with outcomes (primary: progression; secondary: recurrence and cancer-specific survival) was assessed using Cox regression.\n\nResults:\nMedian age was 71 years; 89.5% were males, with 89 (44.5%) cases T1a and 111 (55.5%) T1b. At median follow-up of 71 months, disease progression was observed in 31 (15.5%) and in univariate analysis, substaging, carcinoma in situ, tumour size, and tumour pattern predicted progression. On multivariate analysis only substaging, associated carcinoma in situ, and tumour size remained significant for progression.\n\nConclusions:\nIn HGT1 bladder cancer, the strategy of performing a second TUR only in T1b cases results in a global low progression rate of 15.5%. Tumours deeply invading the lamina propria (HGT1b) showed a three-fold increase in risk of progression. Substaging should be routinely evaluated, with HGT1b cases being thoroughly evaluated for cystectomy. Inclusion in the TNM system should also be carefully considered."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3299,"string":"3,299"},"whole_article_abstract_length":{"kind":"number","value":280,"string":"280"},"other_sections_lengths":{"kind":"list like","value":[],"string":"[]"},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["progression","patients","bc","tur","hgt1","cases","t1b","tumour","cis","recurrence"],"string":"[\n \"progression\",\n \"patients\",\n \"bc\",\n \"tur\",\n \"hgt1\",\n \"cases\",\n \"t1b\",\n \"tumour\",\n \"cis\",\n \"recurrence\"\n]"},"keybert_topics":{"kind":"list like","value":["resection visible tumours","substaging cis tumour","cytology t1a follow","intravesical mitomycin","dose intravesical mitomycin"],"string":"[\n \"resection visible tumours\",\n \"substaging cis tumour\",\n \"cytology t1a follow\",\n \"intravesical mitomycin\",\n \"dose intravesical mitomycin\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"null"},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"null"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] high risk bladder cancer | HGT1 | substaging | reTUR | prognosis [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] high risk bladder cancer | HGT1 | substaging | reTUR | prognosis [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] high risk bladder cancer | HGT1 | substaging | reTUR | prognosis [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"null"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma in Situ | Carcinoma, Squamous Cell | Cohort Studies | Cystectomy | Female | Humans | Male | Middle Aged | Mucous Membrane | Neoplasm Grading | Neoplasm Invasiveness | Neoplasm Recurrence, Local | Reoperation | Urinary Bladder Neoplasms | Urinary Tract [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma in Situ | Carcinoma, Squamous Cell | Cohort Studies | Cystectomy | Female | Humans | Male | Middle Aged | Mucous Membrane | Neoplasm Grading | Neoplasm Invasiveness | Neoplasm Recurrence, Local | Reoperation | Urinary Bladder Neoplasms | Urinary Tract [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma in Situ | Carcinoma, Squamous Cell | Cohort Studies | Cystectomy | Female | Humans | Male | Middle Aged | Mucous Membrane | Neoplasm Grading | Neoplasm Invasiveness | Neoplasm Recurrence, Local | Reoperation | Urinary Bladder Neoplasms | Urinary Tract [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"null"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] resection visible tumours | substaging cis tumour | cytology t1a follow | intravesical mitomycin | dose intravesical mitomycin [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] resection visible tumours | substaging cis tumour | cytology t1a follow | intravesical mitomycin | dose intravesical mitomycin [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] resection visible tumours | substaging cis tumour | cytology t1a follow | intravesical mitomycin | dose intravesical mitomycin [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"null"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] progression | patients | bc | tur | hgt1 | cases | t1b | tumour | cis | recurrence [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] progression | patients | bc | tur | hgt1 | cases | t1b | tumour | cis | recurrence [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] progression | patients | bc | tur | hgt1 | cases | t1b | tumour | cis | recurrence [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"null"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] progression | table | significant | year | t1b | cystectomy | analysis | cases | recurrence | bc [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tur | t1b | progression | strategies | strategies aid selection patients | initial tur mitc | t1a vs t1b | t1a vs t1b tumour | strategy complete initial tur | strategy complete initial [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] progression | t1b | tur | bc | patients | tumour | recurrence | cases | time | hgt1 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"null"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 71 years | 89.5% | 89 | 44.5% | 111 | 55.5% | T1b ||| 71 months | 31 | 15.5% ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] second | T1b | 15.5% ||| three-fold ||| ||| TNM [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| BCG ||| second ||| ||| ||| 71 years | 89.5% | 89 | 44.5% | 111 | 55.5% | T1b ||| 71 months | 31 | 15.5% ||| ||| second | T1b | 15.5% ||| three-fold ||| ||| TNM [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3357,"cells":{"title":{"kind":"string","value":"Current manufactured cigarette smoking and roll-your-own cigarette smoking in Thailand: findings from the 2009 Global Adult Tobacco Survey."},"pmid":{"kind":"string","value":"23530750"},"background_abstract":{"kind":"string","value":"Current smoking prevalence in Thailand decreased from 1991 to 2004 and since that time the prevalence has remained flat. It has been suggested that one of the reasons that the prevalence of current smoking in Thailand has stopped decreasing is due to the use of RYO cigarettes. The aim of this study was to examine characteristics of users of manufactured and RYO cigarettes and dual users in Thailand, in order to determine whether there are differences in the characteristics of users of the different products."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The 2009 Global Adult Tobacco Survey (GATS Thailand) provides detailed information on current smoking patterns. GATS Thailand used a nationally and regionally representative probability sample of 20,566 adults (ages 15 years and above) who were chosen through stratified three-stage cluster sampling and then interviewed face-to-face."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only. 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokers were more likely to live in rural areas. Smokers of manufactured cigarettes appeared to be more knowledgeable about the health risks of tobacco use. However, the difference was confounded with age and education; when demographic variables were controlled, the knowledge differences no longer remained. Smokers of manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appeared to be more addicted than the other two groups as measured by time to first cigarette."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Age Distribution","Aged","China","Female","Health Knowledge, Attitudes, Practice","Health Surveys","Humans","Male","Middle Aged","Multivariate Analysis","Population Surveillance","Prevalence","Product Packaging","Residence Characteristics","Sex Distribution","Smoking","Smoking Cessation","Socioeconomic Factors","Thailand","Tobacco Products"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Age Distribution\",\n \"Aged\",\n \"China\",\n \"Female\",\n \"Health Knowledge, Attitudes, Practice\",\n \"Health Surveys\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Multivariate Analysis\",\n \"Population Surveillance\",\n \"Prevalence\",\n \"Product Packaging\",\n \"Residence Characteristics\",\n \"Sex Distribution\",\n \"Smoking\",\n \"Smoking Cessation\",\n \"Socioeconomic Factors\",\n \"Thailand\",\n \"Tobacco Products\"\n]"},"pmcid":{"kind":"string","value":"3621680"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?"},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"From 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.\nThe target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.\nAll interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.\nThe interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.\nThe questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.\n Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\nUsed as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\n Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\nThe data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"A total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.\n Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\nThe prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\n Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\nThere were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\n Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\nTable 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\n Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nTable 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered."},"other_sections_titles":{"kind":"list like","value":["Background","Definition of variables","Analysis","Prevalence of smoking of tobacco products","Demographic variables","Knowledge","Addiction and quitting","Competing interest","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Definition of variables\",\n \"Analysis\",\n \"Prevalence of smoking of tobacco products\",\n \"Demographic variables\",\n \"Knowledge\",\n \"Addiction and quitting\",\n \"Competing interest\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?","Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.","The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.","The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.","There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.","Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.","Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.","All authors declare to have no competing interest.","SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\n"],"string":"[\n \"In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?\",\n \"Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\",\n \"The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\",\n \"The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\\n1 Smoked manufactured cigarettes only.\\n2 Smoked roll-your-own cigarettes only.\\n3 None or less than primary school.\\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\",\n \"There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\\nMultivariate analysis of type of smokers with demographic variables\\n*p-value of the test for importance of the factor.\",\n \"Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\\n*The number of knowledge questions correctly answered.\",\n \"Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\",\n \"All authors declare to have no competing interest.\",\n \"SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Definition of variables","Analysis","Results","Prevalence of smoking of tobacco products","Demographic variables","Knowledge","Addiction and quitting","Discussion","Conclusions","Competing interest","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Definition of variables\",\n \"Analysis\",\n \"Results\",\n \"Prevalence of smoking of tobacco products\",\n \"Demographic variables\",\n \"Knowledge\",\n \"Addiction and quitting\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interest\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?","From 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.\nThe target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.\nAll interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.\nThe interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.\nThe questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.\n Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\nUsed as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\n Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\nThe data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.","Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.","The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.","A total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.\n Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\nThe prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\n Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\nThere were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\n Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\nTable 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\n Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nTable 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.","The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.","There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.","Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.","Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.","In 2009, we found that the prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only and 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokes were also more likely to live in rural areas. When the demographic variables were controlled, there were no differences between the three groups of smoking either in the percentage who answered all seven questions correctly or in total knowledge score. Respondents who smoked only manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appear to be more addicted than the other two groups as measured by time to first cigarette. These analyses provide recent estimates than have been reported and present information on knowledge and addiction that has not been reported in earlier work.\nWhile comparisons with other surveys such as that National Cigarette Smoking and Alcohol Drinking Survey and the ITC survey can be made, caution should be exercised in that sampling and question wording differed. However, the findings from this study closely mirror those from Young et al. [6] who found that any RYO use was associated with living in rural areas, older average age, lower level of education, male gender, not being in paid work, slightly lower consumption of cigarettes, higher social acceptability of smoking, and positive attitudes toward tobacco regulation.\nThe proportion of RYO cigarette smokers is higher in Thailand than in its neighbors, such Vietnam (1.1%) and China (2.3%) [11,12]. As Young et al. [6] noted there are many factors that may play a role in differences in prevalence in different countries including cultural, legislative and economic factors.\nOne important factor in stimulating the decrease in the prevalence of current smoking in Thailand can be found in the increase in the taxation of manufactured cigarettes [13,14]; the excise tax for manufactured cigarettes has increased from 55% in 1992 to 85% in 2009 [13]. However, the taxation rate for blended shredded tobacco, which is used in RYO cigarettes, has remained as low as 500 Thai Baht (approximately 16.5 U.S. dollars, per kilogram since October 13, 1999), leading to the possibility that some smokers of manufactured cigarettes may have started smoking more RYO cigarettes and reduced or eliminated their smoking of manufactured cigarettes.\nSince there were differences noted in rural areas and for those with lower education and income, it may be that more focused education programs should be developed focusing on RYO smoking. Enhancement of health education programs with information on the harmful effects of both types of cigarettes may provide much needed information for smokers.\nThere are some limitations that should be noted about this survey. Questions on reasons for use of RYO were not included and, therefore, the explanations about economic and cultural facts are speculative. Given that smoking among women does not appear to be acceptable, the estimate of prevalence for women may be subject to some misreporting. Some of the strengths include the rigorous procedures for sampling and interviewing used in GATS Thailand and the high response rate.\nThailand has implemented comprehensive tobacco control interventions, including educational campaigns in the media, legislation to ban smoking in public places, increases in the cigarette excise tax, bans on advertising, and the use of a pictorial warning labels on manufactured cigarettes. Taken together, these legislative measures may have changed social norms and perceptions about tobacco use in Thailand. Meanwhile, lack of comparable policies in the control of RYO cigarettes may be playing a role in the overall prevalence of smoking in Thailand, diluting the success of existing programs.","There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered.","All authors declare to have no competing interest.","SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\n"],"string":"[\n \"In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?\",\n \"From 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.\\nThe target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.\\nAll interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.\\nThe interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.\\nThe questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.\\n Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\\nUsed as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\\n Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\\nThe data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\",\n \"Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\",\n \"The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\",\n \"A total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.\\n Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\\n1 Smoked manufactured cigarettes only.\\n2 Smoked roll-your-own cigarettes only.\\n3 None or less than primary school.\\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\\nThe prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\\n1 Smoked manufactured cigarettes only.\\n2 Smoked roll-your-own cigarettes only.\\n3 None or less than primary school.\\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\\n Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\\nMultivariate analysis of type of smokers with demographic variables\\n*p-value of the test for importance of the factor.\\nThere were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\\nMultivariate analysis of type of smokers with demographic variables\\n*p-value of the test for importance of the factor.\\n Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\\n*The number of knowledge questions correctly answered.\\nTable 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\\n*The number of knowledge questions correctly answered.\\n Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\\nTable 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\",\n \"The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\\n1 Smoked manufactured cigarettes only.\\n2 Smoked roll-your-own cigarettes only.\\n3 None or less than primary school.\\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\",\n \"There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\\nMultivariate analysis of type of smokers with demographic variables\\n*p-value of the test for importance of the factor.\",\n \"Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\\n*The number of knowledge questions correctly answered.\",\n \"Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\",\n \"In 2009, we found that the prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only and 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokes were also more likely to live in rural areas. When the demographic variables were controlled, there were no differences between the three groups of smoking either in the percentage who answered all seven questions correctly or in total knowledge score. Respondents who smoked only manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appear to be more addicted than the other two groups as measured by time to first cigarette. These analyses provide recent estimates than have been reported and present information on knowledge and addiction that has not been reported in earlier work.\\nWhile comparisons with other surveys such as that National Cigarette Smoking and Alcohol Drinking Survey and the ITC survey can be made, caution should be exercised in that sampling and question wording differed. However, the findings from this study closely mirror those from Young et al. [6] who found that any RYO use was associated with living in rural areas, older average age, lower level of education, male gender, not being in paid work, slightly lower consumption of cigarettes, higher social acceptability of smoking, and positive attitudes toward tobacco regulation.\\nThe proportion of RYO cigarette smokers is higher in Thailand than in its neighbors, such Vietnam (1.1%) and China (2.3%) [11,12]. As Young et al. [6] noted there are many factors that may play a role in differences in prevalence in different countries including cultural, legislative and economic factors.\\nOne important factor in stimulating the decrease in the prevalence of current smoking in Thailand can be found in the increase in the taxation of manufactured cigarettes [13,14]; the excise tax for manufactured cigarettes has increased from 55% in 1992 to 85% in 2009 [13]. However, the taxation rate for blended shredded tobacco, which is used in RYO cigarettes, has remained as low as 500 Thai Baht (approximately 16.5 U.S. dollars, per kilogram since October 13, 1999), leading to the possibility that some smokers of manufactured cigarettes may have started smoking more RYO cigarettes and reduced or eliminated their smoking of manufactured cigarettes.\\nSince there were differences noted in rural areas and for those with lower education and income, it may be that more focused education programs should be developed focusing on RYO smoking. Enhancement of health education programs with information on the harmful effects of both types of cigarettes may provide much needed information for smokers.\\nThere are some limitations that should be noted about this survey. Questions on reasons for use of RYO were not included and, therefore, the explanations about economic and cultural facts are speculative. Given that smoking among women does not appear to be acceptable, the estimate of prevalence for women may be subject to some misreporting. Some of the strengths include the rigorous procedures for sampling and interviewing used in GATS Thailand and the high response rate.\\nThailand has implemented comprehensive tobacco control interventions, including educational campaigns in the media, legislation to ban smoking in public places, increases in the cigarette excise tax, bans on advertising, and the use of a pictorial warning labels on manufactured cigarettes. Taken together, these legislative measures may have changed social norms and perceptions about tobacco use in Thailand. Meanwhile, lack of comparable policies in the control of RYO cigarettes may be playing a role in the overall prevalence of smoking in Thailand, diluting the success of existing programs.\",\n \"There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered.\",\n \"All authors declare to have no competing interest.\",\n \"SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,"results",null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Thailand","Manufactured cigarettes","Roll-your-own cigarettes (RYO)","Prevalence of current smoking"],"string":"[\n \"Thailand\",\n \"Manufactured cigarettes\",\n \"Roll-your-own cigarettes (RYO)\",\n \"Prevalence of current smoking\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nIn Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?\n\nMethods:\nFrom 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.\nThe target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.\nAll interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.\nThe interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.\nThe questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.\n Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\nUsed as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\n Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\nThe data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\n\nDefinition of variables:\nUsed as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\n\nAnalysis:\nThe data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\n\nResults:\nA total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.\n Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\nThe prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\n Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\nThere were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\n Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\nTable 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\n Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nTable 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\n\nPrevalence of smoking of tobacco products:\nThe prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\n\nDemographic variables:\nThere were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\n\nKnowledge:\nTable 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\n\nAddiction and quitting:\nTable 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\n\nDiscussion:\nIn 2009, we found that the prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only and 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokes were also more likely to live in rural areas. When the demographic variables were controlled, there were no differences between the three groups of smoking either in the percentage who answered all seven questions correctly or in total knowledge score. Respondents who smoked only manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appear to be more addicted than the other two groups as measured by time to first cigarette. These analyses provide recent estimates than have been reported and present information on knowledge and addiction that has not been reported in earlier work.\nWhile comparisons with other surveys such as that National Cigarette Smoking and Alcohol Drinking Survey and the ITC survey can be made, caution should be exercised in that sampling and question wording differed. However, the findings from this study closely mirror those from Young et al. [6] who found that any RYO use was associated with living in rural areas, older average age, lower level of education, male gender, not being in paid work, slightly lower consumption of cigarettes, higher social acceptability of smoking, and positive attitudes toward tobacco regulation.\nThe proportion of RYO cigarette smokers is higher in Thailand than in its neighbors, such Vietnam (1.1%) and China (2.3%) [11,12]. As Young et al. [6] noted there are many factors that may play a role in differences in prevalence in different countries including cultural, legislative and economic factors.\nOne important factor in stimulating the decrease in the prevalence of current smoking in Thailand can be found in the increase in the taxation of manufactured cigarettes [13,14]; the excise tax for manufactured cigarettes has increased from 55% in 1992 to 85% in 2009 [13]. However, the taxation rate for blended shredded tobacco, which is used in RYO cigarettes, has remained as low as 500 Thai Baht (approximately 16.5 U.S. dollars, per kilogram since October 13, 1999), leading to the possibility that some smokers of manufactured cigarettes may have started smoking more RYO cigarettes and reduced or eliminated their smoking of manufactured cigarettes.\nSince there were differences noted in rural areas and for those with lower education and income, it may be that more focused education programs should be developed focusing on RYO smoking. Enhancement of health education programs with information on the harmful effects of both types of cigarettes may provide much needed information for smokers.\nThere are some limitations that should be noted about this survey. Questions on reasons for use of RYO were not included and, therefore, the explanations about economic and cultural facts are speculative. Given that smoking among women does not appear to be acceptable, the estimate of prevalence for women may be subject to some misreporting. Some of the strengths include the rigorous procedures for sampling and interviewing used in GATS Thailand and the high response rate.\nThailand has implemented comprehensive tobacco control interventions, including educational campaigns in the media, legislation to ban smoking in public places, increases in the cigarette excise tax, bans on advertising, and the use of a pictorial warning labels on manufactured cigarettes. Taken together, these legislative measures may have changed social norms and perceptions about tobacco use in Thailand. Meanwhile, lack of comparable policies in the control of RYO cigarettes may be playing a role in the overall prevalence of smoking in Thailand, diluting the success of existing programs.\n\nConclusions:\nThere are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered.\n\nCompeting interest:\nAll authors declare to have no competing interest.\n\nAuthors’ contributions:\nSB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCurrent smoking prevalence in Thailand decreased from 1991 to 2004 and since that time the prevalence has remained flat. It has been suggested that one of the reasons that the prevalence of current smoking in Thailand has stopped decreasing is due to the use of RYO cigarettes. The aim of this study was to examine characteristics of users of manufactured and RYO cigarettes and dual users in Thailand, in order to determine whether there are differences in the characteristics of users of the different products.\n\nMethods:\nThe 2009 Global Adult Tobacco Survey (GATS Thailand) provides detailed information on current smoking patterns. GATS Thailand used a nationally and regionally representative probability sample of 20,566 adults (ages 15 years and above) who were chosen through stratified three-stage cluster sampling and then interviewed face-to-face.\n\nResults:\nThe prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only. 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokers were more likely to live in rural areas. Smokers of manufactured cigarettes appeared to be more knowledgeable about the health risks of tobacco use. However, the difference was confounded with age and education; when demographic variables were controlled, the knowledge differences no longer remained. Smokers of manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appeared to be more addicted than the other two groups as measured by time to first cigarette.\n\nConclusions:\nThere appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes."},"background_conclusion_text":{"kind":"string","value":"Background:\nIn Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?\n\nConclusions:\nThere are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nCurrent smoking prevalence in Thailand decreased from 1991 to 2004 and since that time the prevalence has remained flat. It has been suggested that one of the reasons that the prevalence of current smoking in Thailand has stopped decreasing is due to the use of RYO cigarettes. The aim of this study was to examine characteristics of users of manufactured and RYO cigarettes and dual users in Thailand, in order to determine whether there are differences in the characteristics of users of the different products.\n\nMethods:\nThe 2009 Global Adult Tobacco Survey (GATS Thailand) provides detailed information on current smoking patterns. GATS Thailand used a nationally and regionally representative probability sample of 20,566 adults (ages 15 years and above) who were chosen through stratified three-stage cluster sampling and then interviewed face-to-face.\n\nResults:\nThe prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only. 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokers were more likely to live in rural areas. Smokers of manufactured cigarettes appeared to be more knowledgeable about the health risks of tobacco use. However, the difference was confounded with age and education; when demographic variables were controlled, the knowledge differences no longer remained. Smokers of manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appeared to be more addicted than the other two groups as measured by time to first cigarette.\n\nConclusions:\nThere appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes."},"whole_article_text_length":{"kind":"number","value":7795,"string":"7,795"},"whole_article_abstract_length":{"kind":"number","value":381,"string":"381"},"other_sections_lengths":{"kind":"list like","value":[472,192,282,309,509,200,326,9,74,16],"string":"[\n 472,\n 192,\n 282,\n 309,\n 509,\n 200,\n 326,\n 9,\n 74,\n 16\n]"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["cigarettes","ryo","manufactured","men","smokers","cigarette","users","smoking","manufactured cigarettes","education"],"string":"[\n \"cigarettes\",\n \"ryo\",\n \"manufactured\",\n \"men\",\n \"smokers\",\n \"cigarette\",\n \"users\",\n \"smoking\",\n \"manufactured cigarettes\",\n \"education\"\n]"},"keybert_topics":{"kind":"list like","value":["smoking thailand diluting","smoking thai adults","smokers thailand malaysia","smoking thailand decreased","tobacco use thailand"],"string":"[\n \"smoking thailand diluting\",\n \"smoking thai adults\",\n \"smokers thailand malaysia\",\n \"smoking thailand decreased\",\n \"tobacco use thailand\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] prevalence | use | smoking | current | period | economic | young | prevalence current | tobacco | decreased [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] selected | use | tobacco | interview | knowledge | cancer | usd | sample | sampling | household [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | men | smokers | manufactured | ryo | women | manufactured cigarettes | cigarette | smoked | users [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ryo | tobacco | ryo cigarettes addition adoption | population subgroups thailand | manufactured ryo cigarettes | thailand include information | thailand include | targeted cessation prevention efforts | targeted cessation prevention | targeted cessation [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | manufactured | ryo | smokers | men | manufactured cigarettes | smoking | cigarette | use | women [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cigarettes | manufactured | ryo | smokers | men | manufactured cigarettes | smoking | cigarette | use | women [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | 1991 | 2004 ||| one | Thailand | RYO ||| RYO | Thailand [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 2009 | Global Adult Tobacco Survey | Thailand ||| 20,566 | (ages 15 years | three [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Thai | 45.6% | 3.1% ||| 18.4% | 1.0% | 15.8% | 1.7% | RYO ||| 11.2% | 0.1% | RYO ||| RYO ||| RYO ||| ||| ||| RYO | the next month ||| RYO | two | first [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | RYO [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | 1991 | 2004 ||| one | Thailand | RYO ||| RYO | Thailand ||| 2009 | Global Adult Tobacco Survey | Thailand ||| 20,566 | (ages 15 years | three ||| ||| Thai | 45.6% | 3.1% ||| 18.4% | 1.0% | 15.8% | 1.7% | RYO ||| 11.2% | 0.1% | RYO ||| RYO ||| RYO ||| ||| ||| RYO | the next month ||| RYO | two | first ||| Thailand | RYO [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Thailand | 1991 | 2004 ||| one | Thailand | RYO ||| RYO | Thailand ||| 2009 | Global Adult Tobacco Survey | Thailand ||| 20,566 | (ages 15 years | three ||| ||| Thai | 45.6% | 3.1% ||| 18.4% | 1.0% | 15.8% | 1.7% | RYO ||| 11.2% | 0.1% | RYO ||| RYO ||| RYO ||| ||| ||| RYO | the next month ||| RYO | two | first ||| Thailand | RYO [SUMMARY]"}}},{"rowIdx":3358,"cells":{"title":{"kind":"string","value":"Clinical Profiles of Asians with NAFLD: A Systematic Review and Meta-Analysis."},"pmid":{"kind":"string","value":"34942625"},"background_abstract":{"kind":"string","value":"NAFLD is increasingly prevalent in Asia, where people suffer more metabolic comorbidities at a lower body mass index (BMI), suggesting potential differences in their clinical profile. Therefore, we attempted to characterize the clinical profile of Asians with NAFLD via a meta-analytic approach."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"We searched PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019. Two authors independently reviewed and selected 104 articles (2,247,754 persons) that identified NAFLD in Asians and reported relevant data, especially BMI and ALT, and excluded individuals with other liver disease and excessive alcohol consumption. Individual patient-level data were obtained from seven cohorts in Asia to complement meta-analyzed data."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Overall, the mean age was 52.07 (95% CI: 51.28-52.85) years, with those from Southeast Asia (42.66, 95% CI: 32.23-53.11) being significantly younger. The mean BMI was 26.2 kg/m2, higher in moderate-severe versus mild hepatic steatosis (28.3 vs. 25.7) patients and NFS ≥ -1.455 versus <-1.455 (27.09 vs. 26.02), with 34% having nonobese NAFLD. The mean ALT was 31.74 U/L, higher in NFS < -1.455 versus ≥-1.455 (33.74 vs. 27.83), though no differences were found by obesity or steatosis severity. The majority of males (85.7%) and females (60.7%) had normal to minimally elevated ALT (1-1.5 × 95% ULN). Individual patient-level data analysis (N = 7,668) demonstrated similar results."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"About one-third of Asians with NAFLD were nonobese, and the majority did not have markedly elevated ALT. Therefore, abnormal ALT or BMI is not recommended as a criterion for NAFLD screening in this population. Additionally, there were significant differences in the clinical profiles of NAFLD among the different regions of Asia."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Male","Female","Humans","Middle Aged","Non-alcoholic Fatty Liver Disease","Body Mass Index","Obesity","Comorbidity"],"string":"[\n \"Male\",\n \"Female\",\n \"Humans\",\n \"Middle Aged\",\n \"Non-alcoholic Fatty Liver Disease\",\n \"Body Mass Index\",\n \"Obesity\",\n \"Comorbidity\"\n]"},"pmcid":{"kind":"string","value":"9808705"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.\nThrough a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Search Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nThis study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nSystemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nTwo authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nIndividual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nIn addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nStatistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\nWe calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Study-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nAs described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nIndividual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\nOverall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5)."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed."},"other_sections_titles":{"kind":"list like","value":["Search Strategy","Systemic Literature Review, Data Extraction, and Quality Assessment","Individual Patient Data","Statistical Analysis","Study-Level Analysis","Individual Patient Data Analysis","Statement of Ethics","Funding Sources","Author Contributions"],"string":"[\n \"Search Strategy\",\n \"Systemic Literature Review, Data Extraction, and Quality Assessment\",\n \"Individual Patient Data\",\n \"Statistical Analysis\",\n \"Study-Level Analysis\",\n \"Individual Patient Data Analysis\",\n \"Statement of Ethics\",\n \"Funding Sources\",\n \"Author Contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).","Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.","In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.","We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).","As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).","Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).","The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).","There is no funding to disclose.","All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision."],"string":"[\n \"This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\",\n \"Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\",\n \"In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\",\n \"We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\",\n \"As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\",\n \"Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\",\n \"The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).\",\n \"There is no funding to disclose.\",\n \"All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Search Strategy","Systemic Literature Review, Data Extraction, and Quality Assessment","Individual Patient Data","Statistical Analysis","Results","Study-Level Analysis","Individual Patient Data Analysis","Discussion","Conclusion","Statement of Ethics","Conflict of Interest Statement","Funding Sources","Author Contributions","Data Availability Statement","Supplementary Material"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Search Strategy\",\n \"Systemic Literature Review, Data Extraction, and Quality Assessment\",\n \"Individual Patient Data\",\n \"Statistical Analysis\",\n \"Results\",\n \"Study-Level Analysis\",\n \"Individual Patient Data Analysis\",\n \"Discussion\",\n \"Conclusion\",\n \"Statement of Ethics\",\n \"Conflict of Interest Statement\",\n \"Funding Sources\",\n \"Author Contributions\",\n \"Data Availability Statement\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.\nThrough a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.","Search Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nThis study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nSystemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nTwo authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nIndividual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nIn addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nStatistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\nWe calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).","This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).","Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.","In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.","We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).","Study-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nAs described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nIndividual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\nOverall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).","As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).","Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).","Our study found that patients with NAFLD in Asia were approximately 52 years old, overweight by Asian cutoffs, about 34% nonobese, with slightly elevated ALT and total cholesterol levels, around 17% diabetic, and 85% with NFS < −1.455 indicating a very low probability of having advanced fibrosis. Males tended to be approximately 4 years younger than females, but their BMIs were similar. As with other populations, neither the absence of obesity nor an elevated ALT should be used to guide the screening or diagnosis of NAFLD for Asians [2, 17, 18].\nWe also found different clinical phenotypes of NAFLD among the various Asian subregions. Those with NAFLD in Southeast Asia were significantly younger, with mean age only 43 years, suggesting that the possibility of NAFLD should not be excluded in young patients. Those from the Western Pacific were older by 10 years and 5 years than those from Southeast Asia or the Eastern Mediterranean, respectively. Those from the Eastern Mediterranean had the highest BMIs though there was no statistical difference among regions. However, all three regions demonstrated only slightly elevated ALT levels without regional differences, again showing that ALT levels should not be a criterion for detecting the presence of NAFLD. Notably, ALT levels were also lower in subgroups with higher likelihood for advanced fibrosis, which is likely due to the increase in the AST/ALT ratio that is part of the NFS, and AST/ALT ratio has been shown to correlate with liver fibrosis in chronic liver disease [19].\nNotably, when we reviewed the characteristics of patients by classification of their hepatic steatosis as mild, moderate, and severe by ultrasound, we found those with severe steatosis on average had BMIs that were 14% greater than those with mild hepatic steatosis and 7% greater than those with moderate steatosis. In addition, those with severe steatosis demonstrated average ALT levels that were increased at 1.5–2 × 95% ULN ALT and were 31% higher than those with mild steatosis. Such a finding suggests that if an elevated ALT level is found in one suspected of having NAFLD, that patient may in fact have severe steatosis and require further assessment.\nTo strengthen our meta-analysis findings, we used individual patient-level data from 7,668 subjects in seven Asian cities in three countries and found that overall the trends of differences between males and females, obese and nonobese, and those with NFS <−1.455 or ≥−1.455 that we reported in our meta-analysis were similar to those in the analyzed individual patient-level data. Of particular interest are our findings on the percent of NAFLD patients with fibrosis, since fibrosis is the single most important predictor of death for those with NAFLD [20, 21]. Since we had individual data, we were able to calculate both the NFS and FIB-4 scores and found similar findings to our meta-analysis in that between 66% and 71% (respectively) fell into the low probability of having advanced fibrosis, while 3.4% and 4.2% (respectively) had a high probability of having advanced fibrosis or cirrhosis. In addition, we found that older, female, obese, and diabetic were more likely to have advanced fibrosis. Therefore, although the percentage of patients with advanced fibrosis is low, the large number of patients with NAFLD amplifies the number of patients negatively affected, especially as the NAFLD burden continues to grow in Asia [22, 23]. Consequently, efforts must continue to raise awareness about NAFLD and its risk factors as well as its management, which includes weight loss through diet and exercise as well as management of patients' cardiometabolic risk factors [24, 25]. Furthermore, efforts must be made to raise awareness about the negative effects of NAFLD among older women as seen in this study and in light of a recent study that found that NASH is now the number one indicator for liver transplant among women [26].\nThis study has its strengths. First, the number of studies included is large, which provided a study population of over 2 million for many of the analyses. As a result, the majority of our reported findings have a good level of precision as noted in our relatively tight 95% confidence intervals. In addition, the quality of our included studies overall was high. Besides published data, we also obtained individual patient data from seven centers for additional patient-level data. We also recognize that there are several limitations. One was the lack of data to determine the NFS >0.676 for the meta-analysis. We are aware that a score between −1.455 and 0.676 is in the indeterminate zone, where certainty of the presence or absence of advanced fibrosis is not available, but we were able to determine the level of fibrosis with data from our patient-level data, which indicate that the vast majority of patients are at low risk for having advanced fibrosis. Another limitation is that the severity of steatosis was determined by ultrasound which may have interobserver variability. To account for this limitation, we included analyses of mild versus moderate versus severe steatosis as well as mild versus moderate-severe steatosis and found that the trends presented were similar. Third, a few of our subanalyses were from only a few studies; however, these studies provided a large number of patients such that our confidence in our reported findings is relatively high, especially as the majority of our findings were replicated using individual data. Lastly, for both our study level and individual patient-level data analyses, the majority of the data were from the Western Pacific region, so our data may be most representative of the Western Pacific region, and thus additional studies are needed for other regions of Asia.","In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed.","The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).","D.Q.H. has received grant support from Exxon Mobil-NUS Research Fellowship for Clinicians and Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship. J.F. is on the advisory board/consulting for Gilead. R.C.C. has received grant support from Gilead. T.K. has received grant support and personal speaker fees from Gilead and AbbVie. M.Y. is on the advisory board/consulting for AbbVie, Arbutus Biopharma, Assembly Biosciences, Bristol Myers Squibb, Dicerna Pharmaceuticals, GlaxoSmithKline, Gilead Sciences, Janssen, Merck Sharp and Dohme, ClearB Therapeutics, and Springbank Pharmaceuticals. H.T. has received grant support from AbbVie, Merck Sharp and Dohme, and Bayer. M.H.N. has received grant support from Pfizer, Gilead, and Enanta and is on the advisory board/consulting for Intercept and Gilead. Other authors have no disclosures.","There is no funding to disclose.","All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision.","The majority of data generated or analyzed during this study are included in this article and its online supplementary files. Further enquiries can be directed to the corresponding author.","Supplementary data\nClick here for additional data file."],"string":"[\n \"Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.\\nThrough a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.\",\n \"Search Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\\nThis study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\\nSystemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\\nTwo authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\\nIndividual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\\nIn addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\\nStatistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\\nWe calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\",\n \"This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\",\n \"Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\",\n \"In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\",\n \"We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\",\n \"Study-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\\nAs described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\\nIndividual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\\nOverall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\",\n \"As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\",\n \"Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\",\n \"Our study found that patients with NAFLD in Asia were approximately 52 years old, overweight by Asian cutoffs, about 34% nonobese, with slightly elevated ALT and total cholesterol levels, around 17% diabetic, and 85% with NFS < −1.455 indicating a very low probability of having advanced fibrosis. Males tended to be approximately 4 years younger than females, but their BMIs were similar. As with other populations, neither the absence of obesity nor an elevated ALT should be used to guide the screening or diagnosis of NAFLD for Asians [2, 17, 18].\\nWe also found different clinical phenotypes of NAFLD among the various Asian subregions. Those with NAFLD in Southeast Asia were significantly younger, with mean age only 43 years, suggesting that the possibility of NAFLD should not be excluded in young patients. Those from the Western Pacific were older by 10 years and 5 years than those from Southeast Asia or the Eastern Mediterranean, respectively. Those from the Eastern Mediterranean had the highest BMIs though there was no statistical difference among regions. However, all three regions demonstrated only slightly elevated ALT levels without regional differences, again showing that ALT levels should not be a criterion for detecting the presence of NAFLD. Notably, ALT levels were also lower in subgroups with higher likelihood for advanced fibrosis, which is likely due to the increase in the AST/ALT ratio that is part of the NFS, and AST/ALT ratio has been shown to correlate with liver fibrosis in chronic liver disease [19].\\nNotably, when we reviewed the characteristics of patients by classification of their hepatic steatosis as mild, moderate, and severe by ultrasound, we found those with severe steatosis on average had BMIs that were 14% greater than those with mild hepatic steatosis and 7% greater than those with moderate steatosis. In addition, those with severe steatosis demonstrated average ALT levels that were increased at 1.5–2 × 95% ULN ALT and were 31% higher than those with mild steatosis. Such a finding suggests that if an elevated ALT level is found in one suspected of having NAFLD, that patient may in fact have severe steatosis and require further assessment.\\nTo strengthen our meta-analysis findings, we used individual patient-level data from 7,668 subjects in seven Asian cities in three countries and found that overall the trends of differences between males and females, obese and nonobese, and those with NFS <−1.455 or ≥−1.455 that we reported in our meta-analysis were similar to those in the analyzed individual patient-level data. Of particular interest are our findings on the percent of NAFLD patients with fibrosis, since fibrosis is the single most important predictor of death for those with NAFLD [20, 21]. Since we had individual data, we were able to calculate both the NFS and FIB-4 scores and found similar findings to our meta-analysis in that between 66% and 71% (respectively) fell into the low probability of having advanced fibrosis, while 3.4% and 4.2% (respectively) had a high probability of having advanced fibrosis or cirrhosis. In addition, we found that older, female, obese, and diabetic were more likely to have advanced fibrosis. Therefore, although the percentage of patients with advanced fibrosis is low, the large number of patients with NAFLD amplifies the number of patients negatively affected, especially as the NAFLD burden continues to grow in Asia [22, 23]. Consequently, efforts must continue to raise awareness about NAFLD and its risk factors as well as its management, which includes weight loss through diet and exercise as well as management of patients' cardiometabolic risk factors [24, 25]. Furthermore, efforts must be made to raise awareness about the negative effects of NAFLD among older women as seen in this study and in light of a recent study that found that NASH is now the number one indicator for liver transplant among women [26].\\nThis study has its strengths. First, the number of studies included is large, which provided a study population of over 2 million for many of the analyses. As a result, the majority of our reported findings have a good level of precision as noted in our relatively tight 95% confidence intervals. In addition, the quality of our included studies overall was high. Besides published data, we also obtained individual patient data from seven centers for additional patient-level data. We also recognize that there are several limitations. One was the lack of data to determine the NFS >0.676 for the meta-analysis. We are aware that a score between −1.455 and 0.676 is in the indeterminate zone, where certainty of the presence or absence of advanced fibrosis is not available, but we were able to determine the level of fibrosis with data from our patient-level data, which indicate that the vast majority of patients are at low risk for having advanced fibrosis. Another limitation is that the severity of steatosis was determined by ultrasound which may have interobserver variability. To account for this limitation, we included analyses of mild versus moderate versus severe steatosis as well as mild versus moderate-severe steatosis and found that the trends presented were similar. Third, a few of our subanalyses were from only a few studies; however, these studies provided a large number of patients such that our confidence in our reported findings is relatively high, especially as the majority of our findings were replicated using individual data. Lastly, for both our study level and individual patient-level data analyses, the majority of the data were from the Western Pacific region, so our data may be most representative of the Western Pacific region, and thus additional studies are needed for other regions of Asia.\",\n \"In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed.\",\n \"The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).\",\n \"D.Q.H. has received grant support from Exxon Mobil-NUS Research Fellowship for Clinicians and Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship. J.F. is on the advisory board/consulting for Gilead. R.C.C. has received grant support from Gilead. T.K. has received grant support and personal speaker fees from Gilead and AbbVie. M.Y. is on the advisory board/consulting for AbbVie, Arbutus Biopharma, Assembly Biosciences, Bristol Myers Squibb, Dicerna Pharmaceuticals, GlaxoSmithKline, Gilead Sciences, Janssen, Merck Sharp and Dohme, ClearB Therapeutics, and Springbank Pharmaceuticals. H.T. has received grant support from AbbVie, Merck Sharp and Dohme, and Bayer. M.H.N. has received grant support from Pfizer, Gilead, and Enanta and is on the advisory board/consulting for Intercept and Gilead. Other authors have no disclosures.\",\n \"There is no funding to disclose.\",\n \"All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision.\",\n \"The majority of data generated or analyzed during this study are included in this article and its online supplementary files. Further enquiries can be directed to the corresponding author.\",\n \"Supplementary data\\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,"results",null,null,"discussion","conclusions",null,"COI-statement",null,null,"data-availability","supplementary-material"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n \"COI-statement\",\n null,\n null,\n \"data-availability\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["Nonalcoholic fatty liver disease","Epidemiology","Liver function tests","Obesity","Fibrosis"],"string":"[\n \"Nonalcoholic fatty liver disease\",\n \"Epidemiology\",\n \"Liver function tests\",\n \"Obesity\",\n \"Fibrosis\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nNonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.\nThrough a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.\n\nMethods:\nSearch Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nThis study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nSystemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nTwo authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nIndividual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nIn addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nStatistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\nWe calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\n\nSearch Strategy:\nThis study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\n\nSystemic Literature Review, Data Extraction, and Quality Assessment:\nTwo authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\n\nIndividual Patient Data:\nIn addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\n\nStatistical Analysis:\nWe calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\n\nResults:\nStudy-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nAs described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nIndividual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\nOverall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\n\nStudy-Level Analysis:\nAs described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\n\nIndividual Patient Data Analysis:\nOverall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\n\nDiscussion:\nOur study found that patients with NAFLD in Asia were approximately 52 years old, overweight by Asian cutoffs, about 34% nonobese, with slightly elevated ALT and total cholesterol levels, around 17% diabetic, and 85% with NFS < −1.455 indicating a very low probability of having advanced fibrosis. Males tended to be approximately 4 years younger than females, but their BMIs were similar. As with other populations, neither the absence of obesity nor an elevated ALT should be used to guide the screening or diagnosis of NAFLD for Asians [2, 17, 18].\nWe also found different clinical phenotypes of NAFLD among the various Asian subregions. Those with NAFLD in Southeast Asia were significantly younger, with mean age only 43 years, suggesting that the possibility of NAFLD should not be excluded in young patients. Those from the Western Pacific were older by 10 years and 5 years than those from Southeast Asia or the Eastern Mediterranean, respectively. Those from the Eastern Mediterranean had the highest BMIs though there was no statistical difference among regions. However, all three regions demonstrated only slightly elevated ALT levels without regional differences, again showing that ALT levels should not be a criterion for detecting the presence of NAFLD. Notably, ALT levels were also lower in subgroups with higher likelihood for advanced fibrosis, which is likely due to the increase in the AST/ALT ratio that is part of the NFS, and AST/ALT ratio has been shown to correlate with liver fibrosis in chronic liver disease [19].\nNotably, when we reviewed the characteristics of patients by classification of their hepatic steatosis as mild, moderate, and severe by ultrasound, we found those with severe steatosis on average had BMIs that were 14% greater than those with mild hepatic steatosis and 7% greater than those with moderate steatosis. In addition, those with severe steatosis demonstrated average ALT levels that were increased at 1.5–2 × 95% ULN ALT and were 31% higher than those with mild steatosis. Such a finding suggests that if an elevated ALT level is found in one suspected of having NAFLD, that patient may in fact have severe steatosis and require further assessment.\nTo strengthen our meta-analysis findings, we used individual patient-level data from 7,668 subjects in seven Asian cities in three countries and found that overall the trends of differences between males and females, obese and nonobese, and those with NFS <−1.455 or ≥−1.455 that we reported in our meta-analysis were similar to those in the analyzed individual patient-level data. Of particular interest are our findings on the percent of NAFLD patients with fibrosis, since fibrosis is the single most important predictor of death for those with NAFLD [20, 21]. Since we had individual data, we were able to calculate both the NFS and FIB-4 scores and found similar findings to our meta-analysis in that between 66% and 71% (respectively) fell into the low probability of having advanced fibrosis, while 3.4% and 4.2% (respectively) had a high probability of having advanced fibrosis or cirrhosis. In addition, we found that older, female, obese, and diabetic were more likely to have advanced fibrosis. Therefore, although the percentage of patients with advanced fibrosis is low, the large number of patients with NAFLD amplifies the number of patients negatively affected, especially as the NAFLD burden continues to grow in Asia [22, 23]. Consequently, efforts must continue to raise awareness about NAFLD and its risk factors as well as its management, which includes weight loss through diet and exercise as well as management of patients' cardiometabolic risk factors [24, 25]. Furthermore, efforts must be made to raise awareness about the negative effects of NAFLD among older women as seen in this study and in light of a recent study that found that NASH is now the number one indicator for liver transplant among women [26].\nThis study has its strengths. First, the number of studies included is large, which provided a study population of over 2 million for many of the analyses. As a result, the majority of our reported findings have a good level of precision as noted in our relatively tight 95% confidence intervals. In addition, the quality of our included studies overall was high. Besides published data, we also obtained individual patient data from seven centers for additional patient-level data. We also recognize that there are several limitations. One was the lack of data to determine the NFS >0.676 for the meta-analysis. We are aware that a score between −1.455 and 0.676 is in the indeterminate zone, where certainty of the presence or absence of advanced fibrosis is not available, but we were able to determine the level of fibrosis with data from our patient-level data, which indicate that the vast majority of patients are at low risk for having advanced fibrosis. Another limitation is that the severity of steatosis was determined by ultrasound which may have interobserver variability. To account for this limitation, we included analyses of mild versus moderate versus severe steatosis as well as mild versus moderate-severe steatosis and found that the trends presented were similar. Third, a few of our subanalyses were from only a few studies; however, these studies provided a large number of patients such that our confidence in our reported findings is relatively high, especially as the majority of our findings were replicated using individual data. Lastly, for both our study level and individual patient-level data analyses, the majority of the data were from the Western Pacific region, so our data may be most representative of the Western Pacific region, and thus additional studies are needed for other regions of Asia.\n\nConclusion:\nIn Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed.\n\nStatement of Ethics:\nThe study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).\n\nConflict of Interest Statement:\nD.Q.H. has received grant support from Exxon Mobil-NUS Research Fellowship for Clinicians and Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship. J.F. is on the advisory board/consulting for Gilead. R.C.C. has received grant support from Gilead. T.K. has received grant support and personal speaker fees from Gilead and AbbVie. M.Y. is on the advisory board/consulting for AbbVie, Arbutus Biopharma, Assembly Biosciences, Bristol Myers Squibb, Dicerna Pharmaceuticals, GlaxoSmithKline, Gilead Sciences, Janssen, Merck Sharp and Dohme, ClearB Therapeutics, and Springbank Pharmaceuticals. H.T. has received grant support from AbbVie, Merck Sharp and Dohme, and Bayer. M.H.N. has received grant support from Pfizer, Gilead, and Enanta and is on the advisory board/consulting for Intercept and Gilead. Other authors have no disclosures.\n\nFunding Sources:\nThere is no funding to disclose.\n\nAuthor Contributions:\nAll authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision.\n\nData Availability Statement:\nThe majority of data generated or analyzed during this study are included in this article and its online supplementary files. Further enquiries can be directed to the corresponding author.\n\nSupplementary Material:\nSupplementary data\nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nNAFLD is increasingly prevalent in Asia, where people suffer more metabolic comorbidities at a lower body mass index (BMI), suggesting potential differences in their clinical profile. Therefore, we attempted to characterize the clinical profile of Asians with NAFLD via a meta-analytic approach.\n\nMethods:\nWe searched PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019. Two authors independently reviewed and selected 104 articles (2,247,754 persons) that identified NAFLD in Asians and reported relevant data, especially BMI and ALT, and excluded individuals with other liver disease and excessive alcohol consumption. Individual patient-level data were obtained from seven cohorts in Asia to complement meta-analyzed data.\n\nResults:\nOverall, the mean age was 52.07 (95% CI: 51.28-52.85) years, with those from Southeast Asia (42.66, 95% CI: 32.23-53.11) being significantly younger. The mean BMI was 26.2 kg/m2, higher in moderate-severe versus mild hepatic steatosis (28.3 vs. 25.7) patients and NFS ≥ -1.455 versus <-1.455 (27.09 vs. 26.02), with 34% having nonobese NAFLD. The mean ALT was 31.74 U/L, higher in NFS < -1.455 versus ≥-1.455 (33.74 vs. 27.83), though no differences were found by obesity or steatosis severity. The majority of males (85.7%) and females (60.7%) had normal to minimally elevated ALT (1-1.5 × 95% ULN). Individual patient-level data analysis (N = 7,668) demonstrated similar results.\n\nConclusions:\nAbout one-third of Asians with NAFLD were nonobese, and the majority did not have markedly elevated ALT. Therefore, abnormal ALT or BMI is not recommended as a criterion for NAFLD screening in this population. Additionally, there were significant differences in the clinical profiles of NAFLD among the different regions of Asia."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nNonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.\nThrough a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.\n\nConclusion:\nIn Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nNAFLD is increasingly prevalent in Asia, where people suffer more metabolic comorbidities at a lower body mass index (BMI), suggesting potential differences in their clinical profile. Therefore, we attempted to characterize the clinical profile of Asians with NAFLD via a meta-analytic approach.\n\nMethods:\nWe searched PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019. Two authors independently reviewed and selected 104 articles (2,247,754 persons) that identified NAFLD in Asians and reported relevant data, especially BMI and ALT, and excluded individuals with other liver disease and excessive alcohol consumption. Individual patient-level data were obtained from seven cohorts in Asia to complement meta-analyzed data.\n\nResults:\nOverall, the mean age was 52.07 (95% CI: 51.28-52.85) years, with those from Southeast Asia (42.66, 95% CI: 32.23-53.11) being significantly younger. The mean BMI was 26.2 kg/m2, higher in moderate-severe versus mild hepatic steatosis (28.3 vs. 25.7) patients and NFS ≥ -1.455 versus <-1.455 (27.09 vs. 26.02), with 34% having nonobese NAFLD. The mean ALT was 31.74 U/L, higher in NFS < -1.455 versus ≥-1.455 (33.74 vs. 27.83), though no differences were found by obesity or steatosis severity. The majority of males (85.7%) and females (60.7%) had normal to minimally elevated ALT (1-1.5 × 95% ULN). Individual patient-level data analysis (N = 7,668) demonstrated similar results.\n\nConclusions:\nAbout one-third of Asians with NAFLD were nonobese, and the majority did not have markedly elevated ALT. Therefore, abnormal ALT or BMI is not recommended as a criterion for NAFLD screening in this population. Additionally, there were significant differences in the clinical profiles of NAFLD among the different regions of Asia."},"whole_article_text_length":{"kind":"number","value":9611,"string":"9,611"},"whole_article_abstract_length":{"kind":"number","value":364,"string":"364"},"other_sections_lengths":{"kind":"list like","value":[136,439,308,339,897,404,129,7,67],"string":"[\n 136,\n 439,\n 308,\n 339,\n 897,\n 404,\n 129,\n 7,\n 67\n]"},"num_sections":{"kind":"number","value":17,"string":"17"},"most_frequent_words":{"kind":"list like","value":["alt","data","steatosis","studies","nafld","95","patients","mean","table","vs"],"string":"[\n \"alt\",\n \"data\",\n \"steatosis\",\n \"studies\",\n \"nafld\",\n \"95\",\n \"patients\",\n \"mean\",\n \"table\",\n \"vs\"\n]"},"keybert_topics":{"kind":"list like","value":["fatty liver disease","liver disease nafld","specific asian patients","bmi asians risk","obesity asians defined"],"string":"[\n \"fatty liver disease\",\n \"liver disease nafld\",\n \"specific asian patients\",\n \"bmi asians risk\",\n \"obesity asians defined\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Nonalcoholic fatty liver disease | Epidemiology | Liver function tests | Obesity | Fibrosis [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Nonalcoholic fatty liver disease | Epidemiology | Liver function tests | Obesity | Fibrosis [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Nonalcoholic fatty liver disease | Epidemiology | Liver function tests | Obesity | Fibrosis [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Nonalcoholic fatty liver disease | Epidemiology | Liver function tests | Obesity | Fibrosis [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Nonalcoholic fatty liver disease | Epidemiology | Liver function tests | Obesity | Fibrosis [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Nonalcoholic fatty liver disease | Epidemiology | Liver function tests | Obesity | Fibrosis [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] nafld | disease | asian | bmi | metabolic | nonalcoholic | asians | lower | liver disease | recent [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | center | data | fibrosis | studies | health | steatosis | japan | data derived | derived [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vs | table | 001 | ci | 95 ci | participants | 26 | 95 | studies | mean [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] asians | bmis | alt | nafld | levels | alt levels | severe | regions southeast asians | asians significantly younger western | alt levels screening [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] data | alt | nafld | studies | steatosis | vs | 001 | funding | disclose | funding disclose [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] data | alt | nafld | studies | steatosis | vs | 001 | funding | disclose | funding disclose [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] NAFLD | Asia | BMI ||| Asians | NAFLD [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] PubMed | EMBASE | Cochrane | January 1, 2000 | January 17, 2019 ||| Two | 104 | 2,247,754 | Asians | BMI | ALT ||| seven | Asia [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 52.07 | 95% | CI | 51.28-52.85 | years | Southeast Asia | 42.66 | 95% | CI | 32.23-53.11 ||| BMI | 26.2 kg | m2 | 28.3 | 25.7 | NFS ≥ | 27.09 | 26.02 | 34% | nonobese NAFLD ||| ALT | 31.74 | U/L | NFS | 33.74 | 27.83 ||| 85.7% | 60.7% | ALT | 1-1.5 | 95% | ULN ||| 7,668 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] About one-third | Asians | NAFLD | ALT ||| ALT | BMI | NAFLD ||| NAFLD | Asia [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] NAFLD | Asia | BMI ||| Asians | NAFLD ||| PubMed | EMBASE | Cochrane | January 1, 2000 | January 17, 2019 ||| Two | 104 | 2,247,754 | Asians | BMI | ALT ||| seven | Asia ||| 52.07 | 95% | CI | 51.28-52.85 | years | Southeast Asia | 42.66 | 95% | CI | 32.23-53.11 ||| BMI | 26.2 kg | m2 | 28.3 | 25.7 | NFS ≥ | 27.09 | 26.02 | 34% | nonobese NAFLD ||| ALT | 31.74 | U/L | NFS | 33.74 | 27.83 ||| 85.7% | 60.7% | ALT | 1-1.5 | 95% | ULN ||| 7,668 ||| About one-third | Asians | NAFLD | ALT ||| ALT | BMI | NAFLD ||| NAFLD | Asia [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] NAFLD | Asia | BMI ||| Asians | NAFLD ||| PubMed | EMBASE | Cochrane | January 1, 2000 | January 17, 2019 ||| Two | 104 | 2,247,754 | Asians | BMI | ALT ||| seven | Asia ||| 52.07 | 95% | CI | 51.28-52.85 | years | Southeast Asia | 42.66 | 95% | CI | 32.23-53.11 ||| BMI | 26.2 kg | m2 | 28.3 | 25.7 | NFS ≥ | 27.09 | 26.02 | 34% | nonobese NAFLD ||| ALT | 31.74 | U/L | NFS | 33.74 | 27.83 ||| 85.7% | 60.7% | ALT | 1-1.5 | 95% | ULN ||| 7,668 ||| About one-third | Asians | NAFLD | ALT ||| ALT | BMI | NAFLD ||| NAFLD | Asia [SUMMARY]"}}},{"rowIdx":3359,"cells":{"title":{"kind":"string","value":"Reliability and Validity of the Tinnitus Handicap Inventory: A Clinical Study of Questionnaires."},"pmid":{"kind":"string","value":"36349675"},"background_abstract":{"kind":"string","value":"The aim of this study is to observe the application of the Chinese version of Tinnitus Handicap Inventory-China in Tinnitus patients and verify its reliability and validity."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"About 1129 patients with tinnitus as the first complaint were selected as subjects. The patients were randomly divided into 2 groups: exploration group (n = 565), whose data were analyzed with reliability analysis method using Statistical Package for the Social Sciences software 19.0, validation group (n = 564), whose data were analyzed with validity analysis method using AMOS21.0."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"(1) Reliability test: The Cronbach's α coefficients of the Tinnitus Handicap Inventory-China scale in both groups were 0.94, among which, the Cronbach's α coefficients of functional factor (F), emotion factor (E), and catastrophic factor (C) in group E were 0.87, 0.90, and 0.78, respectively. The half-reliability of the 2 components is 0.87. The correlation coefficient between items and the scale in group E and group V is 0.36-0.78 and 0.33-0.77, respectively. (2) Content validity: The Kaiser-Meyer-Olkin value of group E is 0.96, a total of 4 common factors were extracted, and the cumulative interpretation rate is 57.844%. The number of factors with load less than 0.4 on the 4 common factors is only 1 (F24), suggesting that this factor had little significance; the number of factors with load more than 0.4 on the 2 common factors is 8 (F1, E6, F9, C11, F15, E21, E22, and C23), suggesting that patients had different understandings of these 8 questions. (3) Structural validity: The root mean square error of approximation value of the AMOS structural model in group V is 0.065, and the root mean square residual value is 0.114, indicating low fitness; the NC value is 3.353, indicating good fitness of the scale, but it still needed to be simplified."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The Chinese version of Tinnitus Handicap Inventory-China has a high reliability when applied in China, but the content validity and structure validity are not high, and the clinical practicability needs to be improved."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Tinnitus","Reproducibility of Results","Surveys and Questionnaires","China","Disease Progression","Psychometrics"],"string":"[\n \"Humans\",\n \"Tinnitus\",\n \"Reproducibility of Results\",\n \"Surveys and Questionnaires\",\n \"China\",\n \"Disease Progression\",\n \"Psychometrics\"\n]"},"pmcid":{"kind":"string","value":"9682740"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.\nData from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3\nBecause of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12"},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Ethical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nThe participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nSubjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nThe Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nStudy Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\nThe patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Tinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nFrequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nReliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nThe Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nExploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nThe KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nConfirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nUsing the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nStatistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nFrequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nReliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nA scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nValidity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nExploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study."},"other_sections_titles":{"kind":"list like","value":["Main Points","Ethical Review","Subjects","Study Design and Statistical Analysis","Tinnitus Handicap Inventory Data","Reliability Analysis: Reliability Detection","Exploratory Factor Analysis: Content Validity","Confirmatory Factor Analysis: Structural Validity","Statistical Analysis","Reliability Analysis","Validity Analysis","Exploratory Factor Analysis","Confirmatory Factor Analysis"],"string":"[\n \"Main Points\",\n \"Ethical Review\",\n \"Subjects\",\n \"Study Design and Statistical Analysis\",\n \"Tinnitus Handicap Inventory Data\",\n \"Reliability Analysis: Reliability Detection\",\n \"Exploratory Factor Analysis: Content Validity\",\n \"Confirmatory Factor Analysis: Structural Validity\",\n \"Statistical Analysis\",\n \"Reliability Analysis\",\n \"Validity Analysis\",\n \"Exploratory Factor Analysis\",\n \"Confirmatory Factor Analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.","The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.","The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. ","The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.","Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.","The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.","The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.","Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.","Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. ","A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.","Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.","Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.","Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model."],"string":"[\n \"The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \\nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.\",\n \"The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\",\n \"The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \",\n \"The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\\n\\nReliability analysis\\n\\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\\n\\nValidity analysis\\n\\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\",\n \"Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\",\n \"The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\",\n \"The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\",\n \"Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\",\n \"Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \",\n \"A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\",\n \"Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\",\n \"Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\",\n \"Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Main Points","Introduction","Methods","Ethical Review","Subjects","Study Design and Statistical Analysis","Results","Tinnitus Handicap Inventory Data","Reliability Analysis: Reliability Detection","Exploratory Factor Analysis: Content Validity","Confirmatory Factor Analysis: Structural Validity","Statistical Analysis","Reliability Analysis","Validity Analysis","Exploratory Factor Analysis","Confirmatory Factor Analysis","Discussion","Conclusions"],"string":"[\n \"Main Points\",\n \"Introduction\",\n \"Methods\",\n \"Ethical Review\",\n \"Subjects\",\n \"Study Design and Statistical Analysis\",\n \"Results\",\n \"Tinnitus Handicap Inventory Data\",\n \"Reliability Analysis: Reliability Detection\",\n \"Exploratory Factor Analysis: Content Validity\",\n \"Confirmatory Factor Analysis: Structural Validity\",\n \"Statistical Analysis\",\n \"Reliability Analysis\",\n \"Validity Analysis\",\n \"Exploratory Factor Analysis\",\n \"Confirmatory Factor Analysis\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.","Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.\nData from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3\nBecause of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12","Ethical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nThe participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nSubjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nThe Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nStudy Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\nThe patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.","The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.","The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. ","The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.","Tinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nFrequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nReliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nThe Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nExploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nThe KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nConfirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nUsing the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nStatistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nFrequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nReliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nA scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nValidity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nExploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.","Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.","The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.","The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.","Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.","Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. ","A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.","Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.","Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.","Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.","The linguistic validation of the Chinese THI from the original THI consisted of 3 phases: (1) The scale was translated and tested by Qiulan Shi14 in 2007. After the full text was translated into English, 3 professionals who were proficient in English and familiar with the scale independently translated it into Chinese and then 1 English professional with no knowledge of the scale and 1 doctor translated it back into Chinese and compared both scales to ensure the Chinese version of the scale was equivalent to the original scale. (2) A cultural adjustment of the scale was then performed. As the original scale was established under a specific cultural background,13 it was discussed among tinnitus patients, otologists, nursing staff, and professionals engaged in quality-of-life research, and the questions were determined to be readily understood by Chinese patients with different education levels. (3) The validation was then finalized, according to the reference method.4\nPrevious studies have reported that the THI performs comparably well in several countries and languages.7,9,15-21 The findings of our study suggest that the Chinese THI has good internal consistency reliability for both the total scale (Cronbach’s alpha = 0.94) and the 3 subscales-functional (Cronbach’s alpha = 0.87), emotional (Cronbach’s alpha = 0.90), and catastrophic (Cronbach’s alpha = 0.78-0.76). The internal consistency reliability of the Chinese THI is similar to the validation of the scale in other languages. For example, Cronbach’s alpha is 0.930 for the United States, Danish, German, Dutch, Lithuanian, and Hebrew THI scales; and it is 0.79-0.95 for the Korean THI, 0.72-0.94 for the Chinese THI (Cantonese), 0.91 for the Italian THI, and 0.94 for the Portuguese THI (Lithuanian). Cronbach’s alpha coefficients for the functional, emotional, and catastrophic subscales corresponded well with those of the original THI version. Our study found that the mean score of the question “Do you feel that your tinnitus problem has placed stress on your relationships with members of your family and friends?” (17E) is 0.6, which is the minimum of all items and consistent with previous Lithuanian research.8 Therefore, Tinnitus appears to have a little emotional impact on family and friends.\nThe THI is widely used in clinical practice and research as a 3-dimensional measure of tinnitus severity. Despite its extensive use, its factor structure remains unclear. Furthermore, the THI can be considered a reliable measure only if both Cronbach’s alpha coefficient and classical test theory are used. Recently, Gos22 analyzed 1115 patients with tinnitus using the more modern and robust item response theory, finding that this bifactor model had the best fit (RMSEA = 0.055; CFI = 0.976; and SRMR = 0.040) and identifying 1 strong general factor and several weak specific factors. Further, Mokken scaling generated a reliable unidimensional scale (Loevinger’s H = 0.463). To refine the THI, these authors proposed that 5 items have to be removed. Their findings support the use of the THI to evaluate tinnitus severity as a reliable, unidimensional scale. However, clinicians and researchers should rely only on its overall score, which reflects global tinnitus severity. Our study confirmed the utility of the 3-factor structure of the THI, which is consistent with an earlier German study of 373 patients with tinnitus.23 On the bases of several combined criteria, we propose that certain THI items be removed to refine the scale, which is consistent with Elżbieta’s study in which THI2, THI8, THI13, THI19, and THI24 were removed. Newman et al24 proposed a shorter version of the THI, consisting of only 10 items, which were selected on the bases of just 3 criteria: a high item/total correlation, the representativeness of the 3 content domains, and face validity. We find that such criteria are insufficient and propose refining the THI instrument by removing just those items with a low degree of fitness. We believe that short-form questionnaires are essential in busy clinical practices and extensive research protocols.\nAdditionally, Kennedy et al25 noted that the THI, compared to other tinnitus-related questionnaires, contains a disproportionately large number of items related to the psychological/emotional aspects of tinnitus. The results of our study also suggest that tinnitus severity, as measured by the Chinese THI, captures mainly the emotional aspects of tinnitus, which have a high Cronbach’s coefficient for the emotional subscale (0.90) in both the E and V groups as shown in Table 2.\nIt should be noted that the THI uses a 3-label nominal or category scale. Many psychophysicists would argue that this makes it inappropriate to average across scores and users. Stevens,26 often referred to as the father of human psychophysics, summarized the different scales (nominal, ordinal, interval, and ratio) and noted their appropriate use and limitations. Numerical scales are appropriate for equal differences and equal ratios. This does not apply to ordinal scales. The sensitivity of the THI has been challenged.27 Other considerations for tinnitus trials designs were proposed by professor Tyler.27,28 He created a questionnaire (developed on emotions, hearing, sleep, and concentration) focused on the primary activities impaired by tinnitus and therefore more sensitive to treatments and the questions and found that the tinnitus primary function questionnaire is valid, reliable, and sensitive and can be used to determine.29 Many scales attempt to quantify the assessment of “quality of life”. However, we believe that understanding the quality of life is complex, and many widely used questionnaires do not capture the broad range of factors that we believe are important. Many do not include questions about communicating. Professor Tyler also developed a preliminary questionnaire designed to measure “The Meaning of Life” from a broader perspective in 2020 year. Four factors were prominent in this initial sample, which called (1) friendship and positive outlook, (2) physical health, (3) hearing and mental health, and (4) satisfaction with life. Participants with tinnitus reported more trouble sleeping than participants with cochlear implants, whereas both groups had lower scores on hearing. Older patients reported more difficulty with remembering things but were more satisfied with their financial situation.30 Therefore, based on the complexity and particularity of tinnitus disease, the evaluation of tinnitus is also complex, and the scale is widely used rather than limited.","The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study."],"string":"[\n \"The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \\nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.\",\n \"Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.\\nData from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3\\nBecause of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12\",\n \"Ethical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\\nThe participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\\nSubjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \\nThe Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \\nStudy Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\\n\\nReliability analysis\\n\\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\\n\\nValidity analysis\\n\\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\\nThe patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\\n\\nReliability analysis\\n\\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\\n\\nValidity analysis\\n\\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\",\n \"The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\",\n \"The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \",\n \"The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\\n\\nReliability analysis\\n\\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\\n\\nValidity analysis\\n\\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\",\n \"Tinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\\nFrequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\\nReliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\\nThe Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\\nExploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\\nThe KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\\nConfirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\\nUsing the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\\nStatistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \\nFrequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \\nReliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\\nA scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\\nValidity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\\nExploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\",\n \"Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\",\n \"The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\",\n \"The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\",\n \"Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\",\n \"Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \",\n \"A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\",\n \"Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\",\n \"Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\",\n \"Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \\nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\",\n \"The linguistic validation of the Chinese THI from the original THI consisted of 3 phases: (1) The scale was translated and tested by Qiulan Shi14 in 2007. After the full text was translated into English, 3 professionals who were proficient in English and familiar with the scale independently translated it into Chinese and then 1 English professional with no knowledge of the scale and 1 doctor translated it back into Chinese and compared both scales to ensure the Chinese version of the scale was equivalent to the original scale. (2) A cultural adjustment of the scale was then performed. As the original scale was established under a specific cultural background,13 it was discussed among tinnitus patients, otologists, nursing staff, and professionals engaged in quality-of-life research, and the questions were determined to be readily understood by Chinese patients with different education levels. (3) The validation was then finalized, according to the reference method.4\\nPrevious studies have reported that the THI performs comparably well in several countries and languages.7,9,15-21 The findings of our study suggest that the Chinese THI has good internal consistency reliability for both the total scale (Cronbach’s alpha = 0.94) and the 3 subscales-functional (Cronbach’s alpha = 0.87), emotional (Cronbach’s alpha = 0.90), and catastrophic (Cronbach’s alpha = 0.78-0.76). The internal consistency reliability of the Chinese THI is similar to the validation of the scale in other languages. For example, Cronbach’s alpha is 0.930 for the United States, Danish, German, Dutch, Lithuanian, and Hebrew THI scales; and it is 0.79-0.95 for the Korean THI, 0.72-0.94 for the Chinese THI (Cantonese), 0.91 for the Italian THI, and 0.94 for the Portuguese THI (Lithuanian). Cronbach’s alpha coefficients for the functional, emotional, and catastrophic subscales corresponded well with those of the original THI version. Our study found that the mean score of the question “Do you feel that your tinnitus problem has placed stress on your relationships with members of your family and friends?” (17E) is 0.6, which is the minimum of all items and consistent with previous Lithuanian research.8 Therefore, Tinnitus appears to have a little emotional impact on family and friends.\\nThe THI is widely used in clinical practice and research as a 3-dimensional measure of tinnitus severity. Despite its extensive use, its factor structure remains unclear. Furthermore, the THI can be considered a reliable measure only if both Cronbach’s alpha coefficient and classical test theory are used. Recently, Gos22 analyzed 1115 patients with tinnitus using the more modern and robust item response theory, finding that this bifactor model had the best fit (RMSEA = 0.055; CFI = 0.976; and SRMR = 0.040) and identifying 1 strong general factor and several weak specific factors. Further, Mokken scaling generated a reliable unidimensional scale (Loevinger’s H = 0.463). To refine the THI, these authors proposed that 5 items have to be removed. Their findings support the use of the THI to evaluate tinnitus severity as a reliable, unidimensional scale. However, clinicians and researchers should rely only on its overall score, which reflects global tinnitus severity. Our study confirmed the utility of the 3-factor structure of the THI, which is consistent with an earlier German study of 373 patients with tinnitus.23 On the bases of several combined criteria, we propose that certain THI items be removed to refine the scale, which is consistent with Elżbieta’s study in which THI2, THI8, THI13, THI19, and THI24 were removed. Newman et al24 proposed a shorter version of the THI, consisting of only 10 items, which were selected on the bases of just 3 criteria: a high item/total correlation, the representativeness of the 3 content domains, and face validity. We find that such criteria are insufficient and propose refining the THI instrument by removing just those items with a low degree of fitness. We believe that short-form questionnaires are essential in busy clinical practices and extensive research protocols.\\nAdditionally, Kennedy et al25 noted that the THI, compared to other tinnitus-related questionnaires, contains a disproportionately large number of items related to the psychological/emotional aspects of tinnitus. The results of our study also suggest that tinnitus severity, as measured by the Chinese THI, captures mainly the emotional aspects of tinnitus, which have a high Cronbach’s coefficient for the emotional subscale (0.90) in both the E and V groups as shown in Table 2.\\nIt should be noted that the THI uses a 3-label nominal or category scale. Many psychophysicists would argue that this makes it inappropriate to average across scores and users. Stevens,26 often referred to as the father of human psychophysics, summarized the different scales (nominal, ordinal, interval, and ratio) and noted their appropriate use and limitations. Numerical scales are appropriate for equal differences and equal ratios. This does not apply to ordinal scales. The sensitivity of the THI has been challenged.27 Other considerations for tinnitus trials designs were proposed by professor Tyler.27,28 He created a questionnaire (developed on emotions, hearing, sleep, and concentration) focused on the primary activities impaired by tinnitus and therefore more sensitive to treatments and the questions and found that the tinnitus primary function questionnaire is valid, reliable, and sensitive and can be used to determine.29 Many scales attempt to quantify the assessment of “quality of life”. However, we believe that understanding the quality of life is complex, and many widely used questionnaires do not capture the broad range of factors that we believe are important. Many do not include questions about communicating. Professor Tyler also developed a preliminary questionnaire designed to measure “The Meaning of Life” from a broader perspective in 2020 year. Four factors were prominent in this initial sample, which called (1) friendship and positive outlook, (2) physical health, (3) hearing and mental health, and (4) satisfaction with life. Participants with tinnitus reported more trouble sleeping than participants with cochlear implants, whereas both groups had lower scores on hearing. Older patients reported more difficulty with remembering things but were more satisfied with their financial situation.30 Therefore, based on the complexity and particularity of tinnitus disease, the evaluation of tinnitus is also complex, and the scale is widely used rather than limited.\",\n \"The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"intro","methods",null,null,null,"results",null,null,null,null,null,null,null,null,null,"discussion","conclusions"],"string":"[\n null,\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Tinnitus","Tinnitus Handicap Inventory","reliability","questionnaire"],"string":"[\n \"Tinnitus\",\n \"Tinnitus Handicap Inventory\",\n \"reliability\",\n \"questionnaire\"\n]"},"whole_article_text":{"kind":"string","value":"Main Points:\nThe Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.\n\nIntroduction:\nTinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.\nData from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3\nBecause of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12\n\nMethods:\nEthical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nThe participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nSubjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nThe Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nStudy Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\nThe patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\n\nEthical Review:\nThe participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\n\nSubjects:\nThe Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \n\nStudy Design and Statistical Analysis:\nThe patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\n\nResults:\nTinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nFrequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nReliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nThe Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nExploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nThe KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nConfirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nUsing the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nStatistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nFrequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nReliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nA scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nValidity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nExploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\n\nTinnitus Handicap Inventory Data:\nFrequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\n\nReliability Analysis: Reliability Detection:\nThe Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\n\nExploratory Factor Analysis: Content Validity:\nThe KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\n\nConfirmatory Factor Analysis: Structural Validity:\nUsing the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\n\nStatistical Analysis:\nFrequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \n\nReliability Analysis:\nA scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\n\nValidity Analysis:\nExploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\n\nExploratory Factor Analysis:\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\n\nConfirmatory Factor Analysis:\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\n\nDiscussion:\nThe linguistic validation of the Chinese THI from the original THI consisted of 3 phases: (1) The scale was translated and tested by Qiulan Shi14 in 2007. After the full text was translated into English, 3 professionals who were proficient in English and familiar with the scale independently translated it into Chinese and then 1 English professional with no knowledge of the scale and 1 doctor translated it back into Chinese and compared both scales to ensure the Chinese version of the scale was equivalent to the original scale. (2) A cultural adjustment of the scale was then performed. As the original scale was established under a specific cultural background,13 it was discussed among tinnitus patients, otologists, nursing staff, and professionals engaged in quality-of-life research, and the questions were determined to be readily understood by Chinese patients with different education levels. (3) The validation was then finalized, according to the reference method.4\nPrevious studies have reported that the THI performs comparably well in several countries and languages.7,9,15-21 The findings of our study suggest that the Chinese THI has good internal consistency reliability for both the total scale (Cronbach’s alpha = 0.94) and the 3 subscales-functional (Cronbach’s alpha = 0.87), emotional (Cronbach’s alpha = 0.90), and catastrophic (Cronbach’s alpha = 0.78-0.76). The internal consistency reliability of the Chinese THI is similar to the validation of the scale in other languages. For example, Cronbach’s alpha is 0.930 for the United States, Danish, German, Dutch, Lithuanian, and Hebrew THI scales; and it is 0.79-0.95 for the Korean THI, 0.72-0.94 for the Chinese THI (Cantonese), 0.91 for the Italian THI, and 0.94 for the Portuguese THI (Lithuanian). Cronbach’s alpha coefficients for the functional, emotional, and catastrophic subscales corresponded well with those of the original THI version. Our study found that the mean score of the question “Do you feel that your tinnitus problem has placed stress on your relationships with members of your family and friends?” (17E) is 0.6, which is the minimum of all items and consistent with previous Lithuanian research.8 Therefore, Tinnitus appears to have a little emotional impact on family and friends.\nThe THI is widely used in clinical practice and research as a 3-dimensional measure of tinnitus severity. Despite its extensive use, its factor structure remains unclear. Furthermore, the THI can be considered a reliable measure only if both Cronbach’s alpha coefficient and classical test theory are used. Recently, Gos22 analyzed 1115 patients with tinnitus using the more modern and robust item response theory, finding that this bifactor model had the best fit (RMSEA = 0.055; CFI = 0.976; and SRMR = 0.040) and identifying 1 strong general factor and several weak specific factors. Further, Mokken scaling generated a reliable unidimensional scale (Loevinger’s H = 0.463). To refine the THI, these authors proposed that 5 items have to be removed. Their findings support the use of the THI to evaluate tinnitus severity as a reliable, unidimensional scale. However, clinicians and researchers should rely only on its overall score, which reflects global tinnitus severity. Our study confirmed the utility of the 3-factor structure of the THI, which is consistent with an earlier German study of 373 patients with tinnitus.23 On the bases of several combined criteria, we propose that certain THI items be removed to refine the scale, which is consistent with Elżbieta’s study in which THI2, THI8, THI13, THI19, and THI24 were removed. Newman et al24 proposed a shorter version of the THI, consisting of only 10 items, which were selected on the bases of just 3 criteria: a high item/total correlation, the representativeness of the 3 content domains, and face validity. We find that such criteria are insufficient and propose refining the THI instrument by removing just those items with a low degree of fitness. We believe that short-form questionnaires are essential in busy clinical practices and extensive research protocols.\nAdditionally, Kennedy et al25 noted that the THI, compared to other tinnitus-related questionnaires, contains a disproportionately large number of items related to the psychological/emotional aspects of tinnitus. The results of our study also suggest that tinnitus severity, as measured by the Chinese THI, captures mainly the emotional aspects of tinnitus, which have a high Cronbach’s coefficient for the emotional subscale (0.90) in both the E and V groups as shown in Table 2.\nIt should be noted that the THI uses a 3-label nominal or category scale. Many psychophysicists would argue that this makes it inappropriate to average across scores and users. Stevens,26 often referred to as the father of human psychophysics, summarized the different scales (nominal, ordinal, interval, and ratio) and noted their appropriate use and limitations. Numerical scales are appropriate for equal differences and equal ratios. This does not apply to ordinal scales. The sensitivity of the THI has been challenged.27 Other considerations for tinnitus trials designs were proposed by professor Tyler.27,28 He created a questionnaire (developed on emotions, hearing, sleep, and concentration) focused on the primary activities impaired by tinnitus and therefore more sensitive to treatments and the questions and found that the tinnitus primary function questionnaire is valid, reliable, and sensitive and can be used to determine.29 Many scales attempt to quantify the assessment of “quality of life”. However, we believe that understanding the quality of life is complex, and many widely used questionnaires do not capture the broad range of factors that we believe are important. Many do not include questions about communicating. Professor Tyler also developed a preliminary questionnaire designed to measure “The Meaning of Life” from a broader perspective in 2020 year. Four factors were prominent in this initial sample, which called (1) friendship and positive outlook, (2) physical health, (3) hearing and mental health, and (4) satisfaction with life. Participants with tinnitus reported more trouble sleeping than participants with cochlear implants, whereas both groups had lower scores on hearing. Older patients reported more difficulty with remembering things but were more satisfied with their financial situation.30 Therefore, based on the complexity and particularity of tinnitus disease, the evaluation of tinnitus is also complex, and the scale is widely used rather than limited.\n\nConclusions:\nThe THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe aim of this study is to observe the application of the Chinese version of Tinnitus Handicap Inventory-China in Tinnitus patients and verify its reliability and validity.\n\nMethods:\nAbout 1129 patients with tinnitus as the first complaint were selected as subjects. The patients were randomly divided into 2 groups: exploration group (n = 565), whose data were analyzed with reliability analysis method using Statistical Package for the Social Sciences software 19.0, validation group (n = 564), whose data were analyzed with validity analysis method using AMOS21.0.\n\nResults:\n(1) Reliability test: The Cronbach's α coefficients of the Tinnitus Handicap Inventory-China scale in both groups were 0.94, among which, the Cronbach's α coefficients of functional factor (F), emotion factor (E), and catastrophic factor (C) in group E were 0.87, 0.90, and 0.78, respectively. The half-reliability of the 2 components is 0.87. The correlation coefficient between items and the scale in group E and group V is 0.36-0.78 and 0.33-0.77, respectively. (2) Content validity: The Kaiser-Meyer-Olkin value of group E is 0.96, a total of 4 common factors were extracted, and the cumulative interpretation rate is 57.844%. The number of factors with load less than 0.4 on the 4 common factors is only 1 (F24), suggesting that this factor had little significance; the number of factors with load more than 0.4 on the 2 common factors is 8 (F1, E6, F9, C11, F15, E21, E22, and C23), suggesting that patients had different understandings of these 8 questions. (3) Structural validity: The root mean square error of approximation value of the AMOS structural model in group V is 0.065, and the root mean square residual value is 0.114, indicating low fitness; the NC value is 3.353, indicating good fitness of the scale, but it still needed to be simplified.\n\nConclusions:\nThe Chinese version of Tinnitus Handicap Inventory-China has a high reliability when applied in China, but the content validity and structure validity are not high, and the clinical practicability needs to be improved."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nTinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.\nData from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3\nBecause of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12\n\nConclusions:\nThe THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe aim of this study is to observe the application of the Chinese version of Tinnitus Handicap Inventory-China in Tinnitus patients and verify its reliability and validity.\n\nMethods:\nAbout 1129 patients with tinnitus as the first complaint were selected as subjects. The patients were randomly divided into 2 groups: exploration group (n = 565), whose data were analyzed with reliability analysis method using Statistical Package for the Social Sciences software 19.0, validation group (n = 564), whose data were analyzed with validity analysis method using AMOS21.0.\n\nResults:\n(1) Reliability test: The Cronbach's α coefficients of the Tinnitus Handicap Inventory-China scale in both groups were 0.94, among which, the Cronbach's α coefficients of functional factor (F), emotion factor (E), and catastrophic factor (C) in group E were 0.87, 0.90, and 0.78, respectively. The half-reliability of the 2 components is 0.87. The correlation coefficient between items and the scale in group E and group V is 0.36-0.78 and 0.33-0.77, respectively. (2) Content validity: The Kaiser-Meyer-Olkin value of group E is 0.96, a total of 4 common factors were extracted, and the cumulative interpretation rate is 57.844%. The number of factors with load less than 0.4 on the 4 common factors is only 1 (F24), suggesting that this factor had little significance; the number of factors with load more than 0.4 on the 2 common factors is 8 (F1, E6, F9, C11, F15, E21, E22, and C23), suggesting that patients had different understandings of these 8 questions. (3) Structural validity: The root mean square error of approximation value of the AMOS structural model in group V is 0.065, and the root mean square residual value is 0.114, indicating low fitness; the NC value is 3.353, indicating good fitness of the scale, but it still needed to be simplified.\n\nConclusions:\nThe Chinese version of Tinnitus Handicap Inventory-China has a high reliability when applied in China, but the content validity and structure validity are not high, and the clinical practicability needs to be improved."},"whole_article_text_length":{"kind":"number","value":12963,"string":"12,963"},"whole_article_abstract_length":{"kind":"number","value":424,"string":"424"},"other_sections_lengths":{"kind":"list like","value":[86,79,204,299,35,89,190,252,75,359,1727,259,600],"string":"[\n 86,\n 79,\n 204,\n 299,\n 35,\n 89,\n 190,\n 252,\n 75,\n 359,\n 1727,\n 259,\n 600\n]"},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list 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[SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi 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degree fitness | group [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | countries | scale | thi scale | research | studies | widely | results | chinese | patients [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | model | scale | tinnitus | correlation | group | patients | value | thi scale | analysis [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] thi | model | scale | tinnitus | correlation | group | patients | value | thi scale | analysis [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Chinese | Tinnitus Handicap Inventory-China | Tinnitus [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] About 1129 | first ||| 2 | 565 | Statistical Package | Social Sciences | 19.0 | 564 [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 1 | Cronbach | the Tinnitus Handicap Inventory-China | 0.94 | Cronbach | F | 0.87 | 0.90 | 0.78 ||| half | 2 | 0.87 ||| 0.36-0.78 | 0.33 ||| 2 | The Kaiser-Meyer-Olkin | 0.96 | 4 | 57.844% ||| less than 0.4 | 4 | only 1 | F24 | more than 0.4 | 2 | 8 | F1 | E6 | F9 | C11 | F15 | E21 | E22 | C23 | 8 ||| 3 | AMOS | 0.065 | 0.114 | NC | 3.353 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Chinese | Tinnitus Handicap Inventory-China | China [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Chinese | Tinnitus Handicap Inventory-China | Tinnitus ||| About 1129 | first ||| 2 | 565 | Statistical Package | Social Sciences | 19.0 | 564 ||| 1 | Cronbach | the Tinnitus Handicap Inventory-China | 0.94 | Cronbach | F | 0.87 | 0.90 | 0.78 ||| half | 2 | 0.87 ||| 0.36-0.78 | 0.33 ||| 2 | The Kaiser-Meyer-Olkin | 0.96 | 4 | 57.844% ||| less than 0.4 | 4 | only 1 | F24 | more than 0.4 | 2 | 8 | F1 | E6 | F9 | C11 | F15 | E21 | E22 | C23 | 8 ||| 3 | AMOS | 0.065 | 0.114 | NC | 3.353 ||| Chinese | Tinnitus Handicap Inventory-China | China [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Chinese | Tinnitus Handicap Inventory-China | Tinnitus ||| About 1129 | first ||| 2 | 565 | Statistical Package | Social Sciences | 19.0 | 564 ||| 1 | Cronbach | the Tinnitus Handicap Inventory-China | 0.94 | Cronbach | F | 0.87 | 0.90 | 0.78 ||| half | 2 | 0.87 ||| 0.36-0.78 | 0.33 ||| 2 | The Kaiser-Meyer-Olkin | 0.96 | 4 | 57.844% ||| less than 0.4 | 4 | only 1 | F24 | more than 0.4 | 2 | 8 | F1 | E6 | F9 | C11 | F15 | E21 | E22 | C23 | 8 ||| 3 | AMOS | 0.065 | 0.114 | NC | 3.353 ||| Chinese | Tinnitus Handicap Inventory-China | China [SUMMARY]"}}},{"rowIdx":3360,"cells":{"title":{"kind":"string","value":"Does intravascular ultrasound provide clinical benefits for percutaneous coronary intervention with bare-metal stent implantation? A meta-analysis of randomized controlled trials."},"pmid":{"kind":"string","value":"22999055"},"background_abstract":{"kind":"string","value":"The role of intravascular ultrasound (IVUS) in percutaneous coronary interventions (PCI) is still controversial despite several previously published meta-analyses. A meta-analysis to evaluate the controversial role of IVUS-guided PCI with bare-metal stenting was performed and a previous published meta-analysis was re-evaluated in order to clarify the discrepancy between results of these studies."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A systematic review was performed by an electronic search of the PubMed, Embase and Web of Knowledge databases and by a manual search of reference lists for randomized controlled trials published until April 2011, with clinical outcomes and, at least, six months of clinical follow-up. A meta-analysis based on the intention to treat was performed with the selected studies."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Five studies and 1,754 patients were included. There were no differences in death (OR = 1.86; 95% CI = 0.88-3.95; p = 0.10), non-fatal myocardial infarction (OR = 0.65; 95% CI = 0.27-1.58; p = 0.35) and major adverse cardiac events (OR = 0.74; 95% CI = 0.49-1.13; p = 0.16). An analysis of the previous published meta-analysis strongly suggested the presence of publication bias."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"There is no evidence to recommend routine IVUS-guided PCI with bare-metal stent implantation. This may be explained by the paucity and heterogeneity of the studies published so far."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Percutaneous Coronary Intervention","Randomized Controlled Trials as Topic","Stents","Treatment Outcome","Ultrasonography, Interventional"],"string":"[\n \"Humans\",\n \"Percutaneous Coronary Intervention\",\n \"Randomized Controlled Trials as Topic\",\n \"Stents\",\n \"Treatment Outcome\",\n \"Ultrasonography, Interventional\"\n]"},"pmcid":{"kind":"string","value":"3534608"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"The protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).\n Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\nWe performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\n Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\nOnly randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\n Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\nThe titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\n Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\nThe intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\nA total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\n Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\nThere were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\n Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\nThe heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\n Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\nWe also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\n Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\nA total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\n Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\nIn order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.\nTherefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.\nThis research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1)."},"other_sections_titles":{"kind":"list like","value":["Background","Strategy search","Eligibility criteria","Study selection","Statistical analysis","Literature search","Qualitative study analysis","Heterogeneity","Publication bias","Meta-analysis results","Reviewing published data","Differences between selected studies","Study biases","Limitations","Competing interest","Authors’ contributions"],"string":"[\n \"Background\",\n \"Strategy search\",\n \"Eligibility criteria\",\n \"Study selection\",\n \"Statistical analysis\",\n \"Literature search\",\n \"Qualitative study analysis\",\n \"Heterogeneity\",\n \"Publication bias\",\n \"Meta-analysis results\",\n \"Reviewing published data\",\n \"Differences between selected studies\",\n \"Study biases\",\n \"Limitations\",\n \"Competing interest\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.","We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.","Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.","The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.","The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.","A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.","There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].","The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).","We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).","A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).","In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.","The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].","The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.","The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.","The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.","LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.\",\n \"We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\",\n \"Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\",\n \"The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\",\n \"The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\",\n \"A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\\nPatient characteristics\\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\",\n \"There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\\nStudy characteristics\\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\",\n \"The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\",\n \"We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\",\n \"A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\\nClinical Outcomes\\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\",\n \"In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\\nOriginal data from the re-evaluated meta-analysis - MACE[13].\\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\",\n \"The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\",\n \"The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\\nN/A, not applicable.\\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\",\n \"The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\",\n \"The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.\",\n \"LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Strategy search","Eligibility criteria","Study selection","Statistical analysis","Results","Literature search","Qualitative study analysis","Heterogeneity","Publication bias","Meta-analysis results","Reviewing published data","Discussion","Differences between selected studies","Study biases","Limitations","Conclusion","Competing interest","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Strategy search\",\n \"Eligibility criteria\",\n \"Study selection\",\n \"Statistical analysis\",\n \"Results\",\n \"Literature search\",\n \"Qualitative study analysis\",\n \"Heterogeneity\",\n \"Publication bias\",\n \"Meta-analysis results\",\n \"Reviewing published data\",\n \"Discussion\",\n \"Differences between selected studies\",\n \"Study biases\",\n \"Limitations\",\n \"Conclusion\",\n \"Competing interest\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.","The protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).\n Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\nWe performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\n Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\nOnly randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\n Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\nThe titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\n Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\nThe intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.","We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.","Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.","The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.","The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method."," Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\nA total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\n Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\nThere were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\n Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\nThe heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\n Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\nWe also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\n Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\nA total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\n Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\nIn order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.","A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.","There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].","The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).","We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).","A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).","In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.","In this rigorously conducted meta-analysis of randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI using bare metal stents, we did not find any advantage of the IVUS-guided strategy over the standard method in clinically relevant outcomes. Indeed, we found evidence of publication bias and of significant heterogeneity among the studies regarding the outcomes MI and MACE. These results diverge from the last two meta-analyses on this topic [12,13], which included studies with provisional stenting, considered surrogate outcomes, and did not evaluate the presence of publication bias. However, they are in concordance with recently published studies of IVUS-guided PCI with drug-eluting stent implantation, which were not associated with significant clinical benefits [30,31].\n Differences between selected studies The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\nThe five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\n Study biases The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\nThe data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\n Limitations The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\nThe paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.","The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].","The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.","The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.","The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.\nTherefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.\nThis research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1).","The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.","LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.\",\n \"The protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).\\n Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\\nWe performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\\n Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\\nOnly randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\\n Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\\nThe titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\\n Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\\nThe intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\",\n \"We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\",\n \"Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\",\n \"The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\",\n \"The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\",\n \" Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\\nPatient characteristics\\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\\nA total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\\nPatient characteristics\\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\\n Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\\nStudy characteristics\\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\\nThere were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\\nStudy characteristics\\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\\n Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\\nThe heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\\n Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\\nWe also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\\n Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\\nClinical Outcomes\\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\\nA total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\\nClinical Outcomes\\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\\n Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\\nOriginal data from the re-evaluated meta-analysis - MACE[13].\\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\\nIn order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\\nOriginal data from the re-evaluated meta-analysis - MACE[13].\\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\",\n \"A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\\nPatient characteristics\\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\",\n \"There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\\nStudy characteristics\\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\",\n \"The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\",\n \"We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\",\n \"A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\\nClinical Outcomes\\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\",\n \"In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\\nOriginal data from the re-evaluated meta-analysis - MACE[13].\\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\",\n \"In this rigorously conducted meta-analysis of randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI using bare metal stents, we did not find any advantage of the IVUS-guided strategy over the standard method in clinically relevant outcomes. Indeed, we found evidence of publication bias and of significant heterogeneity among the studies regarding the outcomes MI and MACE. These results diverge from the last two meta-analyses on this topic [12,13], which included studies with provisional stenting, considered surrogate outcomes, and did not evaluate the presence of publication bias. However, they are in concordance with recently published studies of IVUS-guided PCI with drug-eluting stent implantation, which were not associated with significant clinical benefits [30,31].\\n Differences between selected studies The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\\nThe five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\\n Study biases The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\\nN/A, not applicable.\\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\\nThe data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\\nN/A, not applicable.\\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\\n Limitations The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\\nThe paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\",\n \"The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\",\n \"The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\\nN/A, not applicable.\\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\",\n \"The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\",\n \"The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.\\nTherefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.\\nThis research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1).\",\n \"The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.\",\n \"LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results",null,null,null,null,null,null,"discussion",null,null,null,"conclusions",null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null,\n \"conclusions\",\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Intravascular ultrasound","Meta-analysis","Publication bias","Bare-metal stent","Percutaneous coronary intervention","Coronary artery disease"],"string":"[\n \"Intravascular ultrasound\",\n \"Meta-analysis\",\n \"Publication bias\",\n \"Bare-metal stent\",\n \"Percutaneous coronary intervention\",\n \"Coronary artery disease\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nSince the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.\n\nMethods:\nThe protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).\n Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\nWe performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\n Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\nOnly randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\n Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\nThe titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\n Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\nThe intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\n\nStrategy search:\nWe performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\n\nEligibility criteria:\nOnly randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\n\nStudy selection:\nThe titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\n\nStatistical analysis:\nThe intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\n\nResults:\n Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\nA total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\n Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\nThere were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\n Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\nThe heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\n Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\nWe also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\n Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\nA total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\n Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\nIn order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\n\nLiterature search:\nA total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\n\nQualitative study analysis:\nThere were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\n\nHeterogeneity:\nThe heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\n\nPublication bias:\nWe also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\n\nMeta-analysis results:\nA total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\n\nReviewing published data:\nIn order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\n\nDiscussion:\nIn this rigorously conducted meta-analysis of randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI using bare metal stents, we did not find any advantage of the IVUS-guided strategy over the standard method in clinically relevant outcomes. Indeed, we found evidence of publication bias and of significant heterogeneity among the studies regarding the outcomes MI and MACE. These results diverge from the last two meta-analyses on this topic [12,13], which included studies with provisional stenting, considered surrogate outcomes, and did not evaluate the presence of publication bias. However, they are in concordance with recently published studies of IVUS-guided PCI with drug-eluting stent implantation, which were not associated with significant clinical benefits [30,31].\n Differences between selected studies The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\nThe five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\n Study biases The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\nThe data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\n Limitations The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\nThe paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\n\nDifferences between selected studies:\nThe five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\n\nStudy biases:\nThe data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\n\nLimitations:\nThe paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\n\nConclusion:\nThe clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.\nTherefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.\nThis research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1).\n\nCompeting interest:\nThe authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.\n\nAuthors’ contributions:\nLLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe role of intravascular ultrasound (IVUS) in percutaneous coronary interventions (PCI) is still controversial despite several previously published meta-analyses. A meta-analysis to evaluate the controversial role of IVUS-guided PCI with bare-metal stenting was performed and a previous published meta-analysis was re-evaluated in order to clarify the discrepancy between results of these studies.\n\nMethods:\nA systematic review was performed by an electronic search of the PubMed, Embase and Web of Knowledge databases and by a manual search of reference lists for randomized controlled trials published until April 2011, with clinical outcomes and, at least, six months of clinical follow-up. A meta-analysis based on the intention to treat was performed with the selected studies.\n\nResults:\nFive studies and 1,754 patients were included. There were no differences in death (OR = 1.86; 95% CI = 0.88-3.95; p = 0.10), non-fatal myocardial infarction (OR = 0.65; 95% CI = 0.27-1.58; p = 0.35) and major adverse cardiac events (OR = 0.74; 95% CI = 0.49-1.13; p = 0.16). An analysis of the previous published meta-analysis strongly suggested the presence of publication bias.\n\nConclusions:\nThere is no evidence to recommend routine IVUS-guided PCI with bare-metal stent implantation. This may be explained by the paucity and heterogeneity of the studies published so far."},"background_conclusion_text":{"kind":"string","value":"Background:\nSince the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.\n\nConclusion:\nThe clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.\nTherefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.\nThis research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1)."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe role of intravascular ultrasound (IVUS) in percutaneous coronary interventions (PCI) is still controversial despite several previously published meta-analyses. A meta-analysis to evaluate the controversial role of IVUS-guided PCI with bare-metal stenting was performed and a previous published meta-analysis was re-evaluated in order to clarify the discrepancy between results of these studies.\n\nMethods:\nA systematic review was performed by an electronic search of the PubMed, Embase and Web of Knowledge databases and by a manual search of reference lists for randomized controlled trials published until April 2011, with clinical outcomes and, at least, six months of clinical follow-up. A meta-analysis based on the intention to treat was performed with the selected studies.\n\nResults:\nFive studies and 1,754 patients were included. There were no differences in death (OR = 1.86; 95% CI = 0.88-3.95; p = 0.10), non-fatal myocardial infarction (OR = 0.65; 95% CI = 0.27-1.58; p = 0.35) and major adverse cardiac events (OR = 0.74; 95% CI = 0.49-1.13; p = 0.16). An analysis of the previous published meta-analysis strongly suggested the presence of publication bias.\n\nConclusions:\nThere is no evidence to recommend routine IVUS-guided PCI with bare-metal stent implantation. This may be explained by the paucity and heterogeneity of the studies published so far."},"whole_article_text_length":{"kind":"number","value":8615,"string":"8,615"},"whole_article_abstract_length":{"kind":"number","value":301,"string":"301"},"other_sections_lengths":{"kind":"list like","value":[381,126,148,72,161,179,395,49,154,185,323,209,393,136,20,153],"string":"[\n 381,\n 126,\n 148,\n 72,\n 161,\n 179,\n 395,\n 49,\n 154,\n 185,\n 323,\n 209,\n 393,\n 136,\n 20,\n 153\n]"},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["analysis","study","studies","meta","bias","meta analysis","ivus","mace","coronary","pci"],"string":"[\n \"analysis\",\n \"study\",\n \"studies\",\n \"meta\",\n \"bias\",\n \"meta analysis\",\n \"ivus\",\n \"mace\",\n \"coronary\",\n \"pci\"\n]"},"keybert_topics":{"kind":"list like","value":["ivus interventional ultrasound","coronary intervention pci","pci percutaneous coronary","ivus angio guided","intravascular ultrasound applicable"],"string":"[\n \"ivus interventional ultrasound\",\n \"coronary intervention pci\",\n \"pci percutaneous coronary\",\n \"ivus angio guided\",\n \"intravascular ultrasound applicable\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 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Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] restenosis | ivus | stent | meta | lesions | published | revascularization angiographic restenosis | revascularization angiographic | ivus pci | angiographic restenosis [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes | articles | search | coronary | clinical outcomes | considered | disease | ultraso | clinical | performed [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | studies | study | ci | 95 ci | 95 | figure | mi | mace | meta analysis [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cnpq | ivus guided pci bare | far | guided pci bare | guided pci bare metal | metal stent | metal stent implantation | pci bare metal stent | bare metal stent | bare metal stent implantation [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | meta | studies | meta analysis | coronary | pci | ivus | bias | mi [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] analysis | study | meta | studies | meta analysis | coronary | pci | ivus | bias | mi [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| IVUS [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] PubMed | Embase | April 2011 | six months ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Five | 1,754 ||| 1.86 | 95% | CI | 0.88 | 0.10 | 0.65 | 95% | CI | 0.27-1.58 | 0.35 | 0.74 | 95% | CI | 0.49 | 0.16 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] IVUS ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| IVUS ||| PubMed | Embase | April 2011 | six months ||| ||| Five | 1,754 ||| 1.86 | 95% | CI | 0.88 | 0.10 | 0.65 | 95% | CI | 0.27-1.58 | 0.35 | 0.74 | 95% | CI | 0.49 | 0.16 ||| ||| IVUS ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| IVUS ||| PubMed | Embase | April 2011 | six months ||| ||| Five | 1,754 ||| 1.86 | 95% | CI | 0.88 | 0.10 | 0.65 | 95% | CI | 0.27-1.58 | 0.35 | 0.74 | 95% | CI | 0.49 | 0.16 ||| ||| IVUS ||| [SUMMARY]"}}},{"rowIdx":3361,"cells":{"title":{"kind":"string","value":"Application of Sigma metrics in the quality control strategies of immunology and protein analytes."},"pmid":{"kind":"string","value":"34606652"},"background_abstract":{"kind":"string","value":"Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"While using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Antibodies","Clinical Chemistry Tests","Humans","Proteins","Quality Control","Total Quality Management"],"string":"[\n \"Antibodies\",\n \"Clinical Chemistry Tests\",\n \"Humans\",\n \"Proteins\",\n \"Quality Control\",\n \"Total Quality Management\"\n]"},"pmcid":{"kind":"string","value":"8605144"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\n1\n, \n2\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\n3\n, during‐\n4\n and post‐analytical\n5\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\n1\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI)."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nCumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nDistribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nTable 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nComposition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nWe constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nResetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\nQC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Study design","Instruments and reagents","Evaluation of imprecision","Evaluation of bias","Allowable total error","Sigma metrics calculation","Composition of Sigma Method Decision Charts","Quality goal index ratio","Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes","Distribution of Sigma metrics based on four TEa standards","Composition of Sigma Method Decision Charts","Resetting QC strategies and improvement measures","AUTHOR CONTRIBUTIONS"],"string":"[\n \"INTRODUCTION\",\n \"Study design\",\n \"Instruments and reagents\",\n \"Evaluation of imprecision\",\n \"Evaluation of bias\",\n \"Allowable total error\",\n \"Sigma metrics calculation\",\n \"Composition of Sigma Method Decision Charts\",\n \"Quality goal index ratio\",\n \"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes\",\n \"Distribution of Sigma metrics based on four TEa standards\",\n \"Composition of Sigma Method Decision Charts\",\n \"Resetting QC strategies and improvement measures\",\n \"AUTHOR CONTRIBUTIONS\"\n]"},"other_sections_texts":{"kind":"list like","value":["Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\n1\n, \n2\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\n3\n, during‐\n4\n and post‐analytical\n5\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\n1\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).","This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research","Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.","Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.","Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.","TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.","Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.","Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.","The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n","Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.","Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.","We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels","QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.","YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript."],"string":"[\n \"Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\\n1\\n, \\n2\\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\\n3\\n, during‐\\n4\\n and post‐analytical\\n5\\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\\n1\\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).\",\n \"This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\\n6\\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\\nFlow chart of this research\",\n \"Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\",\n \"Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\\n7\\n, \\n8\\n, \\n9\\n The calculation equation is: RMS CV% = [(CV1\\n2 + CV2\\n2) /2]0.5.\",\n \"Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\\n10\\n, \\n11\\n:\\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\",\n \"TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\\n12\\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\\n(https://biologicalvariation.eu/)\\n\\n13\\n was first adopted, secondly from other researches which EFLM does not cover (RF\\n14\\n and PA\\n15\\n). The TEaBV are calculated using the formula:\\n\\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\\n\\n\\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\\n\\n\\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\\n\\n\\nHere CVI means CV within‐subject, CVG means CV between‐subject.\",\n \"Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\\n6\\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\\nWestgard Sigma multi‐rules\\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\",\n \"Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\",\n \"The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n\\n\",\n \"Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\\nThe TEa source did not cover the TEa of the analyte.\",\n \"Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\\nSigma of 10 analytes based on four different TEa standards\\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\\nNo calculated Sigma obtained.\",\n \"We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\",\n \"QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\",\n \"YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Study design","Instruments and reagents","Evaluation of imprecision","Evaluation of bias","Allowable total error","Sigma metrics calculation","Composition of Sigma Method Decision Charts","Quality goal index ratio","RESULTS","Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes","Distribution of Sigma metrics based on four TEa standards","Composition of Sigma Method Decision Charts","Resetting QC strategies and improvement measures","DISCUSSIONS","CONFLICT OF INTEREST","AUTHOR CONTRIBUTIONS","Supporting information"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Study design\",\n \"Instruments and reagents\",\n \"Evaluation of imprecision\",\n \"Evaluation of bias\",\n \"Allowable total error\",\n \"Sigma metrics calculation\",\n \"Composition of Sigma Method Decision Charts\",\n \"Quality goal index ratio\",\n \"RESULTS\",\n \"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes\",\n \"Distribution of Sigma metrics based on four TEa standards\",\n \"Composition of Sigma Method Decision Charts\",\n \"Resetting QC strategies and improvement measures\",\n \"DISCUSSIONS\",\n \"CONFLICT OF INTEREST\",\n \"AUTHOR CONTRIBUTIONS\",\n \"Supporting information\"\n]"},"all_sections_texts":{"kind":"list like","value":["Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\n1\n, \n2\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\n3\n, during‐\n4\n and post‐analytical\n5\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\n1\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).","Study design This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\nThis study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\nInstruments and reagents Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\nRoche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\nEvaluation of imprecision Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\nImprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\nEvaluation of bias Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\nBias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\nAllowable total error TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\nTEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\nSigma metrics calculation Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\nSigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\nComposition of Sigma Method Decision Charts Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\nLogging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\nQuality goal index ratio The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n\nThe QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n","This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research","Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.","Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.","Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.","TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.","Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.","Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.","The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n","Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nCumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nDistribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nTable 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nComposition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nWe constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nResetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\nQC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.","Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.","Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.","We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels","QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.","In this study, we evaluated the performance of 10 immunology and protein analytes (which named after their BIO‐RAD controls—“Immunology and Protein Control”) at the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine for a one year period based on Sigma metrics. The previous studies mainly focused on the evaluation of analytical performance of clinical biochemistry analytes,\n19\n, \n20\n, \n21\n endocrine analytes,\n10\n, \n22\n tumor marker analytes,\n11\n, \n22\n etc. using Sigma metrics. This study is the first time to focus on the application of Sigma metrics in immunology and protein analytes (IgG, IgA, IgM, C3, C4, CRP, RF, PA, ASO, and Cys C) based on four different sources of TEa. Further the Sigma Method Decision Charts were constructed in terms of TEaNCCL, providing us with a visual view of the analytes’ performance. For analytes with σ<6, the cause for poor performance was evaluated using QGI similar to previous studies.\n10\n, \n11\n\n\nAccording to the equation Sigma = (TEa% ‐ Bias%)/CV%, significantly different σ values are obtained using the same Bias% and CV% but different total allowable error targets similar to the previous studies,\n10\n, \n11\n the greater the TEa is, the greater the Sigma value is. In this study, initially five different TEa targets were included. Most TEa selected from the Clinical Laboratory Improvements Amendments (CLIA)‐88 requirements from the United States are exhibited as 3 times the standard deviation or as semi‐quantitative results\n23\n (data not shown), regardless of whether the standard deviation comes from IQC or the same group variation of the EQA, the variable standard deviations bring difficulties to calculation, and there may be an illusion that analytes with large TEa have better performance and analytes with small TEa have poor performance. Therefore, we would not include TEaCLIA in subsequent calculations. The Sigma metrics of 10 immunology and protein analytes were shown in Table 3 based on four different TEa standards.\nAs mentioned above, surprisingly, while using the TEaNCCL, 9 analytes for IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. On the other hand, while we chose the TEaBV, the Sigma values were quite different, even negative (C3). TEa sources play an important role in the calculation of Sigma value.\n24\n According to the 2015 Strategic Conference on Quality Specifications convened in Milan consensus, there are three more compact levels or models, that is clinical outcomes, biological variabilities, and state‐of‐the‐art, to determine performance specifications in clinical laboratories.\n25\n The actual clinical use of the test results is the gold standard for setting TEa. The optimal TEa should be established depending on the conditions and requirements of the individual laboratory. The laboratory should assess the suitability of the TEa for clinical use and determine if that would allow a larger TEa choice. TEaBV is too strict for our laboratory, after a comprehensive analysis of laboratory status, analytes’ performance, and clinical recognition, therefore, in the subsequent analysis, we chose TEaNCCL as performance specifications.\nAfter calculating the Sigma value, how can we have a more intuitive impression of these results? At this time, Sigma Method Decision Charts are on the stage, which converts all the Sigma metrics into a simple intuitive dashboard.\n25\n Nine Sigma in this research were in the “bull's eye” zone, suggesting so good performance as to world‐class. Cys C fallen in the 3σ‐4σ zone, suggesting there may be more defects. The Sigma Method Decision Charts allows us to get a succinct comprehensive view of an entire instrument's performance, provide us with a visual view of the analytes’ performance. We could intuitively judge the performance of the analytes through this chart. \nThe personalized QC strategies, QGI and Prioritize improvement of 10 analytes according to σNCCL\n\nAnalytes with σ>6 does not need to calculate QGI, and no need for improvement.\nSigma metrics are not just a tool to ascertain the performance of analytes but also a beacon for designing a more cost‐effective QC strategy. Through the assessment of Sigma metrics, we can specify the control rules scientifically, including the number of control materials and the necessary frequency of running those controls. The closer the operating dot of an analyte is to the chart's origin, the easier the QC program is. In our laboratory, we chose 13s/22s QC procedure with two QC levels once per day for the 10 immunology and protein analytes empirically to supervise the performance of analytes in the past. By using Sigma metrics, nine analytes for σ>6 can safely use the 13s procedure with one measurement of two QC levels to gain an appropriate level of analytical quality assurance, avoiding economic costs, and overwork. On the contrary, with the decline in performance, more quality control rules, more different levels of quality controls, and higher quality control frequency are required. As Cys C in this study, that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) were needed and the QC frequency could be increased to one control per 45 clinical samples. As Cys C had 38 average daily measurements in our laboratory,one time QC frequency per day is needed.\nThough the Westgard Sigma multi‐rules provide a scientific and reasonable method for setting QC procedure, it could not fully reflect the precision and accuracy of the method. QGI is a tool to provide easy insights into the reasons for quality errors such as those caused by imprecision, inaccuracy, or both.\n21\n, \n26\n Cys C (QGI = 0.12) had low precision and some action must be taken. We make continuous improvements from five potential causal factors: personnel, machine, material, method, and environment: (Ⅰ) Strengthen personnel training, including QC knowledge and analyzer operation training; (Ⅱ) Perform regular maintenance and calibration of analyzers to ensure the performance of the analyzer, and timely retire the analyzer with poor performance; (Ⅲ) Monitor the temperature of reagent transportation and storage, avoid reagent expiration and excessive on‐board time; (Ⅳ) Revise the operation protocols, perform the machine in strict accordance with the operation protocols; (Ⅴ) Ensure that the working environment of the instrument meets the requirements, such as temperature and humidity. In fact, Cys C used in this research is a third‐party reagent, not Roche original, the reagent batch is updated so quickly, and the difference between batches is large, resulting in a large CV. Aware of this, our laboratory try our best to use the same batch reagents. We would not replace the new batch reagents until the expiration date. In fact, combined with the increase calibration frequency, our laboratory has reduced the average CV of Cys C to below 4% from January to July 2021, leading improvement of precision. Using Westgard Advisor subfunction of BIO‐RAD Unity Real Time, Cys C has higher probability of error detection (Ped), from 0.5 using 13s/22s QC procedure increased to 0.995 using 13s/22s/R4s/41s/6X rules, but also higher probability of false rejection (Pfr), from 0.006 using 13s/22s QC procedure increased to 0.095 using 13s/22s/R4s/41s/6X rules. In future studies, the other aspects should be prioritized to generate more conclusive results.\nNevertheless, there were three aspects of limitations in this research as follows: (Ⅰ).\nBecause the target means in PT/EQA plans were derived from statistical results of peer groups without measurement traceability,\n21\n, \n27\n those using this approach to calculate bias should be aware of possible limitations, including statistical methods used to generate the data and the number of laboratories that participate. There may be increased imprecision due to the small number of laboratories. There also might be concerns about the commutability of PT samples,\n28\n because they are not the same as real patient specimens. (Ⅱ) Another weakness is that the bias evaluation in our research (analyze PT samples at five different concentrations daily for 3 days to obtain 15 results) was not conducted strictly to the method described in the CLSI EP15‐A2 document (analyze one run per day with 3 replicate samples at each of different concentrations daily for 5 days to obtain 15 results), which may have underestimated bias.\n29\n (Ⅲ) The performance specification used to benchmark the methods is the most common limitation in the Sigma researches. Even the Milan consensus details only ideal considerations, there is no one source of analytical goals for all tests, laboratories must choose appropriate practical goals for each individual analytes.\nThis research can provide support for laboratories to select detection systems for these 10 analytes and aid individual laboratories in their choice of proper TEa goals and in working out a detailed troubleshooting action plan as a part of their quality improvement tool. Laboratory staffs can use these tools to help them select high‐quality products, further contributing to the delivery of excellent quality healthcare for patients. The result is more efficient instrument operation, more optimized laboratory workflow, and more reliable test results, ultimately helping clinicians better diagnose and treat their patients.","None declared.","YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript.","Table S1\nClick here for additional data file.\nSupplementary Material\nClick here for additional data file."],"string":"[\n \"Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\\n1\\n, \\n2\\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\\n3\\n, during‐\\n4\\n and post‐analytical\\n5\\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\\n1\\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).\",\n \"Study design This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\\n6\\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\\nFlow chart of this research\\nThis study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\\n6\\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\\nFlow chart of this research\\nInstruments and reagents Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\\nRoche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\\nEvaluation of imprecision Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\\n7\\n, \\n8\\n, \\n9\\n The calculation equation is: RMS CV% = [(CV1\\n2 + CV2\\n2) /2]0.5.\\nImprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\\n7\\n, \\n8\\n, \\n9\\n The calculation equation is: RMS CV% = [(CV1\\n2 + CV2\\n2) /2]0.5.\\nEvaluation of bias Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\\n10\\n, \\n11\\n:\\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\\nBias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\\n10\\n, \\n11\\n:\\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\\nAllowable total error TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\\n12\\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\\n(https://biologicalvariation.eu/)\\n\\n13\\n was first adopted, secondly from other researches which EFLM does not cover (RF\\n14\\n and PA\\n15\\n). The TEaBV are calculated using the formula:\\n\\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\\n\\n\\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\\n\\n\\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\\n\\n\\nHere CVI means CV within‐subject, CVG means CV between‐subject.\\nTEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\\n12\\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\\n(https://biologicalvariation.eu/)\\n\\n13\\n was first adopted, secondly from other researches which EFLM does not cover (RF\\n14\\n and PA\\n15\\n). The TEaBV are calculated using the formula:\\n\\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\\n\\n\\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\\n\\n\\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\\n\\n\\nHere CVI means CV within‐subject, CVG means CV between‐subject.\\nSigma metrics calculation Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\\n6\\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\\nWestgard Sigma multi‐rules\\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\\nSigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\\n6\\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\\nWestgard Sigma multi‐rules\\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\\nComposition of Sigma Method Decision Charts Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\\nLogging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\\nQuality goal index ratio The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n\\n\\nThe QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n\\n\",\n \"This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\\n6\\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\\nFlow chart of this research\",\n \"Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\",\n \"Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\\n7\\n, \\n8\\n, \\n9\\n The calculation equation is: RMS CV% = [(CV1\\n2 + CV2\\n2) /2]0.5.\",\n \"Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\\n10\\n, \\n11\\n:\\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\",\n \"TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\\n12\\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\\n(https://biologicalvariation.eu/)\\n\\n13\\n was first adopted, secondly from other researches which EFLM does not cover (RF\\n14\\n and PA\\n15\\n). The TEaBV are calculated using the formula:\\n\\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\\n\\n\\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\\n\\n\\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\\n\\n\\nHere CVI means CV within‐subject, CVG means CV between‐subject.\",\n \"Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\\n6\\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\\nWestgard Sigma multi‐rules\\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\",\n \"Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\",\n \"The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\\n10\\n, \\n16\\n, \\n17\\n, \\n18\\n\\n\",\n \"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\\nThe TEa source did not cover the TEa of the analyte.\\nCumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\\nThe TEa source did not cover the TEa of the analyte.\\nDistribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\\nSigma of 10 analytes based on four different TEa standards\\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\\nNo calculated Sigma obtained.\\nTable 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\\nSigma of 10 analytes based on four different TEa standards\\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\\nNo calculated Sigma obtained.\\nComposition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\\nWe constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\\nResetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\\nQC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\",\n \"Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\\nThe TEa source did not cover the TEa of the analyte.\",\n \"Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\\nSigma of 10 analytes based on four different TEa standards\\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\\nNo calculated Sigma obtained.\",\n \"We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\",\n \"QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\",\n \"In this study, we evaluated the performance of 10 immunology and protein analytes (which named after their BIO‐RAD controls—“Immunology and Protein Control”) at the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine for a one year period based on Sigma metrics. The previous studies mainly focused on the evaluation of analytical performance of clinical biochemistry analytes,\\n19\\n, \\n20\\n, \\n21\\n endocrine analytes,\\n10\\n, \\n22\\n tumor marker analytes,\\n11\\n, \\n22\\n etc. using Sigma metrics. This study is the first time to focus on the application of Sigma metrics in immunology and protein analytes (IgG, IgA, IgM, C3, C4, CRP, RF, PA, ASO, and Cys C) based on four different sources of TEa. Further the Sigma Method Decision Charts were constructed in terms of TEaNCCL, providing us with a visual view of the analytes’ performance. For analytes with σ<6, the cause for poor performance was evaluated using QGI similar to previous studies.\\n10\\n, \\n11\\n\\n\\nAccording to the equation Sigma = (TEa% ‐ Bias%)/CV%, significantly different σ values are obtained using the same Bias% and CV% but different total allowable error targets similar to the previous studies,\\n10\\n, \\n11\\n the greater the TEa is, the greater the Sigma value is. In this study, initially five different TEa targets were included. Most TEa selected from the Clinical Laboratory Improvements Amendments (CLIA)‐88 requirements from the United States are exhibited as 3 times the standard deviation or as semi‐quantitative results\\n23\\n (data not shown), regardless of whether the standard deviation comes from IQC or the same group variation of the EQA, the variable standard deviations bring difficulties to calculation, and there may be an illusion that analytes with large TEa have better performance and analytes with small TEa have poor performance. Therefore, we would not include TEaCLIA in subsequent calculations. The Sigma metrics of 10 immunology and protein analytes were shown in Table 3 based on four different TEa standards.\\nAs mentioned above, surprisingly, while using the TEaNCCL, 9 analytes for IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. On the other hand, while we chose the TEaBV, the Sigma values were quite different, even negative (C3). TEa sources play an important role in the calculation of Sigma value.\\n24\\n According to the 2015 Strategic Conference on Quality Specifications convened in Milan consensus, there are three more compact levels or models, that is clinical outcomes, biological variabilities, and state‐of‐the‐art, to determine performance specifications in clinical laboratories.\\n25\\n The actual clinical use of the test results is the gold standard for setting TEa. The optimal TEa should be established depending on the conditions and requirements of the individual laboratory. The laboratory should assess the suitability of the TEa for clinical use and determine if that would allow a larger TEa choice. TEaBV is too strict for our laboratory, after a comprehensive analysis of laboratory status, analytes’ performance, and clinical recognition, therefore, in the subsequent analysis, we chose TEaNCCL as performance specifications.\\nAfter calculating the Sigma value, how can we have a more intuitive impression of these results? At this time, Sigma Method Decision Charts are on the stage, which converts all the Sigma metrics into a simple intuitive dashboard.\\n25\\n Nine Sigma in this research were in the “bull's eye” zone, suggesting so good performance as to world‐class. Cys C fallen in the 3σ‐4σ zone, suggesting there may be more defects. The Sigma Method Decision Charts allows us to get a succinct comprehensive view of an entire instrument's performance, provide us with a visual view of the analytes’ performance. We could intuitively judge the performance of the analytes through this chart. \\nThe personalized QC strategies, QGI and Prioritize improvement of 10 analytes according to σNCCL\\n\\nAnalytes with σ>6 does not need to calculate QGI, and no need for improvement.\\nSigma metrics are not just a tool to ascertain the performance of analytes but also a beacon for designing a more cost‐effective QC strategy. Through the assessment of Sigma metrics, we can specify the control rules scientifically, including the number of control materials and the necessary frequency of running those controls. The closer the operating dot of an analyte is to the chart's origin, the easier the QC program is. In our laboratory, we chose 13s/22s QC procedure with two QC levels once per day for the 10 immunology and protein analytes empirically to supervise the performance of analytes in the past. By using Sigma metrics, nine analytes for σ>6 can safely use the 13s procedure with one measurement of two QC levels to gain an appropriate level of analytical quality assurance, avoiding economic costs, and overwork. On the contrary, with the decline in performance, more quality control rules, more different levels of quality controls, and higher quality control frequency are required. As Cys C in this study, that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) were needed and the QC frequency could be increased to one control per 45 clinical samples. As Cys C had 38 average daily measurements in our laboratory,one time QC frequency per day is needed.\\nThough the Westgard Sigma multi‐rules provide a scientific and reasonable method for setting QC procedure, it could not fully reflect the precision and accuracy of the method. QGI is a tool to provide easy insights into the reasons for quality errors such as those caused by imprecision, inaccuracy, or both.\\n21\\n, \\n26\\n Cys C (QGI = 0.12) had low precision and some action must be taken. We make continuous improvements from five potential causal factors: personnel, machine, material, method, and environment: (Ⅰ) Strengthen personnel training, including QC knowledge and analyzer operation training; (Ⅱ) Perform regular maintenance and calibration of analyzers to ensure the performance of the analyzer, and timely retire the analyzer with poor performance; (Ⅲ) Monitor the temperature of reagent transportation and storage, avoid reagent expiration and excessive on‐board time; (Ⅳ) Revise the operation protocols, perform the machine in strict accordance with the operation protocols; (Ⅴ) Ensure that the working environment of the instrument meets the requirements, such as temperature and humidity. In fact, Cys C used in this research is a third‐party reagent, not Roche original, the reagent batch is updated so quickly, and the difference between batches is large, resulting in a large CV. Aware of this, our laboratory try our best to use the same batch reagents. We would not replace the new batch reagents until the expiration date. In fact, combined with the increase calibration frequency, our laboratory has reduced the average CV of Cys C to below 4% from January to July 2021, leading improvement of precision. Using Westgard Advisor subfunction of BIO‐RAD Unity Real Time, Cys C has higher probability of error detection (Ped), from 0.5 using 13s/22s QC procedure increased to 0.995 using 13s/22s/R4s/41s/6X rules, but also higher probability of false rejection (Pfr), from 0.006 using 13s/22s QC procedure increased to 0.095 using 13s/22s/R4s/41s/6X rules. In future studies, the other aspects should be prioritized to generate more conclusive results.\\nNevertheless, there were three aspects of limitations in this research as follows: (Ⅰ).\\nBecause the target means in PT/EQA plans were derived from statistical results of peer groups without measurement traceability,\\n21\\n, \\n27\\n those using this approach to calculate bias should be aware of possible limitations, including statistical methods used to generate the data and the number of laboratories that participate. There may be increased imprecision due to the small number of laboratories. There also might be concerns about the commutability of PT samples,\\n28\\n because they are not the same as real patient specimens. (Ⅱ) Another weakness is that the bias evaluation in our research (analyze PT samples at five different concentrations daily for 3 days to obtain 15 results) was not conducted strictly to the method described in the CLSI EP15‐A2 document (analyze one run per day with 3 replicate samples at each of different concentrations daily for 5 days to obtain 15 results), which may have underestimated bias.\\n29\\n (Ⅲ) The performance specification used to benchmark the methods is the most common limitation in the Sigma researches. Even the Milan consensus details only ideal considerations, there is no one source of analytical goals for all tests, laboratories must choose appropriate practical goals for each individual analytes.\\nThis research can provide support for laboratories to select detection systems for these 10 analytes and aid individual laboratories in their choice of proper TEa goals and in working out a detailed troubleshooting action plan as a part of their quality improvement tool. Laboratory staffs can use these tools to help them select high‐quality products, further contributing to the delivery of excellent quality healthcare for patients. The result is more efficient instrument operation, more optimized laboratory workflow, and more reliable test results, ultimately helping clinicians better diagnose and treat their patients.\",\n \"None declared.\",\n \"YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript.\",\n \"Table S1\\nClick here for additional data file.\\nSupplementary Material\\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials-and-methods",null,null,null,null,null,null,null,null,"results",null,null,null,null,"discussion","COI-statement",null,"supplementary-material"],"string":"[\n null,\n \"materials-and-methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"COI-statement\",\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["allowable total error","immunology and protein analytes","quality goal index","Sigma Method Decision Charts","Sigma metrics"],"string":"[\n \"allowable total error\",\n \"immunology and protein analytes\",\n \"quality goal index\",\n \"Sigma Method Decision Charts\",\n \"Sigma metrics\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nDetection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\n1\n, \n2\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\n3\n, during‐\n4\n and post‐analytical\n5\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\n1\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).\n\nMATERIALS AND METHODS:\nStudy design This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\nThis study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\nInstruments and reagents Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\nRoche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\nEvaluation of imprecision Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\nImprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\nEvaluation of bias Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\nBias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\nAllowable total error TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\nTEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\nSigma metrics calculation Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\nSigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\nComposition of Sigma Method Decision Charts Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\nLogging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\nQuality goal index ratio The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n\nThe QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n\n\nStudy design:\nThis study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\n\nInstruments and reagents:\nRoche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\n\nEvaluation of imprecision:\nImprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\n\nEvaluation of bias:\nBias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\n\nAllowable total error:\nTEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\n\nSigma metrics calculation:\nSigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\n\nComposition of Sigma Method Decision Charts:\nLogging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\n\nQuality goal index ratio:\nThe QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n\n\nRESULTS:\nCumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nCumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nDistribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nTable 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nComposition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nWe constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nResetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\nQC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\n\nCumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes:\nCumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\n\nDistribution of Sigma metrics based on four TEa standards:\nTable 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\n\nComposition of Sigma Method Decision Charts:\nWe constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\n\nResetting QC strategies and improvement measures:\nQC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\n\nDISCUSSIONS:\nIn this study, we evaluated the performance of 10 immunology and protein analytes (which named after their BIO‐RAD controls—“Immunology and Protein Control”) at the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine for a one year period based on Sigma metrics. The previous studies mainly focused on the evaluation of analytical performance of clinical biochemistry analytes,\n19\n, \n20\n, \n21\n endocrine analytes,\n10\n, \n22\n tumor marker analytes,\n11\n, \n22\n etc. using Sigma metrics. This study is the first time to focus on the application of Sigma metrics in immunology and protein analytes (IgG, IgA, IgM, C3, C4, CRP, RF, PA, ASO, and Cys C) based on four different sources of TEa. Further the Sigma Method Decision Charts were constructed in terms of TEaNCCL, providing us with a visual view of the analytes’ performance. For analytes with σ<6, the cause for poor performance was evaluated using QGI similar to previous studies.\n10\n, \n11\n\n\nAccording to the equation Sigma = (TEa% ‐ Bias%)/CV%, significantly different σ values are obtained using the same Bias% and CV% but different total allowable error targets similar to the previous studies,\n10\n, \n11\n the greater the TEa is, the greater the Sigma value is. In this study, initially five different TEa targets were included. Most TEa selected from the Clinical Laboratory Improvements Amendments (CLIA)‐88 requirements from the United States are exhibited as 3 times the standard deviation or as semi‐quantitative results\n23\n (data not shown), regardless of whether the standard deviation comes from IQC or the same group variation of the EQA, the variable standard deviations bring difficulties to calculation, and there may be an illusion that analytes with large TEa have better performance and analytes with small TEa have poor performance. Therefore, we would not include TEaCLIA in subsequent calculations. The Sigma metrics of 10 immunology and protein analytes were shown in Table 3 based on four different TEa standards.\nAs mentioned above, surprisingly, while using the TEaNCCL, 9 analytes for IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. On the other hand, while we chose the TEaBV, the Sigma values were quite different, even negative (C3). TEa sources play an important role in the calculation of Sigma value.\n24\n According to the 2015 Strategic Conference on Quality Specifications convened in Milan consensus, there are three more compact levels or models, that is clinical outcomes, biological variabilities, and state‐of‐the‐art, to determine performance specifications in clinical laboratories.\n25\n The actual clinical use of the test results is the gold standard for setting TEa. The optimal TEa should be established depending on the conditions and requirements of the individual laboratory. The laboratory should assess the suitability of the TEa for clinical use and determine if that would allow a larger TEa choice. TEaBV is too strict for our laboratory, after a comprehensive analysis of laboratory status, analytes’ performance, and clinical recognition, therefore, in the subsequent analysis, we chose TEaNCCL as performance specifications.\nAfter calculating the Sigma value, how can we have a more intuitive impression of these results? At this time, Sigma Method Decision Charts are on the stage, which converts all the Sigma metrics into a simple intuitive dashboard.\n25\n Nine Sigma in this research were in the “bull's eye” zone, suggesting so good performance as to world‐class. Cys C fallen in the 3σ‐4σ zone, suggesting there may be more defects. The Sigma Method Decision Charts allows us to get a succinct comprehensive view of an entire instrument's performance, provide us with a visual view of the analytes’ performance. We could intuitively judge the performance of the analytes through this chart. \nThe personalized QC strategies, QGI and Prioritize improvement of 10 analytes according to σNCCL\n\nAnalytes with σ>6 does not need to calculate QGI, and no need for improvement.\nSigma metrics are not just a tool to ascertain the performance of analytes but also a beacon for designing a more cost‐effective QC strategy. Through the assessment of Sigma metrics, we can specify the control rules scientifically, including the number of control materials and the necessary frequency of running those controls. The closer the operating dot of an analyte is to the chart's origin, the easier the QC program is. In our laboratory, we chose 13s/22s QC procedure with two QC levels once per day for the 10 immunology and protein analytes empirically to supervise the performance of analytes in the past. By using Sigma metrics, nine analytes for σ>6 can safely use the 13s procedure with one measurement of two QC levels to gain an appropriate level of analytical quality assurance, avoiding economic costs, and overwork. On the contrary, with the decline in performance, more quality control rules, more different levels of quality controls, and higher quality control frequency are required. As Cys C in this study, that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) were needed and the QC frequency could be increased to one control per 45 clinical samples. As Cys C had 38 average daily measurements in our laboratory,one time QC frequency per day is needed.\nThough the Westgard Sigma multi‐rules provide a scientific and reasonable method for setting QC procedure, it could not fully reflect the precision and accuracy of the method. QGI is a tool to provide easy insights into the reasons for quality errors such as those caused by imprecision, inaccuracy, or both.\n21\n, \n26\n Cys C (QGI = 0.12) had low precision and some action must be taken. We make continuous improvements from five potential causal factors: personnel, machine, material, method, and environment: (Ⅰ) Strengthen personnel training, including QC knowledge and analyzer operation training; (Ⅱ) Perform regular maintenance and calibration of analyzers to ensure the performance of the analyzer, and timely retire the analyzer with poor performance; (Ⅲ) Monitor the temperature of reagent transportation and storage, avoid reagent expiration and excessive on‐board time; (Ⅳ) Revise the operation protocols, perform the machine in strict accordance with the operation protocols; (Ⅴ) Ensure that the working environment of the instrument meets the requirements, such as temperature and humidity. In fact, Cys C used in this research is a third‐party reagent, not Roche original, the reagent batch is updated so quickly, and the difference between batches is large, resulting in a large CV. Aware of this, our laboratory try our best to use the same batch reagents. We would not replace the new batch reagents until the expiration date. In fact, combined with the increase calibration frequency, our laboratory has reduced the average CV of Cys C to below 4% from January to July 2021, leading improvement of precision. Using Westgard Advisor subfunction of BIO‐RAD Unity Real Time, Cys C has higher probability of error detection (Ped), from 0.5 using 13s/22s QC procedure increased to 0.995 using 13s/22s/R4s/41s/6X rules, but also higher probability of false rejection (Pfr), from 0.006 using 13s/22s QC procedure increased to 0.095 using 13s/22s/R4s/41s/6X rules. In future studies, the other aspects should be prioritized to generate more conclusive results.\nNevertheless, there were three aspects of limitations in this research as follows: (Ⅰ).\nBecause the target means in PT/EQA plans were derived from statistical results of peer groups without measurement traceability,\n21\n, \n27\n those using this approach to calculate bias should be aware of possible limitations, including statistical methods used to generate the data and the number of laboratories that participate. There may be increased imprecision due to the small number of laboratories. There also might be concerns about the commutability of PT samples,\n28\n because they are not the same as real patient specimens. (Ⅱ) Another weakness is that the bias evaluation in our research (analyze PT samples at five different concentrations daily for 3 days to obtain 15 results) was not conducted strictly to the method described in the CLSI EP15‐A2 document (analyze one run per day with 3 replicate samples at each of different concentrations daily for 5 days to obtain 15 results), which may have underestimated bias.\n29\n (Ⅲ) The performance specification used to benchmark the methods is the most common limitation in the Sigma researches. Even the Milan consensus details only ideal considerations, there is no one source of analytical goals for all tests, laboratories must choose appropriate practical goals for each individual analytes.\nThis research can provide support for laboratories to select detection systems for these 10 analytes and aid individual laboratories in their choice of proper TEa goals and in working out a detailed troubleshooting action plan as a part of their quality improvement tool. Laboratory staffs can use these tools to help them select high‐quality products, further contributing to the delivery of excellent quality healthcare for patients. The result is more efficient instrument operation, more optimized laboratory workflow, and more reliable test results, ultimately helping clinicians better diagnose and treat their patients.\n\nCONFLICT OF INTEREST:\nNone declared.\n\nAUTHOR CONTRIBUTIONS:\nYL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript.\n\nSupporting information:\nTable S1\nClick here for additional data file.\nSupplementary Material\nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSix Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma.\n\nMethods:\nAssays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values.\n\nResults:\nWhile using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12).\n\nConclusions:\nThe laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":9241,"string":"9,241"},"whole_article_abstract_length":{"kind":"number","value":375,"string":"375"},"other_sections_lengths":{"kind":"list like","value":[454,112,298,111,258,154,168,150,137,219,269,172,188,70],"string":"[\n 454,\n 112,\n 298,\n 111,\n 258,\n 154,\n 168,\n 150,\n 137,\n 219,\n 269,\n 172,\n 188,\n 70\n]"},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["sigma","analytes","tea","cv","performance","bias","mean","qc","quality","nccl"],"string":"[\n \"sigma\",\n \"analytes\",\n \"tea\",\n \"cv\",\n \"performance\",\n \"bias\",\n \"mean\",\n \"qc\",\n \"quality\",\n \"nccl\"\n]"},"keybert_topics":{"kind":"list like","value":["analytes displayed sigma","combining sigma quality","medical laboratories sigma","laboratories sigma metrics","quality control sigma"],"string":"[\n \"analytes displayed sigma\",\n \"combining sigma quality\",\n \"medical laboratories sigma\",\n \"laboratories sigma metrics\",\n \"quality control sigma\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] allowable total error | immunology and protein analytes | quality goal index | Sigma Method Decision Charts | Sigma metrics [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] allowable total error | immunology and protein analytes | quality goal index | Sigma Method Decision Charts | Sigma metrics [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] allowable total error | immunology and protein analytes | quality goal index | Sigma Method Decision Charts | Sigma metrics [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Antibodies | Clinical Chemistry Tests | Humans | Proteins | Quality Control | Total Quality Management [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Antibodies | Clinical Chemistry Tests | Humans | Proteins | Quality Control | Total Quality Management [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Antibodies | Clinical Chemistry Tests | Humans | Proteins | Quality Control | Total Quality Management [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] analytes displayed sigma | combining sigma quality | medical laboratories sigma | laboratories sigma metrics | quality control sigma [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] analytes displayed sigma | combining sigma quality | medical laboratories sigma | laboratories sigma metrics | quality control sigma [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] analytes displayed sigma | combining sigma quality | medical laboratories sigma | laboratories sigma metrics | quality control sigma [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] sigma | analytes | tea | cv | performance | bias | mean | qc | quality | nccl [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] sigma | analytes | tea | cv | performance | bias | mean | qc | quality | nccl [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] sigma | analytes | tea | cv | performance | bias | mean | qc | quality | nccl [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] quality | sigma | laboratories | quality control | control | management | medical laboratories | medical | defect | million [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] analytes | tea | sigma | qc | performance | mean | variation | cys | teanccl | biological variation [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] sigma | analytes | declared | tea | cv | mean | performance | qc | qgi | bias [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Six Sigma | 6σ ||| Six Sigma [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] TEaNCCL | 90% | Cys C ||| only three | IgG | CRP | CRP | CRP | 6σ ||| TEaNCCL | Sigma Method Decision Charts ||| Cys C | five | 13s ||| ||| 6 | 1 | Batch | 45 | QC ||| one | QC | 13s | 2 | 1 | Batch | 1000 ||| Cys C | 0.12 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Six Sigma | 6σ ||| Six Sigma ||| Assays | 10 | Immunoglobulin G | IgG | Immunoglobulin A | Immunoglobulin M | 3 | 4 | C4 | Rheumatoid | ASO | CRP | Cystatin C | Cys ||| Sigma | four | CV | QC | 1 | 2020 ||| ||| Sigma ||| TEaNCCL | 90% | Cys C ||| only three | IgG | CRP | CRP | CRP | 6σ ||| TEaNCCL | Sigma Method Decision Charts ||| Cys C | five | 13s ||| ||| 6 | 1 | Batch | 45 | QC ||| one | QC | 13s | 2 | 1 | Batch | 1000 ||| Cys C ||| Sigma [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3362,"cells":{"title":{"kind":"string","value":"Comparing Results of Five Glomerular Filtration Rate-Estimating Equations in the Korean General Population: MDRD Study, Revised Lund-Malmö, and Three CKD-EPI Equations."},"pmid":{"kind":"string","value":"27578504"},"background_abstract":{"kind":"string","value":"Estimated glomerular filtration rate (eGFR) is a widely used index of kidney function. Recently, new formulas such as the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations or the Lund-Malmö equation were introduced for assessing eGFR. We compared them with the Modification of Diet in Renal Disease (MDRD) Study equation in the Korean adult population."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The study population comprised 1,482 individuals (median age 51 [42-59] yr, 48.9% males) who received annual physical check-ups during the year 2014. Serum creatinine (Cr) and cystatin C (CysC) were measured. We conducted a retrospective analysis using five GFR estimating equations (MDRD Study, revised Lund-Malmö, and Cr and/or CysC-based CKD-EPI equations). Reduced GFR was defined as eGFR <60 mL/min/1.73 m²."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"For the GFR category distribution, large discrepancies were observed depending on the equation used; category G1 (≥90 mL/min/1.73 m²) ranged from 7.4-81.8%. Compared with the MDRD Study equation, the other four equations overestimated GFR, and CysC-based equations showed a greater difference (-31.3 for CKD-EPI(CysC) and -20.5 for CKD-EPI(Cr-CysC)). CysC-based equations decreased the prevalence of reduced GFR by one third (9.4% in the MDRD Study and 2.4% in CKD-EPI(CysC))."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our data shows that there are remarkable differences in eGFR assessment in the Korean population depending on the equation used, especially in normal or mildly decreased categories. Further prospective studies are necessary in various clinical settings."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Algorithms","Creatinine","Cystatin C","Female","Glomerular Filtration Rate","Humans","Male","Middle Aged","Renal Insufficiency, Chronic","Retrospective Studies"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Algorithms\",\n \"Creatinine\",\n \"Cystatin C\",\n \"Female\",\n \"Glomerular Filtration Rate\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Renal Insufficiency, Chronic\",\n \"Retrospective Studies\"\n]"},"pmcid":{"kind":"string","value":"5011104"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Early detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.\nThere have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":" 1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\nDuring the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\n 2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\nGFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\n 3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\nGFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\n 4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\nThe eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" 1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\nThe baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\n 2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\nCategorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\n 3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\nThe prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["1. Study population","2. Estimation of GFR","3. GFR categories","4. Statistical analyses","1. GFR category distribution in the study population","2. Concordance between MDRD Study equation and other equations","3. Prevalence of reduced eGFR"],"string":"[\n \"1. Study population\",\n \"2. Estimation of GFR\",\n \"3. GFR categories\",\n \"4. Statistical analyses\",\n \"1. GFR category distribution in the study population\",\n \"2. Concordance between MDRD Study equation and other equations\",\n \"3. Prevalence of reduced eGFR\"\n]"},"other_sections_texts":{"kind":"list like","value":["During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.","GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.","GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].","The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.","The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).","Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).","The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation."],"string":"[\n \"During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\",\n \"GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\",\n \"GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\",\n \"The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\",\n \"The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\",\n \"Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\",\n \"The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","1. Study population","2. Estimation of GFR","3. GFR categories","4. Statistical analyses","RESULTS","1. GFR category distribution in the study population","2. Concordance between MDRD Study equation and other equations","3. Prevalence of reduced eGFR","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"1. Study population\",\n \"2. Estimation of GFR\",\n \"3. GFR categories\",\n \"4. Statistical analyses\",\n \"RESULTS\",\n \"1. GFR category distribution in the study population\",\n \"2. Concordance between MDRD Study equation and other equations\",\n \"3. Prevalence of reduced eGFR\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Early detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.\nThere have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults."," 1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\nDuring the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\n 2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\nGFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\n 3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\nGFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\n 4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\nThe eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.","During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.","GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.","GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].","The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant."," 1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\nThe baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\n 2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\nCategorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\n 3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\nThe prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.","The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).","Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).","The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.","The Cr-based CKD-EPI equation is recommended for the initial assessment of GFR, and CysC-based CKD-EPI equations can be used for confirmation of kidney disease according to the KDIGO guidelines [423]. In this study, we compared five eGFR equations, including CysC-based formulas, in the Korean population. The prevalence of reduced GFR by CysC-based CKD-EPI equations has not been reported in Korea yet.\nThe eGFR classification differed considerably according to the equation used for estimation, especially between CKD-EPICysC or CKD-EPICr-CysC equations compared with the MDRD Study equation. Most of the study population (>80%) were in the G2 category (60-89 mL/min/1.73 m2) according to the MDRD Study equation, but in G1 (≥90 mL/min/1.73 m2) according to the CKD-EPICysC equation. The reason for this discrepancy might be related to the eGFR distribution of the study population. Mean eGFRMDRD was 73.5 mL/min/1.73 m2 with a standard deviation of 12.2 mL/min/1.73 m2; thus, there were many results around the cutoff value of 90 mL/min/1.73 m2. In addition, the CKD-EPICysC and CKD-EPICr-CysC equations yielded systematically higher eGFR results (mean difference 31.4 mL/min/1.73 m2 and 20.6 mL/min/1.73 m2, respectively) in comparison with the MDRD Study equation. This finding was in line with previous studies. In the US National Health and Nutrition Examination Survey (NHANES) 1999-2002 data, the distribution of eGFRCKD-EPI CysC was broader and shifted to the right compared with that of eGFRMDRD [24]. Thus, upward reclassification might be common in CysC-based equations.\nAll equations, except for the CKD-EPICysC equation, showed good correlation with the MDRD Study equation. CKD-EPICysC showed only a moderate correlation (r=0.49). The three different CKD-EPI equations showed an overall low prevalence of reduced GFR compared with the MDRD Study equation, especially according to the two CysC-containing equations. The LMRevised equation was recently reported to outperform the MDRD Study and CKD-EPI equations in a Swedish population [15]; however, there has been no evaluation of this equation in the Asian population. It yielded similar mean eGFR results compared with the MDRD Study equation; these two equations showed a very high correlation and a similar prevalence of reduced GFR. However, eGFR was underestimated in patients ≥ 60 yr, when using the LMRevised equation. This observation needs to be subjected to further studies because of the increased possibility of co-morbidities in older patients. The LMRevised equation was generated only from the Swedish population; hence, ethnic differences might have influenced GFR estimation as well.\nAlthough the CKD-EPICr equation is recommended by KDIGO for initial GFR assessment, newer GFR estimating equations have been developed and validated, including a Korean version of the CKD-EPICr equation [25], a serum Cr-based full age spectrum equation [26], and a CysC-based equation based on a Caucasian, Asian, pediatric, and adult population (CAPA) [27]. Our study did not aim to compare all recent equations; however, we analyzed the CysC-based CAPA equation briefly. The mean eGFRCAPA (106.3 mL/min/1.73 m2), eGFR difference in comparison with MDRD Study equation (-32.9), and prevalence of eGFR less than 60 mL/min/1.73 m2 (2.7%) were similar when compared with CKD-EPICysC equation.\nThe prevalence of reduced GFR has been reported differently depending on the study population and the GFR-estimating equation used. In several previous studies, there were clinically significant differences in the prevalence of stage 3 or higher CKD depending on the equation used to estimate GFR. Delanaye et al [20] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRMDRD (13%), intermediate for eGFRCKD-EPI Cr (9.8%), and the lowest for eGFRCKD-EPI Cr-CysC (5%) and eGFRCKD-EPI CysC (4.7%) in 4,189 Belgian patients over 50 yr old. This prevalence trend was similar to ours. One Japanese study showed a 2-fold difference of prevalence between the MDRD Study and CKD-EPICr equations (12.8 vs 6.5%), by studying over 26,000 participants who underwent annual health check-ups [16]. Lujambio et al [28] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRCKD-EPI CysC (21.8%), intermediate for eGFRCKD-EPI Cr-CysC (11.8%), and the lowest for eGFRMDRD (5.9%) and eGFRCKD-EPI Cr (3.4%) in 119 Uruguayans.\nIn the US, the prevalence of reduced GFR (eGFR <60 mL/min/1.73 m2) by CKD-EPICr equation was reported to be 4.7% from 1988-1994 and 6.5% from 1999-2002 from NHANES data [29]. In Korea, the prevalence of reduced GFR by the CKD-EPICr equation has been reported as 7.7% in 2007 and 2.6% in 2010 from Korea NHANES data [1718]. In this study, it was 5.1% of the whole study population. This difference could be due to the following reasons. First, the proportion of younger individuals under 40 yr old was lower than that in other studies (23% vs 30-32%). Compared with 2010 Korea NHANES data, the mean eGFR was relatively lower in this study (81.4 mL/min/1.73 m2 vs 95.9-96.8 mL/min/1.73 m2), resulting in higher prevalence of reduced GFR compared with other studies. Second, the study period was different (2014 vs 2007-2010), although the impact of this on the prevalence is still uncertain.\nOur retrospective study has several limitations. First, we com-pared all the eGFR equations to the MDRD Study equation, because of the absence of GFR data measured by the gold standard method [18]. Therefore, it was impossible to evaluate the accuracy. The magnitude of bias, calculated by measured GFR-calculated GFR was previously reported to be 2.5-5.8 mL/min/1.73 m2, and this difference could influence the prevalence of CKD stages [1724]. Second, our population might not be representative of the entire population of Korea. Third, we could not analyze albuminuria data or other markers of kidney damage. The classification of CKD was not performed, which is based on both GFR category and albuminuria category. Thus, there could be the CKD patients among the subjects with eGFR ≥60 mL/min/1.73 m2.\nIn conclusion, this is the first study that compared five eGFR equations in the Korean population. Our data demonstrated remarkable differences in GFR assessment depending on the equation used. The proportion of each GFR category varied considerably, and CysC-containing equations yielded higher eGFRs and showed larger differences compared with the MDRD Study equation. The prevalence of reduced GFR was lowered by the CKD-EPI equations. Further studies using prospective design and in various ethnicities are necessary."],"string":"[\n \"Early detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.\\nThere have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults.\",\n \" 1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\\nDuring the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\\n 2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\\nGFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\\n 3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\\nGFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\\n 4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\\nThe eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\",\n \"During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\",\n \"GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\",\n \"GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\",\n \"The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\",\n \" 1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\\nThe baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\\n 2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\\nCategorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\\n 3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\\nThe prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\",\n \"The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\",\n \"Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\",\n \"The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\",\n \"The Cr-based CKD-EPI equation is recommended for the initial assessment of GFR, and CysC-based CKD-EPI equations can be used for confirmation of kidney disease according to the KDIGO guidelines [423]. In this study, we compared five eGFR equations, including CysC-based formulas, in the Korean population. The prevalence of reduced GFR by CysC-based CKD-EPI equations has not been reported in Korea yet.\\nThe eGFR classification differed considerably according to the equation used for estimation, especially between CKD-EPICysC or CKD-EPICr-CysC equations compared with the MDRD Study equation. Most of the study population (>80%) were in the G2 category (60-89 mL/min/1.73 m2) according to the MDRD Study equation, but in G1 (≥90 mL/min/1.73 m2) according to the CKD-EPICysC equation. The reason for this discrepancy might be related to the eGFR distribution of the study population. Mean eGFRMDRD was 73.5 mL/min/1.73 m2 with a standard deviation of 12.2 mL/min/1.73 m2; thus, there were many results around the cutoff value of 90 mL/min/1.73 m2. In addition, the CKD-EPICysC and CKD-EPICr-CysC equations yielded systematically higher eGFR results (mean difference 31.4 mL/min/1.73 m2 and 20.6 mL/min/1.73 m2, respectively) in comparison with the MDRD Study equation. This finding was in line with previous studies. In the US National Health and Nutrition Examination Survey (NHANES) 1999-2002 data, the distribution of eGFRCKD-EPI CysC was broader and shifted to the right compared with that of eGFRMDRD [24]. Thus, upward reclassification might be common in CysC-based equations.\\nAll equations, except for the CKD-EPICysC equation, showed good correlation with the MDRD Study equation. CKD-EPICysC showed only a moderate correlation (r=0.49). The three different CKD-EPI equations showed an overall low prevalence of reduced GFR compared with the MDRD Study equation, especially according to the two CysC-containing equations. The LMRevised equation was recently reported to outperform the MDRD Study and CKD-EPI equations in a Swedish population [15]; however, there has been no evaluation of this equation in the Asian population. It yielded similar mean eGFR results compared with the MDRD Study equation; these two equations showed a very high correlation and a similar prevalence of reduced GFR. However, eGFR was underestimated in patients ≥ 60 yr, when using the LMRevised equation. This observation needs to be subjected to further studies because of the increased possibility of co-morbidities in older patients. The LMRevised equation was generated only from the Swedish population; hence, ethnic differences might have influenced GFR estimation as well.\\nAlthough the CKD-EPICr equation is recommended by KDIGO for initial GFR assessment, newer GFR estimating equations have been developed and validated, including a Korean version of the CKD-EPICr equation [25], a serum Cr-based full age spectrum equation [26], and a CysC-based equation based on a Caucasian, Asian, pediatric, and adult population (CAPA) [27]. Our study did not aim to compare all recent equations; however, we analyzed the CysC-based CAPA equation briefly. The mean eGFRCAPA (106.3 mL/min/1.73 m2), eGFR difference in comparison with MDRD Study equation (-32.9), and prevalence of eGFR less than 60 mL/min/1.73 m2 (2.7%) were similar when compared with CKD-EPICysC equation.\\nThe prevalence of reduced GFR has been reported differently depending on the study population and the GFR-estimating equation used. In several previous studies, there were clinically significant differences in the prevalence of stage 3 or higher CKD depending on the equation used to estimate GFR. Delanaye et al [20] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRMDRD (13%), intermediate for eGFRCKD-EPI Cr (9.8%), and the lowest for eGFRCKD-EPI Cr-CysC (5%) and eGFRCKD-EPI CysC (4.7%) in 4,189 Belgian patients over 50 yr old. This prevalence trend was similar to ours. One Japanese study showed a 2-fold difference of prevalence between the MDRD Study and CKD-EPICr equations (12.8 vs 6.5%), by studying over 26,000 participants who underwent annual health check-ups [16]. Lujambio et al [28] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRCKD-EPI CysC (21.8%), intermediate for eGFRCKD-EPI Cr-CysC (11.8%), and the lowest for eGFRMDRD (5.9%) and eGFRCKD-EPI Cr (3.4%) in 119 Uruguayans.\\nIn the US, the prevalence of reduced GFR (eGFR <60 mL/min/1.73 m2) by CKD-EPICr equation was reported to be 4.7% from 1988-1994 and 6.5% from 1999-2002 from NHANES data [29]. In Korea, the prevalence of reduced GFR by the CKD-EPICr equation has been reported as 7.7% in 2007 and 2.6% in 2010 from Korea NHANES data [1718]. In this study, it was 5.1% of the whole study population. This difference could be due to the following reasons. First, the proportion of younger individuals under 40 yr old was lower than that in other studies (23% vs 30-32%). Compared with 2010 Korea NHANES data, the mean eGFR was relatively lower in this study (81.4 mL/min/1.73 m2 vs 95.9-96.8 mL/min/1.73 m2), resulting in higher prevalence of reduced GFR compared with other studies. Second, the study period was different (2014 vs 2007-2010), although the impact of this on the prevalence is still uncertain.\\nOur retrospective study has several limitations. First, we com-pared all the eGFR equations to the MDRD Study equation, because of the absence of GFR data measured by the gold standard method [18]. Therefore, it was impossible to evaluate the accuracy. The magnitude of bias, calculated by measured GFR-calculated GFR was previously reported to be 2.5-5.8 mL/min/1.73 m2, and this difference could influence the prevalence of CKD stages [1724]. Second, our population might not be representative of the entire population of Korea. Third, we could not analyze albuminuria data or other markers of kidney damage. The classification of CKD was not performed, which is based on both GFR category and albuminuria category. Thus, there could be the CKD patients among the subjects with eGFR ≥60 mL/min/1.73 m2.\\nIn conclusion, this is the first study that compared five eGFR equations in the Korean population. Our data demonstrated remarkable differences in GFR assessment depending on the equation used. The proportion of each GFR category varied considerably, and CysC-containing equations yielded higher eGFRs and showed larger differences compared with the MDRD Study equation. The prevalence of reduced GFR was lowered by the CKD-EPI equations. Further studies using prospective design and in various ethnicities are necessary.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Estimated glomerular filtration rate","MDRD Study equation","CKD-EPI equation","Revised Lund-Malmö equation"],"string":"[\n \"Estimated glomerular filtration rate\",\n \"MDRD Study equation\",\n \"CKD-EPI equation\",\n \"Revised Lund-Malmö equation\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nEarly detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.\nThere have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults.\n\nMETHODS:\n 1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\nDuring the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\n 2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\nGFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\n 3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\nGFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\n 4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\nThe eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\n\n1. Study population:\nDuring the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\n\n2. Estimation of GFR:\nGFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\n\n3. GFR categories:\nGFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\n\n4. Statistical analyses:\nThe eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\n\nRESULTS:\n 1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\nThe baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\n 2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\nCategorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\n 3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\nThe prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\n\n1. GFR category distribution in the study population:\nThe baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\n\n2. Concordance between MDRD Study equation and other equations:\nCategorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\n\n3. Prevalence of reduced eGFR:\nThe prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\n\nDISCUSSION:\nThe Cr-based CKD-EPI equation is recommended for the initial assessment of GFR, and CysC-based CKD-EPI equations can be used for confirmation of kidney disease according to the KDIGO guidelines [423]. In this study, we compared five eGFR equations, including CysC-based formulas, in the Korean population. The prevalence of reduced GFR by CysC-based CKD-EPI equations has not been reported in Korea yet.\nThe eGFR classification differed considerably according to the equation used for estimation, especially between CKD-EPICysC or CKD-EPICr-CysC equations compared with the MDRD Study equation. Most of the study population (>80%) were in the G2 category (60-89 mL/min/1.73 m2) according to the MDRD Study equation, but in G1 (≥90 mL/min/1.73 m2) according to the CKD-EPICysC equation. The reason for this discrepancy might be related to the eGFR distribution of the study population. Mean eGFRMDRD was 73.5 mL/min/1.73 m2 with a standard deviation of 12.2 mL/min/1.73 m2; thus, there were many results around the cutoff value of 90 mL/min/1.73 m2. In addition, the CKD-EPICysC and CKD-EPICr-CysC equations yielded systematically higher eGFR results (mean difference 31.4 mL/min/1.73 m2 and 20.6 mL/min/1.73 m2, respectively) in comparison with the MDRD Study equation. This finding was in line with previous studies. In the US National Health and Nutrition Examination Survey (NHANES) 1999-2002 data, the distribution of eGFRCKD-EPI CysC was broader and shifted to the right compared with that of eGFRMDRD [24]. Thus, upward reclassification might be common in CysC-based equations.\nAll equations, except for the CKD-EPICysC equation, showed good correlation with the MDRD Study equation. CKD-EPICysC showed only a moderate correlation (r=0.49). The three different CKD-EPI equations showed an overall low prevalence of reduced GFR compared with the MDRD Study equation, especially according to the two CysC-containing equations. The LMRevised equation was recently reported to outperform the MDRD Study and CKD-EPI equations in a Swedish population [15]; however, there has been no evaluation of this equation in the Asian population. It yielded similar mean eGFR results compared with the MDRD Study equation; these two equations showed a very high correlation and a similar prevalence of reduced GFR. However, eGFR was underestimated in patients ≥ 60 yr, when using the LMRevised equation. This observation needs to be subjected to further studies because of the increased possibility of co-morbidities in older patients. The LMRevised equation was generated only from the Swedish population; hence, ethnic differences might have influenced GFR estimation as well.\nAlthough the CKD-EPICr equation is recommended by KDIGO for initial GFR assessment, newer GFR estimating equations have been developed and validated, including a Korean version of the CKD-EPICr equation [25], a serum Cr-based full age spectrum equation [26], and a CysC-based equation based on a Caucasian, Asian, pediatric, and adult population (CAPA) [27]. Our study did not aim to compare all recent equations; however, we analyzed the CysC-based CAPA equation briefly. The mean eGFRCAPA (106.3 mL/min/1.73 m2), eGFR difference in comparison with MDRD Study equation (-32.9), and prevalence of eGFR less than 60 mL/min/1.73 m2 (2.7%) were similar when compared with CKD-EPICysC equation.\nThe prevalence of reduced GFR has been reported differently depending on the study population and the GFR-estimating equation used. In several previous studies, there were clinically significant differences in the prevalence of stage 3 or higher CKD depending on the equation used to estimate GFR. Delanaye et al [20] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRMDRD (13%), intermediate for eGFRCKD-EPI Cr (9.8%), and the lowest for eGFRCKD-EPI Cr-CysC (5%) and eGFRCKD-EPI CysC (4.7%) in 4,189 Belgian patients over 50 yr old. This prevalence trend was similar to ours. One Japanese study showed a 2-fold difference of prevalence between the MDRD Study and CKD-EPICr equations (12.8 vs 6.5%), by studying over 26,000 participants who underwent annual health check-ups [16]. Lujambio et al [28] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRCKD-EPI CysC (21.8%), intermediate for eGFRCKD-EPI Cr-CysC (11.8%), and the lowest for eGFRMDRD (5.9%) and eGFRCKD-EPI Cr (3.4%) in 119 Uruguayans.\nIn the US, the prevalence of reduced GFR (eGFR <60 mL/min/1.73 m2) by CKD-EPICr equation was reported to be 4.7% from 1988-1994 and 6.5% from 1999-2002 from NHANES data [29]. In Korea, the prevalence of reduced GFR by the CKD-EPICr equation has been reported as 7.7% in 2007 and 2.6% in 2010 from Korea NHANES data [1718]. In this study, it was 5.1% of the whole study population. This difference could be due to the following reasons. First, the proportion of younger individuals under 40 yr old was lower than that in other studies (23% vs 30-32%). Compared with 2010 Korea NHANES data, the mean eGFR was relatively lower in this study (81.4 mL/min/1.73 m2 vs 95.9-96.8 mL/min/1.73 m2), resulting in higher prevalence of reduced GFR compared with other studies. Second, the study period was different (2014 vs 2007-2010), although the impact of this on the prevalence is still uncertain.\nOur retrospective study has several limitations. First, we com-pared all the eGFR equations to the MDRD Study equation, because of the absence of GFR data measured by the gold standard method [18]. Therefore, it was impossible to evaluate the accuracy. The magnitude of bias, calculated by measured GFR-calculated GFR was previously reported to be 2.5-5.8 mL/min/1.73 m2, and this difference could influence the prevalence of CKD stages [1724]. Second, our population might not be representative of the entire population of Korea. Third, we could not analyze albuminuria data or other markers of kidney damage. The classification of CKD was not performed, which is based on both GFR category and albuminuria category. Thus, there could be the CKD patients among the subjects with eGFR ≥60 mL/min/1.73 m2.\nIn conclusion, this is the first study that compared five eGFR equations in the Korean population. Our data demonstrated remarkable differences in GFR assessment depending on the equation used. The proportion of each GFR category varied considerably, and CysC-containing equations yielded higher eGFRs and showed larger differences compared with the MDRD Study equation. The prevalence of reduced GFR was lowered by the CKD-EPI equations. Further studies using prospective design and in various ethnicities are necessary.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nEstimated glomerular filtration rate (eGFR) is a widely used index of kidney function. Recently, new formulas such as the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations or the Lund-Malmö equation were introduced for assessing eGFR. We compared them with the Modification of Diet in Renal Disease (MDRD) Study equation in the Korean adult population.\n\nMethods:\nThe study population comprised 1,482 individuals (median age 51 [42-59] yr, 48.9% males) who received annual physical check-ups during the year 2014. Serum creatinine (Cr) and cystatin C (CysC) were measured. We conducted a retrospective analysis using five GFR estimating equations (MDRD Study, revised Lund-Malmö, and Cr and/or CysC-based CKD-EPI equations). Reduced GFR was defined as eGFR <60 mL/min/1.73 m².\n\nResults:\nFor the GFR category distribution, large discrepancies were observed depending on the equation used; category G1 (≥90 mL/min/1.73 m²) ranged from 7.4-81.8%. Compared with the MDRD Study equation, the other four equations overestimated GFR, and CysC-based equations showed a greater difference (-31.3 for CKD-EPI(CysC) and -20.5 for CKD-EPI(Cr-CysC)). CysC-based equations decreased the prevalence of reduced GFR by one third (9.4% in the MDRD Study and 2.4% in CKD-EPI(CysC)).\n\nConclusions:\nOur data shows that there are remarkable differences in eGFR assessment in the Korean population depending on the equation used, especially in normal or mildly decreased categories. Further prospective studies are necessary in various clinical settings."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":7502,"string":"7,502"},"whole_article_abstract_length":{"kind":"number","value":316,"string":"316"},"other_sections_lengths":{"kind":"list like","value":[162,619,103,320,268,296,109],"string":"[\n 162,\n 619,\n 103,\n 320,\n 268,\n 296,\n 109\n]"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["ckd","gfr","equation","equations","min","cysc","73","study","min 73","ml"],"string":"[\n \"ckd\",\n \"gfr\",\n \"equation\",\n \"equations\",\n \"min\",\n \"cysc\",\n \"73\",\n \"study\",\n \"min 73\",\n \"ml\"\n]"},"keybert_topics":{"kind":"list like","value":["estimating gfr egfr","diet renal","index assessing kidney","gfr estimation ckd","kidney disease epidemiology"],"string":"[\n \"estimating gfr egfr\",\n \"diet renal\",\n \"index assessing kidney\",\n \"gfr estimation ckd\",\n \"kidney disease epidemiology\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | equation | kidney | equations | gfr | disease | epi | ckd epi | cysc ckd | kidney function [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] scr | scysc | serum | males | laboratory | max | females | gfr | min | 150 [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | epicr | ckd epicr | equations | equation | 73 | mdrd | ml min | min 73 m2 | min 73 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ckd | equation | gfr | equations | min | 73 | scr | cysc | 73 m2 | ml [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| the Chronic Kidney Disease Epidemiology Collaboration ||| the Modification of Diet | Korean [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 1,482 | age 51 ||| 42 | 48.9% | annual | the year 2014 ||| Cr ||| five | GFR | MDRD Study | Lund-Malmö | Cr and/or CysC | CKD-EPI ||| 60 mL [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] GFR | G1 | 7.4-81.8% ||| MDRD Study | four | GFR ||| GFR | one third | 9.4% | the MDRD Study | 2.4% | CKD-EPI(CysC [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| the Chronic Kidney Disease Epidemiology Collaboration ||| the Modification of Diet | Korean ||| 1,482 | age 51 ||| 42 | 48.9% | annual | the year 2014 ||| Cr ||| five | GFR | MDRD Study | Lund-Malmö | Cr and/or CysC | CKD-EPI ||| 60 mL ||| GFR | G1 | 7.4-81.8% ||| MDRD Study | four | GFR ||| GFR | one third | 9.4% | the MDRD Study | 2.4% | CKD-EPI(CysC ||| Korean ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3363,"cells":{"title":{"kind":"string","value":"Acceptability of provider-initiated HIV testing as an intervention for prevention of mother to child transmission of HIV and associated factors among pregnant women attending at Public Health Facilities in Assosa town, Northwest Ethiopia."},"pmid":{"kind":"string","value":"26553035"},"background_abstract":{"kind":"string","value":"Despite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Institutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Acceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Cross-Sectional Studies","Educational Status","Ethiopia","Female","HIV Infections","Health Knowledge, Attitudes, Practice","Humans","Infectious Disease Transmission, Vertical","Mass Screening","Middle Aged","Multivariate Analysis","Patient Acceptance of Health Care","Pregnancy","Pregnancy Complications, Infectious","Prenatal Care","Public Facilities","Regression Analysis","Social Class","Surveys and Questionnaires","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Cross-Sectional Studies\",\n \"Educational Status\",\n \"Ethiopia\",\n \"Female\",\n \"HIV Infections\",\n \"Health Knowledge, Attitudes, Practice\",\n \"Humans\",\n \"Infectious Disease Transmission, Vertical\",\n \"Mass Screening\",\n \"Middle Aged\",\n \"Multivariate Analysis\",\n \"Patient Acceptance of Health Care\",\n \"Pregnancy\",\n \"Pregnancy Complications, Infectious\",\n \"Prenatal Care\",\n \"Public Facilities\",\n \"Regression Analysis\",\n \"Social Class\",\n \"Surveys and Questionnaires\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"4638027"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Of the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)\nAge\n≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)\nResidence\nUrban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)\nReligion\nOrthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)\nEthnicity\nBerta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa\n48 (84.2)9 (15.8)57 (14.7)\nEducational status\nNot able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)\nOccupation\nMerchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb\n105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)\nMarital status\nUnmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)\nHousehold expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\nbEmployed include government employed and private employed\nSocio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\n\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\n\nbEmployed include government employed and private employed\n Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2)."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.\nHousewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study."},"other_sections_titles":{"kind":"list like","value":["Background","Operational definitions","Data collection and quality control","Ethical consideration","Data management and analysis","Association between acceptance of PITC and each explanatory variable","Socio-demographic factors","Association between knowledge, attitudes and partner communication variables with PITC acceptance"],"string":"[\n \"Background\",\n \"Operational definitions\",\n \"Data collection and quality control\",\n \"Ethical consideration\",\n \"Data management and analysis\",\n \"Association between acceptance of PITC and each explanatory variable\",\n \"Socio-demographic factors\",\n \"Association between knowledge, attitudes and partner communication variables with PITC acceptance\"\n]"},"other_sections_texts":{"kind":"list like","value":["Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.","Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.","The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.","Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.","All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness."," Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).","There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used","Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2)."],"string":"[\n \"Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.\",\n \"Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\",\n \"The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\",\n \"Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\",\n \"All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\",\n \" Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\",\n \"There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\",\n \"Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Operational definitions","Data collection and quality control","Ethical consideration","Data management and analysis","Results","Association between acceptance of PITC and each explanatory variable","Socio-demographic factors","Association between knowledge, attitudes and partner communication variables with PITC acceptance","Discussion","Conclusion"],"string":"[\n \"Background\",\n \"Methods\",\n \"Operational definitions\",\n \"Data collection and quality control\",\n \"Ethical consideration\",\n \"Data management and analysis\",\n \"Results\",\n \"Association between acceptance of PITC and each explanatory variable\",\n \"Socio-demographic factors\",\n \"Association between knowledge, attitudes and partner communication variables with PITC acceptance\",\n \"Discussion\",\n \"Conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.","Health facility based cross sectional study was conducted from February to March, 2014 in Assosa town. Assosa town is the capital city of Benshangul Gumuz National, Regional State, located Northwest of Ethiopia at 680 km from Addis Ababa. According to the 2007 central statistics agency report of Ethiopia, a total population of the town was 24,214 (12,463 were males and 11,751 were females) [19].\nAdministratively, the town is structured into four urban kebeles. It has one general hospital and one health centre. Both health facilities currently provide ANC, PMTCT and PITC for pregnant mothers. PITC is given under informed consent to all pregnant mothers attending to the facilities based on national testing strategy guideline [13]. Based on the record review prior to data collection, the average flow of pregnant mothers in the hospital and the heal center were 450 and 250 for all ANC visits (ANC visit one up to four) per month, respectively.\nThe source population was all pregnant women attended ANC in the health facilities. The study population was 15–49 years’ old pregnant women who attended ANC services during the study period. The study units were 15–49 years’ old pregnant women who attended ANC services during the study period in Assosa town public health facilities and who selected by sampling procedures. Pregnant women, Women who were critically sick, unable to hear, or had another disability that impaired their ability to communicate were excluded.\nThe sample size was calculated using single population proportion formula considering the following assumptions; 95 % confidence interval with the corresponding value 1.96, P = 56 % (proportion of pregnant women had comprehensive knowledge on HIV/AIDS [15]), the margin of error 0.05 and with the addition of 5 % non-response rate, the total sample size became 398.\nThe sample size was proportionally allocated to the two health facilities based on the flow of ANC clients. After routine ANC visits, participants were approached for consent using a systematic random sampling method. During selection, the first pregnant woman was selected randomly by lottery method and then every other ANC follower was included in the study after obtaining their consent. Finally, data was collected using interviewer administered questionnaires until the required sample size was achieved.\n Operational definitions Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\nAcceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\n Data collection and quality control The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\nThe questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\n Ethical consideration Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\nEthical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\n Data management and analysis All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\nAll collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.","Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.","The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.","Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.","All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.","Of the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)\nAge\n≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)\nResidence\nUrban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)\nReligion\nOrthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)\nEthnicity\nBerta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa\n48 (84.2)9 (15.8)57 (14.7)\nEducational status\nNot able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)\nOccupation\nMerchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb\n105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)\nMarital status\nUnmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)\nHousehold expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\nbEmployed include government employed and private employed\nSocio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\n\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\n\nbEmployed include government employed and private employed\n Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2)."," Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).","There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used","Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).","Provider-initiated HIV testing and counselling process is crucial to achieve high coverage of HIV testing among pregnant women attending ANC for the PMTCT. The overall acceptance of PITC in this study was (80.8 %) which is consistent with the acceptance rates of PITC reported by a study done in Gondar, North West Ethiopia (82.5 %) [15]. This also much higher than the acceptance rates of VCT studies conducted Southwest parts of Ethiopia; in Illubabor (27.0 %) and in Arba-Minch (74.4 %) [16, 17]. The higher acceptance rate could be due to pregnant women consider that PITC as a standard care for the prevention of mother to child HIV transmission or it may be due to the government gives highest emphasis about the availability of comprehensive HIV/AIDS care (availability of rapid testing kits and increased access of ART drugs in ANC clinics).\nHowever, this finding was less than a study done in Nekemte, West part of Ethiopia (87.7 %) [17]. Acceptability of PITC also differs for studies conducted in different parts of Africa. This finding was relatively comparable to a study conducted in two rural districts of Zimbabwe (79 %), but lower than studies in Cameroon (88.3 %), in Botswana (95 %), in rural Ugandan hospital (97 %) and in Urban Zimbabwe 99.9 % [21–25].\nIn the present study rural women acceptability of PITC were more likely than urban residents. In contrary to this a study conducted in Gondar showed that urban residents were more likely to accept PITC than rural residents [15]. The difference in this study could be many reasons. In our context, majority of rural women were farmer, no or less educated and may not get information from different sources so, rural women might not refuse PITC because they expect that their health care provider will react negatively for their next visit, delivery or they fear that they will receive inappropriate care as a result of their refusal.\nThe other possible reason could be rural women may consider refusing of testing may not be the right of pregnant women or they may consider testing is a must because they simply accept what the health provider told them without question. These possible explanations are in line with a study conducted in Kenya where high coverage of HIV testing at ANC may be achieved at the cost of women not understanding that testing is optional and the current set-up makes most women believe that HIV testing is a prerequisite for obtaining other ANC services [26].\nIn this finding; merchants, employed women and students were more likely to accept PITC as compared to housewives. It is in line with a study done in Dre Dawa where employed women were more likely to accept testing than unemployed ones [27]. This finding also indicated that women who had higher expenditure were more likely to accept PITC than women who had lower expenditure. In line with this, a study conducted in Northwest part of Ethiopia indicated that acceptance rate of PITC were higher among those who had higher expenditure [15]. But when expenditure was adjusted with other variables in this study, it did not retain its significance. The possible reasons for association between occupation and acceptance of PITC might be for those women who had been employed, merchants and students had got access to information about PMTCT, PITC and MTCT from their respective occupation places and friends. This information might lead to independently influence their decision for accept PITC.\nIn this study 84 and 65 % pregnant women had sufficient knowledge about HIV/AIDS and PMTCT respectively. These findings are higher than a study conducted in Northwest of Ethiopia where 60 and 59 % had knowledge about HIV/AIDS and PMTCT respectively [14]. Unlike studies conducted other parts of Ethiopia [15, 27], knowledge on HIV/AIDS and PMTCT was not significantly associated for the acceptance of PITC. It indicated that knowledge about HIV/AIDS and PMTCT may not sufficient to guarantee for behavioural change. According to Fisher and colleagues, as cited by Kalich-man and Simbayi, factual based education about HIV transmission is found to be necessary but insufficient in promoting HIV testing [28].\nPositive association was reported between acceptability of PITC during ANC and more favourable attitude towards PITC. This finding was consistent with studies conducted other parts of Ethiopia [15, 27]. This could be due to the reason that pregnant women who understand the importance of testing HIV for the prevention of mother to child transmission of HIV.\nNo risk perception of acquiring HIV was observed among 60 % of the respondents in the present study. This finding was much higher than Dire Dawa (30.3 %) and much lower than Nekemte (87.5 %) findings [18, 27]. Perceived risk of getting HIV was not found to be associated with acceptance of HIV testing. A similar finding was reported from a study conducted in Ethiopia [15]. This could be due to no risk perception observed among the majority of the study participants.\nThe other independently associated factor for acceptance of testing was stigmatizing attitudes towards people having HIV/AIDS. Stigmatization attitude was a negative impact for acceptability of PITC. In line with this, a study in Humera (Ethiopia) showed that people having stigmatized attitude were decline of testing [29]. Other studies showed that AIDS related stigma may be categorized into two types of reactions to people with HIV: “instrumental AIDS stigma” (originating from a desire to protect oneself from this illness) and “symbolic AIDS stigma” (related to moral judgment) [30] and in this study stigma index included both these aspects. Unwillingness to care for relatives with AIDS, unwillingness to buy vegetables from shop sellers with AIDS, unwillingness to eat together and not allowing a teacher with AIDS to continue teaching can be categorized as instrumental AIDS stigma. Keeping HIV/AIDS infection secret can be categorized under symbolic AIDS stigma.\nStigma in this study area could have emerged due to different reasons. In the early period of the HIV epidemic in Ethiopia, many of the educational materials contained pictures of emaciated persons with AIDS. These types of educational messages might have created fears about the disease which resulted in deep rooted stigma towards people living with HIV. But similar study conducted in Gondar (Ethiopia) showed that stigmatized attitude was not significantly associated for the acceptability of PITC [15]. In Gondar study PITC may result in an atmosphere of open discussion about HIV in health care settings. This openness may spread to communities where pregnant women feel comfortable to ask and learn about HIV transmission and prevention.\nAccording to this study women who did not plan to disclose their test results of HIV from their partner were declined testing. Similarly a study conducted in Gambella (Ethiopia) showed that those who did not want to disclose test results to partner were 6.9 times more likely to refused testing [20]. The possible reasons for not disclosing to their partners might be due to fear of divorce, demand for money from her partner, cultural influence or perceived lack of acceptance. These possible reasons were consistent with a study done in Gondar where 78.5 % pregnant women expect negative reaction like insult her, psychological harassment, physical violence, disruption of marriage and stop financial support [31]. Similarly a study in Gambella showed that cultural factors like the role of polygamy marriage in creating rival wives who want to keep their test result as secret from their husband for fear of losing him and the demand for compensation payment from the one assumed to bring the disease may enforce not to disclose test results [20].\nAnother important finding in this study was those who perceived pre-test counselling service as well were more likely to accept testing. In line for this a study done in Kenya showed that good quality HIV pre-test counselling is central for making pregnant women understand and accept the reasons for testing and encourage consent to HIV testing [26]. Another study done in Gambella showed that the pre-test counselling service being fair were 6 times more likely to refuse HIV testing than those who stated their impression on the pre-test counselling service being very good [20]. Now a day’s increased number of pregnant women flow and the strategy of testing that a country follows “testing followed by counselling” may not result full course of counselling service. Due to this reason pregnant women might be loose their confidence to accept testing and they perceive pre-test counselling service quality as poor and may end up with decline of testing.\nThe first limitation was since information in the survey was self reported; some degree of underreporting of socially unacceptable behaviours and attitudes such as stigma was likely. The second limitation may be since interview was made at the end of ANC service, they might be in stressful to wait extra time and they might be respond questions without understanding. As much as possible social desirability biases were reduced by using other than health professionals for data collection and presenting study aims to the respondents clearly. This study assessed many aspects for PITC acceptability of pregnant women so, the study provides useful information that will inform the implementation and strengthening of PMTCT in Benshangul Gumuz Region and areas with similar set ups.","In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.\nHousewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study."],"string":"[\n \"Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.\",\n \"Health facility based cross sectional study was conducted from February to March, 2014 in Assosa town. Assosa town is the capital city of Benshangul Gumuz National, Regional State, located Northwest of Ethiopia at 680 km from Addis Ababa. According to the 2007 central statistics agency report of Ethiopia, a total population of the town was 24,214 (12,463 were males and 11,751 were females) [19].\\nAdministratively, the town is structured into four urban kebeles. It has one general hospital and one health centre. Both health facilities currently provide ANC, PMTCT and PITC for pregnant mothers. PITC is given under informed consent to all pregnant mothers attending to the facilities based on national testing strategy guideline [13]. Based on the record review prior to data collection, the average flow of pregnant mothers in the hospital and the heal center were 450 and 250 for all ANC visits (ANC visit one up to four) per month, respectively.\\nThe source population was all pregnant women attended ANC in the health facilities. The study population was 15–49 years’ old pregnant women who attended ANC services during the study period. The study units were 15–49 years’ old pregnant women who attended ANC services during the study period in Assosa town public health facilities and who selected by sampling procedures. Pregnant women, Women who were critically sick, unable to hear, or had another disability that impaired their ability to communicate were excluded.\\nThe sample size was calculated using single population proportion formula considering the following assumptions; 95 % confidence interval with the corresponding value 1.96, P = 56 % (proportion of pregnant women had comprehensive knowledge on HIV/AIDS [15]), the margin of error 0.05 and with the addition of 5 % non-response rate, the total sample size became 398.\\nThe sample size was proportionally allocated to the two health facilities based on the flow of ANC clients. After routine ANC visits, participants were approached for consent using a systematic random sampling method. During selection, the first pregnant woman was selected randomly by lottery method and then every other ANC follower was included in the study after obtaining their consent. Finally, data was collected using interviewer administered questionnaires until the required sample size was achieved.\\n Operational definitions Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\\nAcceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\\n Data collection and quality control The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\\nThe questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\\n Ethical consideration Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\\nEthical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\\n Data management and analysis All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\\nAll collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\",\n \"Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\",\n \"The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\",\n \"Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\",\n \"All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\",\n \"Of the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)\\nAge\\n≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)\\nResidence\\nUrban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)\\nReligion\\nOrthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)\\nEthnicity\\nBerta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa\\n48 (84.2)9 (15.8)57 (14.7)\\nEducational status\\nNot able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)\\nOccupation\\nMerchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb\\n105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)\\nMarital status\\nUnmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)\\nHousehold expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)\\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\\nbEmployed include government employed and private employed\\nSocio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\n\\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\\n\\nbEmployed include government employed and private employed\\n Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\\n Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\",\n \" Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\",\n \"There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\\nResidence\\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\\nEthnicity\\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\\nOccupation\\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\\nAttitude towards PITC\\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\\nStigmatization\\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\\nPlanned to disclose results to partner after testing\\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\\nPerceived the pre- test counselling service\\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\\nPartner reaction to positive result\\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\\nFor explanatory variables having more than 2 categories, the overall significance of P value used\",\n \"Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\",\n \"Provider-initiated HIV testing and counselling process is crucial to achieve high coverage of HIV testing among pregnant women attending ANC for the PMTCT. The overall acceptance of PITC in this study was (80.8 %) which is consistent with the acceptance rates of PITC reported by a study done in Gondar, North West Ethiopia (82.5 %) [15]. This also much higher than the acceptance rates of VCT studies conducted Southwest parts of Ethiopia; in Illubabor (27.0 %) and in Arba-Minch (74.4 %) [16, 17]. The higher acceptance rate could be due to pregnant women consider that PITC as a standard care for the prevention of mother to child HIV transmission or it may be due to the government gives highest emphasis about the availability of comprehensive HIV/AIDS care (availability of rapid testing kits and increased access of ART drugs in ANC clinics).\\nHowever, this finding was less than a study done in Nekemte, West part of Ethiopia (87.7 %) [17]. Acceptability of PITC also differs for studies conducted in different parts of Africa. This finding was relatively comparable to a study conducted in two rural districts of Zimbabwe (79 %), but lower than studies in Cameroon (88.3 %), in Botswana (95 %), in rural Ugandan hospital (97 %) and in Urban Zimbabwe 99.9 % [21–25].\\nIn the present study rural women acceptability of PITC were more likely than urban residents. In contrary to this a study conducted in Gondar showed that urban residents were more likely to accept PITC than rural residents [15]. The difference in this study could be many reasons. In our context, majority of rural women were farmer, no or less educated and may not get information from different sources so, rural women might not refuse PITC because they expect that their health care provider will react negatively for their next visit, delivery or they fear that they will receive inappropriate care as a result of their refusal.\\nThe other possible reason could be rural women may consider refusing of testing may not be the right of pregnant women or they may consider testing is a must because they simply accept what the health provider told them without question. These possible explanations are in line with a study conducted in Kenya where high coverage of HIV testing at ANC may be achieved at the cost of women not understanding that testing is optional and the current set-up makes most women believe that HIV testing is a prerequisite for obtaining other ANC services [26].\\nIn this finding; merchants, employed women and students were more likely to accept PITC as compared to housewives. It is in line with a study done in Dre Dawa where employed women were more likely to accept testing than unemployed ones [27]. This finding also indicated that women who had higher expenditure were more likely to accept PITC than women who had lower expenditure. In line with this, a study conducted in Northwest part of Ethiopia indicated that acceptance rate of PITC were higher among those who had higher expenditure [15]. But when expenditure was adjusted with other variables in this study, it did not retain its significance. The possible reasons for association between occupation and acceptance of PITC might be for those women who had been employed, merchants and students had got access to information about PMTCT, PITC and MTCT from their respective occupation places and friends. This information might lead to independently influence their decision for accept PITC.\\nIn this study 84 and 65 % pregnant women had sufficient knowledge about HIV/AIDS and PMTCT respectively. These findings are higher than a study conducted in Northwest of Ethiopia where 60 and 59 % had knowledge about HIV/AIDS and PMTCT respectively [14]. Unlike studies conducted other parts of Ethiopia [15, 27], knowledge on HIV/AIDS and PMTCT was not significantly associated for the acceptance of PITC. It indicated that knowledge about HIV/AIDS and PMTCT may not sufficient to guarantee for behavioural change. According to Fisher and colleagues, as cited by Kalich-man and Simbayi, factual based education about HIV transmission is found to be necessary but insufficient in promoting HIV testing [28].\\nPositive association was reported between acceptability of PITC during ANC and more favourable attitude towards PITC. This finding was consistent with studies conducted other parts of Ethiopia [15, 27]. This could be due to the reason that pregnant women who understand the importance of testing HIV for the prevention of mother to child transmission of HIV.\\nNo risk perception of acquiring HIV was observed among 60 % of the respondents in the present study. This finding was much higher than Dire Dawa (30.3 %) and much lower than Nekemte (87.5 %) findings [18, 27]. Perceived risk of getting HIV was not found to be associated with acceptance of HIV testing. A similar finding was reported from a study conducted in Ethiopia [15]. This could be due to no risk perception observed among the majority of the study participants.\\nThe other independently associated factor for acceptance of testing was stigmatizing attitudes towards people having HIV/AIDS. Stigmatization attitude was a negative impact for acceptability of PITC. In line with this, a study in Humera (Ethiopia) showed that people having stigmatized attitude were decline of testing [29]. Other studies showed that AIDS related stigma may be categorized into two types of reactions to people with HIV: “instrumental AIDS stigma” (originating from a desire to protect oneself from this illness) and “symbolic AIDS stigma” (related to moral judgment) [30] and in this study stigma index included both these aspects. Unwillingness to care for relatives with AIDS, unwillingness to buy vegetables from shop sellers with AIDS, unwillingness to eat together and not allowing a teacher with AIDS to continue teaching can be categorized as instrumental AIDS stigma. Keeping HIV/AIDS infection secret can be categorized under symbolic AIDS stigma.\\nStigma in this study area could have emerged due to different reasons. In the early period of the HIV epidemic in Ethiopia, many of the educational materials contained pictures of emaciated persons with AIDS. These types of educational messages might have created fears about the disease which resulted in deep rooted stigma towards people living with HIV. But similar study conducted in Gondar (Ethiopia) showed that stigmatized attitude was not significantly associated for the acceptability of PITC [15]. In Gondar study PITC may result in an atmosphere of open discussion about HIV in health care settings. This openness may spread to communities where pregnant women feel comfortable to ask and learn about HIV transmission and prevention.\\nAccording to this study women who did not plan to disclose their test results of HIV from their partner were declined testing. Similarly a study conducted in Gambella (Ethiopia) showed that those who did not want to disclose test results to partner were 6.9 times more likely to refused testing [20]. The possible reasons for not disclosing to their partners might be due to fear of divorce, demand for money from her partner, cultural influence or perceived lack of acceptance. These possible reasons were consistent with a study done in Gondar where 78.5 % pregnant women expect negative reaction like insult her, psychological harassment, physical violence, disruption of marriage and stop financial support [31]. Similarly a study in Gambella showed that cultural factors like the role of polygamy marriage in creating rival wives who want to keep their test result as secret from their husband for fear of losing him and the demand for compensation payment from the one assumed to bring the disease may enforce not to disclose test results [20].\\nAnother important finding in this study was those who perceived pre-test counselling service as well were more likely to accept testing. In line for this a study done in Kenya showed that good quality HIV pre-test counselling is central for making pregnant women understand and accept the reasons for testing and encourage consent to HIV testing [26]. Another study done in Gambella showed that the pre-test counselling service being fair were 6 times more likely to refuse HIV testing than those who stated their impression on the pre-test counselling service being very good [20]. Now a day’s increased number of pregnant women flow and the strategy of testing that a country follows “testing followed by counselling” may not result full course of counselling service. Due to this reason pregnant women might be loose their confidence to accept testing and they perceive pre-test counselling service quality as poor and may end up with decline of testing.\\nThe first limitation was since information in the survey was self reported; some degree of underreporting of socially unacceptable behaviours and attitudes such as stigma was likely. The second limitation may be since interview was made at the end of ANC service, they might be in stressful to wait extra time and they might be respond questions without understanding. As much as possible social desirability biases were reduced by using other than health professionals for data collection and presenting study aims to the respondents clearly. This study assessed many aspects for PITC acceptability of pregnant women so, the study provides useful information that will inform the implementation and strengthening of PMTCT in Benshangul Gumuz Region and areas with similar set ups.\",\n \"In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.\\nHousewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,"results",null,null,null,"discussion","conclusion"],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]"},"keywords":{"kind":"list like","value":["Pregnant women","Provider-initiated HIV testing and counselling"],"string":"[\n \"Pregnant women\",\n \"Provider-initiated HIV testing and counselling\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nWorldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.\n\nMethods:\nHealth facility based cross sectional study was conducted from February to March, 2014 in Assosa town. Assosa town is the capital city of Benshangul Gumuz National, Regional State, located Northwest of Ethiopia at 680 km from Addis Ababa. According to the 2007 central statistics agency report of Ethiopia, a total population of the town was 24,214 (12,463 were males and 11,751 were females) [19].\nAdministratively, the town is structured into four urban kebeles. It has one general hospital and one health centre. Both health facilities currently provide ANC, PMTCT and PITC for pregnant mothers. PITC is given under informed consent to all pregnant mothers attending to the facilities based on national testing strategy guideline [13]. Based on the record review prior to data collection, the average flow of pregnant mothers in the hospital and the heal center were 450 and 250 for all ANC visits (ANC visit one up to four) per month, respectively.\nThe source population was all pregnant women attended ANC in the health facilities. The study population was 15–49 years’ old pregnant women who attended ANC services during the study period. The study units were 15–49 years’ old pregnant women who attended ANC services during the study period in Assosa town public health facilities and who selected by sampling procedures. Pregnant women, Women who were critically sick, unable to hear, or had another disability that impaired their ability to communicate were excluded.\nThe sample size was calculated using single population proportion formula considering the following assumptions; 95 % confidence interval with the corresponding value 1.96, P = 56 % (proportion of pregnant women had comprehensive knowledge on HIV/AIDS [15]), the margin of error 0.05 and with the addition of 5 % non-response rate, the total sample size became 398.\nThe sample size was proportionally allocated to the two health facilities based on the flow of ANC clients. After routine ANC visits, participants were approached for consent using a systematic random sampling method. During selection, the first pregnant woman was selected randomly by lottery method and then every other ANC follower was included in the study after obtaining their consent. Finally, data was collected using interviewer administered questionnaires until the required sample size was achieved.\n Operational definitions Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\nAcceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\n Data collection and quality control The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\nThe questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\n Ethical consideration Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\nEthical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\n Data management and analysis All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\nAll collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\n\nOperational definitions:\nAcceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\n\nData collection and quality control:\nThe questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\n\nEthical consideration:\nEthical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\n\nData management and analysis:\nAll collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\n\nResults:\nOf the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)\nAge\n≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)\nResidence\nUrban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)\nReligion\nOrthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)\nEthnicity\nBerta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa\n48 (84.2)9 (15.8)57 (14.7)\nEducational status\nNot able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)\nOccupation\nMerchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb\n105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)\nMarital status\nUnmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)\nHousehold expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\nbEmployed include government employed and private employed\nSocio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\n\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\n\nbEmployed include government employed and private employed\n Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n\nAssociation between acceptance of PITC and each explanatory variable:\n Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n\nSocio-demographic factors:\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n\nAssociation between knowledge, attitudes and partner communication variables with PITC acceptance:\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n\nDiscussion:\nProvider-initiated HIV testing and counselling process is crucial to achieve high coverage of HIV testing among pregnant women attending ANC for the PMTCT. The overall acceptance of PITC in this study was (80.8 %) which is consistent with the acceptance rates of PITC reported by a study done in Gondar, North West Ethiopia (82.5 %) [15]. This also much higher than the acceptance rates of VCT studies conducted Southwest parts of Ethiopia; in Illubabor (27.0 %) and in Arba-Minch (74.4 %) [16, 17]. The higher acceptance rate could be due to pregnant women consider that PITC as a standard care for the prevention of mother to child HIV transmission or it may be due to the government gives highest emphasis about the availability of comprehensive HIV/AIDS care (availability of rapid testing kits and increased access of ART drugs in ANC clinics).\nHowever, this finding was less than a study done in Nekemte, West part of Ethiopia (87.7 %) [17]. Acceptability of PITC also differs for studies conducted in different parts of Africa. This finding was relatively comparable to a study conducted in two rural districts of Zimbabwe (79 %), but lower than studies in Cameroon (88.3 %), in Botswana (95 %), in rural Ugandan hospital (97 %) and in Urban Zimbabwe 99.9 % [21–25].\nIn the present study rural women acceptability of PITC were more likely than urban residents. In contrary to this a study conducted in Gondar showed that urban residents were more likely to accept PITC than rural residents [15]. The difference in this study could be many reasons. In our context, majority of rural women were farmer, no or less educated and may not get information from different sources so, rural women might not refuse PITC because they expect that their health care provider will react negatively for their next visit, delivery or they fear that they will receive inappropriate care as a result of their refusal.\nThe other possible reason could be rural women may consider refusing of testing may not be the right of pregnant women or they may consider testing is a must because they simply accept what the health provider told them without question. These possible explanations are in line with a study conducted in Kenya where high coverage of HIV testing at ANC may be achieved at the cost of women not understanding that testing is optional and the current set-up makes most women believe that HIV testing is a prerequisite for obtaining other ANC services [26].\nIn this finding; merchants, employed women and students were more likely to accept PITC as compared to housewives. It is in line with a study done in Dre Dawa where employed women were more likely to accept testing than unemployed ones [27]. This finding also indicated that women who had higher expenditure were more likely to accept PITC than women who had lower expenditure. In line with this, a study conducted in Northwest part of Ethiopia indicated that acceptance rate of PITC were higher among those who had higher expenditure [15]. But when expenditure was adjusted with other variables in this study, it did not retain its significance. The possible reasons for association between occupation and acceptance of PITC might be for those women who had been employed, merchants and students had got access to information about PMTCT, PITC and MTCT from their respective occupation places and friends. This information might lead to independently influence their decision for accept PITC.\nIn this study 84 and 65 % pregnant women had sufficient knowledge about HIV/AIDS and PMTCT respectively. These findings are higher than a study conducted in Northwest of Ethiopia where 60 and 59 % had knowledge about HIV/AIDS and PMTCT respectively [14]. Unlike studies conducted other parts of Ethiopia [15, 27], knowledge on HIV/AIDS and PMTCT was not significantly associated for the acceptance of PITC. It indicated that knowledge about HIV/AIDS and PMTCT may not sufficient to guarantee for behavioural change. According to Fisher and colleagues, as cited by Kalich-man and Simbayi, factual based education about HIV transmission is found to be necessary but insufficient in promoting HIV testing [28].\nPositive association was reported between acceptability of PITC during ANC and more favourable attitude towards PITC. This finding was consistent with studies conducted other parts of Ethiopia [15, 27]. This could be due to the reason that pregnant women who understand the importance of testing HIV for the prevention of mother to child transmission of HIV.\nNo risk perception of acquiring HIV was observed among 60 % of the respondents in the present study. This finding was much higher than Dire Dawa (30.3 %) and much lower than Nekemte (87.5 %) findings [18, 27]. Perceived risk of getting HIV was not found to be associated with acceptance of HIV testing. A similar finding was reported from a study conducted in Ethiopia [15]. This could be due to no risk perception observed among the majority of the study participants.\nThe other independently associated factor for acceptance of testing was stigmatizing attitudes towards people having HIV/AIDS. Stigmatization attitude was a negative impact for acceptability of PITC. In line with this, a study in Humera (Ethiopia) showed that people having stigmatized attitude were decline of testing [29]. Other studies showed that AIDS related stigma may be categorized into two types of reactions to people with HIV: “instrumental AIDS stigma” (originating from a desire to protect oneself from this illness) and “symbolic AIDS stigma” (related to moral judgment) [30] and in this study stigma index included both these aspects. Unwillingness to care for relatives with AIDS, unwillingness to buy vegetables from shop sellers with AIDS, unwillingness to eat together and not allowing a teacher with AIDS to continue teaching can be categorized as instrumental AIDS stigma. Keeping HIV/AIDS infection secret can be categorized under symbolic AIDS stigma.\nStigma in this study area could have emerged due to different reasons. In the early period of the HIV epidemic in Ethiopia, many of the educational materials contained pictures of emaciated persons with AIDS. These types of educational messages might have created fears about the disease which resulted in deep rooted stigma towards people living with HIV. But similar study conducted in Gondar (Ethiopia) showed that stigmatized attitude was not significantly associated for the acceptability of PITC [15]. In Gondar study PITC may result in an atmosphere of open discussion about HIV in health care settings. This openness may spread to communities where pregnant women feel comfortable to ask and learn about HIV transmission and prevention.\nAccording to this study women who did not plan to disclose their test results of HIV from their partner were declined testing. Similarly a study conducted in Gambella (Ethiopia) showed that those who did not want to disclose test results to partner were 6.9 times more likely to refused testing [20]. The possible reasons for not disclosing to their partners might be due to fear of divorce, demand for money from her partner, cultural influence or perceived lack of acceptance. These possible reasons were consistent with a study done in Gondar where 78.5 % pregnant women expect negative reaction like insult her, psychological harassment, physical violence, disruption of marriage and stop financial support [31]. Similarly a study in Gambella showed that cultural factors like the role of polygamy marriage in creating rival wives who want to keep their test result as secret from their husband for fear of losing him and the demand for compensation payment from the one assumed to bring the disease may enforce not to disclose test results [20].\nAnother important finding in this study was those who perceived pre-test counselling service as well were more likely to accept testing. In line for this a study done in Kenya showed that good quality HIV pre-test counselling is central for making pregnant women understand and accept the reasons for testing and encourage consent to HIV testing [26]. Another study done in Gambella showed that the pre-test counselling service being fair were 6 times more likely to refuse HIV testing than those who stated their impression on the pre-test counselling service being very good [20]. Now a day’s increased number of pregnant women flow and the strategy of testing that a country follows “testing followed by counselling” may not result full course of counselling service. Due to this reason pregnant women might be loose their confidence to accept testing and they perceive pre-test counselling service quality as poor and may end up with decline of testing.\nThe first limitation was since information in the survey was self reported; some degree of underreporting of socially unacceptable behaviours and attitudes such as stigma was likely. The second limitation may be since interview was made at the end of ANC service, they might be in stressful to wait extra time and they might be respond questions without understanding. As much as possible social desirability biases were reduced by using other than health professionals for data collection and presenting study aims to the respondents clearly. This study assessed many aspects for PITC acceptability of pregnant women so, the study provides useful information that will inform the implementation and strengthening of PMTCT in Benshangul Gumuz Region and areas with similar set ups.\n\nConclusion:\nIn conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.\nHousewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nDespite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors.\n\nMethods:\nInstitutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20.\n\nResults:\nA total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted.\n\nConclusions:\nAcceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests."},"background_conclusion_text":{"kind":"string","value":"Background:\nWorldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.\n\nConclusion:\nIn conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.\nHousewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nDespite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors.\n\nMethods:\nInstitutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20.\n\nResults:\nA total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted.\n\nConclusions:\nAcceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests."},"whole_article_text_length":{"kind":"number","value":10248,"string":"10,248"},"whole_article_abstract_length":{"kind":"number","value":391,"string":"391"},"other_sections_lengths":{"kind":"list like","value":[703,198,152,76,108,1342,424,236],"string":"[\n 703,\n 198,\n 152,\n 76,\n 108,\n 1342,\n 424,\n 236\n]"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["pitc","hiv","testing","95","ci","95 ci","women","aor","00","pregnant"],"string":"[\n \"pitc\",\n \"hiv\",\n \"testing\",\n \"95\",\n \"ci\",\n \"95 ci\",\n \"women\",\n \"aor\",\n \"00\",\n \"pregnant\"\n]"},"keybert_topics":{"kind":"list like","value":["2011 prevalence hiv","mother child hiv","hiv prevalence pregnant","hiv ethiopia year","hiv epidemic ethiopia"],"string":"[\n \"2011 prevalence hiv\",\n \"mother child hiv\",\n \"hiv prevalence pregnant\",\n \"hiv ethiopia year\",\n \"hiv epidemic ethiopia\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Pregnant women | Provider-initiated HIV testing and counselling [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Pregnant women | Provider-initiated HIV testing and counselling [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Pregnant women | Provider-initiated HIV testing and counselling [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Pregnant women | Provider-initiated HIV testing and counselling [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Pregnant women | Provider-initiated HIV testing and counselling [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | transmission | child | people | mother child | mother | ethiopia | living hiv | mother child transmission | people living hiv [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ci | 95 ci | 00 | aor | 95 | 24 | 001 | pitc | 001 00 | likely [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] women | community | increase | test | pregnant women | pregnant | pitc | especially | acceptability pitc pregnant | acceptability pitc pregnant women [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | pitc | ci | 95 ci | testing | women | 95 | aor | attitude | study [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | pitc | ci | 95 ci | testing | women | 95 | aor | attitude | study [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 386 | 97 % | 312 | 80.8 % ||| AOR 4.04 | 95 % | CI | 1.24-13.11 ||| AOR | 6.00 | 95 % | CI | 1.45-24.75 | AOR 4.43 | 95 % | CI | 1.18-16.68 | 2.15 | 95 % | CI | 1.08-4.30 ||| AOR | 95 % | CI | 1.23-10.16 ||| AOR | 14.85 | 95 % | 4.60-47.94 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 398 | February to March 2014 | two | Assosa ||| ||| Bivariate | SPSS | 20 ||| 386 | 97 % | 312 | 80.8 % ||| AOR 4.04 | 95 % | CI | 1.24-13.11 ||| AOR | 6.00 | 95 % | CI | 1.45-24.75 | AOR 4.43 | 95 % | CI | 1.18-16.68 | 2.15 | 95 % | CI | 1.08-4.30 ||| AOR | 95 % | CI | 1.23-10.16 ||| AOR | 14.85 | 95 % | 4.60-47.94 ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| 398 | February to March 2014 | two | Assosa ||| ||| Bivariate | SPSS | 20 ||| 386 | 97 % | 312 | 80.8 % ||| AOR 4.04 | 95 % | CI | 1.24-13.11 ||| AOR | 6.00 | 95 % | CI | 1.45-24.75 | AOR 4.43 | 95 % | CI | 1.18-16.68 | 2.15 | 95 % | CI | 1.08-4.30 ||| AOR | 95 % | CI | 1.23-10.16 ||| AOR | 14.85 | 95 % | 4.60-47.94 ||| ||| ||| [SUMMARY]"}}},{"rowIdx":3364,"cells":{"title":{"kind":"string","value":"Molecular characterization of Blastocystis subtypes isolated in the city of Uberaba, Minas Gerais State, Brazil."},"pmid":{"kind":"string","value":"34431950"},"background_abstract":{"kind":"string","value":"Blastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Blastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Polymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Blastocystis","Blastocystis Infections","Brazil","Cattle","Feces","Phylogeny","Swine"],"string":"[\n \"Animals\",\n \"Blastocystis\",\n \"Blastocystis Infections\",\n \"Brazil\",\n \"Cattle\",\n \"Feces\",\n \"Phylogeny\",\n \"Swine\"\n]"},"pmcid":{"kind":"string","value":"8405216"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1\n-\n4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4. \nAlthough underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1\n,\n5\n,\n6. \nThe molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7\n-\n11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124\n,\n10\n,\n12. Other subtypes have been found in pets, livestock, and zoo animals12. \nSeveral studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13\n,\n14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94\n,\n12\n,\n15\n-\n19\n,\n20.\nIn distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21\n-\n25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6\n,\n26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.\nThis study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"\nBlastocystis samples\n In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nIn this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nDNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nTotal DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nDNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nAll amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nEthical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \nThe project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. "},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates. \nPCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.\n\nTABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.\nBlastocystis isolates\nPCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†\nIdentity (%)PCR-STSSSU-rDNA amplificationSpeI digestion\n\n\n\nH09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡\nKX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.\n\n*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).\n†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.\n‡Sample classified as ST1 in the study of Moura et al. (2018). \nND: Not determined.\nSeven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined. \nAmplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.\nIn this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.\nSequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1). \n\nFIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.\n\nIn summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.\nPhylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1). "},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["\nBlastocystis samples\n","DNA extraction and PCR","DNA sequencing and analysis","Ethical approval"],"string":"[\n \"\\nBlastocystis samples\\n\",\n \"DNA extraction and PCR\",\n \"DNA sequencing and analysis\",\n \"Ethical approval\"\n]"},"other_sections_texts":{"kind":"list like","value":["In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.","Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n","All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n","The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. "],"string":"[\n \"In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\",\n \"Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\\n,\\n29.\\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\\n\\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\\n\\n\\n\",\n \"All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \\nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \\nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\\n\",\n \"The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null],"string":"[\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","\nBlastocystis samples\n","DNA extraction and PCR","DNA sequencing and analysis","Ethical approval","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"\\nBlastocystis samples\\n\",\n \"DNA extraction and PCR\",\n \"DNA sequencing and analysis\",\n \"Ethical approval\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1\n-\n4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4. \nAlthough underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1\n,\n5\n,\n6. \nThe molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7\n-\n11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124\n,\n10\n,\n12. Other subtypes have been found in pets, livestock, and zoo animals12. \nSeveral studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13\n,\n14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94\n,\n12\n,\n15\n-\n19\n,\n20.\nIn distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21\n-\n25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6\n,\n26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.\nThis study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region.","\nBlastocystis samples\n In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nIn this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nDNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nTotal DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nDNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nAll amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nEthical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \nThe project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. ","In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.","Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n","All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n","The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. ","PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates. \nPCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.\n\nTABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.\nBlastocystis isolates\nPCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†\nIdentity (%)PCR-STSSSU-rDNA amplificationSpeI digestion\n\n\n\nH09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡\nKX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.\n\n*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).\n†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.\n‡Sample classified as ST1 in the study of Moura et al. (2018). \nND: Not determined.\nSeven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined. \nAmplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.\nIn this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.\nSequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1). \n\nFIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.\n\nIn summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.\nPhylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1). ","In the present study, we confirmed that the SSU rDNA gene is polymorphic, as DNA products of different sizes from the described 1.1 kb fragment29 were amplified in human feces, indicating a similarity of approximately 99% with the Blastocystis sequences deposited in GenBank. Several indel events were observed in the generated sequences. \nThese results showed ST1-ST3, ST5, and ST10 in the city of Uberaba, with ST5 and ST10 present only in pigs and cattle, respectively. ST1 and ST3 were isolated from both humans and pigs, demonstrating the zoonotic potential of transmission of Blastocystis species, as reported by other authors31\n-\n34.\nFew studies have been carried out in Brazil regarding the geographic distribution of Blastocystis subtypes and their hosts35. ST1-ST4 have been reported to date, with a predominance of ST1 and ST321\n,\n26\n,\n35\n-\n36. In the world panorama, the most prevalent subtypes of Blastocystis spp. are ST1-ST4, but ST5-ST9 and ST12 have also been described and have different regional prevalence37\n-\n39. Mixed infections have also been reported11, showing that complex genotypes may occur in various regions of the world.\nPhylogenetic analysis showed that sequences originating from humans formed two distinct groups, one of which contained the sequences of the isolates characterized as ST1 and the other with the isolates characterized as ST3. In recent studies22\n,\n35, the grouping of subtypes into separate clades was shown by phylogenetic analysis of the different Blastocystis subtypes observed in Brazil in the South, Southeast, and Midwest regions. Pig isolates, although more similar to each other than human isolates, were divided into clades of the dendrogram, either isolated or belonging to one of the groups of human isolates. These data reinforce the potential for zoonotic transmission22\n,\n35\n,\n40. \nIn our study, 4 of the 18 water samples amplified with Blastocystis-specific primers followed by sequencing did not correspond to sequences of this parasite. Other studies in Brazil using PCR with primers other than those we used showed that amplified DNA fragments of water samples from the Tietê River, State of São Paulo, also did not correspond to the Blastocystis sequences deposited in GenBank35. These results show that the primers directed to rDNA targets, which are highly conserved regions among eukaryotes, are inadequate for investigating Blastocystis in environmental samples. Consequently, the use of primers directed to these regions is a limitation for investigating Blastocystis in environmental samples; their use in PCR tests may contribute to false-positive results if other techniques, such as DNA sequencing, are not employed. These observations point to the need for developing new specific primers and/or new techniques to investigate the presence of Blastocystis in environmental samples. \nBased on our study, we concluded that the SSU rDNA gene is polymorphic and may define intraspecific variations leading to the grouping of the isolates according to their genetic characteristics. In addition, it was verified that in the studied region, the subtypes of Blastocystis were ST1, ST3, and ST2, and zoonotic transmission is possible, as ST1 was found in both humans and pigs in the region. Finally, it was observed that the PCR of the SSU rDNA gene was not useful for the detection of Blastocystis in environmental samples, as it amplified the non-specific DNA of algae."],"string":"[\n \"Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1\\n-\\n4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4. \\nAlthough underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1\\n,\\n5\\n,\\n6. \\nThe molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7\\n-\\n11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124\\n,\\n10\\n,\\n12. Other subtypes have been found in pets, livestock, and zoo animals12. \\nSeveral studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13\\n,\\n14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94\\n,\\n12\\n,\\n15\\n-\\n19\\n,\\n20.\\nIn distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21\\n-\\n25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6\\n,\\n26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.\\nThis study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region.\",\n \"\\nBlastocystis samples\\n In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\\nIn this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\\nDNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\\n,\\n29.\\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\\n\\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\\n\\n\\n\\nTotal DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\\n,\\n29.\\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\\n\\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\\n\\n\\n\\nDNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \\nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \\nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\\n\\nAll amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \\nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \\nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\\n\\nEthical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \\nThe project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \",\n \"In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\",\n \"Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\\n,\\n29.\\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\\n\\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\\n\\n\\n\",\n \"All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \\nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \\nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\\n\",\n \"The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \",\n \"PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates. \\nPCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.\\n\\nTABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.\\nBlastocystis isolates\\nPCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†\\nIdentity (%)PCR-STSSSU-rDNA amplificationSpeI digestion\\n\\n\\n\\nH09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡\\nKX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.\\n\\n*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).\\n†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.\\n‡Sample classified as ST1 in the study of Moura et al. (2018). \\nND: Not determined.\\nSeven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined. \\nAmplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.\\nIn this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.\\nSequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1). \\n\\nFIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.\\n\\nIn summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.\\nPhylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1). \",\n \"In the present study, we confirmed that the SSU rDNA gene is polymorphic, as DNA products of different sizes from the described 1.1 kb fragment29 were amplified in human feces, indicating a similarity of approximately 99% with the Blastocystis sequences deposited in GenBank. Several indel events were observed in the generated sequences. \\nThese results showed ST1-ST3, ST5, and ST10 in the city of Uberaba, with ST5 and ST10 present only in pigs and cattle, respectively. ST1 and ST3 were isolated from both humans and pigs, demonstrating the zoonotic potential of transmission of Blastocystis species, as reported by other authors31\\n-\\n34.\\nFew studies have been carried out in Brazil regarding the geographic distribution of Blastocystis subtypes and their hosts35. ST1-ST4 have been reported to date, with a predominance of ST1 and ST321\\n,\\n26\\n,\\n35\\n-\\n36. In the world panorama, the most prevalent subtypes of Blastocystis spp. are ST1-ST4, but ST5-ST9 and ST12 have also been described and have different regional prevalence37\\n-\\n39. Mixed infections have also been reported11, showing that complex genotypes may occur in various regions of the world.\\nPhylogenetic analysis showed that sequences originating from humans formed two distinct groups, one of which contained the sequences of the isolates characterized as ST1 and the other with the isolates characterized as ST3. In recent studies22\\n,\\n35, the grouping of subtypes into separate clades was shown by phylogenetic analysis of the different Blastocystis subtypes observed in Brazil in the South, Southeast, and Midwest regions. Pig isolates, although more similar to each other than human isolates, were divided into clades of the dendrogram, either isolated or belonging to one of the groups of human isolates. These data reinforce the potential for zoonotic transmission22\\n,\\n35\\n,\\n40. \\nIn our study, 4 of the 18 water samples amplified with Blastocystis-specific primers followed by sequencing did not correspond to sequences of this parasite. Other studies in Brazil using PCR with primers other than those we used showed that amplified DNA fragments of water samples from the Tietê River, State of São Paulo, also did not correspond to the Blastocystis sequences deposited in GenBank35. These results show that the primers directed to rDNA targets, which are highly conserved regions among eukaryotes, are inadequate for investigating Blastocystis in environmental samples. Consequently, the use of primers directed to these regions is a limitation for investigating Blastocystis in environmental samples; their use in PCR tests may contribute to false-positive results if other techniques, such as DNA sequencing, are not employed. These observations point to the need for developing new specific primers and/or new techniques to investigate the presence of Blastocystis in environmental samples. \\nBased on our study, we concluded that the SSU rDNA gene is polymorphic and may define intraspecific variations leading to the grouping of the isolates according to their genetic characteristics. In addition, it was verified that in the studied region, the subtypes of Blastocystis were ST1, ST3, and ST2, and zoonotic transmission is possible, as ST1 was found in both humans and pigs in the region. Finally, it was observed that the PCR of the SSU rDNA gene was not useful for the detection of Blastocystis in environmental samples, as it amplified the non-specific DNA of algae.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Blastocystis species","Subtype","Genetic characterization","Brazil"],"string":"[\n \"Blastocystis species\",\n \"Subtype\",\n \"Genetic characterization\",\n \"Brazil\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nBlastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1\n-\n4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4. \nAlthough underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1\n,\n5\n,\n6. \nThe molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7\n-\n11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124\n,\n10\n,\n12. Other subtypes have been found in pets, livestock, and zoo animals12. \nSeveral studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13\n,\n14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94\n,\n12\n,\n15\n-\n19\n,\n20.\nIn distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21\n-\n25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6\n,\n26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.\nThis study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region.\n\nMETHODS:\n\nBlastocystis samples\n In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nIn this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nDNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nTotal DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nDNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nAll amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nEthical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \nThe project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \n\n\nBlastocystis samples\n:\nIn this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\n\nDNA extraction and PCR:\nTotal DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\n\nDNA sequencing and analysis:\nAll amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\n\nEthical approval:\nThe project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: cep@uftm.edu.br, under protocol number 1804. \n\nRESULTS:\nPCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates. \nPCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.\n\nTABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.\nBlastocystis isolates\nPCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†\nIdentity (%)PCR-STSSSU-rDNA amplificationSpeI digestion\n\n\n\nH09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡\nKX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.\n\n*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).\n†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.\n‡Sample classified as ST1 in the study of Moura et al. (2018). \nND: Not determined.\nSeven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined. \nAmplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.\nIn this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.\nSequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1). \n\nFIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.\n\nIn summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.\nPhylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1). \n\nDISCUSSION:\nIn the present study, we confirmed that the SSU rDNA gene is polymorphic, as DNA products of different sizes from the described 1.1 kb fragment29 were amplified in human feces, indicating a similarity of approximately 99% with the Blastocystis sequences deposited in GenBank. Several indel events were observed in the generated sequences. \nThese results showed ST1-ST3, ST5, and ST10 in the city of Uberaba, with ST5 and ST10 present only in pigs and cattle, respectively. ST1 and ST3 were isolated from both humans and pigs, demonstrating the zoonotic potential of transmission of Blastocystis species, as reported by other authors31\n-\n34.\nFew studies have been carried out in Brazil regarding the geographic distribution of Blastocystis subtypes and their hosts35. ST1-ST4 have been reported to date, with a predominance of ST1 and ST321\n,\n26\n,\n35\n-\n36. In the world panorama, the most prevalent subtypes of Blastocystis spp. are ST1-ST4, but ST5-ST9 and ST12 have also been described and have different regional prevalence37\n-\n39. Mixed infections have also been reported11, showing that complex genotypes may occur in various regions of the world.\nPhylogenetic analysis showed that sequences originating from humans formed two distinct groups, one of which contained the sequences of the isolates characterized as ST1 and the other with the isolates characterized as ST3. In recent studies22\n,\n35, the grouping of subtypes into separate clades was shown by phylogenetic analysis of the different Blastocystis subtypes observed in Brazil in the South, Southeast, and Midwest regions. Pig isolates, although more similar to each other than human isolates, were divided into clades of the dendrogram, either isolated or belonging to one of the groups of human isolates. These data reinforce the potential for zoonotic transmission22\n,\n35\n,\n40. \nIn our study, 4 of the 18 water samples amplified with Blastocystis-specific primers followed by sequencing did not correspond to sequences of this parasite. Other studies in Brazil using PCR with primers other than those we used showed that amplified DNA fragments of water samples from the Tietê River, State of São Paulo, also did not correspond to the Blastocystis sequences deposited in GenBank35. These results show that the primers directed to rDNA targets, which are highly conserved regions among eukaryotes, are inadequate for investigating Blastocystis in environmental samples. Consequently, the use of primers directed to these regions is a limitation for investigating Blastocystis in environmental samples; their use in PCR tests may contribute to false-positive results if other techniques, such as DNA sequencing, are not employed. These observations point to the need for developing new specific primers and/or new techniques to investigate the presence of Blastocystis in environmental samples. \nBased on our study, we concluded that the SSU rDNA gene is polymorphic and may define intraspecific variations leading to the grouping of the isolates according to their genetic characteristics. In addition, it was verified that in the studied region, the subtypes of Blastocystis were ST1, ST3, and ST2, and zoonotic transmission is possible, as ST1 was found in both humans and pigs in the region. Finally, it was observed that the PCR of the SSU rDNA gene was not useful for the detection of Blastocystis in environmental samples, as it amplified the non-specific DNA of algae.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nBlastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates.\n\nMethods:\nBlastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification.\n\nResults:\nPolymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific.\n\nConclusions:\nThe data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":4248,"string":"4,248"},"whole_article_abstract_length":{"kind":"number","value":329,"string":"329"},"other_sections_lengths":{"kind":"list like","value":[61,237,339,59],"string":"[\n 61,\n 237,\n 339,\n 59\n]"},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["blastocystis","pcr","samples","isolates","st1","rdna","ssu","subtypes","sequences","dna"],"string":"[\n \"blastocystis\",\n \"pcr\",\n \"samples\",\n \"isolates\",\n \"st1\",\n \"rdna\",\n \"ssu\",\n \"subtypes\",\n \"sequences\",\n \"dna\"\n]"},"keybert_topics":{"kind":"list like","value":["gene blastocystis isolates","seven blastocystis isolates","characterize isolates blastocystis","prevalence blastocystis spp","brazil blastocystis isolates"],"string":"[\n \"gene blastocystis isolates\",\n \"seven blastocystis isolates\",\n \"characterize isolates blastocystis\",\n \"prevalence blastocystis spp\",\n \"brazil blastocystis isolates\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] blastocystis | relationship | 17 | st1 | epidemiological profile | epidemiological | relationship blastocystis | subtypes st1 | 12 | remains [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] pcr | sequence | blastocystis | samples | sequenced | performed | rdna | ssu | analysis | dna [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 99 | pcr | isolates | blastocystis | ref | 100 | sequences | restriction | 98 | samples [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] blastocystis | pcr | samples | isolates | st1 | sequences | rdna | sequence | subtypes | dna [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Blastocystis ||| Blastocystis | Uberaba | Minas Gerais | Brazil [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 26 | Uberaba ||| GenBank [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 21 | 18 ||| ST1 | 53.3% | 40.0% | 6.7% | 100% | 50.0% | 25% | 25% ||| 98%-99% | Blastocystis | GenBank ||| Blastocystis | two | one | ST1 ||| Blastocystis [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Blastocystis | Uberaba | Minas Gerais | Brazil ||| 26 | Uberaba ||| GenBank ||| ||| 21 | 18 ||| ST1 | 53.3% | 40.0% | 6.7% | 100% | 50.0% | 25% | 25% ||| 98%-99% | Blastocystis | GenBank ||| Blastocystis | two | one | ST1 ||| Blastocystis ||| Blastocystis | Uberaba | Blastocystis [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3365,"cells":{"title":{"kind":"string","value":"The relationship between cam morphology and hip and groin symptoms and signs in young male football players."},"pmid":{"kind":"string","value":"32201993"},"background_abstract":{"kind":"string","value":"Conflicting and limited high-quality prospective data are available on the associations between cam morphology and hip and groin symptoms and range of motion (ROM)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Academy male football players (n = 49, 17-24 years) were included. Standardized antero-posterior pelvic and frog-leg lateral radiographs were obtained at baseline, 2.5- and 5-year follow-up. The femoral head-neck junction was quantified by: Visual score. Cam morphology (flattening or prominence), large cam (prominence). Alpha angle. Cam morphology (≥60°), large cam (≥78°). Cam morphology duration was defined as long (first present at baseline) or short (only from 2.5- to 5-year follow-up). Current symptoms at 5-year follow-up were assessed using a hip and groin pain question and by the \"Hip and Groin Outcome Score\" (HAGOS). HAGOS scores were categorized into: most symptoms (≥2 domains in lowest interquartile range [IQR]), least symptoms (≥2 domains in highest IQR). Hip ROM was measured by goniometry at 5-year follow-up."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Large cam morphology based on visual score was associated with hip and groin pain (23.8% vs. 7.1%, OR: 3.17, CI: [1.15-8.70], P = .026), but not with HAGOS scores. Cam morphology presence, size, and duration were associated with limited flexion of around 6° and/or 3° to 6° for internal rotation."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Cam morphology presence, size, and duration were associated with limited hip flexion and/or internal rotation, but differences might not exceed the minimal clinical important difference. Whether cam morphology results in symptoms is uncertain."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Athletes","Groin","Hip Joint","Humans","Male","Musculoskeletal Pain","Pain Measurement","Range of Motion, Articular","Soccer","Surveys and Questionnaires","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Athletes\",\n \"Groin\",\n \"Hip Joint\",\n \"Humans\",\n \"Male\",\n \"Musculoskeletal Pain\",\n \"Pain Measurement\",\n \"Range of Motion, Articular\",\n \"Soccer\",\n \"Surveys and Questionnaires\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"7317829"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":" Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\nAt baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\n Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\nThree radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\n Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\nThe femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\n Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\nThe alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\n Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\nThe independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\n Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\nThe third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\n Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\nThe researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\n Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\nThe association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\nDemographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\n Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\nNine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\n Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\nHip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\n Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nThe average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference."},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Study participants","Radiographs","Visual scores","Alpha angle","Definition of cam morphology and large cam morphology","Cam morphology duration","Hip and groin pain/symptoms","Questionnaire on hip pain and participant characteristics","Hip and groin outcome score","Hip range of motion","Statistical analysis","Participant characteristics","Cam morphology and hip and groin pain","Cam morphology and hip and groin symptoms","Cam morphology and range of motion","Cam morphology and hip and groin pain/symptoms","Cam morphology and range of motion","Limitations","Range of motion","PERSPECTIVES",""],"string":"[\n \"INTRODUCTION\",\n \"Study participants\",\n \"Radiographs\",\n \"Visual scores\",\n \"Alpha angle\",\n \"Definition of cam morphology and large cam morphology\",\n \"Cam morphology duration\",\n \"Hip and groin pain/symptoms\",\n \"Questionnaire on hip pain and participant characteristics\",\n \"Hip and groin outcome score\",\n \"Hip range of motion\",\n \"Statistical analysis\",\n \"Participant characteristics\",\n \"Cam morphology and hip and groin pain\",\n \"Cam morphology and hip and groin symptoms\",\n \"Cam morphology and range of motion\",\n \"Cam morphology and hip and groin pain/symptoms\",\n \"Cam morphology and range of motion\",\n \"Limitations\",\n \"Range of motion\",\n \"PERSPECTIVES\",\n \"\"\n]"},"other_sections_texts":{"kind":"list like","value":["Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.","At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.","Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.","The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n","The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.","The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n","The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up."," Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.","Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.","The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.","The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.","The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.","Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.","Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).","Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n","The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.","Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n","Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.","Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.","The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n","Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.","Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points"],"string":"[\n \"Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\\n1\\n while the incidence varies between 4% and 19%.\\n2\\n, \\n3\\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\\n4\\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\\n4\\n\\n\\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\\n5\\n, \\n6\\n, \\n7\\n, \\n8\\n, \\n9\\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\\n10\\n, \\n11\\n\\n\\nCam morphology prevalence in football players is high.\\n5\\n, \\n6\\n, \\n7\\n, \\n9\\n, \\n12\\n Although there is an association between cam morphology and hip osteoarthritis (OA),\\n12\\n, \\n13\\n, \\n14\\n, \\n15\\n, \\n16\\n, \\n17\\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\\n18\\n, \\n19\\n, \\n20\\n, \\n21\\n, \\n22\\n One available longitudinal case‐control study\\n23\\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\\n24\\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\\n13\\n and cartilage damage.\\n25\\n, \\n26\\n, \\n27\\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.\",\n \"At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\\n5\\n, \\n6\\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\\nParticipant characteristics at 5‐year follow‐up\\nLeft\\nRight\\nPain\\nSymptoms\\nActivities of daily living\\nSports and recreational activity\\nPhysical activity\\nQuality of life\\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\",\n \"Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\\n5\\n, \\n6\\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\",\n \"The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\\n5\\n, \\n6\\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\\n5\\n, \\n7\\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\\n5\\n\\n\",\n \"The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\\n28\\n and was used previously.\\n5\\n, \\n6\\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\\n13\\n The standard error of measurement (SEM) was 3.45.\",\n \"The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\\n29\\n\\n\",\n \"The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\",\n \" Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\",\n \"Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\",\n \"The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\",\n \"The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\\n5\\n, \\n6\\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\",\n \"The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\",\n \"Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\\n9\\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\\na\\n\\n\\nBaseline (n = 49)\\n5‐year follow‐up participants\\nBaseline (n = 40)\\n5‐year follow‐up dropouts\\nVisual score\\nAlpha angle\\nFlexion\\nLeft\\nRight\\nAbduction\\nLeft\\nRight\\nAdduction\\nLeft\\nRight\\nInternal rotation\\nLeft\\nRight\\nExternal rotation\\nLeft\\nRight\\nExtension\\nLeft\\nRight\\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\\nValues are expressed as mean ± standard deviation.\\nData of n = 38 are presented, due to missing data.\\nBolded P‐values indicate a statistically significant difference.\",\n \"Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\\nYes\\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\\nLarge cam morphology vs having no large cam morphology.\\nBolded P‐values indicate a statistically significant difference.\\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\",\n \"Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\\na\\n\\n\\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\\nValues are expressed as median (IQR, 25th‐75th centile).\\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\\n\",\n \"The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\",\n \"Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\\n22\\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\\n19\\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\\n20\\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\\n24\\n could not identify an association between cam morphology and groin injuries in professional athletes.\\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\\n23\\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\\n21\\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\\n18\\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\\n34\\n\\n\\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\\n12\\n, \\n29\\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\\n5\\n, \\n6\\n, \\n7\\n, \\n8\\n, \\n9\\n, \\n35\\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\\n11\\n\\n\",\n \"Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\\n36\\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\\n37\\n found a significant association between alpha angle and limited internal rotation. Mosler et al\\n38\\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\\n39\\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\\n33\\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\\n9\\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\",\n \"Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\\n12\\n, \\n35\\n and might have influenced symptoms.\\n40\\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\\n4\\n the risk of false‐negative measurements was minimalized.\",\n \"The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\\n41\\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\\n42\\n\\n\",\n \"Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.\",\n \"Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Study participants","Radiographs","Visual scores","Alpha angle","Definition of cam morphology and large cam morphology","Cam morphology duration","Hip and groin pain/symptoms","Questionnaire on hip pain and participant characteristics","Hip and groin outcome score","Hip range of motion","Statistical analysis","RESULTS","Participant characteristics","Cam morphology and hip and groin pain","Cam morphology and hip and groin symptoms","Cam morphology and range of motion","DISCUSSION","Cam morphology and hip and groin pain/symptoms","Cam morphology and range of motion","Limitations","Range of motion","CONCLUSION","PERSPECTIVES","","Supporting information"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Study participants\",\n \"Radiographs\",\n \"Visual scores\",\n \"Alpha angle\",\n \"Definition of cam morphology and large cam morphology\",\n \"Cam morphology duration\",\n \"Hip and groin pain/symptoms\",\n \"Questionnaire on hip pain and participant characteristics\",\n \"Hip and groin outcome score\",\n \"Hip range of motion\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Participant characteristics\",\n \"Cam morphology and hip and groin pain\",\n \"Cam morphology and hip and groin symptoms\",\n \"Cam morphology and range of motion\",\n \"DISCUSSION\",\n \"Cam morphology and hip and groin pain/symptoms\",\n \"Cam morphology and range of motion\",\n \"Limitations\",\n \"Range of motion\",\n \"CONCLUSION\",\n \"PERSPECTIVES\",\n \"\",\n \"Supporting information\"\n]"},"all_sections_texts":{"kind":"list like","value":["Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up."," Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\nAt baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\n Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\nThree radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\n Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\nThe femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\n Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\nThe alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\n Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\nThe independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\n Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\nThe third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\n Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\nThe researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\n Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\nThe association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.","At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.","Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.","The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n","The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.","The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n","The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up."," Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.","Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.","The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.","The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.","The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used."," Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\nDemographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\n Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\nNine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\n Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\nHip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\n Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nThe average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.","Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.","Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).","Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n","The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.","The relationship between cam morphology and hip and groin symptoms is inconsistent. A large cam morphology based on the visual score in young male academy football players showed an association with hip and groin pain, but not with more hip and groin symptoms as defined by the HAGOS score. A longer cam morphology duration was not significantly associated with more hip and groin symptoms. Cam morphology presence and size were associated with limited flexion and internal rotation, whereas a longer cam morphology duration was only associated with limited flexion.\n Cam morphology and hip and groin pain/symptoms Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\nLarge cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\n Cam morphology and range of motion Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\nSignificant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\n Limitations Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\nSome limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\n Range of motion The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n\nThe ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n","Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n","Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.","Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.","The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n","Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings.","Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.","Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points","Table S1\nClick here for additional data file."],"string":"[\n \"Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\\n1\\n while the incidence varies between 4% and 19%.\\n2\\n, \\n3\\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\\n4\\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\\n4\\n\\n\\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\\n5\\n, \\n6\\n, \\n7\\n, \\n8\\n, \\n9\\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\\n10\\n, \\n11\\n\\n\\nCam morphology prevalence in football players is high.\\n5\\n, \\n6\\n, \\n7\\n, \\n9\\n, \\n12\\n Although there is an association between cam morphology and hip osteoarthritis (OA),\\n12\\n, \\n13\\n, \\n14\\n, \\n15\\n, \\n16\\n, \\n17\\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\\n18\\n, \\n19\\n, \\n20\\n, \\n21\\n, \\n22\\n One available longitudinal case‐control study\\n23\\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\\n24\\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\\n13\\n and cartilage damage.\\n25\\n, \\n26\\n, \\n27\\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.\",\n \" Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\\n5\\n, \\n6\\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\\nParticipant characteristics at 5‐year follow‐up\\nLeft\\nRight\\nPain\\nSymptoms\\nActivities of daily living\\nSports and recreational activity\\nPhysical activity\\nQuality of life\\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\\nAt baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\\n5\\n, \\n6\\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\\nParticipant characteristics at 5‐year follow‐up\\nLeft\\nRight\\nPain\\nSymptoms\\nActivities of daily living\\nSports and recreational activity\\nPhysical activity\\nQuality of life\\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\\n Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\\n5\\n, \\n6\\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\\nThree radiographs were obtained during this study by the same standardized radiographic protocol as described previously\\n5\\n, \\n6\\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\\n Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\\n5\\n, \\n6\\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\\n5\\n, \\n7\\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\\n5\\n\\n\\nThe femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\\n5\\n, \\n6\\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\\n5\\n, \\n7\\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\\n5\\n\\n\\n Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\\n28\\n and was used previously.\\n5\\n, \\n6\\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\\n13\\n The standard error of measurement (SEM) was 3.45.\\nThe alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\\n28\\n and was used previously.\\n5\\n, \\n6\\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\\n13\\n The standard error of measurement (SEM) was 3.45.\\n Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\\n29\\n\\n\\nThe independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\\n29\\n\\n\\n Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\\nThe third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\\n Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\\n Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\\n Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\\n5\\n, \\n6\\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\\nThe researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\\n5\\n, \\n6\\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\\n Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\\nThe association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\",\n \"At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\\n5\\n, \\n6\\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\\nParticipant characteristics at 5‐year follow‐up\\nLeft\\nRight\\nPain\\nSymptoms\\nActivities of daily living\\nSports and recreational activity\\nPhysical activity\\nQuality of life\\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\",\n \"Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\\n5\\n, \\n6\\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\",\n \"The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\\n5\\n, \\n6\\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\\n5\\n, \\n7\\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\\n5\\n\\n\",\n \"The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\\n28\\n and was used previously.\\n5\\n, \\n6\\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\\n13\\n The standard error of measurement (SEM) was 3.45.\",\n \"The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\\n29\\n\\n\",\n \"The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\",\n \" Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\",\n \"Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\",\n \"The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\\n30\\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\\n30\\n, \\n31\\n, \\n32\\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\\n32\\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\\n33\\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\",\n \"The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\\n5\\n, \\n6\\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\",\n \"The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\",\n \" Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\\n9\\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\\na\\n\\n\\nBaseline (n = 49)\\n5‐year follow‐up participants\\nBaseline (n = 40)\\n5‐year follow‐up dropouts\\nVisual score\\nAlpha angle\\nFlexion\\nLeft\\nRight\\nAbduction\\nLeft\\nRight\\nAdduction\\nLeft\\nRight\\nInternal rotation\\nLeft\\nRight\\nExternal rotation\\nLeft\\nRight\\nExtension\\nLeft\\nRight\\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\\nValues are expressed as mean ± standard deviation.\\nData of n = 38 are presented, due to missing data.\\nBolded P‐values indicate a statistically significant difference.\\nDemographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\\n9\\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\\na\\n\\n\\nBaseline (n = 49)\\n5‐year follow‐up participants\\nBaseline (n = 40)\\n5‐year follow‐up dropouts\\nVisual score\\nAlpha angle\\nFlexion\\nLeft\\nRight\\nAbduction\\nLeft\\nRight\\nAdduction\\nLeft\\nRight\\nInternal rotation\\nLeft\\nRight\\nExternal rotation\\nLeft\\nRight\\nExtension\\nLeft\\nRight\\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\\nValues are expressed as mean ± standard deviation.\\nData of n = 38 are presented, due to missing data.\\nBolded P‐values indicate a statistically significant difference.\\n Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\\nYes\\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\\nLarge cam morphology vs having no large cam morphology.\\nBolded P‐values indicate a statistically significant difference.\\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\\nNine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\\nYes\\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\\nLarge cam morphology vs having no large cam morphology.\\nBolded P‐values indicate a statistically significant difference.\\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\\n Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\\na\\n\\n\\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\\nValues are expressed as median (IQR, 25th‐75th centile).\\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\\n\\nHip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\\na\\n\\n\\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\\nValues are expressed as median (IQR, 25th‐75th centile).\\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\\n\\n Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\\nThe average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\",\n \"Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\\n9\\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\\na\\n\\n\\nBaseline (n = 49)\\n5‐year follow‐up participants\\nBaseline (n = 40)\\n5‐year follow‐up dropouts\\nVisual score\\nAlpha angle\\nFlexion\\nLeft\\nRight\\nAbduction\\nLeft\\nRight\\nAdduction\\nLeft\\nRight\\nInternal rotation\\nLeft\\nRight\\nExternal rotation\\nLeft\\nRight\\nExtension\\nLeft\\nRight\\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\\nValues are expressed as mean ± standard deviation.\\nData of n = 38 are presented, due to missing data.\\nBolded P‐values indicate a statistically significant difference.\",\n \"Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\\nYes\\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\\nLarge cam morphology vs having no large cam morphology.\\nBolded P‐values indicate a statistically significant difference.\\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\",\n \"Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\\na\\n\\n\\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\\nValues are expressed as median (IQR, 25th‐75th centile).\\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\\n\",\n \"The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\\nValues are expressed as mean ± standard deviation.\\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\\nBolded P‐values indicate a statistically significant difference.\",\n \"The relationship between cam morphology and hip and groin symptoms is inconsistent. A large cam morphology based on the visual score in young male academy football players showed an association with hip and groin pain, but not with more hip and groin symptoms as defined by the HAGOS score. A longer cam morphology duration was not significantly associated with more hip and groin symptoms. Cam morphology presence and size were associated with limited flexion and internal rotation, whereas a longer cam morphology duration was only associated with limited flexion.\\n Cam morphology and hip and groin pain/symptoms Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\\n22\\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\\n19\\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\\n20\\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\\n24\\n could not identify an association between cam morphology and groin injuries in professional athletes.\\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\\n23\\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\\n21\\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\\n18\\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\\n34\\n\\n\\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\\n12\\n, \\n29\\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\\n5\\n, \\n6\\n, \\n7\\n, \\n8\\n, \\n9\\n, \\n35\\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\\n11\\n\\n\\nLarge cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\\n22\\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\\n19\\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\\n20\\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\\n24\\n could not identify an association between cam morphology and groin injuries in professional athletes.\\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\\n23\\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\\n21\\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\\n18\\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\\n34\\n\\n\\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\\n12\\n, \\n29\\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\\n5\\n, \\n6\\n, \\n7\\n, \\n8\\n, \\n9\\n, \\n35\\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\\n11\\n\\n\\n Cam morphology and range of motion Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\\n36\\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\\n37\\n found a significant association between alpha angle and limited internal rotation. Mosler et al\\n38\\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\\n39\\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\\n33\\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\\n9\\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\\nSignificant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\\n36\\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\\n37\\n found a significant association between alpha angle and limited internal rotation. Mosler et al\\n38\\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\\n39\\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\\n33\\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\\n9\\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\\n Limitations Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\\n12\\n, \\n35\\n and might have influenced symptoms.\\n40\\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\\n4\\n the risk of false‐negative measurements was minimalized.\\nSome limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\\n12\\n, \\n35\\n and might have influenced symptoms.\\n40\\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\\n4\\n the risk of false‐negative measurements was minimalized.\\n Range of motion The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\\n41\\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\\n42\\n\\n\\nThe ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\\n41\\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\\n42\\n\\n\",\n \"Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\\n22\\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\\n19\\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\\n20\\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\\n24\\n could not identify an association between cam morphology and groin injuries in professional athletes.\\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\\n23\\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\\n21\\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\\n18\\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\\n34\\n\\n\\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\\n12\\n, \\n29\\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\\n5\\n, \\n6\\n, \\n7\\n, \\n8\\n, \\n9\\n, \\n35\\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\\n11\\n\\n\",\n \"Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\\n36\\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\\n37\\n found a significant association between alpha angle and limited internal rotation. Mosler et al\\n38\\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\\n39\\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\\n33\\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\\n9\\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\",\n \"Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\\n12\\n, \\n35\\n and might have influenced symptoms.\\n40\\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\\n4\\n the risk of false‐negative measurements was minimalized.\",\n \"The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\\n41\\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\\n42\\n\\n\",\n \"Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings.\",\n \"Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.\",\n \"Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points\",\n \"Table S1\\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,"results",null,null,null,null,"discussion",null,null,null,null,"conclusions",null,null,"supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null,\n null,\n \"conclusions\",\n null,\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["FAI syndrome","femoroacetabular impingement","pain","range of motion"],"string":"[\n \"FAI syndrome\",\n \"femoroacetabular impingement\",\n \"pain\",\n \"range of motion\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nHip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.\n\nMETHODS:\n Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\nAt baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\n Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\nThree radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\n Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\nThe femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\n Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\nThe alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\n Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\nThe independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\n Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\nThe third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\n Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\nThe researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\n Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\nThe association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\n\nStudy participants:\nAt baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\n\nRadiographs:\nThree radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\n\nVisual scores:\nThe femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\n\nAlpha angle:\nThe alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\n\nDefinition of cam morphology and large cam morphology:\nThe independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\n\nCam morphology duration:\nThe third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\n\nHip and groin pain/symptoms:\n Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n\nQuestionnaire on hip pain and participant characteristics:\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n\nHip and groin outcome score:\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n\nHip range of motion:\nThe researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\n\nStatistical analysis:\nThe association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\n\nRESULTS:\n Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\nDemographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\n Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\nNine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\n Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\nHip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\n Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nThe average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\n\nParticipant characteristics:\nDemographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\n\nCam morphology and hip and groin pain:\nNine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\n\nCam morphology and hip and groin symptoms:\nHip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\n\nCam morphology and range of motion:\nThe average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\n\nDISCUSSION:\nThe relationship between cam morphology and hip and groin symptoms is inconsistent. A large cam morphology based on the visual score in young male academy football players showed an association with hip and groin pain, but not with more hip and groin symptoms as defined by the HAGOS score. A longer cam morphology duration was not significantly associated with more hip and groin symptoms. Cam morphology presence and size were associated with limited flexion and internal rotation, whereas a longer cam morphology duration was only associated with limited flexion.\n Cam morphology and hip and groin pain/symptoms Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\nLarge cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\n Cam morphology and range of motion Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\nSignificant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\n Limitations Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\nSome limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\n Range of motion The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n\nThe ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n\n\nCam morphology and hip and groin pain/symptoms:\nLarge cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\n\nCam morphology and range of motion:\nSignificant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\n\nLimitations:\nSome limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\n\nRange of motion:\nThe ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n\n\nCONCLUSION:\nData of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings.\n\nPERSPECTIVES:\nOur study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.\n\n:\nFlowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points\n\nSupporting information:\nTable S1\nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nConflicting and limited high-quality prospective data are available on the associations between cam morphology and hip and groin symptoms and range of motion (ROM).\n\nMethods:\nAcademy male football players (n = 49, 17-24 years) were included. Standardized antero-posterior pelvic and frog-leg lateral radiographs were obtained at baseline, 2.5- and 5-year follow-up. The femoral head-neck junction was quantified by: Visual score. Cam morphology (flattening or prominence), large cam (prominence). Alpha angle. Cam morphology (≥60°), large cam (≥78°). Cam morphology duration was defined as long (first present at baseline) or short (only from 2.5- to 5-year follow-up). Current symptoms at 5-year follow-up were assessed using a hip and groin pain question and by the \"Hip and Groin Outcome Score\" (HAGOS). HAGOS scores were categorized into: most symptoms (≥2 domains in lowest interquartile range [IQR]), least symptoms (≥2 domains in highest IQR). Hip ROM was measured by goniometry at 5-year follow-up.\n\nResults:\nLarge cam morphology based on visual score was associated with hip and groin pain (23.8% vs. 7.1%, OR: 3.17, CI: [1.15-8.70], P = .026), but not with HAGOS scores. Cam morphology presence, size, and duration were associated with limited flexion of around 6° and/or 3° to 6° for internal rotation.\n\nConclusions:\nCam morphology presence, size, and duration were associated with limited hip flexion and/or internal rotation, but differences might not exceed the minimal clinical important difference. Whether cam morphology results in symptoms is uncertain."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nHip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.\n\nCONCLUSION:\nData of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nConflicting and limited high-quality prospective data are available on the associations between cam morphology and hip and groin symptoms and range of motion (ROM).\n\nMethods:\nAcademy male football players (n = 49, 17-24 years) were included. Standardized antero-posterior pelvic and frog-leg lateral radiographs were obtained at baseline, 2.5- and 5-year follow-up. The femoral head-neck junction was quantified by: Visual score. Cam morphology (flattening or prominence), large cam (prominence). Alpha angle. Cam morphology (≥60°), large cam (≥78°). Cam morphology duration was defined as long (first present at baseline) or short (only from 2.5- to 5-year follow-up). Current symptoms at 5-year follow-up were assessed using a hip and groin pain question and by the \"Hip and Groin Outcome Score\" (HAGOS). HAGOS scores were categorized into: most symptoms (≥2 domains in lowest interquartile range [IQR]), least symptoms (≥2 domains in highest IQR). Hip ROM was measured by goniometry at 5-year follow-up.\n\nResults:\nLarge cam morphology based on visual score was associated with hip and groin pain (23.8% vs. 7.1%, OR: 3.17, CI: [1.15-8.70], P = .026), but not with HAGOS scores. Cam morphology presence, size, and duration were associated with limited flexion of around 6° and/or 3° to 6° for internal rotation.\n\nConclusions:\nCam morphology presence, size, and duration were associated with limited hip flexion and/or internal rotation, but differences might not exceed the minimal clinical important difference. Whether cam morphology results in symptoms is uncertain."},"whole_article_text_length":{"kind":"number","value":16550,"string":"16,550"},"whole_article_abstract_length":{"kind":"number","value":348,"string":"348"},"other_sections_lengths":{"kind":"list like","value":[463,235,38,110,149,118,62,662,114,208,119,227,335,458,584,477,793,292,307,88,147,15],"string":"[\n 463,\n 235,\n 38,\n 110,\n 149,\n 118,\n 62,\n 662,\n 114,\n 208,\n 119,\n 227,\n 335,\n 458,\n 584,\n 477,\n 793,\n 292,\n 307,\n 88,\n 147,\n 15\n]"},"num_sections":{"kind":"number","value":27,"string":"27"},"most_frequent_words":{"kind":"list like","value":["cam","morphology","cam morphology","hip","hips","symptoms","groin","group","pain","hip groin"],"string":"[\n \"cam\",\n \"morphology\",\n \"cam morphology\",\n \"hip\",\n \"hips\",\n \"symptoms\",\n \"groin\",\n \"group\",\n \"pain\",\n \"hip groin\"\n]"},"keybert_topics":{"kind":"list like","value":["hip groin symptoms","causes hip groin","groin symptoms athletes","athletes femoroacetabular","athletes femoroacetabular impingement"],"string":"[\n \"hip groin symptoms\",\n \"causes hip groin\",\n \"groin symptoms athletes\",\n \"athletes femoroacetabular\",\n \"athletes femoroacetabular impingement\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] FAI syndrome | femoroacetabular impingement | pain | 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[SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | symptoms | association cam | association | association cam morphology | hip | football | groin [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hip | groin | year | pain | participants | year follow | follow | outcome | hip groin | group [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | hips | cam morphology | morphology | group | persons | group symptoms | vs | cam morphology based | morphology based [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] morphology | cam morphology | cam | cohort | develop football | size duration bony cam | suggest presence size | suggest presence size duration | years cam morphology development | cohort study suggest presence [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | group | groin | pain | follow [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | group | groin | pain | follow [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 49 | 17-24 years ||| 2.5- | 5-year ||| ||| ||| ||| ||| first | 2.5- | 5-year ||| 5-year ||| IQR ||| ROM | 5-year [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 23.8% | 7.1% | 3.17 | CI ||| 1.15 | .026 | HAGOS ||| 6° and/or 3° to 6 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 49 | 17-24 years ||| 2.5- | 5-year ||| ||| ||| ||| ||| first | 2.5- | 5-year ||| 5-year ||| IQR ||| ROM | 5-year ||| 23.8% | 7.1% | 3.17 | CI ||| 1.15 | .026 | HAGOS ||| 6° and/or 3° to 6 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 49 | 17-24 years ||| 2.5- | 5-year ||| ||| ||| ||| ||| first | 2.5- | 5-year ||| 5-year ||| IQR ||| ROM | 5-year ||| 23.8% | 7.1% | 3.17 | CI ||| 1.15 | .026 | HAGOS ||| 6° and/or 3° to 6 ||| ||| [SUMMARY]"}}},{"rowIdx":3366,"cells":{"title":{"kind":"string","value":"Complications Associated With Nasopharyngeal COVID-19 Testing: An Analysis of the MAUDE Database and Literature Review."},"pmid":{"kind":"string","value":"34547903"},"background_abstract":{"kind":"string","value":"Nasopharyngeal swab testing, which has greatly increased in utilization due to the COVID-19 pandemic, is generally safe and well-tolerated, although it may be rarely associated with adverse events."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Publicly reported adverse events associated with nasopharyngeal COVID-19 testing within the Manufacturer and User Facility Device Experience (MAUDE) database and the published literature were queried."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 129 adverse events were reported, including 66 from the MAUDE database and 63 from literature review. The most common complications were swab fracture resulting in retained foreign body (47%), followed by epistaxis (17%), and headache (11%). Seven (12%) of the reported retained foreign body cases required removal under general anesthesia, while 1 (5%) of the epistaxis cases required surgical intervention. The most serious adverse event was meningitis following cerebrospinal fluid leak."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Patients and healthcare providers should be aware of the potential risks associated with testing, with attention to ensuring proper technique, and be prepared to recognize and manage adverse events."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["COVID-19","COVID-19 Testing","Databases, Factual","Humans","Nasopharynx","Pandemics","SARS-CoV-2"],"string":"[\n \"COVID-19\",\n \"COVID-19 Testing\",\n \"Databases, Factual\",\n \"Humans\",\n \"Nasopharynx\",\n \"Pandemics\",\n \"SARS-CoV-2\"\n]"},"pmcid":{"kind":"string","value":"8808139"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"This study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8\nSummary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.\nIncidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.\nMAUDE, Manufacturer and User Facility Device Experience."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery."},"other_sections_titles":{"kind":"list like","value":["Conclusion"],"string":"[\n \"Conclusion\"\n]"},"other_sections_texts":{"kind":"list like","value":["Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery."],"string":"[\n \"Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Results","Discussion","Conclusion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.","This study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test.","The MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8\nSummary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.\nIncidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.\nMAUDE, Manufacturer and User Facility Device Experience.","Nasopharyngeal testing for COVID-19 is generally safe and well-tolerated, with the risk of any adverse event ranging from 0.02% to 0.16% in previous single-center retrospective reviews.9,10 Nevertheless, healthcare providers should be cognizant of and well-equipped to manage complications associated with testing. In general, most of these adverse events may likely be mitigated by proper sampling technique, where the swab is inserted parallel to the nasal floor as opposed to a more superiorly oriented axis.\nSwab fracture resulting in a retained foreign body was the most common adverse event reported both in the MAUDE database and the literature. Swabs have an inherent breakpoint mechanism to aid in easy transfer to the transport vial. However, this breakpoint is vulnerable to accidental fragmentation during sample collection, especially in uncooperative patients or sedated patients upon whom undue force is applied.11 Furthermore, when not inserted along the nasal floor axis, the swab may contact structures that can increase the risk of fracture, such as septal spurs and the inferior and middle turbinates. Retrieval was generally performed with or without local anesthesia under direct visualization with direct rhinoscopy or nasal endoscopy. However, if the fragmented swab is not visualized, patients must be carefully monitored for foreign body ingestion or aspiration. One report discussed the need for an upper GI endoscopy to avoid possible perforation given the ingested swab's body length and sharp pointed form.12\nAlthough epistaxis was a rare and largely self-limited adverse event, its incidence warrants caution when testing individuals at increased bleeding risk. Mucosal trauma is the most likely etiology, which may be avoided by staying low in the nose. COVID-19 disproportionally affects the elderly, many of whom are treated with oral anticoagulants. Studies examining epistaxis rates following nasopharyngeal testing among such at-risk patients are needed, and a risk assessment should be considered prior to testing. Alternatively, less invasive testing methods including throat swabbing or saliva sampling can be considered.\nIatrogenic CSF leak is an unusual yet potentially fatal complication following testing. Whereas one case involved a patient with a pre-existing encephalocele7, 4 patients did not have radiographic or visual evidence of a skull base defect before testing.8–10,20 These reports illustrate the importance of proper nasopharyngeal swab technique which has been described through simulation models,13 videos,14 and graphics.15 Furthermore, providers should exhibit increased caution in patients with history of sinus/skull base surgery and consider alternative testing methods including throat swabbing or saliva sampling. As many nasopharyngeal swabs are performed in a drive-through format, patients should be queried about prior endonasal endoscopic sinus or skull base operations prior to testing.\nWe recognize that the MAUDE database is limited, especially in describing patient characteristics and comorbidities which may help identify patients who are more susceptible to experiencing a nasopharyngeal swab-related adverse event. Reports of device-related complications are mandatory for industry and voluntary for providers and patients, thus data may be under-reported. Additionally, MAUDE reports may contain erroneous narrative information. There is also a possibility that some of the complications published in the literature may have also been submitted via the MAUDE database and therefore duplicated; however, the rate of complications found through the MAUDE database and the literature review do not suggest that this happened at a significant degree. While future peer-reviewed studies will continue to provide the strongest evidence on this topic, the MAUDE database helps fill the current gap in knowledge, making this study the most comprehensive overview of adverse events following nasopharyngeal COVID-19 testing.","Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery."],"string":"[\n \"Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.\",\n \"This study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test.\",\n \"The MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8\\nSummary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.\\nIncidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.\\nMAUDE, Manufacturer and User Facility Device Experience.\",\n \"Nasopharyngeal testing for COVID-19 is generally safe and well-tolerated, with the risk of any adverse event ranging from 0.02% to 0.16% in previous single-center retrospective reviews.9,10 Nevertheless, healthcare providers should be cognizant of and well-equipped to manage complications associated with testing. In general, most of these adverse events may likely be mitigated by proper sampling technique, where the swab is inserted parallel to the nasal floor as opposed to a more superiorly oriented axis.\\nSwab fracture resulting in a retained foreign body was the most common adverse event reported both in the MAUDE database and the literature. Swabs have an inherent breakpoint mechanism to aid in easy transfer to the transport vial. However, this breakpoint is vulnerable to accidental fragmentation during sample collection, especially in uncooperative patients or sedated patients upon whom undue force is applied.11 Furthermore, when not inserted along the nasal floor axis, the swab may contact structures that can increase the risk of fracture, such as septal spurs and the inferior and middle turbinates. Retrieval was generally performed with or without local anesthesia under direct visualization with direct rhinoscopy or nasal endoscopy. However, if the fragmented swab is not visualized, patients must be carefully monitored for foreign body ingestion or aspiration. One report discussed the need for an upper GI endoscopy to avoid possible perforation given the ingested swab's body length and sharp pointed form.12\\nAlthough epistaxis was a rare and largely self-limited adverse event, its incidence warrants caution when testing individuals at increased bleeding risk. Mucosal trauma is the most likely etiology, which may be avoided by staying low in the nose. COVID-19 disproportionally affects the elderly, many of whom are treated with oral anticoagulants. Studies examining epistaxis rates following nasopharyngeal testing among such at-risk patients are needed, and a risk assessment should be considered prior to testing. Alternatively, less invasive testing methods including throat swabbing or saliva sampling can be considered.\\nIatrogenic CSF leak is an unusual yet potentially fatal complication following testing. Whereas one case involved a patient with a pre-existing encephalocele7, 4 patients did not have radiographic or visual evidence of a skull base defect before testing.8–10,20 These reports illustrate the importance of proper nasopharyngeal swab technique which has been described through simulation models,13 videos,14 and graphics.15 Furthermore, providers should exhibit increased caution in patients with history of sinus/skull base surgery and consider alternative testing methods including throat swabbing or saliva sampling. As many nasopharyngeal swabs are performed in a drive-through format, patients should be queried about prior endonasal endoscopic sinus or skull base operations prior to testing.\\nWe recognize that the MAUDE database is limited, especially in describing patient characteristics and comorbidities which may help identify patients who are more susceptible to experiencing a nasopharyngeal swab-related adverse event. Reports of device-related complications are mandatory for industry and voluntary for providers and patients, thus data may be under-reported. Additionally, MAUDE reports may contain erroneous narrative information. There is also a possibility that some of the complications published in the literature may have also been submitted via the MAUDE database and therefore duplicated; however, the rate of complications found through the MAUDE database and the literature review do not suggest that this happened at a significant degree. While future peer-reviewed studies will continue to provide the strongest evidence on this topic, the MAUDE database helps fill the current gap in knowledge, making this study the most comprehensive overview of adverse events following nasopharyngeal COVID-19 testing.\",\n \"Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results","discussion",null],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n \"discussion\",\n null\n]"},"keywords":{"kind":"list like","value":["COVID-19","nasopharyngeal swab","MAUDE","adverse events","complications"],"string":"[\n \"COVID-19\",\n \"nasopharyngeal swab\",\n \"MAUDE\",\n \"adverse events\",\n \"complications\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nDiagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.\n\nMethods:\nThis study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test.\n\nResults:\nThe MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8\nSummary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.\nIncidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.\nMAUDE, Manufacturer and User Facility Device Experience.\n\nDiscussion:\nNasopharyngeal testing for COVID-19 is generally safe and well-tolerated, with the risk of any adverse event ranging from 0.02% to 0.16% in previous single-center retrospective reviews.9,10 Nevertheless, healthcare providers should be cognizant of and well-equipped to manage complications associated with testing. In general, most of these adverse events may likely be mitigated by proper sampling technique, where the swab is inserted parallel to the nasal floor as opposed to a more superiorly oriented axis.\nSwab fracture resulting in a retained foreign body was the most common adverse event reported both in the MAUDE database and the literature. Swabs have an inherent breakpoint mechanism to aid in easy transfer to the transport vial. However, this breakpoint is vulnerable to accidental fragmentation during sample collection, especially in uncooperative patients or sedated patients upon whom undue force is applied.11 Furthermore, when not inserted along the nasal floor axis, the swab may contact structures that can increase the risk of fracture, such as septal spurs and the inferior and middle turbinates. Retrieval was generally performed with or without local anesthesia under direct visualization with direct rhinoscopy or nasal endoscopy. However, if the fragmented swab is not visualized, patients must be carefully monitored for foreign body ingestion or aspiration. One report discussed the need for an upper GI endoscopy to avoid possible perforation given the ingested swab's body length and sharp pointed form.12\nAlthough epistaxis was a rare and largely self-limited adverse event, its incidence warrants caution when testing individuals at increased bleeding risk. Mucosal trauma is the most likely etiology, which may be avoided by staying low in the nose. COVID-19 disproportionally affects the elderly, many of whom are treated with oral anticoagulants. Studies examining epistaxis rates following nasopharyngeal testing among such at-risk patients are needed, and a risk assessment should be considered prior to testing. Alternatively, less invasive testing methods including throat swabbing or saliva sampling can be considered.\nIatrogenic CSF leak is an unusual yet potentially fatal complication following testing. Whereas one case involved a patient with a pre-existing encephalocele7, 4 patients did not have radiographic or visual evidence of a skull base defect before testing.8–10,20 These reports illustrate the importance of proper nasopharyngeal swab technique which has been described through simulation models,13 videos,14 and graphics.15 Furthermore, providers should exhibit increased caution in patients with history of sinus/skull base surgery and consider alternative testing methods including throat swabbing or saliva sampling. As many nasopharyngeal swabs are performed in a drive-through format, patients should be queried about prior endonasal endoscopic sinus or skull base operations prior to testing.\nWe recognize that the MAUDE database is limited, especially in describing patient characteristics and comorbidities which may help identify patients who are more susceptible to experiencing a nasopharyngeal swab-related adverse event. Reports of device-related complications are mandatory for industry and voluntary for providers and patients, thus data may be under-reported. Additionally, MAUDE reports may contain erroneous narrative information. There is also a possibility that some of the complications published in the literature may have also been submitted via the MAUDE database and therefore duplicated; however, the rate of complications found through the MAUDE database and the literature review do not suggest that this happened at a significant degree. While future peer-reviewed studies will continue to provide the strongest evidence on this topic, the MAUDE database helps fill the current gap in knowledge, making this study the most comprehensive overview of adverse events following nasopharyngeal COVID-19 testing.\n\nConclusion:\nNasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nNasopharyngeal swab testing, which has greatly increased in utilization due to the COVID-19 pandemic, is generally safe and well-tolerated, although it may be rarely associated with adverse events.\n\nMethods:\nPublicly reported adverse events associated with nasopharyngeal COVID-19 testing within the Manufacturer and User Facility Device Experience (MAUDE) database and the published literature were queried.\n\nResults:\nA total of 129 adverse events were reported, including 66 from the MAUDE database and 63 from literature review. The most common complications were swab fracture resulting in retained foreign body (47%), followed by epistaxis (17%), and headache (11%). Seven (12%) of the reported retained foreign body cases required removal under general anesthesia, while 1 (5%) of the epistaxis cases required surgical intervention. The most serious adverse event was meningitis following cerebrospinal fluid leak.\n\nConclusions:\nPatients and healthcare providers should be aware of the potential risks associated with testing, with attention to ensuring proper technique, and be prepared to recognize and manage adverse events."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nDiagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.\n\nConclusion:\nNasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nNasopharyngeal swab testing, which has greatly increased in utilization due to the COVID-19 pandemic, is generally safe and well-tolerated, although it may be rarely associated with adverse events.\n\nMethods:\nPublicly reported adverse events associated with nasopharyngeal COVID-19 testing within the Manufacturer and User Facility Device Experience (MAUDE) database and the published literature were queried.\n\nResults:\nA total of 129 adverse events were reported, including 66 from the MAUDE database and 63 from literature review. The most common complications were swab fracture resulting in retained foreign body (47%), followed by epistaxis (17%), and headache (11%). Seven (12%) of the reported retained foreign body cases required removal under general anesthesia, while 1 (5%) of the epistaxis cases required surgical intervention. The most serious adverse event was meningitis following cerebrospinal fluid leak.\n\nConclusions:\nPatients and healthcare providers should be aware of the potential risks associated with testing, with attention to ensuring proper technique, and be prepared to recognize and manage adverse events."},"whole_article_text_length":{"kind":"number","value":1333,"string":"1,333"},"whole_article_abstract_length":{"kind":"number","value":206,"string":"206"},"other_sections_lengths":{"kind":"list like","value":[71],"string":"[\n 71\n]"},"num_sections":{"kind":"number","value":5,"string":"5"},"most_frequent_words":{"kind":"list like","value":["testing","swab","nasopharyngeal","adverse","19","covid 19","covid","complications","maude","patients"],"string":"[\n \"testing\",\n \"swab\",\n \"nasopharyngeal\",\n \"adverse\",\n \"19\",\n \"covid 19\",\n \"covid\",\n \"complications\",\n \"maude\",\n \"patients\"\n]"},"keybert_topics":{"kind":"list like","value":["nasopharyngeal testing risk","covid 19 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nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 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The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Amino Acids","Cysteine","Cardiomyopathy, Hypertrophic","Biomarkers","Proline","Arginine","Ornithine","Glycine","Choline"],"string":"[\n \"Humans\",\n \"Amino Acids\",\n \"Cysteine\",\n \"Cardiomyopathy, Hypertrophic\",\n \"Biomarkers\",\n \"Proline\",\n \"Arginine\",\n \"Ornithine\",\n \"Glycine\",\n \"Choline\"\n]"},"pmcid":{"kind":"string","value":"9746752"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]\nTo date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.\nCirculatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.\nIn the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Ethical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nThis prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nStudy population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nA total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nInclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nThe inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nTargeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nTo exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nStatistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\nContinuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Baseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nA total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nPlasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nTargeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nScreening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nIntegrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nScreening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\nAnother major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care."},"other_sections_titles":{"kind":"list like","value":["Ethical approval","Study population","Inclusion and exclusion criteria","Targeted AA metabolomics in plasma","Statistical analysis","Baseline characteristics","Plasma AA and derivatives levels and their correlations with baseline characteristics","Screening model to discriminate HCM from NC","Screening model to discriminate HOCM from HNCM","Limitations","Funding"],"string":"[\n \"Ethical approval\",\n \"Study population\",\n \"Inclusion and exclusion criteria\",\n \"Targeted AA metabolomics in plasma\",\n \"Statistical analysis\",\n \"Baseline characteristics\",\n \"Plasma AA and derivatives levels and their correlations with baseline characteristics\",\n \"Screening model to discriminate HCM from NC\",\n \"Screening model to discriminate HOCM from HNCM\",\n \"Limitations\",\n \"Funding\"\n]"},"other_sections_texts":{"kind":"list like","value":["This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.","A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]","The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.","To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.","Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).","A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.","Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.","Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.","Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.","The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.","This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08)."],"string":"[\n \"This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\",\n \"A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\",\n \"The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\",\n \"To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\",\n \"Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\",\n \"A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\",\n \"Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\",\n \"Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\",\n \"Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\",\n \"The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.\",\n \"This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Ethical approval","Study population","Inclusion and exclusion criteria","Targeted AA metabolomics in plasma","Statistical analysis","Results","Baseline characteristics","Plasma AA and derivatives levels and their correlations with baseline characteristics","Screening model to discriminate HCM from NC","Screening model to discriminate HOCM from HNCM","Discussion","Limitations","Conclusions","Funding","Conflicts of interest","Supplementary Material","Supplementary Material"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Ethical approval\",\n \"Study population\",\n \"Inclusion and exclusion criteria\",\n \"Targeted AA metabolomics in plasma\",\n \"Statistical analysis\",\n \"Results\",\n \"Baseline characteristics\",\n \"Plasma AA and derivatives levels and their correlations with baseline characteristics\",\n \"Screening model to discriminate HCM from NC\",\n \"Screening model to discriminate HOCM from HNCM\",\n \"Discussion\",\n \"Limitations\",\n \"Conclusions\",\n \"Funding\",\n \"Conflicts of interest\",\n \"Supplementary Material\",\n \"Supplementary Material\"\n]"},"all_sections_texts":{"kind":"list like","value":["Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]\nTo date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.\nCirculatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.\nIn the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.","Ethical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nThis prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nStudy population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nA total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nInclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nThe inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nTargeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nTo exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nStatistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\nContinuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).","This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.","A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]","The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.","To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.","Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).","Baseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nA total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nPlasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nTargeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nScreening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nIntegrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nScreening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\nAnother major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.","A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.","Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.","Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.","Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.","HCM is an underdiagnosed monogenetic cardiovascular disease accompanied by metabolic disorders. Growing evidence indicates that aberrations in myocardial energy metabolism contribute to the development of hypertrophy and heart failure.[27,28] In the present study, we profiled plasma AAs and derivatives, and identified AA metabolite panels with high discriminative accuracy to distinguish HCM individuals. Furthermore, multiple AAs and derivatives were significantly correlated with clinical parameters, such as the MWT and LVOTGrest. These findings provide an opportunity for developing panels of plasma biomarkers to help physicians initially identify HCM.\nHCM may be defined clinically, genetically, and histologically with high clinical heterogeneity. Some individuals remain asymptomatic throughout their lifetime with little need for treatment, while some may exhibit a progressive course, such as severe heart failure, stroke, chest pain, or even SCD.[29] It remains challenging to diagnose HCM promptly in clinical practice. Thus, a tremendous clinical challenge exists to differentiate or predict which individuals experience HCM. Even though HCM is diagnosed, it is still difficult to discern HOCM and HNCM, owing to the vulnerable LVOTG. Developing novel non-invasive, objective, and available screening biomarkers may help identify HCM in the population and improve care quality, especially in medically underserved patients. Metabolites are promising tools to understand the pathophysiological changes involved in disease onset and progression, and they can be used as biomarkers for disease prediction, diagnosis, and prognosis.[30,31] Metabolomic technologies, such as ultra-performance liquid chromatography–mass spectrometry, (semi)quantitatively measure hundreds of unique metabolites, identifying a broad range of metabolic pathways, and only small volumes of biofluids are needed.[32] Moreover, sophisticated machine learning algorithms are utilized to analyze heterogeneous datasets to discover useful patterns that would be difficult for even well-trained professionals to identify, and they have been generally recognized as an effective approach in clinical diagnostics, precision treatments, and health monitoring.[33]\nSpecifically, aberrations in cardiac AA metabolism have been shown to be associated with cardiac hypertrophy and heart failure progression.[27,28] Emerging evidence has demonstrated that metabolic aberrations may participate in the initiation and development of HCM, even in preclinical HCM.[34–36] By utilizing targeted AA metabolomics, the present study investigated the circulating AA and derivatives profile in HCM patients. Interestingly, plasma arginine, phenylalanine, tyrosine, ornithine, proline, alanine, asparagine, creatine, tryptophan, and choline levels correlated with LVOTGrest, and ornithine levels correlated with MWT, indicating that the above metabolites are associated with the severity of HCM. These results suggested that systemic AA and derivatives dysmetabolism may be involved in the pathogenesis of HCM and that changes in the plasma AA and derivatives profile may have the potential to detect or diagnose HCM.\nUnivariate analysis showed that 10 AAs and derivatives (serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid) in the HCM groups were significantly different from those in the NC group. After systematically profiling AA metabolic alterations in HCM, four metabolites (proline, glycine, cysteine, and choline) were selected as a potential AA and derivatives panel for discerning HCM from the NC, considering discriminative efficacy and clinical utility. In the training and validation sets, the AUC values of the ROC analysis were 0.83 and 0.79, respectively, demonstrating the good value of detecing HCM.\nTo minimize the confounding factors that may affect the metabolomic differences, we included a “pure” NC group accompanied by no common comorbidities. The mean age of the “pure” NC group was 34 years, which may limit generalizability to the general population. Among the different factors that may influence systemic metabolism, age and sex were not matched between HCM patients and their NC counterparts. In the past 10 years, the average age of patients diagnosed with HCM has increased by 10 years to 51 years, and male patients account for 60% of the cases.[37] In the present study, the average age of HCM patients was nearly 47 years, and males accounted for 72% of the cases. Although the baseline characteristics were mismatched in the NC and HCM groups, the sensitivity analysis stratified by age, sex, and BMI showed a consistent AUC value of approximately 0.80 across all the subgroups. Similarly, the previous history of HP had a limited effect on the discriminative performance. These results indicated that age, sex, BMI, and previous history of HP exerted a limited influence on the establishment and discriminating efficacy of the model.\nSeveral AAs and derivatives have been reported to be involved in the regulation of cardiovascular pathology. Plasma homocysteine levels are associated with the incidence of cardiovascular diseases. Homocysteine reduction improves cardiac function and ameliorates pathological structural remodeling.[38] Choline ameliorates cardiac dysfunction by regulating the expression of genes involved in the ketone body and fatty acid metabolism. This process may be involved in the activation of the sirtuin 3/adenosine monophosphate (AMP)-activated protein kinase pathway.[39] Accumulating evidence demonstrates that choline supplementation prevents myocardial hypertrophy, fibrosis, and the inflammatory response induced by pressure overload.[40,41] The elevation in plasma choline levels is associated with the incidence of heart failure.[42] Glycine promotes the synthesis of the antioxidant metabolite, glutathione, and it reduces the production of mitochondrial oxidized protein and increases adenosine triphosphate, thereby improving cardiac function and ventricular remodeling.[43] The decrease in plasma glycine levels observed in HCM patients indicates that HCM patients are in a state of oxidative stress. Proline improves myocardial remodeling and reduces infarct size and cardiomyocyte death after myocardial infarction. Proline plays a protective role in postischemic heart failure by promoting oxidative phosphorylation and improving the redox state.[44] The above studies suggest that cysteine, choline, glycine, and proline may play an important role in the pathogenesis of HCM, and more research is needed to confirm these findings. The panel comprised of the four AAs and derivatives has the potential to be used for broad population-based screening of HCM.\nLeft ventricular outflow tract obstruction (LVOTO) has malignant effects on patients. A previous study has reported that HNCM patients have higher morbidity and arrhythmic risk than HOCM patients, while HOCM patients, who are generally much older, have a higher BMI, New York Heart Association class, and overall risk.[45] Additionally, therapy choice and prognosis are quite different between HOCM and HNCM patients, and septal incision therapy may be recommended for medical refractory patients with LVOTG ≥50 mmHg. Therefore, provocative maneuvers, despite their relatively high cost and limited availability, should be considered in patients with low peak resting gradients (ie, <30 mmHg) to induce the presence of LVOTO, particularly for those with symptoms, even if provocative tests have some lethal risks.\nIn the present study, among the 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between the HOCM and HNCM groups, three metabolites (arginine, proline, and ornithine) were validated as potential biomarkers to further differentiate HOCM from HNCM with similar AUC values of 0.83 and 0.82 in the training and validation sets, respectively. Arginine levels have been linked with heart failure in a rat model.[46] HCM patients show specific features of endothelial dysfunction detectable in peripheral blood that are more severe in HOCM than HNCM.[47] In the present study, the level of plasma arginine increased in HCM, particularly in HOCM, and the levels of proline and ornithine, as the main products of arginine metabolism, decreased, indicating that arginine metabolism disorders occur in HCM, particularly for those with HOCM. Therefore, this panel can be used as a supplement to distinguish whether there is an obstruction for HCM patients, especially those with inconclusive Echo results or contraindications to stress Echo. Recognizing the existence of obstruction is helpful to guide clinical treatment strategies.\nThe present study developed two AA and derivatives panels to discriminate HCM patients from NCs as well as HOCM from HNCM patients with high sensitivity and specificity. It should be noted that the AAs and derivatives selected in these panels are different, which may indicate that distinct pathological mechanisms exist in AA metabolism during HNCM and HOCM progression.","The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.","The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care.","This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08).","None.","",""],"string":"[\n \"Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]\\nTo date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.\\nCirculatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.\\nIn the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.\",\n \"Ethical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\\nThis prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\\nStudy population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\\nA total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\\nInclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\\nThe inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\\nTargeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\\nTo exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\\nStatistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\\nContinuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\",\n \"This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\",\n \"A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\",\n \"The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\",\n \"To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\",\n \"Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\",\n \"Baseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\\nA total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\\nPlasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\\nTargeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\\nScreening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\\nIntegrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\\nScreening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\\nAnother major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\",\n \"A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\",\n \"Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\",\n \"Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\",\n \"Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\",\n \"HCM is an underdiagnosed monogenetic cardiovascular disease accompanied by metabolic disorders. Growing evidence indicates that aberrations in myocardial energy metabolism contribute to the development of hypertrophy and heart failure.[27,28] In the present study, we profiled plasma AAs and derivatives, and identified AA metabolite panels with high discriminative accuracy to distinguish HCM individuals. Furthermore, multiple AAs and derivatives were significantly correlated with clinical parameters, such as the MWT and LVOTGrest. These findings provide an opportunity for developing panels of plasma biomarkers to help physicians initially identify HCM.\\nHCM may be defined clinically, genetically, and histologically with high clinical heterogeneity. Some individuals remain asymptomatic throughout their lifetime with little need for treatment, while some may exhibit a progressive course, such as severe heart failure, stroke, chest pain, or even SCD.[29] It remains challenging to diagnose HCM promptly in clinical practice. Thus, a tremendous clinical challenge exists to differentiate or predict which individuals experience HCM. Even though HCM is diagnosed, it is still difficult to discern HOCM and HNCM, owing to the vulnerable LVOTG. Developing novel non-invasive, objective, and available screening biomarkers may help identify HCM in the population and improve care quality, especially in medically underserved patients. Metabolites are promising tools to understand the pathophysiological changes involved in disease onset and progression, and they can be used as biomarkers for disease prediction, diagnosis, and prognosis.[30,31] Metabolomic technologies, such as ultra-performance liquid chromatography–mass spectrometry, (semi)quantitatively measure hundreds of unique metabolites, identifying a broad range of metabolic pathways, and only small volumes of biofluids are needed.[32] Moreover, sophisticated machine learning algorithms are utilized to analyze heterogeneous datasets to discover useful patterns that would be difficult for even well-trained professionals to identify, and they have been generally recognized as an effective approach in clinical diagnostics, precision treatments, and health monitoring.[33]\\nSpecifically, aberrations in cardiac AA metabolism have been shown to be associated with cardiac hypertrophy and heart failure progression.[27,28] Emerging evidence has demonstrated that metabolic aberrations may participate in the initiation and development of HCM, even in preclinical HCM.[34–36] By utilizing targeted AA metabolomics, the present study investigated the circulating AA and derivatives profile in HCM patients. Interestingly, plasma arginine, phenylalanine, tyrosine, ornithine, proline, alanine, asparagine, creatine, tryptophan, and choline levels correlated with LVOTGrest, and ornithine levels correlated with MWT, indicating that the above metabolites are associated with the severity of HCM. These results suggested that systemic AA and derivatives dysmetabolism may be involved in the pathogenesis of HCM and that changes in the plasma AA and derivatives profile may have the potential to detect or diagnose HCM.\\nUnivariate analysis showed that 10 AAs and derivatives (serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid) in the HCM groups were significantly different from those in the NC group. After systematically profiling AA metabolic alterations in HCM, four metabolites (proline, glycine, cysteine, and choline) were selected as a potential AA and derivatives panel for discerning HCM from the NC, considering discriminative efficacy and clinical utility. In the training and validation sets, the AUC values of the ROC analysis were 0.83 and 0.79, respectively, demonstrating the good value of detecing HCM.\\nTo minimize the confounding factors that may affect the metabolomic differences, we included a “pure” NC group accompanied by no common comorbidities. The mean age of the “pure” NC group was 34 years, which may limit generalizability to the general population. Among the different factors that may influence systemic metabolism, age and sex were not matched between HCM patients and their NC counterparts. In the past 10 years, the average age of patients diagnosed with HCM has increased by 10 years to 51 years, and male patients account for 60% of the cases.[37] In the present study, the average age of HCM patients was nearly 47 years, and males accounted for 72% of the cases. Although the baseline characteristics were mismatched in the NC and HCM groups, the sensitivity analysis stratified by age, sex, and BMI showed a consistent AUC value of approximately 0.80 across all the subgroups. Similarly, the previous history of HP had a limited effect on the discriminative performance. These results indicated that age, sex, BMI, and previous history of HP exerted a limited influence on the establishment and discriminating efficacy of the model.\\nSeveral AAs and derivatives have been reported to be involved in the regulation of cardiovascular pathology. Plasma homocysteine levels are associated with the incidence of cardiovascular diseases. Homocysteine reduction improves cardiac function and ameliorates pathological structural remodeling.[38] Choline ameliorates cardiac dysfunction by regulating the expression of genes involved in the ketone body and fatty acid metabolism. This process may be involved in the activation of the sirtuin 3/adenosine monophosphate (AMP)-activated protein kinase pathway.[39] Accumulating evidence demonstrates that choline supplementation prevents myocardial hypertrophy, fibrosis, and the inflammatory response induced by pressure overload.[40,41] The elevation in plasma choline levels is associated with the incidence of heart failure.[42] Glycine promotes the synthesis of the antioxidant metabolite, glutathione, and it reduces the production of mitochondrial oxidized protein and increases adenosine triphosphate, thereby improving cardiac function and ventricular remodeling.[43] The decrease in plasma glycine levels observed in HCM patients indicates that HCM patients are in a state of oxidative stress. Proline improves myocardial remodeling and reduces infarct size and cardiomyocyte death after myocardial infarction. Proline plays a protective role in postischemic heart failure by promoting oxidative phosphorylation and improving the redox state.[44] The above studies suggest that cysteine, choline, glycine, and proline may play an important role in the pathogenesis of HCM, and more research is needed to confirm these findings. The panel comprised of the four AAs and derivatives has the potential to be used for broad population-based screening of HCM.\\nLeft ventricular outflow tract obstruction (LVOTO) has malignant effects on patients. A previous study has reported that HNCM patients have higher morbidity and arrhythmic risk than HOCM patients, while HOCM patients, who are generally much older, have a higher BMI, New York Heart Association class, and overall risk.[45] Additionally, therapy choice and prognosis are quite different between HOCM and HNCM patients, and septal incision therapy may be recommended for medical refractory patients with LVOTG ≥50 mmHg. Therefore, provocative maneuvers, despite their relatively high cost and limited availability, should be considered in patients with low peak resting gradients (ie, <30 mmHg) to induce the presence of LVOTO, particularly for those with symptoms, even if provocative tests have some lethal risks.\\nIn the present study, among the 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between the HOCM and HNCM groups, three metabolites (arginine, proline, and ornithine) were validated as potential biomarkers to further differentiate HOCM from HNCM with similar AUC values of 0.83 and 0.82 in the training and validation sets, respectively. Arginine levels have been linked with heart failure in a rat model.[46] HCM patients show specific features of endothelial dysfunction detectable in peripheral blood that are more severe in HOCM than HNCM.[47] In the present study, the level of plasma arginine increased in HCM, particularly in HOCM, and the levels of proline and ornithine, as the main products of arginine metabolism, decreased, indicating that arginine metabolism disorders occur in HCM, particularly for those with HOCM. Therefore, this panel can be used as a supplement to distinguish whether there is an obstruction for HCM patients, especially those with inconclusive Echo results or contraindications to stress Echo. Recognizing the existence of obstruction is helpful to guide clinical treatment strategies.\\nThe present study developed two AA and derivatives panels to discriminate HCM patients from NCs as well as HOCM from HNCM patients with high sensitivity and specificity. It should be noted that the AAs and derivatives selected in these panels are different, which may indicate that distinct pathological mechanisms exist in AA metabolism during HNCM and HOCM progression.\",\n \"The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.\",\n \"The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care.\",\n \"This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08).\",\n \"None.\",\n \"\",\n \"\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,"results",null,null,null,null,"discussion",null,"conclusion",null,"COI-statement","supplementary-material","supplementary-material"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusion\",\n null,\n \"COI-statement\",\n \"supplementary-material\",\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["Hypertrophic cardiomyopathy","Amino acids","Targeted metabolomics","Biomarkers","Algorithm"],"string":"[\n \"Hypertrophic cardiomyopathy\",\n \"Amino acids\",\n \"Targeted metabolomics\",\n \"Biomarkers\",\n \"Algorithm\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nHypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]\nTo date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.\nCirculatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.\nIn the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.\n\nMethods:\nEthical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nThis prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nStudy population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nA total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nInclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nThe inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nTargeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nTo exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nStatistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\nContinuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\n\nEthical approval:\nThis prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\n\nStudy population:\nA total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\n\nInclusion and exclusion criteria:\nThe inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\n\nTargeted AA metabolomics in plasma:\nTo exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\n\nStatistical analysis:\nContinuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\n\nResults:\nBaseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nA total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nPlasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nTargeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nScreening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nIntegrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nScreening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\nAnother major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\n\nBaseline characteristics:\nA total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\n\nPlasma AA and derivatives levels and their correlations with baseline characteristics:\nTargeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\n\nScreening model to discriminate HCM from NC:\nIntegrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\n\nScreening model to discriminate HOCM from HNCM:\nAnother major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\n\nDiscussion:\nHCM is an underdiagnosed monogenetic cardiovascular disease accompanied by metabolic disorders. Growing evidence indicates that aberrations in myocardial energy metabolism contribute to the development of hypertrophy and heart failure.[27,28] In the present study, we profiled plasma AAs and derivatives, and identified AA metabolite panels with high discriminative accuracy to distinguish HCM individuals. Furthermore, multiple AAs and derivatives were significantly correlated with clinical parameters, such as the MWT and LVOTGrest. These findings provide an opportunity for developing panels of plasma biomarkers to help physicians initially identify HCM.\nHCM may be defined clinically, genetically, and histologically with high clinical heterogeneity. Some individuals remain asymptomatic throughout their lifetime with little need for treatment, while some may exhibit a progressive course, such as severe heart failure, stroke, chest pain, or even SCD.[29] It remains challenging to diagnose HCM promptly in clinical practice. Thus, a tremendous clinical challenge exists to differentiate or predict which individuals experience HCM. Even though HCM is diagnosed, it is still difficult to discern HOCM and HNCM, owing to the vulnerable LVOTG. Developing novel non-invasive, objective, and available screening biomarkers may help identify HCM in the population and improve care quality, especially in medically underserved patients. Metabolites are promising tools to understand the pathophysiological changes involved in disease onset and progression, and they can be used as biomarkers for disease prediction, diagnosis, and prognosis.[30,31] Metabolomic technologies, such as ultra-performance liquid chromatography–mass spectrometry, (semi)quantitatively measure hundreds of unique metabolites, identifying a broad range of metabolic pathways, and only small volumes of biofluids are needed.[32] Moreover, sophisticated machine learning algorithms are utilized to analyze heterogeneous datasets to discover useful patterns that would be difficult for even well-trained professionals to identify, and they have been generally recognized as an effective approach in clinical diagnostics, precision treatments, and health monitoring.[33]\nSpecifically, aberrations in cardiac AA metabolism have been shown to be associated with cardiac hypertrophy and heart failure progression.[27,28] Emerging evidence has demonstrated that metabolic aberrations may participate in the initiation and development of HCM, even in preclinical HCM.[34–36] By utilizing targeted AA metabolomics, the present study investigated the circulating AA and derivatives profile in HCM patients. Interestingly, plasma arginine, phenylalanine, tyrosine, ornithine, proline, alanine, asparagine, creatine, tryptophan, and choline levels correlated with LVOTGrest, and ornithine levels correlated with MWT, indicating that the above metabolites are associated with the severity of HCM. These results suggested that systemic AA and derivatives dysmetabolism may be involved in the pathogenesis of HCM and that changes in the plasma AA and derivatives profile may have the potential to detect or diagnose HCM.\nUnivariate analysis showed that 10 AAs and derivatives (serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid) in the HCM groups were significantly different from those in the NC group. After systematically profiling AA metabolic alterations in HCM, four metabolites (proline, glycine, cysteine, and choline) were selected as a potential AA and derivatives panel for discerning HCM from the NC, considering discriminative efficacy and clinical utility. In the training and validation sets, the AUC values of the ROC analysis were 0.83 and 0.79, respectively, demonstrating the good value of detecing HCM.\nTo minimize the confounding factors that may affect the metabolomic differences, we included a “pure” NC group accompanied by no common comorbidities. The mean age of the “pure” NC group was 34 years, which may limit generalizability to the general population. Among the different factors that may influence systemic metabolism, age and sex were not matched between HCM patients and their NC counterparts. In the past 10 years, the average age of patients diagnosed with HCM has increased by 10 years to 51 years, and male patients account for 60% of the cases.[37] In the present study, the average age of HCM patients was nearly 47 years, and males accounted for 72% of the cases. Although the baseline characteristics were mismatched in the NC and HCM groups, the sensitivity analysis stratified by age, sex, and BMI showed a consistent AUC value of approximately 0.80 across all the subgroups. Similarly, the previous history of HP had a limited effect on the discriminative performance. These results indicated that age, sex, BMI, and previous history of HP exerted a limited influence on the establishment and discriminating efficacy of the model.\nSeveral AAs and derivatives have been reported to be involved in the regulation of cardiovascular pathology. Plasma homocysteine levels are associated with the incidence of cardiovascular diseases. Homocysteine reduction improves cardiac function and ameliorates pathological structural remodeling.[38] Choline ameliorates cardiac dysfunction by regulating the expression of genes involved in the ketone body and fatty acid metabolism. This process may be involved in the activation of the sirtuin 3/adenosine monophosphate (AMP)-activated protein kinase pathway.[39] Accumulating evidence demonstrates that choline supplementation prevents myocardial hypertrophy, fibrosis, and the inflammatory response induced by pressure overload.[40,41] The elevation in plasma choline levels is associated with the incidence of heart failure.[42] Glycine promotes the synthesis of the antioxidant metabolite, glutathione, and it reduces the production of mitochondrial oxidized protein and increases adenosine triphosphate, thereby improving cardiac function and ventricular remodeling.[43] The decrease in plasma glycine levels observed in HCM patients indicates that HCM patients are in a state of oxidative stress. Proline improves myocardial remodeling and reduces infarct size and cardiomyocyte death after myocardial infarction. Proline plays a protective role in postischemic heart failure by promoting oxidative phosphorylation and improving the redox state.[44] The above studies suggest that cysteine, choline, glycine, and proline may play an important role in the pathogenesis of HCM, and more research is needed to confirm these findings. The panel comprised of the four AAs and derivatives has the potential to be used for broad population-based screening of HCM.\nLeft ventricular outflow tract obstruction (LVOTO) has malignant effects on patients. A previous study has reported that HNCM patients have higher morbidity and arrhythmic risk than HOCM patients, while HOCM patients, who are generally much older, have a higher BMI, New York Heart Association class, and overall risk.[45] Additionally, therapy choice and prognosis are quite different between HOCM and HNCM patients, and septal incision therapy may be recommended for medical refractory patients with LVOTG ≥50 mmHg. Therefore, provocative maneuvers, despite their relatively high cost and limited availability, should be considered in patients with low peak resting gradients (ie, <30 mmHg) to induce the presence of LVOTO, particularly for those with symptoms, even if provocative tests have some lethal risks.\nIn the present study, among the 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between the HOCM and HNCM groups, three metabolites (arginine, proline, and ornithine) were validated as potential biomarkers to further differentiate HOCM from HNCM with similar AUC values of 0.83 and 0.82 in the training and validation sets, respectively. Arginine levels have been linked with heart failure in a rat model.[46] HCM patients show specific features of endothelial dysfunction detectable in peripheral blood that are more severe in HOCM than HNCM.[47] In the present study, the level of plasma arginine increased in HCM, particularly in HOCM, and the levels of proline and ornithine, as the main products of arginine metabolism, decreased, indicating that arginine metabolism disorders occur in HCM, particularly for those with HOCM. Therefore, this panel can be used as a supplement to distinguish whether there is an obstruction for HCM patients, especially those with inconclusive Echo results or contraindications to stress Echo. Recognizing the existence of obstruction is helpful to guide clinical treatment strategies.\nThe present study developed two AA and derivatives panels to discriminate HCM patients from NCs as well as HOCM from HNCM patients with high sensitivity and specificity. It should be noted that the AAs and derivatives selected in these panels are different, which may indicate that distinct pathological mechanisms exist in AA metabolism during HNCM and HOCM progression.\n\nLimitations:\nThe present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.\n\nConclusions:\nThe present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care.\n\nFunding:\nThis research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08).\n\nConflicts of interest:\nNone.\n\nSupplementary Material:\n\n\nSupplementary Material:\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM.\n\nMethods:\nPlasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.\n\nResults:\nThe univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively.\n\nConclusions:\nThe plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nHypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]\nTo date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.\nCirculatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.\nIn the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.\n\nConclusions:\nThe present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nHypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM.\n\nMethods:\nPlasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.\n\nResults:\nThe univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively.\n\nConclusions:\nThe plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive."},"whole_article_text_length":{"kind":"number","value":13656,"string":"13,656"},"whole_article_abstract_length":{"kind":"number","value":454,"string":"454"},"other_sections_lengths":{"kind":"list like","value":[48,260,196,265,368,879,468,866,338,214,38],"string":"[\n 48,\n 260,\n 196,\n 265,\n 368,\n 879,\n 468,\n 866,\n 338,\n 214,\n 38\n]"},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["hcm","hocm","hncm","00","nc","patients","screening","aa","figure","auc"],"string":"[\n \"hcm\",\n \"hocm\",\n \"hncm\",\n \"00\",\n \"nc\",\n \"patients\",\n \"screening\",\n \"aa\",\n \"figure\",\n \"auc\"\n]"},"keybert_topics":{"kind":"list like","value":["hypertrophic cardiomyopathy hncm","cardiomyopathy hncm non","hcm left ventricular","hcm normal ventricular","echocardiographic characteristics hcm"],"string":"[\n \"hypertrophic cardiomyopathy hncm\",\n \"cardiomyopathy hncm non\",\n \"hcm left ventricular\",\n \"hcm normal ventricular\",\n \"echocardiographic characteristics hcm\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] high | hcm | screening | biomarkers | imaging | aa | myocardial | circulating | lvotg | hocm [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | samples | variables | exercise | study | usa | exercise stress | model | lvotg | potential biomarkers [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 00 | hcm | 001 | nc | figure | vs | supplementary | auc | diastolic | mitral [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] diagnosis | hcm patients | hcm | patients | aa derivatives | derivatives | screening | algorithms separate aa derivatives | prompt | easily available screening [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | patients | screening | 00 | aa | hncm | samples | figure | study [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hcm | hocm | patients | screening | 00 | aa | hncm | samples | figure | study [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] HCM ||| HCM | HCM | two ||| HCM ||| HCM [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Plasma | 166 | 57 | HOCM | 52 | 57 | first | the International Cooperation Center for HCM | Xijing Hospital | between December 2019 and September 2020 | AA metabolomics ||| Three | HCM | HCM | NC | HNCM [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] HCM | NC ||| Four | Panel A | HCM ||| ROC | Panel A | ROC | 0.83 | 0.75-0.91 | 0.79 | 0.65 ||| 10 | HOCM | HNCM | 3 | two ||| AUC | 0.83 | 0.74-0.93 | 0.82 | 0.66 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] HCM | NC ||| two | HCM [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] HCM ||| HCM | HCM | two ||| HCM ||| HCM | 166 | 57 | HOCM | 52 | 57 | first | the International Cooperation Center for HCM | Xijing Hospital | between December 2019 and September 2020 | AA metabolomics ||| Three | HCM | HCM | NC | HNCM ||| ||| HCM | NC ||| Four | Panel A | HCM ||| ROC | Panel A | ROC | 0.83 | 0.75-0.91 | 0.79 | 0.65 ||| 10 | HOCM | HNCM | 3 | two ||| AUC | 0.83 | 0.74-0.93 | 0.82 | 0.66 ||| HCM | NC ||| two | HCM [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] HCM ||| HCM | HCM | two ||| HCM ||| HCM | 166 | 57 | HOCM | 52 | 57 | first | the International Cooperation Center for HCM | Xijing Hospital | between December 2019 and September 2020 | AA metabolomics ||| Three | HCM | HCM | NC | HNCM ||| ||| HCM | NC ||| Four | Panel A | HCM ||| ROC | Panel A | ROC | 0.83 | 0.75-0.91 | 0.79 | 0.65 ||| 10 | HOCM | HNCM | 3 | two ||| AUC | 0.83 | 0.74-0.93 | 0.82 | 0.66 ||| HCM | NC ||| two | HCM [SUMMARY]"}}},{"rowIdx":3368,"cells":{"title":{"kind":"string","value":"Prevalence survey of healthcare-associated infections and antimicrobial use at the University Hospital \"Paolo Giaccone\", Palermo, Italy."},"pmid":{"kind":"string","value":"24779280"},"background_abstract":{"kind":"string","value":"Healthcare-associated infections (HAIs) and antimicrobial resistance are well known major public health threats. The first goal of our study was to describe the prevalence of HAI, while the second goal was to describe the antibiotic consumption at our University Hospital, \"P. Giaccone\" in Palermo, Italy."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"A standardized methodology for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use in European acute care hospital developed by the European Centre for Disease Prevention and Control (ECDC) was piloted across Europe. The teaching Hospital \"P. Giaccone\" in Palermo, Italy, participated in the study."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Out of 328 surveyed patients, 12 (3.6%) had an HAI and 159 (48.5%) were receiving at least one antimicrobial agent. Prevalence results were highest in intensive care units, with 17.6% patients with HAI. Bloodstream infections represented the most common type (50%) of HAI. Surgical prophylaxis was the indication for antimicrobial prescribing in 59 (37.1%) out of 159 patients and exceeded 24 hours in 54 (91.5%) cases."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The results suggest that in our hospital there was a frequent and inappropriate use of antimicrobials, especially in the setting of surgical prophylaxis."},"conclusions_abstract_label":{"kind":"string","value":"DISCUSSION"},"mesh_descriptor_names":{"kind":"list like","value":["Anti-Bacterial Agents","Antibiotic Prophylaxis","Bacteremia","Cross Infection","Drug Resistance, Bacterial","Drug Utilization","Female","Hospitals, University","Humans","Intensive Care Units","Italy","Male","Middle Aged","Prevalence"],"string":"[\n \"Anti-Bacterial Agents\",\n \"Antibiotic Prophylaxis\",\n \"Bacteremia\",\n \"Cross Infection\",\n \"Drug Resistance, Bacterial\",\n \"Drug Utilization\",\n \"Female\",\n \"Hospitals, University\",\n \"Humans\",\n \"Intensive Care Units\",\n \"Italy\",\n \"Male\",\n \"Middle Aged\",\n \"Prevalence\"\n]"},"pmcid":{"kind":"string","value":"4718321"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Healthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.\nExcessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.\nOn September 2011, the teaching Hospital \"Paolo Giaccone\" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.\nThe specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" SETTING The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\nThe Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\n POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\nThe standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\n CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\nEuropean case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\n DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.\nThe antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"A total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).\nTwelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).\nOverall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).\nPattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.\nECDC: European Centre for Disease Prevention and Control.\nTotal of patients treated for relative indication.\nPercent of the total.\nPatients receiving the indicated number of antimicrobial agents for each indication of treatment.\nPercent each within category.\nIn 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.\nDistribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).\nAnatomical Therapeutic Chemical\nIn MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).\nData on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["SETTING","POINT PREVALENCE SURVEY (PPS)","CASE DEFINITION","DEFINITIONS OF ANTIMICROBIAL USE DATA"],"string":"[\n \"SETTING\",\n \"POINT PREVALENCE SURVEY (PPS)\",\n \"CASE DEFINITION\",\n \"DEFINITIONS OF ANTIMICROBIAL USE DATA\"\n]"},"other_sections_texts":{"kind":"list like","value":["The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.","The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.","European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.","The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS."],"string":"[\n \"The Teaching Hospital \\\"P. Giaccone\\\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\",\n \"The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \\\"Hospital data form\\\" including general information on the type of surveyed hospital and \\\"Patient data form\\\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\",\n \"European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\\nor\\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\",\n \"The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\\nother indications (e.g. use of erythromycin as a prokinetic agent);\\nunknown indication/reason (assessed during PPS);\\nunknown/missing information on indication no verified during PPS.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null],"string":"[\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","SETTING","POINT PREVALENCE SURVEY (PPS)","CASE DEFINITION","DEFINITIONS OF ANTIMICROBIAL USE DATA","Results","Discussion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"SETTING\",\n \"POINT PREVALENCE SURVEY (PPS)\",\n \"CASE DEFINITION\",\n \"DEFINITIONS OF ANTIMICROBIAL USE DATA\",\n \"Results\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Healthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.\nExcessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.\nOn September 2011, the teaching Hospital \"Paolo Giaccone\" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.\nThe specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level."," SETTING The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\nThe Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\n POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\nThe standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\n CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\nEuropean case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\n DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.\nThe antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.","The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.","The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.","European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.","The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.","A total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).\nTwelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).\nOverall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).\nPattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.\nECDC: European Centre for Disease Prevention and Control.\nTotal of patients treated for relative indication.\nPercent of the total.\nPatients receiving the indicated number of antimicrobial agents for each indication of treatment.\nPercent each within category.\nIn 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.\nDistribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).\nAnatomical Therapeutic Chemical\nIn MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).\nData on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae.","HAIs are a serious problem, contributing heavily to the burden of the hospital costs [7]. These infections have a negative impact on patient because of the worsening of underlying medical condition and the increased morbidity and mortality [7].\nThe rapidly escalating prevalence of antimicrobial resistance is a global concern. The reduced susceptibility to most current of available antimicrobial agents coupled with the progressive shortage of newly approved compounds is a worrisome situation [7]. Major problems are encountered for a growing number of Gram-positive organisms (i.e., Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus spp.) and Gram-negative pathogens (i.e., Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae) [7]. Serious infections caused by resistant bacteria do not respond to therapy and are often associated with worse outcomes, including increased rates of complications, additional costs, higher associated mortality rates and prolonged hospital stays [8].\nHAIs' prevalence of 3.6% observed in our survey is lower than that reported in other European studies and that observed in 2008 (6.7%) in the same hospital. The limits of the HAI's estimate by PPS are well known. However, because case-mix and admission rules are not changed in the last years, it can be presumed that some prevention and control interventions have been to some extent effective. In particular\nsome guidelines were issued to implement infection control procedures, such as Antibiotic perisurgical prophylaxis in adults, Ambulances hygiene, handwashing (Clean care is safer care), Isolation measures in the AOUP P. Giaccone, Palermo, isolation of patients with colonization/infection by multi-resistant pathogens. So healthcare workers training programs were carried out as well.\nThe European prevalence has been estimated to be 7.1% by ECDC, based on a review of 30 national or multicentre PPS in 19 countries in its annual epidemiological report for 2008 [4, 6]. However an important issue that should be considered for the interpretation of the epidemiological results of this and future surveys is the standardization of data collection in participating hospitals to compare results between hospitals, regions and countries. Indeed, HAI prevalence may depend on differences in methodology and patients case-mix and not only on performance variations.\nHigh rates of antibiotic usage were observed in our hospital. In particular our survey revealed use of broad spectrum antibiotics, sometimes combined in multidrug protocols, because of emergence of antibiotic-resistant microorganisms [9]. On the other hand, due to the widespread diffusion of MDR organisms in our healthcare settings, it is often necessary to start an initial antibiotic therapy with multidrug protocols or last resource molecules, such as colistin. New diagnostic methods that allow to obtain microbiological confirmation in short time could be very useful. It is known that the excessive and unnecessary use of antibiotics is the main driving force of the high rates of drug resistant infections we are seeing in recent years [10, 11].\nThe results of this survey confirm those of a parallel analysis with another classical approach in which the antibiotic use in the hospital was measured by evaluating the numbers of defined daily doses (DDD) (Malta R et al., 2010) for 100 bed days or for number of admissions. The DDD consumed in 2011 were in fact 82.06 as per 100 bed-days and 5.41 per admission. For the surgical wards, overall the DDD were 94.00 per100 bed-days and 5.98 per admission; quinolones accounted for 25.6% of the DDD consumed, cephalosporins for 23.7%, penicillins for 16.2% and metronidazole for 14.4%. Moreover, DDD consumed for 100 bed-days in our hospital proved to be higher than those observed in a sample of five hospitals in Emilia Romagna despite increasing by 18% in the three years period, 64.9 DDD/100 bed days in 2002 to 76.7 in 2004 [9, 12, 14].\nResults showed also that a significant area of antibacterial overuse was SP. According to the international consensus, antibiotic prophylaxis in surgery involves the administration of an antibacterial agent for a very short time, temporally located just before the beginning of intervention [12]. However, our study revealed that the antibiotics were used for longer intervals of times. A reason of inappropriate antibacterial drug use was the frequent administration as SP of drugs, such as 3rd generation cephalosporins, which are not recommended for surgical prophylaxis and should be reserved to the treatment of severe active infections.\nOur study has some limitation. Indeed the PP study design has some inherent limits, including reduced periods of observation and the possibility of obtaining biased results. It has the advantage to be very economical in terms of both time and human and financial resources. Repeated PPS represent a more feasible alternative for hospital-wide surveillance of all HAIs, while still allowing the estimation of HAIs burden in acute hospitals, and helping to prioritize areas requiring interventions [1]. Therefore, although it was carried out in full compliance with the European protocol, the study has likely some limitations inherent to the study design. Continuous surveillance, especially prospective active surveillance, is the gold standard to improve patient safety.\nThe HAI prevalence rate detected in our PPS was lower than expected. However, our investigation highlighted a large misuse of antibacterial drugs, in particular in the area of SP. Prophylactic use of antibiotics in surgery is an example of how a potentially useful practice can be misused. More in general, inappropriate antibiotic use can lead to increased morbidity, prolonged hospital stay and need for more expensive drugs [13, 14]. The main aim of antibiotic stewardship is to bring a change in prescribing which could lead to control of drug resistance, decreased costs and improved quality of antibiotic use. Approaches that have to be, also locally, adopted include educational programs, development of prescribing guidelines (e.g. Clean Care is Safer Care, WHO) [15], monitoring of drug resistance patterns, turning to restrictive hospital formularies [16, 17]."],"string":"[\n \"Healthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.\\nExcessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.\\nOn September 2011, the teaching Hospital \\\"Paolo Giaccone\\\" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.\\nThe specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level.\",\n \" SETTING The Teaching Hospital \\\"P. Giaccone\\\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\\nThe Teaching Hospital \\\"P. Giaccone\\\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\\n POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \\\"Hospital data form\\\" including general information on the type of surveyed hospital and \\\"Patient data form\\\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\\nThe standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \\\"Hospital data form\\\" including general information on the type of surveyed hospital and \\\"Patient data form\\\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\\n CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\\nor\\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\\nEuropean case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\\nor\\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\\n DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\\nother indications (e.g. use of erythromycin as a prokinetic agent);\\nunknown indication/reason (assessed during PPS);\\nunknown/missing information on indication no verified during PPS.\\nThe antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\\nother indications (e.g. use of erythromycin as a prokinetic agent);\\nunknown indication/reason (assessed during PPS);\\nunknown/missing information on indication no verified during PPS.\",\n \"The Teaching Hospital \\\"P. Giaccone\\\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\",\n \"The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \\\"Hospital data form\\\" including general information on the type of surveyed hospital and \\\"Patient data form\\\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\",\n \"European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\\nor\\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\",\n \"The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\\nother indications (e.g. use of erythromycin as a prokinetic agent);\\nunknown indication/reason (assessed during PPS);\\nunknown/missing information on indication no verified during PPS.\",\n \"A total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).\\nTwelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).\\nOverall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).\\nPattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.\\nECDC: European Centre for Disease Prevention and Control.\\nTotal of patients treated for relative indication.\\nPercent of the total.\\nPatients receiving the indicated number of antimicrobial agents for each indication of treatment.\\nPercent each within category.\\nIn 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.\\nDistribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).\\nAnatomical Therapeutic Chemical\\nIn MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).\\nData on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae.\",\n \"HAIs are a serious problem, contributing heavily to the burden of the hospital costs [7]. These infections have a negative impact on patient because of the worsening of underlying medical condition and the increased morbidity and mortality [7].\\nThe rapidly escalating prevalence of antimicrobial resistance is a global concern. The reduced susceptibility to most current of available antimicrobial agents coupled with the progressive shortage of newly approved compounds is a worrisome situation [7]. Major problems are encountered for a growing number of Gram-positive organisms (i.e., Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus spp.) and Gram-negative pathogens (i.e., Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae) [7]. Serious infections caused by resistant bacteria do not respond to therapy and are often associated with worse outcomes, including increased rates of complications, additional costs, higher associated mortality rates and prolonged hospital stays [8].\\nHAIs' prevalence of 3.6% observed in our survey is lower than that reported in other European studies and that observed in 2008 (6.7%) in the same hospital. The limits of the HAI's estimate by PPS are well known. However, because case-mix and admission rules are not changed in the last years, it can be presumed that some prevention and control interventions have been to some extent effective. In particular\\nsome guidelines were issued to implement infection control procedures, such as Antibiotic perisurgical prophylaxis in adults, Ambulances hygiene, handwashing (Clean care is safer care), Isolation measures in the AOUP P. Giaccone, Palermo, isolation of patients with colonization/infection by multi-resistant pathogens. So healthcare workers training programs were carried out as well.\\nThe European prevalence has been estimated to be 7.1% by ECDC, based on a review of 30 national or multicentre PPS in 19 countries in its annual epidemiological report for 2008 [4, 6]. However an important issue that should be considered for the interpretation of the epidemiological results of this and future surveys is the standardization of data collection in participating hospitals to compare results between hospitals, regions and countries. Indeed, HAI prevalence may depend on differences in methodology and patients case-mix and not only on performance variations.\\nHigh rates of antibiotic usage were observed in our hospital. In particular our survey revealed use of broad spectrum antibiotics, sometimes combined in multidrug protocols, because of emergence of antibiotic-resistant microorganisms [9]. On the other hand, due to the widespread diffusion of MDR organisms in our healthcare settings, it is often necessary to start an initial antibiotic therapy with multidrug protocols or last resource molecules, such as colistin. New diagnostic methods that allow to obtain microbiological confirmation in short time could be very useful. It is known that the excessive and unnecessary use of antibiotics is the main driving force of the high rates of drug resistant infections we are seeing in recent years [10, 11].\\nThe results of this survey confirm those of a parallel analysis with another classical approach in which the antibiotic use in the hospital was measured by evaluating the numbers of defined daily doses (DDD) (Malta R et al., 2010) for 100 bed days or for number of admissions. The DDD consumed in 2011 were in fact 82.06 as per 100 bed-days and 5.41 per admission. For the surgical wards, overall the DDD were 94.00 per100 bed-days and 5.98 per admission; quinolones accounted for 25.6% of the DDD consumed, cephalosporins for 23.7%, penicillins for 16.2% and metronidazole for 14.4%. Moreover, DDD consumed for 100 bed-days in our hospital proved to be higher than those observed in a sample of five hospitals in Emilia Romagna despite increasing by 18% in the three years period, 64.9 DDD/100 bed days in 2002 to 76.7 in 2004 [9, 12, 14].\\nResults showed also that a significant area of antibacterial overuse was SP. According to the international consensus, antibiotic prophylaxis in surgery involves the administration of an antibacterial agent for a very short time, temporally located just before the beginning of intervention [12]. However, our study revealed that the antibiotics were used for longer intervals of times. A reason of inappropriate antibacterial drug use was the frequent administration as SP of drugs, such as 3rd generation cephalosporins, which are not recommended for surgical prophylaxis and should be reserved to the treatment of severe active infections.\\nOur study has some limitation. Indeed the PP study design has some inherent limits, including reduced periods of observation and the possibility of obtaining biased results. It has the advantage to be very economical in terms of both time and human and financial resources. Repeated PPS represent a more feasible alternative for hospital-wide surveillance of all HAIs, while still allowing the estimation of HAIs burden in acute hospitals, and helping to prioritize areas requiring interventions [1]. Therefore, although it was carried out in full compliance with the European protocol, the study has likely some limitations inherent to the study design. Continuous surveillance, especially prospective active surveillance, is the gold standard to improve patient safety.\\nThe HAI prevalence rate detected in our PPS was lower than expected. However, our investigation highlighted a large misuse of antibacterial drugs, in particular in the area of SP. Prophylactic use of antibiotics in surgery is an example of how a potentially useful practice can be misused. More in general, inappropriate antibiotic use can lead to increased morbidity, prolonged hospital stay and need for more expensive drugs [13, 14]. The main aim of antibiotic stewardship is to bring a change in prescribing which could lead to control of drug resistance, decreased costs and improved quality of antibiotic use. Approaches that have to be, also locally, adopted include educational programs, development of prescribing guidelines (e.g. Clean Care is Safer Care, WHO) [15], monitoring of drug resistance patterns, turning to restrictive hospital formularies [16, 17].\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","methods",null,null,null,null,"results","discussion"],"string":"[\n \"introduction\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Healthcare-associated infections","Antimicrobials drug resistance","Point Prevalence Survey"],"string":"[\n \"Healthcare-associated infections\",\n \"Antimicrobials drug resistance\",\n \"Point Prevalence Survey\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nHealthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.\nExcessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.\nOn September 2011, the teaching Hospital \"Paolo Giaccone\" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.\nThe specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level.\n\nMethods:\n SETTING The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\nThe Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\n POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\nThe standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\n CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\nEuropean case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\n DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.\nThe antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.\n\nSETTING:\nThe Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\n\nPOINT PREVALENCE SURVEY (PPS):\nThe standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\n\nCASE DEFINITION:\nEuropean case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\n\nDEFINITIONS OF ANTIMICROBIAL USE DATA:\nThe antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.\n\nResults:\nA total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).\nTwelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).\nOverall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).\nPattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.\nECDC: European Centre for Disease Prevention and Control.\nTotal of patients treated for relative indication.\nPercent of the total.\nPatients receiving the indicated number of antimicrobial agents for each indication of treatment.\nPercent each within category.\nIn 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.\nDistribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).\nAnatomical Therapeutic Chemical\nIn MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).\nData on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae.\n\nDiscussion:\nHAIs are a serious problem, contributing heavily to the burden of the hospital costs [7]. These infections have a negative impact on patient because of the worsening of underlying medical condition and the increased morbidity and mortality [7].\nThe rapidly escalating prevalence of antimicrobial resistance is a global concern. The reduced susceptibility to most current of available antimicrobial agents coupled with the progressive shortage of newly approved compounds is a worrisome situation [7]. Major problems are encountered for a growing number of Gram-positive organisms (i.e., Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus spp.) and Gram-negative pathogens (i.e., Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae) [7]. Serious infections caused by resistant bacteria do not respond to therapy and are often associated with worse outcomes, including increased rates of complications, additional costs, higher associated mortality rates and prolonged hospital stays [8].\nHAIs' prevalence of 3.6% observed in our survey is lower than that reported in other European studies and that observed in 2008 (6.7%) in the same hospital. The limits of the HAI's estimate by PPS are well known. However, because case-mix and admission rules are not changed in the last years, it can be presumed that some prevention and control interventions have been to some extent effective. In particular\nsome guidelines were issued to implement infection control procedures, such as Antibiotic perisurgical prophylaxis in adults, Ambulances hygiene, handwashing (Clean care is safer care), Isolation measures in the AOUP P. Giaccone, Palermo, isolation of patients with colonization/infection by multi-resistant pathogens. So healthcare workers training programs were carried out as well.\nThe European prevalence has been estimated to be 7.1% by ECDC, based on a review of 30 national or multicentre PPS in 19 countries in its annual epidemiological report for 2008 [4, 6]. However an important issue that should be considered for the interpretation of the epidemiological results of this and future surveys is the standardization of data collection in participating hospitals to compare results between hospitals, regions and countries. Indeed, HAI prevalence may depend on differences in methodology and patients case-mix and not only on performance variations.\nHigh rates of antibiotic usage were observed in our hospital. In particular our survey revealed use of broad spectrum antibiotics, sometimes combined in multidrug protocols, because of emergence of antibiotic-resistant microorganisms [9]. On the other hand, due to the widespread diffusion of MDR organisms in our healthcare settings, it is often necessary to start an initial antibiotic therapy with multidrug protocols or last resource molecules, such as colistin. New diagnostic methods that allow to obtain microbiological confirmation in short time could be very useful. It is known that the excessive and unnecessary use of antibiotics is the main driving force of the high rates of drug resistant infections we are seeing in recent years [10, 11].\nThe results of this survey confirm those of a parallel analysis with another classical approach in which the antibiotic use in the hospital was measured by evaluating the numbers of defined daily doses (DDD) (Malta R et al., 2010) for 100 bed days or for number of admissions. The DDD consumed in 2011 were in fact 82.06 as per 100 bed-days and 5.41 per admission. For the surgical wards, overall the DDD were 94.00 per100 bed-days and 5.98 per admission; quinolones accounted for 25.6% of the DDD consumed, cephalosporins for 23.7%, penicillins for 16.2% and metronidazole for 14.4%. Moreover, DDD consumed for 100 bed-days in our hospital proved to be higher than those observed in a sample of five hospitals in Emilia Romagna despite increasing by 18% in the three years period, 64.9 DDD/100 bed days in 2002 to 76.7 in 2004 [9, 12, 14].\nResults showed also that a significant area of antibacterial overuse was SP. According to the international consensus, antibiotic prophylaxis in surgery involves the administration of an antibacterial agent for a very short time, temporally located just before the beginning of intervention [12]. However, our study revealed that the antibiotics were used for longer intervals of times. A reason of inappropriate antibacterial drug use was the frequent administration as SP of drugs, such as 3rd generation cephalosporins, which are not recommended for surgical prophylaxis and should be reserved to the treatment of severe active infections.\nOur study has some limitation. Indeed the PP study design has some inherent limits, including reduced periods of observation and the possibility of obtaining biased results. It has the advantage to be very economical in terms of both time and human and financial resources. Repeated PPS represent a more feasible alternative for hospital-wide surveillance of all HAIs, while still allowing the estimation of HAIs burden in acute hospitals, and helping to prioritize areas requiring interventions [1]. Therefore, although it was carried out in full compliance with the European protocol, the study has likely some limitations inherent to the study design. Continuous surveillance, especially prospective active surveillance, is the gold standard to improve patient safety.\nThe HAI prevalence rate detected in our PPS was lower than expected. However, our investigation highlighted a large misuse of antibacterial drugs, in particular in the area of SP. Prophylactic use of antibiotics in surgery is an example of how a potentially useful practice can be misused. More in general, inappropriate antibiotic use can lead to increased morbidity, prolonged hospital stay and need for more expensive drugs [13, 14]. The main aim of antibiotic stewardship is to bring a change in prescribing which could lead to control of drug resistance, decreased costs and improved quality of antibiotic use. Approaches that have to be, also locally, adopted include educational programs, development of prescribing guidelines (e.g. Clean Care is Safer Care, WHO) [15], monitoring of drug resistance patterns, turning to restrictive hospital formularies [16, 17].\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHealthcare-associated infections (HAIs) and antimicrobial resistance are well known major public health threats. The first goal of our study was to describe the prevalence of HAI, while the second goal was to describe the antibiotic consumption at our University Hospital, \"P. Giaccone\" in Palermo, Italy.\n\nMethods:\nA standardized methodology for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use in European acute care hospital developed by the European Centre for Disease Prevention and Control (ECDC) was piloted across Europe. The teaching Hospital \"P. Giaccone\" in Palermo, Italy, participated in the study.\n\nResults:\nOut of 328 surveyed patients, 12 (3.6%) had an HAI and 159 (48.5%) were receiving at least one antimicrobial agent. Prevalence results were highest in intensive care units, with 17.6% patients with HAI. Bloodstream infections represented the most common type (50%) of HAI. Surgical prophylaxis was the indication for antimicrobial prescribing in 59 (37.1%) out of 159 patients and exceeded 24 hours in 54 (91.5%) cases.\n\nConclusions:\nThe results suggest that in our hospital there was a frequent and inappropriate use of antimicrobials, especially in the setting of surgical prophylaxis."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":4818,"string":"4,818"},"whole_article_abstract_length":{"kind":"number","value":247,"string":"247"},"other_sections_lengths":{"kind":"list like","value":[71,333,145,252],"string":"[\n 71,\n 333,\n 145,\n 252\n]"},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["survey","hai","data","hospital","use","patients","antimicrobial","infections","care","prevalence"],"string":"[\n \"survey\",\n \"hai\",\n \"data\",\n \"hospital\",\n \"use\",\n \"patients\",\n \"antimicrobial\",\n \"infections\",\n \"care\",\n \"prevalence\"\n]"},"keybert_topics":{"kind":"list like","value":["hai antibiotic use","antibiotic use hospital","rates antibiotic usage","escalating prevalence antimicrobial","antimicrobial use data"],"string":"[\n \"hai antibiotic use\",\n \"antibiotic use hospital\",\n \"rates antibiotic usage\",\n \"escalating prevalence antimicrobial\",\n \"antimicrobial use data\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Healthcare-associated infections | Antimicrobials drug resistance | Point Prevalence Survey [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Healthcare-associated infections | Antimicrobials drug resistance | Point Prevalence Survey [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Healthcare-associated infections | Antimicrobials drug resistance | Point Prevalence Survey [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Healthcare-associated infections | Antimicrobials drug resistance | Point Prevalence Survey [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Anti-Bacterial Agents | Antibiotic Prophylaxis | Bacteremia | Cross Infection | Drug Resistance, Bacterial | Drug Utilization | Female | Hospitals, University | Humans | Intensive Care Units | Italy | Male | Middle Aged | Prevalence [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Anti-Bacterial Agents | Antibiotic Prophylaxis | Bacteremia | Cross Infection | Drug Resistance, Bacterial | Drug Utilization | Female | Hospitals, University | Humans | Intensive Care Units | Italy | Male | Middle Aged | Prevalence [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Anti-Bacterial Agents | Antibiotic Prophylaxis | Bacteremia | Cross Infection | Drug Resistance, Bacterial | Drug Utilization | Female | Hospitals, University | Humans | Intensive Care Units | Italy | Male | Middle Aged | Prevalence [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Anti-Bacterial Agents | Antibiotic Prophylaxis | Bacteremia | Cross Infection | Drug Resistance, Bacterial | Drug Utilization | Female | Hospitals, University | Humans | Intensive Care Units | Italy | Male | Middle Aged | Prevalence [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hai antibiotic use | antibiotic use hospital | rates antibiotic usage | escalating prevalence antimicrobial | antimicrobial use data [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hai antibiotic use | antibiotic use hospital | rates antibiotic usage | escalating prevalence antimicrobial | antimicrobial use data [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hai antibiotic use | antibiotic use hospital | rates antibiotic usage | escalating prevalence antimicrobial | antimicrobial use data [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hai antibiotic use | antibiotic use hospital | rates antibiotic usage | escalating prevalence antimicrobial | antimicrobial use data [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] survey | hai | data | hospital | use | patients | antimicrobial | infections | care | prevalence [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] survey | hai | data | hospital | use | patients | antimicrobial | infections | care | prevalence [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] survey | hai | data | hospital | use | patients | antimicrobial | infections | care | prevalence [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] survey | hai | data | hospital | use | patients | antimicrobial | infections | care | prevalence [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] use | infections | antibiotic use | hai | standardized | 000 | tract infections | worldwide | prevalence | antibiotic [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] data | survey | hospital | hai | day | acquired | collected | prophylaxis | definitions | antimicrobial [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | cases | agents | 37 | 14 | 328 | remaining | patients hai | medicine | decubitus [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | survey | hai | data | use | patients | infections | prophylaxis | antimicrobial | care [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Healthcare ||| first | HAI | second | University Hospital | Palermo | Italy [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] PPS | European | the European Centre for Disease Prevention and Control | ECDC | Europe ||| Palermo | Italy [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 328 | 12 | 3.6% | HAI | 159 | 48.5% | at least one ||| 17.6% | HAI ||| 50% | HAI ||| 59 | 37.1% | 159 | 24 hours | 54 | 91.5% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Healthcare ||| first | HAI | second | University Hospital | Palermo | Italy ||| PPS | European | the European Centre for Disease Prevention and Control | ECDC | Europe ||| Palermo | Italy ||| 328 | 12 | 3.6% | HAI | 159 | 48.5% | at least one ||| 17.6% | HAI ||| 50% | HAI ||| 59 | 37.1% | 159 | 24 hours | 54 | 91.5% ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3369,"cells":{"title":{"kind":"string","value":"Clinical assessment of liver metabolism during hypothermic oxygenated machine perfusion using microdialysis."},"pmid":{"kind":"string","value":"34516020"},"background_abstract":{"kind":"string","value":"While growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"To assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Median (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2nd-hour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN. These correlations were not apparent when perfusate levels were considered. In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"MD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Cold Ischemia","Female","Glucose","Graft Survival","Humans","Lactic Acid","Liver","Liver Transplantation","Male","Microdialysis","Middle Aged","Organ Preservation","Perfusion","Pilot Projects","Prospective Studies"],"string":"[\n \"Aged\",\n \"Cold Ischemia\",\n \"Female\",\n \"Glucose\",\n \"Graft Survival\",\n \"Humans\",\n \"Lactic Acid\",\n \"Liver\",\n \"Liver Transplantation\",\n \"Male\",\n \"Microdialysis\",\n \"Middle Aged\",\n \"Organ Preservation\",\n \"Perfusion\",\n \"Pilot Projects\",\n \"Prospective Studies\"\n]"},"pmcid":{"kind":"string","value":"9292750"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\n1\n, \n2\n, \n3\n extended‐criteria donors after brain death (DBD)\n4\n, \n5\n, and steatotic grafts.\n6\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\n7\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\n8\n, \n9\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\n10\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\n11\n, \n12\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\n13\n, \n14\n, \n15\n, \n16\n, \n17\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\n18\n, \n19\n, \n20\n, \n21\n, \n22\n and explored as a tool for early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\n30\n MD has been occasionally used in the setting of kidney machine perfusion\n31\n, \n32\n, \n33\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":" D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\nRecipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\n Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\nFigure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\n Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\nFigure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\n Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\nHistological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]"},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","PATIENTS AND METHODS","Study design","Outcome measures","Statistical analysis","D‐HOPE procedure and clinical outcome","Microdialysate and perfusate metabolites","Microdialysate and perfusate FMN","Histology and expression of inflammatory cytokines","AUTHOR CONTRIBUTIONS"],"string":"[\n \"INTRODUCTION\",\n \"PATIENTS AND METHODS\",\n \"Study design\",\n \"Outcome measures\",\n \"Statistical analysis\",\n \"D‐HOPE procedure and clinical outcome\",\n \"Microdialysate and perfusate metabolites\",\n \"Microdialysate and perfusate FMN\",\n \"Histology and expression of inflammatory cytokines\",\n \"AUTHOR CONTRIBUTIONS\"\n]"},"other_sections_texts":{"kind":"list like","value":["Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\n1\n, \n2\n, \n3\n extended‐criteria donors after brain death (DBD)\n4\n, \n5\n, and steatotic grafts.\n6\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\n7\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\n8\n, \n9\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\n10\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\n11\n, \n12\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\n13\n, \n14\n, \n15\n, \n16\n, \n17\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\n18\n, \n19\n, \n20\n, \n21\n, \n22\n and explored as a tool for early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\n30\n MD has been occasionally used in the setting of kidney machine perfusion\n31\n, \n32\n, \n33\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT."," Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\nPrimary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).","This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.","Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.","Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).","Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.","Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.","Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]","Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]","Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision."],"string":"[\n \"Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\\n1\\n, \\n2\\n, \\n3\\n extended‐criteria donors after brain death (DBD)\\n4\\n, \\n5\\n, and steatotic grafts.\\n6\\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\\n7\\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\\n8\\n, \\n9\\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\\n10\\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\\n11\\n, \\n12\\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\\n13\\n, \\n14\\n, \\n15\\n, \\n16\\n, \\n17\\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\\n18\\n, \\n19\\n, \\n20\\n, \\n21\\n, \\n22\\n and explored as a tool for early detection of ischemic complications and acute rejection.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n, \\n27\\n, \\n28\\n, \\n29\\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\\n30\\n MD has been occasionally used in the setting of kidney machine perfusion\\n31\\n, \\n32\\n, \\n33\\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT.\",\n \" Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\\n34\\n\\n\\nOur machine perfusion and LT protocols have been previously described.\\n4\\n, \\n34\\n, \\n35\\n, \\n36\\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\\n37\\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\\n13\\n, \\n38\\n, \\n39\\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\\n34\\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\\n40\\n, \\n41\\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\\n34\\n\\n\\nOur machine perfusion and LT protocols have been previously described.\\n4\\n, \\n34\\n, \\n35\\n, \\n36\\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\\n37\\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\\n13\\n, \\n38\\n, \\n39\\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\\n34\\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\\n40\\n, \\n41\\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\\n42\\n and L‐GrAFT score.\\n43\\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\\n44\\n and comprehensive complication index\\n45\\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\\nPrimary outcome measures were early allograft dysfunction (EAD)\\n42\\n and L‐GrAFT score.\\n43\\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\\n44\\n and comprehensive complication index\\n45\\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\",\n \"This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\\n34\\n\\n\\nOur machine perfusion and LT protocols have been previously described.\\n4\\n, \\n34\\n, \\n35\\n, \\n36\\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\\n37\\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\\n13\\n, \\n38\\n, \\n39\\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\\n34\\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\\n40\\n, \\n41\\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\",\n \"Primary outcome measures were early allograft dysfunction (EAD)\\n42\\n and L‐GrAFT score.\\n43\\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\\n44\\n and comprehensive complication index\\n45\\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\",\n \"Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\",\n \"Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\\nBaseline characteristics of recipients and donors\\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\\nDeveloped early allograft dysfunction.\\nDeveloped graft failure.\\nOperational characteristics of liver grafts\\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\\nStudy variables in the whole series and according to the onset of early allograft dysfunction\\nData are presented as median [IQR] or number (%), as appropriate.\\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\",\n \"Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\",\n \"Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\",\n \"Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\",\n \"Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","PATIENTS AND METHODS","Study design","Outcome measures","Statistical analysis","RESULTS","D‐HOPE procedure and clinical outcome","Microdialysate and perfusate metabolites","Microdialysate and perfusate FMN","Histology and expression of inflammatory cytokines","DISCUSSION","CONFLICT OF INTEREST","AUTHOR CONTRIBUTIONS","Supporting information"],"string":"[\n \"INTRODUCTION\",\n \"PATIENTS AND METHODS\",\n \"Study design\",\n \"Outcome measures\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"D‐HOPE procedure and clinical outcome\",\n \"Microdialysate and perfusate metabolites\",\n \"Microdialysate and perfusate FMN\",\n \"Histology and expression of inflammatory cytokines\",\n \"DISCUSSION\",\n \"CONFLICT OF INTEREST\",\n \"AUTHOR CONTRIBUTIONS\",\n \"Supporting information\"\n]"},"all_sections_texts":{"kind":"list like","value":["Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\n1\n, \n2\n, \n3\n extended‐criteria donors after brain death (DBD)\n4\n, \n5\n, and steatotic grafts.\n6\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\n7\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\n8\n, \n9\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\n10\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\n11\n, \n12\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\n13\n, \n14\n, \n15\n, \n16\n, \n17\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\n18\n, \n19\n, \n20\n, \n21\n, \n22\n and explored as a tool for early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\n30\n MD has been occasionally used in the setting of kidney machine perfusion\n31\n, \n32\n, \n33\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT."," Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\nPrimary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).","This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.","Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.","Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/)."," D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\nRecipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\n Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\nFigure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\n Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\nFigure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\n Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\nHistological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]","Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.","Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.","Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]","Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]","In the setting of clinical LT, the use of MD has been explored mainly as a tool for monitoring and early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n However, one fascinating feature of this technology is the possibility of evaluating liver metabolism during different phases of organ retrieval, static cold storage, implantation, and early postoperative course.\n18\n, \n19\n, \n21\n, \n22\n In particular, the studies by Nowak et al.\n19\n and Silva et al.\n21\n have clearly shown that both glucose and lactate slowly build up during SCS, whereas their levels increase steeply during implantation and organ reperfusion, to return progressively to baseline levels within 3 h following reperfusion. Perera et al.\n46\n have shown that end‐ischemic interstitial lactate level and lactate/pyruvate ratio are higher in grafts from DCD donors, this finding being associated with more severe glycogen depletion. Similarly, glutamate also appears to accumulate during SCS, reaching end‐ischemic levels of approximately 300 μmol/L, which progressively normalize in the hours following graft reperfusion.\n20\n In contrast, pyruvate quickly becomes undetectable during SCS (reflecting lack of metabolic activity during this phase), rises above pre‐SCS values upon reperfusion (suggesting a phase of hypermetabolism) to return to baseline in the following hours.\n19\n\n\nThe release of glucose in the extracellular space represents a hepatic‐specific response to ischemia and it has been interpreted as a result of glycogen breakdown.\n19\n In other tissues, ischemic events are associated with very low interstitial glucose. Interestingly, the rate of glucose and lactate accumulation increases during implantation\n19\n and back table preparation,\n21\n suggesting a potential role for temperature during these phases. Previous studies have suggested a prognostic value of MD metabolites, as patients developing initial poor function had higher MD lactate level during back table preparation and delayed clearance after graft reperfusion.\n21\n, \n46\n\n\nIn this study, we sought to use MD to deepen our understanding of liver metabolism during SCS and D‐HOPE. Our main finding is that glucose and lactate are released in the extracellular space upon initiation of D‐HOPE and their levels correlate with other known predictors of graft function (cold ischemia time and macrosteatosis), FMN perfusate level, graft dysfunction, and worse clinical outcome.\nIn our study, D‐HOPE did not alter pyruvate and glutamate levels, as compared to the expected kinetics during SCS. In particular, pyruvate levels were almost undetectable and glutamate levels were constantly high, in keeping with findings from previous studies.\n20\n The lower end‐ischemic lactate (~1900 μmol/L) and glutamate (~200 μmol/L) concentrations observed in our study (~200 μmol/L), as compared to those observed in previous studies by Silva et al.\n20\n, \n21\n can be explained by the relatively short ischemia time before D‐HOPE (5 h 44 min) in our series. Interestingly, the only graft that failed in our series had constantly very low levels of MD glutamate during back table and D‐HOPE.\nWhy D‐HOPE was associated with a significant increase of glucose and lactate into microdialysate is open to several interpretations. Temperature may have played a role, as the device employed for machine perfusion operates at ~10°C. During rewarming, it is possible that increased metabolism enhanced glycogenolysis, which in turn resulted in increased glucose release. The composition of the perfusion fluid used for D‐HOPE, containing 180 mg/ml of dextrose, may also have influenced MD glucose level. However, it should be noted that glucose levels in microdialysate were lower than those in perfusate and their kinetics was different (Figure 3). In addition, perfusion fluid composition was unlikely to affect lactate levels and progressive accumulation of lactate in the renal cortex has also been observed in a study using MD in a model of hypothermic perfusion of porcine kidneys.\n33\n\n\nWhatever the mechanism, glucose and lactate released in the extracellular space during D‐HOPE can be interpreted as markers of graft injury. The rise of MD glucose and lactate during the first 2 h of D‐HOPE was more important in grafts that developed early dysfunction, whereas it was mild or absent in those exhibiting primary function (Figure 3). However, in the few cases that had a 3‐h MD sample available, glucose and lactate levels started to decrease also in EAD group (Table S2). Consequently, it seems that restoration of aerobic metabolism during D‐HOPE could take longer in more severely damaged grafts.\nImportantly, there was a significant discrepancy between perfusate and MD kinetics of glucose and lactate (Figure 3). Perfusate analysis, as a measure of larger and global compartment, did not seem to entirely capture metabolic changes happening at a cellular level, a finding that was more evident for glucose and which is in keeping with our previous study on ex‐vivo lung perfusion.\n30\n Perfusate glucose level increased during D‐HOPE independently of graft function, whereas MD levels of glucose were higher in EAD patients, with a significant difference during the 2nd hour of machine perfusion. As lactate is produced during anaerobic glycolysis and glucose is released during ischemia due to hepatocytes glycogenolysis, we might hypothesize that the grafts that developed EAD did not fully recover after SCS and continued to experience a certain degree of hypoxia during machine perfusion, as demonstrated by their metabolic profile. In line with this observation, glycogen content was reduced in EAD livers, likely due to glycogen depletion during cold preservation, an observation which is in keeping with findings from Perera et al.\n46\n\n\nThe next logical step was comparing these findings with perfusate and MD levels of FMN, a marker of mitochondrial injury that recently emerged as a promising tool for graft viability assessment.\n40\n, \n41\n As opposed to glucose and lactate, D‐HOPE start determined a steep decrease of MD FMN levels, suggesting its rapid washout from the extracellular space and confirming protection of mitochondria during D‐HOPE. In contrast, FMN appeared to accumulate in perfusate, with an incremental trend in grafts who developed EAD and with the highest levels observed in the only failing graft of this series (Figure 5). This finding, which is in keeping with those from Mueller et al.,\n40\n also suggests a mechanistic interpretation to the association between microdialysate metabolites and graft dysfunction. Taken as a whole, our data seem to point to the same direction: mitochondrial injury sustained during SCS, which is proportional to cold ischemia time and macrosteatosis, is reflected by the accumulation of FMN in perfusate and release of glucose and lactate in the extracellular space during D‐HOPE, highlighting their potential as viability markers. Noteworthy, these metabolites can be measured point‐of‐care using MD equipment during D‐HOPE.\nThis study has numerous implications. First, it confirms that precious information on graft function and injury can be gathered also during hypothermic perfusion, challenging the concept that normothermic perfusion is a prerequisite for graft viability assessment.\n47\n, \n48\n Second, while perfusate analysis appears handier, this study and previous experiences\n23\n, \n24\n, \n27\n, \n28\n suggest that MD is feasible without major modifications of routine practice. In our opinion, microdialysate and perfusate analysis should be seen as complementary rather than alternative techniques, as metabolites kinetics appears to be different in these two compartments. Real‐time assessment of graft function could drive not only graft acceptance but also influence the duration of machine perfusion, as more severely damaged grafts may require longer perfusion time to recover from ischemic injury.\nThird, this pilot study represents a baseline based on which other potential applications of MD during machine perfusion could be explored. In a recent study on perfusate analysis during D‐HOPE,\n34\n we identified alanine aminotransferase as the most reliable predictor of EAD. Unfortunately, MD ALT could not be evaluated due to its molecular weight (100 kDa). Further studies on the subject should ideally employ available 100 kDa MD catheters to allow measurement of other molecules, including inflammatory cytokines, as also suggested by a recent study.\n49\n Finally, MD could be used to assess liver metabolism and explore new viability criteria during normothermic machine perfusion, a setting mimicking normal physiology and characterized by longer perfusion times.\n50\n\n\nLimitations of our study include the small sample size of 10 grafts with rather heterogeneous characteristics, the lack of a power analysis, and the choice of EAD as an endpoint, which has been discouraged in machine perfusion trials as it strongly relies on transaminase level after transplant and could be influenced by the washout phenomenon during machine perfusion.\n51\n\n\nIn this study, microdialysis time was limited to less than 4 h by design, allowing complete measurement of MD metabolites at only 3 time points. As previous studies have shown a quick recovery of aerobic metabolism after liver reperfusion\n21\n, \n46\n and due to concerns about sterility breaches, we did not consider leaving the MD in place during this phase, possibly missing relevant metabolic changes.\n31\n Rapid sampling MD\n33\n could improve the yield of MD as applied to procedures of limited duration, whereas the use of an MD catheter with higher molecular weight cutoff membrane would allow measuring MD concentration of other potentially important molecules.\nIn conclusion, this study expands previous knowledge on liver metabolism during D‐HOPE and confirms that liver graft injury and function can be assessed during hypothermic machine perfusion. These preliminary findings require validation in larger studies allowing correlation of MD parameters with clinically relevant endpoints and possibly exploring further applications of MD in machine perfusion.","The authors of this manuscript have no conflicts of interest to disclose.","Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision.","Supplementary Material\nClick here for additional data file."],"string":"[\n \"Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\\n1\\n, \\n2\\n, \\n3\\n extended‐criteria donors after brain death (DBD)\\n4\\n, \\n5\\n, and steatotic grafts.\\n6\\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\\n7\\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\\n8\\n, \\n9\\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\\n10\\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\\n11\\n, \\n12\\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\\n13\\n, \\n14\\n, \\n15\\n, \\n16\\n, \\n17\\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\\n18\\n, \\n19\\n, \\n20\\n, \\n21\\n, \\n22\\n and explored as a tool for early detection of ischemic complications and acute rejection.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n, \\n27\\n, \\n28\\n, \\n29\\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\\n30\\n MD has been occasionally used in the setting of kidney machine perfusion\\n31\\n, \\n32\\n, \\n33\\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT.\",\n \" Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\\n34\\n\\n\\nOur machine perfusion and LT protocols have been previously described.\\n4\\n, \\n34\\n, \\n35\\n, \\n36\\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\\n37\\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\\n13\\n, \\n38\\n, \\n39\\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\\n34\\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\\n40\\n, \\n41\\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\\n34\\n\\n\\nOur machine perfusion and LT protocols have been previously described.\\n4\\n, \\n34\\n, \\n35\\n, \\n36\\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\\n37\\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\\n13\\n, \\n38\\n, \\n39\\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\\n34\\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\\n40\\n, \\n41\\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\\n42\\n and L‐GrAFT score.\\n43\\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\\n44\\n and comprehensive complication index\\n45\\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\\nPrimary outcome measures were early allograft dysfunction (EAD)\\n42\\n and L‐GrAFT score.\\n43\\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\\n44\\n and comprehensive complication index\\n45\\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\",\n \"This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\\n34\\n\\n\\nOur machine perfusion and LT protocols have been previously described.\\n4\\n, \\n34\\n, \\n35\\n, \\n36\\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\\n37\\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\\n13\\n, \\n38\\n, \\n39\\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\\n34\\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\\n40\\n, \\n41\\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\",\n \"Primary outcome measures were early allograft dysfunction (EAD)\\n42\\n and L‐GrAFT score.\\n43\\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\\n44\\n and comprehensive complication index\\n45\\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\",\n \"Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\",\n \" D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\\nBaseline characteristics of recipients and donors\\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\\nDeveloped early allograft dysfunction.\\nDeveloped graft failure.\\nOperational characteristics of liver grafts\\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\\nStudy variables in the whole series and according to the onset of early allograft dysfunction\\nData are presented as median [IQR] or number (%), as appropriate.\\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\\nRecipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\\nBaseline characteristics of recipients and donors\\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\\nDeveloped early allograft dysfunction.\\nDeveloped graft failure.\\nOperational characteristics of liver grafts\\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\\nStudy variables in the whole series and according to the onset of early allograft dysfunction\\nData are presented as median [IQR] or number (%), as appropriate.\\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\\n Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\\nFigure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\\n Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\\nFigure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\\n Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\\nHistological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\",\n \"Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\\nBaseline characteristics of recipients and donors\\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\\nDeveloped early allograft dysfunction.\\nDeveloped graft failure.\\nOperational characteristics of liver grafts\\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\\nStudy variables in the whole series and according to the onset of early allograft dysfunction\\nData are presented as median [IQR] or number (%), as appropriate.\\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\",\n \"Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\",\n \"Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\",\n \"Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\",\n \"In the setting of clinical LT, the use of MD has been explored mainly as a tool for monitoring and early detection of ischemic complications and acute rejection.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n, \\n27\\n, \\n28\\n, \\n29\\n However, one fascinating feature of this technology is the possibility of evaluating liver metabolism during different phases of organ retrieval, static cold storage, implantation, and early postoperative course.\\n18\\n, \\n19\\n, \\n21\\n, \\n22\\n In particular, the studies by Nowak et al.\\n19\\n and Silva et al.\\n21\\n have clearly shown that both glucose and lactate slowly build up during SCS, whereas their levels increase steeply during implantation and organ reperfusion, to return progressively to baseline levels within 3 h following reperfusion. Perera et al.\\n46\\n have shown that end‐ischemic interstitial lactate level and lactate/pyruvate ratio are higher in grafts from DCD donors, this finding being associated with more severe glycogen depletion. Similarly, glutamate also appears to accumulate during SCS, reaching end‐ischemic levels of approximately 300 μmol/L, which progressively normalize in the hours following graft reperfusion.\\n20\\n In contrast, pyruvate quickly becomes undetectable during SCS (reflecting lack of metabolic activity during this phase), rises above pre‐SCS values upon reperfusion (suggesting a phase of hypermetabolism) to return to baseline in the following hours.\\n19\\n\\n\\nThe release of glucose in the extracellular space represents a hepatic‐specific response to ischemia and it has been interpreted as a result of glycogen breakdown.\\n19\\n In other tissues, ischemic events are associated with very low interstitial glucose. Interestingly, the rate of glucose and lactate accumulation increases during implantation\\n19\\n and back table preparation,\\n21\\n suggesting a potential role for temperature during these phases. Previous studies have suggested a prognostic value of MD metabolites, as patients developing initial poor function had higher MD lactate level during back table preparation and delayed clearance after graft reperfusion.\\n21\\n, \\n46\\n\\n\\nIn this study, we sought to use MD to deepen our understanding of liver metabolism during SCS and D‐HOPE. Our main finding is that glucose and lactate are released in the extracellular space upon initiation of D‐HOPE and their levels correlate with other known predictors of graft function (cold ischemia time and macrosteatosis), FMN perfusate level, graft dysfunction, and worse clinical outcome.\\nIn our study, D‐HOPE did not alter pyruvate and glutamate levels, as compared to the expected kinetics during SCS. In particular, pyruvate levels were almost undetectable and glutamate levels were constantly high, in keeping with findings from previous studies.\\n20\\n The lower end‐ischemic lactate (~1900 μmol/L) and glutamate (~200 μmol/L) concentrations observed in our study (~200 μmol/L), as compared to those observed in previous studies by Silva et al.\\n20\\n, \\n21\\n can be explained by the relatively short ischemia time before D‐HOPE (5 h 44 min) in our series. Interestingly, the only graft that failed in our series had constantly very low levels of MD glutamate during back table and D‐HOPE.\\nWhy D‐HOPE was associated with a significant increase of glucose and lactate into microdialysate is open to several interpretations. Temperature may have played a role, as the device employed for machine perfusion operates at ~10°C. During rewarming, it is possible that increased metabolism enhanced glycogenolysis, which in turn resulted in increased glucose release. The composition of the perfusion fluid used for D‐HOPE, containing 180 mg/ml of dextrose, may also have influenced MD glucose level. However, it should be noted that glucose levels in microdialysate were lower than those in perfusate and their kinetics was different (Figure 3). In addition, perfusion fluid composition was unlikely to affect lactate levels and progressive accumulation of lactate in the renal cortex has also been observed in a study using MD in a model of hypothermic perfusion of porcine kidneys.\\n33\\n\\n\\nWhatever the mechanism, glucose and lactate released in the extracellular space during D‐HOPE can be interpreted as markers of graft injury. The rise of MD glucose and lactate during the first 2 h of D‐HOPE was more important in grafts that developed early dysfunction, whereas it was mild or absent in those exhibiting primary function (Figure 3). However, in the few cases that had a 3‐h MD sample available, glucose and lactate levels started to decrease also in EAD group (Table S2). Consequently, it seems that restoration of aerobic metabolism during D‐HOPE could take longer in more severely damaged grafts.\\nImportantly, there was a significant discrepancy between perfusate and MD kinetics of glucose and lactate (Figure 3). Perfusate analysis, as a measure of larger and global compartment, did not seem to entirely capture metabolic changes happening at a cellular level, a finding that was more evident for glucose and which is in keeping with our previous study on ex‐vivo lung perfusion.\\n30\\n Perfusate glucose level increased during D‐HOPE independently of graft function, whereas MD levels of glucose were higher in EAD patients, with a significant difference during the 2nd hour of machine perfusion. As lactate is produced during anaerobic glycolysis and glucose is released during ischemia due to hepatocytes glycogenolysis, we might hypothesize that the grafts that developed EAD did not fully recover after SCS and continued to experience a certain degree of hypoxia during machine perfusion, as demonstrated by their metabolic profile. In line with this observation, glycogen content was reduced in EAD livers, likely due to glycogen depletion during cold preservation, an observation which is in keeping with findings from Perera et al.\\n46\\n\\n\\nThe next logical step was comparing these findings with perfusate and MD levels of FMN, a marker of mitochondrial injury that recently emerged as a promising tool for graft viability assessment.\\n40\\n, \\n41\\n As opposed to glucose and lactate, D‐HOPE start determined a steep decrease of MD FMN levels, suggesting its rapid washout from the extracellular space and confirming protection of mitochondria during D‐HOPE. In contrast, FMN appeared to accumulate in perfusate, with an incremental trend in grafts who developed EAD and with the highest levels observed in the only failing graft of this series (Figure 5). This finding, which is in keeping with those from Mueller et al.,\\n40\\n also suggests a mechanistic interpretation to the association between microdialysate metabolites and graft dysfunction. Taken as a whole, our data seem to point to the same direction: mitochondrial injury sustained during SCS, which is proportional to cold ischemia time and macrosteatosis, is reflected by the accumulation of FMN in perfusate and release of glucose and lactate in the extracellular space during D‐HOPE, highlighting their potential as viability markers. Noteworthy, these metabolites can be measured point‐of‐care using MD equipment during D‐HOPE.\\nThis study has numerous implications. First, it confirms that precious information on graft function and injury can be gathered also during hypothermic perfusion, challenging the concept that normothermic perfusion is a prerequisite for graft viability assessment.\\n47\\n, \\n48\\n Second, while perfusate analysis appears handier, this study and previous experiences\\n23\\n, \\n24\\n, \\n27\\n, \\n28\\n suggest that MD is feasible without major modifications of routine practice. In our opinion, microdialysate and perfusate analysis should be seen as complementary rather than alternative techniques, as metabolites kinetics appears to be different in these two compartments. Real‐time assessment of graft function could drive not only graft acceptance but also influence the duration of machine perfusion, as more severely damaged grafts may require longer perfusion time to recover from ischemic injury.\\nThird, this pilot study represents a baseline based on which other potential applications of MD during machine perfusion could be explored. In a recent study on perfusate analysis during D‐HOPE,\\n34\\n we identified alanine aminotransferase as the most reliable predictor of EAD. Unfortunately, MD ALT could not be evaluated due to its molecular weight (100 kDa). Further studies on the subject should ideally employ available 100 kDa MD catheters to allow measurement of other molecules, including inflammatory cytokines, as also suggested by a recent study.\\n49\\n Finally, MD could be used to assess liver metabolism and explore new viability criteria during normothermic machine perfusion, a setting mimicking normal physiology and characterized by longer perfusion times.\\n50\\n\\n\\nLimitations of our study include the small sample size of 10 grafts with rather heterogeneous characteristics, the lack of a power analysis, and the choice of EAD as an endpoint, which has been discouraged in machine perfusion trials as it strongly relies on transaminase level after transplant and could be influenced by the washout phenomenon during machine perfusion.\\n51\\n\\n\\nIn this study, microdialysis time was limited to less than 4 h by design, allowing complete measurement of MD metabolites at only 3 time points. As previous studies have shown a quick recovery of aerobic metabolism after liver reperfusion\\n21\\n, \\n46\\n and due to concerns about sterility breaches, we did not consider leaving the MD in place during this phase, possibly missing relevant metabolic changes.\\n31\\n Rapid sampling MD\\n33\\n could improve the yield of MD as applied to procedures of limited duration, whereas the use of an MD catheter with higher molecular weight cutoff membrane would allow measuring MD concentration of other potentially important molecules.\\nIn conclusion, this study expands previous knowledge on liver metabolism during D‐HOPE and confirms that liver graft injury and function can be assessed during hypothermic machine perfusion. These preliminary findings require validation in larger studies allowing correlation of MD parameters with clinically relevant endpoints and possibly exploring further applications of MD in machine perfusion.\",\n \"The authors of this manuscript have no conflicts of interest to disclose.\",\n \"Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision.\",\n \"Supplementary Material\\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,"results",null,null,null,null,"discussion","COI-statement",null,"supplementary-material"],"string":"[\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"COI-statement\",\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["extracellular fluid","flavin mononucleotide","liver metabolism","liver viability assessment","machine perfusion","microdialysis"],"string":"[\n \"extracellular fluid\",\n \"flavin mononucleotide\",\n \"liver metabolism\",\n \"liver viability assessment\",\n \"machine perfusion\",\n \"microdialysis\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nHypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\n1\n, \n2\n, \n3\n extended‐criteria donors after brain death (DBD)\n4\n, \n5\n, and steatotic grafts.\n6\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\n7\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\n8\n, \n9\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\n10\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\n11\n, \n12\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\n13\n, \n14\n, \n15\n, \n16\n, \n17\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\n18\n, \n19\n, \n20\n, \n21\n, \n22\n and explored as a tool for early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\n30\n MD has been occasionally used in the setting of kidney machine perfusion\n31\n, \n32\n, \n33\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT.\n\nPATIENTS AND METHODS:\n Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\nPrimary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\n\nStudy design:\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\n\nOutcome measures:\nPrimary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\n\nStatistical analysis:\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\n\nRESULTS:\n D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\nRecipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\n Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\nFigure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\n Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\nFigure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\n Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\nHistological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\n\nD‐HOPE procedure and clinical outcome:\nRecipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\n\nMicrodialysate and perfusate metabolites:\nFigure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\n\nMicrodialysate and perfusate FMN:\nFigure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\n\nHistology and expression of inflammatory cytokines:\nHistological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\n\nDISCUSSION:\nIn the setting of clinical LT, the use of MD has been explored mainly as a tool for monitoring and early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n However, one fascinating feature of this technology is the possibility of evaluating liver metabolism during different phases of organ retrieval, static cold storage, implantation, and early postoperative course.\n18\n, \n19\n, \n21\n, \n22\n In particular, the studies by Nowak et al.\n19\n and Silva et al.\n21\n have clearly shown that both glucose and lactate slowly build up during SCS, whereas their levels increase steeply during implantation and organ reperfusion, to return progressively to baseline levels within 3 h following reperfusion. Perera et al.\n46\n have shown that end‐ischemic interstitial lactate level and lactate/pyruvate ratio are higher in grafts from DCD donors, this finding being associated with more severe glycogen depletion. Similarly, glutamate also appears to accumulate during SCS, reaching end‐ischemic levels of approximately 300 μmol/L, which progressively normalize in the hours following graft reperfusion.\n20\n In contrast, pyruvate quickly becomes undetectable during SCS (reflecting lack of metabolic activity during this phase), rises above pre‐SCS values upon reperfusion (suggesting a phase of hypermetabolism) to return to baseline in the following hours.\n19\n\n\nThe release of glucose in the extracellular space represents a hepatic‐specific response to ischemia and it has been interpreted as a result of glycogen breakdown.\n19\n In other tissues, ischemic events are associated with very low interstitial glucose. Interestingly, the rate of glucose and lactate accumulation increases during implantation\n19\n and back table preparation,\n21\n suggesting a potential role for temperature during these phases. Previous studies have suggested a prognostic value of MD metabolites, as patients developing initial poor function had higher MD lactate level during back table preparation and delayed clearance after graft reperfusion.\n21\n, \n46\n\n\nIn this study, we sought to use MD to deepen our understanding of liver metabolism during SCS and D‐HOPE. Our main finding is that glucose and lactate are released in the extracellular space upon initiation of D‐HOPE and their levels correlate with other known predictors of graft function (cold ischemia time and macrosteatosis), FMN perfusate level, graft dysfunction, and worse clinical outcome.\nIn our study, D‐HOPE did not alter pyruvate and glutamate levels, as compared to the expected kinetics during SCS. In particular, pyruvate levels were almost undetectable and glutamate levels were constantly high, in keeping with findings from previous studies.\n20\n The lower end‐ischemic lactate (~1900 μmol/L) and glutamate (~200 μmol/L) concentrations observed in our study (~200 μmol/L), as compared to those observed in previous studies by Silva et al.\n20\n, \n21\n can be explained by the relatively short ischemia time before D‐HOPE (5 h 44 min) in our series. Interestingly, the only graft that failed in our series had constantly very low levels of MD glutamate during back table and D‐HOPE.\nWhy D‐HOPE was associated with a significant increase of glucose and lactate into microdialysate is open to several interpretations. Temperature may have played a role, as the device employed for machine perfusion operates at ~10°C. During rewarming, it is possible that increased metabolism enhanced glycogenolysis, which in turn resulted in increased glucose release. The composition of the perfusion fluid used for D‐HOPE, containing 180 mg/ml of dextrose, may also have influenced MD glucose level. However, it should be noted that glucose levels in microdialysate were lower than those in perfusate and their kinetics was different (Figure 3). In addition, perfusion fluid composition was unlikely to affect lactate levels and progressive accumulation of lactate in the renal cortex has also been observed in a study using MD in a model of hypothermic perfusion of porcine kidneys.\n33\n\n\nWhatever the mechanism, glucose and lactate released in the extracellular space during D‐HOPE can be interpreted as markers of graft injury. The rise of MD glucose and lactate during the first 2 h of D‐HOPE was more important in grafts that developed early dysfunction, whereas it was mild or absent in those exhibiting primary function (Figure 3). However, in the few cases that had a 3‐h MD sample available, glucose and lactate levels started to decrease also in EAD group (Table S2). Consequently, it seems that restoration of aerobic metabolism during D‐HOPE could take longer in more severely damaged grafts.\nImportantly, there was a significant discrepancy between perfusate and MD kinetics of glucose and lactate (Figure 3). Perfusate analysis, as a measure of larger and global compartment, did not seem to entirely capture metabolic changes happening at a cellular level, a finding that was more evident for glucose and which is in keeping with our previous study on ex‐vivo lung perfusion.\n30\n Perfusate glucose level increased during D‐HOPE independently of graft function, whereas MD levels of glucose were higher in EAD patients, with a significant difference during the 2nd hour of machine perfusion. As lactate is produced during anaerobic glycolysis and glucose is released during ischemia due to hepatocytes glycogenolysis, we might hypothesize that the grafts that developed EAD did not fully recover after SCS and continued to experience a certain degree of hypoxia during machine perfusion, as demonstrated by their metabolic profile. In line with this observation, glycogen content was reduced in EAD livers, likely due to glycogen depletion during cold preservation, an observation which is in keeping with findings from Perera et al.\n46\n\n\nThe next logical step was comparing these findings with perfusate and MD levels of FMN, a marker of mitochondrial injury that recently emerged as a promising tool for graft viability assessment.\n40\n, \n41\n As opposed to glucose and lactate, D‐HOPE start determined a steep decrease of MD FMN levels, suggesting its rapid washout from the extracellular space and confirming protection of mitochondria during D‐HOPE. In contrast, FMN appeared to accumulate in perfusate, with an incremental trend in grafts who developed EAD and with the highest levels observed in the only failing graft of this series (Figure 5). This finding, which is in keeping with those from Mueller et al.,\n40\n also suggests a mechanistic interpretation to the association between microdialysate metabolites and graft dysfunction. Taken as a whole, our data seem to point to the same direction: mitochondrial injury sustained during SCS, which is proportional to cold ischemia time and macrosteatosis, is reflected by the accumulation of FMN in perfusate and release of glucose and lactate in the extracellular space during D‐HOPE, highlighting their potential as viability markers. Noteworthy, these metabolites can be measured point‐of‐care using MD equipment during D‐HOPE.\nThis study has numerous implications. First, it confirms that precious information on graft function and injury can be gathered also during hypothermic perfusion, challenging the concept that normothermic perfusion is a prerequisite for graft viability assessment.\n47\n, \n48\n Second, while perfusate analysis appears handier, this study and previous experiences\n23\n, \n24\n, \n27\n, \n28\n suggest that MD is feasible without major modifications of routine practice. In our opinion, microdialysate and perfusate analysis should be seen as complementary rather than alternative techniques, as metabolites kinetics appears to be different in these two compartments. Real‐time assessment of graft function could drive not only graft acceptance but also influence the duration of machine perfusion, as more severely damaged grafts may require longer perfusion time to recover from ischemic injury.\nThird, this pilot study represents a baseline based on which other potential applications of MD during machine perfusion could be explored. In a recent study on perfusate analysis during D‐HOPE,\n34\n we identified alanine aminotransferase as the most reliable predictor of EAD. Unfortunately, MD ALT could not be evaluated due to its molecular weight (100 kDa). Further studies on the subject should ideally employ available 100 kDa MD catheters to allow measurement of other molecules, including inflammatory cytokines, as also suggested by a recent study.\n49\n Finally, MD could be used to assess liver metabolism and explore new viability criteria during normothermic machine perfusion, a setting mimicking normal physiology and characterized by longer perfusion times.\n50\n\n\nLimitations of our study include the small sample size of 10 grafts with rather heterogeneous characteristics, the lack of a power analysis, and the choice of EAD as an endpoint, which has been discouraged in machine perfusion trials as it strongly relies on transaminase level after transplant and could be influenced by the washout phenomenon during machine perfusion.\n51\n\n\nIn this study, microdialysis time was limited to less than 4 h by design, allowing complete measurement of MD metabolites at only 3 time points. As previous studies have shown a quick recovery of aerobic metabolism after liver reperfusion\n21\n, \n46\n and due to concerns about sterility breaches, we did not consider leaving the MD in place during this phase, possibly missing relevant metabolic changes.\n31\n Rapid sampling MD\n33\n could improve the yield of MD as applied to procedures of limited duration, whereas the use of an MD catheter with higher molecular weight cutoff membrane would allow measuring MD concentration of other potentially important molecules.\nIn conclusion, this study expands previous knowledge on liver metabolism during D‐HOPE and confirms that liver graft injury and function can be assessed during hypothermic machine perfusion. These preliminary findings require validation in larger studies allowing correlation of MD parameters with clinically relevant endpoints and possibly exploring further applications of MD in machine perfusion.\n\nCONFLICT OF INTEREST:\nThe authors of this manuscript have no conflicts of interest to disclose.\n\nAUTHOR CONTRIBUTIONS:\nDamiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision.\n\nSupporting information:\nSupplementary Material\nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWhile growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood.\n\nMethods:\nTo assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome.\n\nResults:\nMedian (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2nd-hour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN. These correlations were not apparent when perfusate levels were considered. In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts.\n\nConclusions:\nMD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":14165,"string":"14,165"},"whole_article_abstract_length":{"kind":"number","value":301,"string":"301"},"other_sections_lengths":{"kind":"list like","value":[563,2695,1112,80,148,972,859,242,418,111],"string":"[\n 563,\n 2695,\n 1112,\n 80,\n 148,\n 972,\n 859,\n 242,\n 418,\n 111\n]"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["hope","md","graft","levels","perfusion","ead","perfusate","machine","lactate","machine perfusion"],"string":"[\n \"hope\",\n \"md\",\n \"graft\",\n \"levels\",\n \"perfusion\",\n \"ead\",\n \"perfusate\",\n \"machine\",\n \"lactate\",\n \"machine perfusion\"\n]"},"keybert_topics":{"kind":"list like","value":["protection mitochondria hope","ischemia reperfusion","metabolism liver reperfusion","transfer organ reperfusion","perfusion donor hope"],"string":"[\n \"protection mitochondria hope\",\n \"ischemia reperfusion\",\n \"metabolism liver reperfusion\",\n \"transfer organ reperfusion\",\n \"perfusion donor hope\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] extracellular fluid | flavin mononucleotide | liver metabolism | liver viability assessment | machine perfusion | microdialysis [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] extracellular fluid | flavin mononucleotide | liver metabolism | liver viability assessment | machine perfusion | microdialysis [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] extracellular fluid | flavin mononucleotide | liver metabolism | liver viability assessment | machine perfusion | microdialysis [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Cold Ischemia | Female | Glucose | Graft Survival | Humans | Lactic Acid | Liver | Liver Transplantation | Male | Microdialysis | Middle Aged | Organ Preservation | Perfusion | Pilot Projects | Prospective Studies [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Cold Ischemia | Female | Glucose | Graft Survival | Humans | Lactic Acid | Liver | Liver Transplantation | Male | Microdialysis | Middle Aged | Organ Preservation | Perfusion | Pilot Projects | Prospective Studies [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Cold Ischemia | Female | Glucose | Graft Survival | Humans | Lactic Acid | Liver | Liver Transplantation | Male | Microdialysis | Middle Aged | Organ Preservation | Perfusion | Pilot Projects | Prospective Studies [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] protection mitochondria hope | ischemia reperfusion | metabolism liver reperfusion | transfer organ reperfusion | perfusion donor hope [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] protection mitochondria hope | ischemia reperfusion | metabolism liver reperfusion | transfer organ reperfusion | perfusion donor hope [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] protection mitochondria hope | ischemia reperfusion | metabolism liver reperfusion | transfer organ reperfusion | perfusion donor hope [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hope | md | graft | levels | perfusion | ead | perfusate | machine | lactate | machine perfusion [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hope | md | graft | levels | perfusion | ead | perfusate | machine | lactate | machine perfusion [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hope | md | graft | levels | perfusion | ead | perfusate | machine | lactate | machine perfusion [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hope | metabolism | md | liver | assess | liver metabolism | mitochondrial | tool | potential | scs [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] graft | ead | hope | levels | developed | hour | 2nd | 2nd hour | figure | time [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hope | md | graft | levels | ead | perfusion | liver | lactate | perfusate | machine [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] IQR | MD | 228 | 210 | 245 | 116 | 103 | 143 | Three | EAD ||| MD | 9.88 ||| 0.01 | 3.71 | 0.08 ||| 2nd-hour | EAD ||| 2nd-hour | MD | FMN ||| ||| MD FMN | FMN [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| MD | 10 ||| ||| IQR | MD | 228 | 210 | 245 | 116 | 103 | 143 | Three | EAD ||| MD | 9.88 ||| 0.01 | 3.71 | 0.08 ||| 2nd-hour | EAD ||| 2nd-hour | MD | FMN ||| ||| MD FMN | FMN ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3370,"cells":{"title":{"kind":"string","value":"Awareness of HIV functional cure and willingness in participating in related clinical trials: comparison between antiretroviral naïve and experienced men who have sex with men living with HIV."},"pmid":{"kind":"string","value":"35428275"},"background_abstract":{"kind":"string","value":"Human immunodeficiency virus (HIV) functional cure is a novel biomedical strategy characterized by sustained viral suppression without the need for life-long medications. The attitude of people living with HIV (PLHIV) towards functional cure and clinical trials are understudied. We aimed to examine the awareness and levels of anticipation for HIV functional cure among men who have sex with men (MSM) living with HIV, and their willingness to join trials as differentiated by their antiretroviral treatment status."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"MSM living with HIV with and those without treatment history were recruited from Hong Kong's HIV specialist clinics. Self-administered questionnaires covering behavioral profile, perceived impact of HIV cure, attitude towards HIV functional cure and related clinical trials were collected. Clinical data were separately transcribed. Determinants of perceptions and attitudes were identified by logistic regression models."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Of 356 MSM living with HIV recruited, less than half (42%) were aware of HIV functional cure, but they had a high level of anticipation for it. Treatment-experienced participants were more likely to be aware of HIV functional cure. Awareness was associated with continued engagement in sexual activities after HIV diagnosis and sexually transmitted infection (STI) diagnosis. Higher anticipation was observed among older MSM living with HIV but it was negatively associated with one's awareness. Over 90% were willing to join functional cure trials, especially those who had previously been diagnosed with STI and had engaged in chemsex in the past year. Advice from healthcare professional was an important factor considered by those willing to join clinical trials. Younger, better educated MSM, and those with lower CD4 counts were more concerned about potential risk of AIDS and potential complications upon trial participation."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"MSM living with HIV, especially those sexually active, showed positive attitude towards functional cure and willingness to join related clinical trials despite low awareness. To enhance preparedness for HIV functional cure trials, community education, updated information and appropriate medical advice would be needed. Safety is a major concern for potential enrollees in HIV functional cure trials."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Cross-Sectional Studies","Female","HIV Infections","Homosexuality, Male","Humans","Male","Sexual Behavior","Sexual and Gender Minorities","Sexually Transmitted Diseases"],"string":"[\n \"Cross-Sectional Studies\",\n \"Female\",\n \"HIV Infections\",\n \"Homosexuality, Male\",\n \"Humans\",\n \"Male\",\n \"Sexual Behavior\",\n \"Sexual and Gender Minorities\",\n \"Sexually Transmitted Diseases\"\n]"},"pmcid":{"kind":"string","value":"9013029"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Demographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nBetween March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nPerceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nPerceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAwareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nOverall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAnticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAmong all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAcceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\nShould there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment."},"other_sections_titles":{"kind":"list like","value":["Background","Methods","Demographics","Perceived benefits of HIV cure","Awareness of HIV functional cure","Anticipation of HIV functional cure","Acceptance of an HIV functional cure trial",""],"string":"[\n \"Background\",\n \"Methods\",\n \"Demographics\",\n \"Perceived benefits of HIV cure\",\n \"Awareness of HIV functional cure\",\n \"Anticipation of HIV functional cure\",\n \"Acceptance of an HIV functional cure trial\",\n \"\"\n]"},"other_sections_texts":{"kind":"list like","value":["Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.","Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.","Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data","Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001","Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001","Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).","Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).","\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356)."],"string":"[\n \"Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.\",\n \"Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.\",\n \"Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\\n\\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\\naTotal number for each variable may not add up to N = 356 due to missing data\",\n \"Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\\n\\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\\nn\\n\\n%\\n\\nn\\n\\n%\\n\\nOR (95% CI)\\n\\n(a) Perceived impacts of HIV cure\\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\\n(b) Perception and attitude towards HIV functional cure\\n\\nAwareness of HIV functional cure\\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\\nLevel of anticipation for HIV functional cure\\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\\nWillingness in joining a functional cure trial\\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\\n(c) Considerations about HIV functional cure trial\\n\\nImportant factors to be considered about the trial\\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\\nConcerns about consequences of joining a trial\\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\\n*p < 0.05 ** p < 0.01 *** p < 0.001\\n\\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\\nIMP1a\\nIMP2aIMP3aIMP4aIMP5a\\nIMP6aIMP7a\\nAge group\\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\\nEducation level\\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \\nSTI history in the past year\\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\\nSexual activity after HIV diagnosis\\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \\nEngagement in chemsex\\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\\nAbout Functional cure\\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\\n* p < 0.05 ** p < 0.01 *** p < 0.001\",\n \"Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\\n\\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nAge group\\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\\nEducation level\\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \\nSTI history in the past year\\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\\n Sexual activity after HIV diagnosis\\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\\n Engagement in chemsex\\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\\nAbout functional cure\\n Awareness\\n–\\n\\n–\\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\\n–\\n\\n–\\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\\n* p < 0.05 ** p < 0.01 *** p < 0.001\",\n \"Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\",\n \"Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\",\n \"\\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\\nAdditional file 1. Questionnaire.\\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results","Demographics","Perceived benefits of HIV cure","Awareness of HIV functional cure","Anticipation of HIV functional cure","Acceptance of an HIV functional cure trial","Discussion","Conclusions","Supplementary Information",""],"string":"[\n \"Background\",\n \"Methods\",\n \"Results\",\n \"Demographics\",\n \"Perceived benefits of HIV cure\",\n \"Awareness of HIV functional cure\",\n \"Anticipation of HIV functional cure\",\n \"Acceptance of an HIV functional cure trial\",\n \"Discussion\",\n \"Conclusions\",\n \"Supplementary Information\",\n \"\"\n]"},"all_sections_texts":{"kind":"list like","value":["Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.","Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.","Demographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nBetween March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nPerceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nPerceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAwareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nOverall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAnticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAmong all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAcceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\nShould there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).","Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data","Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001","Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001","Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).","Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).","HIV functional cure is a relatively new concept for PLHIV. Despite their generally low awareness, MSM living with HIV had, as shown in this study, a high level of anticipation for HIV functional cure, especially those who had been diagnosed and put on ART for some years. The effective achievement of functional cure carries the potential prospect of getting rid of repeated clinic visits and taking long-term medications [11]. The benefit of being untransmittable may, however, not be regarded as an important impact for HIV cure because in the current era of “undetectable equals untransmittable”, adherence to ART is already effective in minimizing the risk of transmission. In our cohorts of MSM living with HIV, the attitudes differed by one’s ART treatment experience. Treatment-naïve participants were more concerned about their immune function and AIDS progression than those ART-experienced, as deteriorated health was perceived by newly diagnosed patients an imminent consequence brought on by HIV [12]. Anticipation for HIV functional cure was apparently lower among those who were aware of it. Although some 42% had heard about functional cure, most had limited knowledge of it and cared little about the potential benefit of restoring their immune function and potential adverse effects arising from the new therapy. With the increasing likelihood that functional cure may soon become the next major advance in HIV treatment [7], there is the need for promoting community education on the rationale and strategy for HIV functional cure. As in many countries, MSM accounted for a high proportion of PLHIV in Hong Kong [13], and who continued to be significantly impacted by the epidemic [14]. The MSM community, especially those living with HIV would need to be targeted in anticipation of the introduction of functional cure therapy. In our study, MSM engaging in higher risk sexual behaviors were more likely to be aware of the recent advancement on HIV science, such as pre-exposure prophylaxis [15]. This echoed our results that sexually active PLHIV, particularly those engaged in condomless sex as indicated by their STI history, were more likely to be aware of HIV functional cure.\nThere are diverse reasons for PLHIV to look forward to receiving functional cure therapy. It is intuitive that PLHIV with a weaker immune system were less likely to be sexually active and they wished to restore and stabilize their immune function and were at the same time more concerned about adverse HIV outcomes after joining functional cure trials. The fact that younger PLHIV cared more about disease development and transmittability was likely because they were newly diagnosed and would remain sexually active for a longer time in the future. PLHIV with higher education level regarded de-labelling of HIV an important impact of HIV cure. In Hong Kong, public stigma against PLHIV [16] and perceived discrimination [17] have been reported. It is therefore an ideal vision for them to remove the label of HIV after achieving some sort of cure. Discrimination in workplace and difficulty in career development could be experienced by PLHIV [18]. Although highly educated PLHIV were found to deal better with workplace discrimination and improve employment quality than those with lower education level [19], they were more concerned with their career development that removing stigma means removing a barrier in their career paths. From the perspective of the general public, increased awareness and knowledge in HIV science advancement might be able to reduce stigma. A previous study showed that heterosexually active adults who were aware of the untransmittable nature of PLHIV achieving undetectable viral load had a lower level of anticipated HIV stigma [20]. As this study focuses on PLHIV, a follow-up study in the general population and the MSM community would be needed to assess the impact of functional cure on general, and dating- and sex-related enacted stigmas.\nOverall, the idea of an HIV functional cure trial was well accepted by our cohorts of MSM living with HIV with over 90% indicating their willingness to join, similar to the high acceptance rate of 95% in a previous study [11]. Potential trial participants were more likely to have engaged in higher risk sexual activities as reflected by their report of STI in the preceding year. This might be precipitated by a prospect of continuation or re-engagement in adventurous condomless behaviors upon cure [21]. Safety is a major concern for clinical trials involving the use of novel agents like neutralizing antibodies or therapeutic vaccines. Careful monitoring of PLHIV in the trial would be warranted not just for protecting participants’ health but also easing their concerns of side effects and adverse HIV outcomes. Hesitation would arise if the trial requires interruption of ART or if it takes a long time, in relation to the risk of rebound and potential adverse effects [11]. Trusting relationship between PLHIV, researchers and healthcare workers could be beneficial for ensuring the delivery of optimal health outcomes [22]. Based on potential participants’ trust on healthcare professional advice, collaboration with HIV clinics on explaining the details and safety issues of the trial would be paramount.\nThis study carries some limitations. Like other epidemiological studies, there were inherent recall and social desirability biases in the administration of behavioral questionnaires. We had minimized these biases by adopting a shorter recall period and omitting fine details about sexual activities. The adoption of a self-administering approach should have reduced embarrassments in response to interviewers’ questions. Incorporation of clinical data by transcription benefited the study by providing objective measurements of participants’ HIV outcomes. In the absence of any functional cure trials, the exploration of one’s willingness to participate in a hypothetical trial without having one in place could be postulational. Nevertheless, the systematic inquiries into the awareness and anticipation of functional HIV cure among MSM living with HIV and their inclination in joining a hypothetical study have generated useful results which could be of reference for the planning of a functional cure trial.","To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment."," \nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\n\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).","\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356)."],"string":"[\n \"Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.\",\n \"Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.\",\n \"Demographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\\n\\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\\naTotal number for each variable may not add up to N = 356 due to missing data\\nBetween March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\\n\\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\\naTotal number for each variable may not add up to N = 356 due to missing data\\nPerceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\\n\\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\\nn\\n\\n%\\n\\nn\\n\\n%\\n\\nOR (95% CI)\\n\\n(a) Perceived impacts of HIV cure\\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\\n(b) Perception and attitude towards HIV functional cure\\n\\nAwareness of HIV functional cure\\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\\nLevel of anticipation for HIV functional cure\\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\\nWillingness in joining a functional cure trial\\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\\n(c) Considerations about HIV functional cure trial\\n\\nImportant factors to be considered about the trial\\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\\nConcerns about consequences of joining a trial\\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\\n*p < 0.05 ** p < 0.01 *** p < 0.001\\n\\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\\nIMP1a\\nIMP2aIMP3aIMP4aIMP5a\\nIMP6aIMP7a\\nAge group\\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\\nEducation level\\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \\nSTI history in the past year\\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\\nSexual activity after HIV diagnosis\\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \\nEngagement in chemsex\\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\\nAbout Functional cure\\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\\n* p < 0.05 ** p < 0.01 *** p < 0.001\\nPerceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\\n\\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\\nn\\n\\n%\\n\\nn\\n\\n%\\n\\nOR (95% CI)\\n\\n(a) Perceived impacts of HIV cure\\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\\n(b) Perception and attitude towards HIV functional cure\\n\\nAwareness of HIV functional cure\\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\\nLevel of anticipation for HIV functional cure\\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\\nWillingness in joining a functional cure trial\\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\\n(c) Considerations about HIV functional cure trial\\n\\nImportant factors to be considered about the trial\\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\\nConcerns about consequences of joining a trial\\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\\n*p < 0.05 ** p < 0.01 *** p < 0.001\\n\\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\\nIMP1a\\nIMP2aIMP3aIMP4aIMP5a\\nIMP6aIMP7a\\nAge group\\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\\nEducation level\\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \\nSTI history in the past year\\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\\nSexual activity after HIV diagnosis\\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \\nEngagement in chemsex\\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\\nAbout Functional cure\\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\\n* p < 0.05 ** p < 0.01 *** p < 0.001\\nAwareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\\n\\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nAge group\\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\\nEducation level\\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \\nSTI history in the past year\\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\\n Sexual activity after HIV diagnosis\\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\\n Engagement in chemsex\\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\\nAbout functional cure\\n Awareness\\n–\\n\\n–\\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\\n–\\n\\n–\\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\\n* p < 0.05 ** p < 0.01 *** p < 0.001\\nOverall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\\n\\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nAge group\\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\\nEducation level\\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \\nSTI history in the past year\\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\\n Sexual activity after HIV diagnosis\\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\\n Engagement in chemsex\\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\\nAbout functional cure\\n Awareness\\n–\\n\\n–\\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\\n–\\n\\n–\\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\\n* p < 0.05 ** p < 0.01 *** p < 0.001\\nAnticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\\nAmong all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\\nAcceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\\nShould there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\",\n \"Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\\n\\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\\naTotal number for each variable may not add up to N = 356 due to missing data\",\n \"Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\\n\\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\\nn\\n\\n%\\n\\nn\\n\\n%\\n\\nOR (95% CI)\\n\\n(a) Perceived impacts of HIV cure\\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\\n(b) Perception and attitude towards HIV functional cure\\n\\nAwareness of HIV functional cure\\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\\nLevel of anticipation for HIV functional cure\\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\\nWillingness in joining a functional cure trial\\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\\n(c) Considerations about HIV functional cure trial\\n\\nImportant factors to be considered about the trial\\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\\nConcerns about consequences of joining a trial\\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\\n*p < 0.05 ** p < 0.01 *** p < 0.001\\n\\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\\nIMP1a\\nIMP2aIMP3aIMP4aIMP5a\\nIMP6aIMP7a\\nAge group\\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\\nEducation level\\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \\nSTI history in the past year\\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\\nSexual activity after HIV diagnosis\\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \\nEngagement in chemsex\\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\\nAbout Functional cure\\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\\n* p < 0.05 ** p < 0.01 *** p < 0.001\",\n \"Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\\n\\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nCrude OR\\n\\nAdjusted OR\\n\\nAge group\\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\\nEducation level\\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \\nSTI history in the past year\\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\\n Sexual activity after HIV diagnosis\\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\\n Engagement in chemsex\\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\\nCD4 cell count (cells/uL)\\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\\nAbout functional cure\\n Awareness\\n–\\n\\n–\\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\\n–\\n\\n–\\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\\n* p < 0.05 ** p < 0.01 *** p < 0.001\",\n \"Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\",\n \"Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\",\n \"HIV functional cure is a relatively new concept for PLHIV. Despite their generally low awareness, MSM living with HIV had, as shown in this study, a high level of anticipation for HIV functional cure, especially those who had been diagnosed and put on ART for some years. The effective achievement of functional cure carries the potential prospect of getting rid of repeated clinic visits and taking long-term medications [11]. The benefit of being untransmittable may, however, not be regarded as an important impact for HIV cure because in the current era of “undetectable equals untransmittable”, adherence to ART is already effective in minimizing the risk of transmission. In our cohorts of MSM living with HIV, the attitudes differed by one’s ART treatment experience. Treatment-naïve participants were more concerned about their immune function and AIDS progression than those ART-experienced, as deteriorated health was perceived by newly diagnosed patients an imminent consequence brought on by HIV [12]. Anticipation for HIV functional cure was apparently lower among those who were aware of it. Although some 42% had heard about functional cure, most had limited knowledge of it and cared little about the potential benefit of restoring their immune function and potential adverse effects arising from the new therapy. With the increasing likelihood that functional cure may soon become the next major advance in HIV treatment [7], there is the need for promoting community education on the rationale and strategy for HIV functional cure. As in many countries, MSM accounted for a high proportion of PLHIV in Hong Kong [13], and who continued to be significantly impacted by the epidemic [14]. The MSM community, especially those living with HIV would need to be targeted in anticipation of the introduction of functional cure therapy. In our study, MSM engaging in higher risk sexual behaviors were more likely to be aware of the recent advancement on HIV science, such as pre-exposure prophylaxis [15]. This echoed our results that sexually active PLHIV, particularly those engaged in condomless sex as indicated by their STI history, were more likely to be aware of HIV functional cure.\\nThere are diverse reasons for PLHIV to look forward to receiving functional cure therapy. It is intuitive that PLHIV with a weaker immune system were less likely to be sexually active and they wished to restore and stabilize their immune function and were at the same time more concerned about adverse HIV outcomes after joining functional cure trials. The fact that younger PLHIV cared more about disease development and transmittability was likely because they were newly diagnosed and would remain sexually active for a longer time in the future. PLHIV with higher education level regarded de-labelling of HIV an important impact of HIV cure. In Hong Kong, public stigma against PLHIV [16] and perceived discrimination [17] have been reported. It is therefore an ideal vision for them to remove the label of HIV after achieving some sort of cure. Discrimination in workplace and difficulty in career development could be experienced by PLHIV [18]. Although highly educated PLHIV were found to deal better with workplace discrimination and improve employment quality than those with lower education level [19], they were more concerned with their career development that removing stigma means removing a barrier in their career paths. From the perspective of the general public, increased awareness and knowledge in HIV science advancement might be able to reduce stigma. A previous study showed that heterosexually active adults who were aware of the untransmittable nature of PLHIV achieving undetectable viral load had a lower level of anticipated HIV stigma [20]. As this study focuses on PLHIV, a follow-up study in the general population and the MSM community would be needed to assess the impact of functional cure on general, and dating- and sex-related enacted stigmas.\\nOverall, the idea of an HIV functional cure trial was well accepted by our cohorts of MSM living with HIV with over 90% indicating their willingness to join, similar to the high acceptance rate of 95% in a previous study [11]. Potential trial participants were more likely to have engaged in higher risk sexual activities as reflected by their report of STI in the preceding year. This might be precipitated by a prospect of continuation or re-engagement in adventurous condomless behaviors upon cure [21]. Safety is a major concern for clinical trials involving the use of novel agents like neutralizing antibodies or therapeutic vaccines. Careful monitoring of PLHIV in the trial would be warranted not just for protecting participants’ health but also easing their concerns of side effects and adverse HIV outcomes. Hesitation would arise if the trial requires interruption of ART or if it takes a long time, in relation to the risk of rebound and potential adverse effects [11]. Trusting relationship between PLHIV, researchers and healthcare workers could be beneficial for ensuring the delivery of optimal health outcomes [22]. Based on potential participants’ trust on healthcare professional advice, collaboration with HIV clinics on explaining the details and safety issues of the trial would be paramount.\\nThis study carries some limitations. Like other epidemiological studies, there were inherent recall and social desirability biases in the administration of behavioral questionnaires. We had minimized these biases by adopting a shorter recall period and omitting fine details about sexual activities. The adoption of a self-administering approach should have reduced embarrassments in response to interviewers’ questions. Incorporation of clinical data by transcription benefited the study by providing objective measurements of participants’ HIV outcomes. In the absence of any functional cure trials, the exploration of one’s willingness to participate in a hypothetical trial without having one in place could be postulational. Nevertheless, the systematic inquiries into the awareness and anticipation of functional HIV cure among MSM living with HIV and their inclination in joining a hypothetical study have generated useful results which could be of reference for the planning of a functional cure trial.\",\n \"To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment.\",\n \" \\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\\nAdditional file 1. Questionnaire.\\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\\n\\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\\nAdditional file 1. Questionnaire.\\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\",\n \"\\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\\nAdditional file 1. Questionnaire.\\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,"results",null,null,null,null,null,"discussion","conclusion","supplementary-material",null],"string":"[\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\",\n \"supplementary-material\",\n null\n]"},"keywords":{"kind":"list like","value":["HIV","Functional cure","Men who have sex with men","Awareness"],"string":"[\n \"HIV\",\n \"Functional cure\",\n \"Men who have sex with men\",\n \"Awareness\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nLife-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.\n\nMethods:\nData for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.\n\nResults:\nDemographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nBetween March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nPerceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nPerceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAwareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nOverall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAnticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAmong all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAcceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\nShould there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\n\nDemographics:\nBetween March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\n\nPerceived benefits of HIV cure:\nPerceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\n\nAwareness of HIV functional cure:\nOverall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\n\nAnticipation of HIV functional cure:\nAmong all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\n\nAcceptance of an HIV functional cure trial:\nShould there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\n\nDiscussion:\nHIV functional cure is a relatively new concept for PLHIV. Despite their generally low awareness, MSM living with HIV had, as shown in this study, a high level of anticipation for HIV functional cure, especially those who had been diagnosed and put on ART for some years. The effective achievement of functional cure carries the potential prospect of getting rid of repeated clinic visits and taking long-term medications [11]. The benefit of being untransmittable may, however, not be regarded as an important impact for HIV cure because in the current era of “undetectable equals untransmittable”, adherence to ART is already effective in minimizing the risk of transmission. In our cohorts of MSM living with HIV, the attitudes differed by one’s ART treatment experience. Treatment-naïve participants were more concerned about their immune function and AIDS progression than those ART-experienced, as deteriorated health was perceived by newly diagnosed patients an imminent consequence brought on by HIV [12]. Anticipation for HIV functional cure was apparently lower among those who were aware of it. Although some 42% had heard about functional cure, most had limited knowledge of it and cared little about the potential benefit of restoring their immune function and potential adverse effects arising from the new therapy. With the increasing likelihood that functional cure may soon become the next major advance in HIV treatment [7], there is the need for promoting community education on the rationale and strategy for HIV functional cure. As in many countries, MSM accounted for a high proportion of PLHIV in Hong Kong [13], and who continued to be significantly impacted by the epidemic [14]. The MSM community, especially those living with HIV would need to be targeted in anticipation of the introduction of functional cure therapy. In our study, MSM engaging in higher risk sexual behaviors were more likely to be aware of the recent advancement on HIV science, such as pre-exposure prophylaxis [15]. This echoed our results that sexually active PLHIV, particularly those engaged in condomless sex as indicated by their STI history, were more likely to be aware of HIV functional cure.\nThere are diverse reasons for PLHIV to look forward to receiving functional cure therapy. It is intuitive that PLHIV with a weaker immune system were less likely to be sexually active and they wished to restore and stabilize their immune function and were at the same time more concerned about adverse HIV outcomes after joining functional cure trials. The fact that younger PLHIV cared more about disease development and transmittability was likely because they were newly diagnosed and would remain sexually active for a longer time in the future. PLHIV with higher education level regarded de-labelling of HIV an important impact of HIV cure. In Hong Kong, public stigma against PLHIV [16] and perceived discrimination [17] have been reported. It is therefore an ideal vision for them to remove the label of HIV after achieving some sort of cure. Discrimination in workplace and difficulty in career development could be experienced by PLHIV [18]. Although highly educated PLHIV were found to deal better with workplace discrimination and improve employment quality than those with lower education level [19], they were more concerned with their career development that removing stigma means removing a barrier in their career paths. From the perspective of the general public, increased awareness and knowledge in HIV science advancement might be able to reduce stigma. A previous study showed that heterosexually active adults who were aware of the untransmittable nature of PLHIV achieving undetectable viral load had a lower level of anticipated HIV stigma [20]. As this study focuses on PLHIV, a follow-up study in the general population and the MSM community would be needed to assess the impact of functional cure on general, and dating- and sex-related enacted stigmas.\nOverall, the idea of an HIV functional cure trial was well accepted by our cohorts of MSM living with HIV with over 90% indicating their willingness to join, similar to the high acceptance rate of 95% in a previous study [11]. Potential trial participants were more likely to have engaged in higher risk sexual activities as reflected by their report of STI in the preceding year. This might be precipitated by a prospect of continuation or re-engagement in adventurous condomless behaviors upon cure [21]. Safety is a major concern for clinical trials involving the use of novel agents like neutralizing antibodies or therapeutic vaccines. Careful monitoring of PLHIV in the trial would be warranted not just for protecting participants’ health but also easing their concerns of side effects and adverse HIV outcomes. Hesitation would arise if the trial requires interruption of ART or if it takes a long time, in relation to the risk of rebound and potential adverse effects [11]. Trusting relationship between PLHIV, researchers and healthcare workers could be beneficial for ensuring the delivery of optimal health outcomes [22]. Based on potential participants’ trust on healthcare professional advice, collaboration with HIV clinics on explaining the details and safety issues of the trial would be paramount.\nThis study carries some limitations. Like other epidemiological studies, there were inherent recall and social desirability biases in the administration of behavioral questionnaires. We had minimized these biases by adopting a shorter recall period and omitting fine details about sexual activities. The adoption of a self-administering approach should have reduced embarrassments in response to interviewers’ questions. Incorporation of clinical data by transcription benefited the study by providing objective measurements of participants’ HIV outcomes. In the absence of any functional cure trials, the exploration of one’s willingness to participate in a hypothetical trial without having one in place could be postulational. Nevertheless, the systematic inquiries into the awareness and anticipation of functional HIV cure among MSM living with HIV and their inclination in joining a hypothetical study have generated useful results which could be of reference for the planning of a functional cure trial.\n\nConclusions:\nTo PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment.\n\nSupplementary Information:\n \nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\n\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\n\n:\n\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHuman immunodeficiency virus (HIV) functional cure is a novel biomedical strategy characterized by sustained viral suppression without the need for life-long medications. The attitude of people living with HIV (PLHIV) towards functional cure and clinical trials are understudied. We aimed to examine the awareness and levels of anticipation for HIV functional cure among men who have sex with men (MSM) living with HIV, and their willingness to join trials as differentiated by their antiretroviral treatment status.\n\nMethods:\nMSM living with HIV with and those without treatment history were recruited from Hong Kong's HIV specialist clinics. Self-administered questionnaires covering behavioral profile, perceived impact of HIV cure, attitude towards HIV functional cure and related clinical trials were collected. Clinical data were separately transcribed. Determinants of perceptions and attitudes were identified by logistic regression models.\n\nResults:\nOf 356 MSM living with HIV recruited, less than half (42%) were aware of HIV functional cure, but they had a high level of anticipation for it. Treatment-experienced participants were more likely to be aware of HIV functional cure. Awareness was associated with continued engagement in sexual activities after HIV diagnosis and sexually transmitted infection (STI) diagnosis. Higher anticipation was observed among older MSM living with HIV but it was negatively associated with one's awareness. Over 90% were willing to join functional cure trials, especially those who had previously been diagnosed with STI and had engaged in chemsex in the past year. Advice from healthcare professional was an important factor considered by those willing to join clinical trials. Younger, better educated MSM, and those with lower CD4 counts were more concerned about potential risk of AIDS and potential complications upon trial participation.\n\nConclusions:\nMSM living with HIV, especially those sexually active, showed positive attitude towards functional cure and willingness to join related clinical trials despite low awareness. To enhance preparedness for HIV functional cure trials, community education, updated information and appropriate medical advice would be needed. Safety is a major concern for potential enrollees in HIV functional cure trials."},"background_conclusion_text":{"kind":"string","value":"Background:\nLife-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.\n\nConclusions:\nTo PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nHuman immunodeficiency virus (HIV) functional cure is a novel biomedical strategy characterized by sustained viral suppression without the need for life-long medications. The attitude of people living with HIV (PLHIV) towards functional cure and clinical trials are understudied. We aimed to examine the awareness and levels of anticipation for HIV functional cure among men who have sex with men (MSM) living with HIV, and their willingness to join trials as differentiated by their antiretroviral treatment status.\n\nMethods:\nMSM living with HIV with and those without treatment history were recruited from Hong Kong's HIV specialist clinics. Self-administered questionnaires covering behavioral profile, perceived impact of HIV cure, attitude towards HIV functional cure and related clinical trials were collected. Clinical data were separately transcribed. Determinants of perceptions and attitudes were identified by logistic regression models.\n\nResults:\nOf 356 MSM living with HIV recruited, less than half (42%) were aware of HIV functional cure, but they had a high level of anticipation for it. Treatment-experienced participants were more likely to be aware of HIV functional cure. Awareness was associated with continued engagement in sexual activities after HIV diagnosis and sexually transmitted infection (STI) diagnosis. Higher anticipation was observed among older MSM living with HIV but it was negatively associated with one's awareness. Over 90% were willing to join functional cure trials, especially those who had previously been diagnosed with STI and had engaged in chemsex in the past year. Advice from healthcare professional was an important factor considered by those willing to join clinical trials. Younger, better educated MSM, and those with lower CD4 counts were more concerned about potential risk of AIDS and potential complications upon trial participation.\n\nConclusions:\nMSM living with HIV, especially those sexually active, showed positive attitude towards functional cure and willingness to join related clinical trials despite low awareness. To enhance preparedness for HIV functional cure trials, community education, updated information and appropriate medical advice would be needed. Safety is a major concern for potential enrollees in HIV functional cure trials."},"whole_article_text_length":{"kind":"number","value":12661,"string":"12,661"},"whole_article_abstract_length":{"kind":"number","value":393,"string":"393"},"other_sections_lengths":{"kind":"list like","value":[523,903,549,1343,531,184,549,143],"string":"[\n 523,\n 903,\n 549,\n 1343,\n 531,\n 184,\n 549,\n 143\n]"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["hiv","cure","functional","functional cure","95","ci","95 ci","trial","001","hiv functional"],"string":"[\n \"hiv\",\n \"cure\",\n \"functional\",\n \"functional cure\",\n \"95\",\n \"ci\",\n \"95 ci\",\n \"trial\",\n \"001\",\n \"hiv functional\"\n]"},"keybert_topics":{"kind":"list like","value":["hiv cure studies","cure hiv functional","cure consideration hiv","hiv cure functional","plhiv hiv cure"],"string":"[\n \"hiv cure studies\",\n \"cure hiv functional\",\n \"cure consideration hiv\",\n \"hiv cure functional\",\n \"plhiv hiv cure\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] HIV | Functional cure | Men who have sex with men | Awareness [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] HIV | Functional cure | Men who have sex with men | Awareness [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] HIV | Functional cure | Men who have sex with men | Awareness [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] HIV | Functional cure | Men who have sex with men | Awareness [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] HIV | Functional cure | Men who have sex with men | Awareness [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | HIV Infections | Homosexuality, Male | Humans | Male | Sexual Behavior | Sexual and Gender Minorities | Sexually Transmitted Diseases [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | HIV Infections | Homosexuality, Male | Humans | Male | Sexual Behavior | Sexual and Gender Minorities | Sexually Transmitted Diseases [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | HIV Infections | Homosexuality, Male | Humans | Male | Sexual Behavior | Sexual and Gender Minorities | Sexually Transmitted Diseases [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | HIV Infections | Homosexuality, Male | Humans | Male | Sexual Behavior | Sexual and Gender Minorities | Sexually Transmitted Diseases [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Cross-Sectional Studies | Female | HIV Infections | Homosexuality, Male | Humans | Male | Sexual Behavior | Sexual and Gender Minorities | Sexually Transmitted Diseases [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv cure studies | cure hiv functional | cure consideration hiv | hiv cure functional | plhiv hiv cure 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functional cure | 95 | ci | 95 ci | trial | 001 | hiv functional [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | functional | functional cure | 95 | ci | 95 ci | trial | 001 | hiv functional [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | functional | functional cure | 95 | ci | 95 ci | trial | 001 | hiv functional [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | functional | functional cure | 95 | ci | 95 ci | trial | 001 | hiv functional [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | functional | functional cure | 95 | ci | 95 ci | trial | 001 | hiv functional [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cure | plhiv | hiv | functional cure | functional | art | functional cure research | cure research | research | hiv infection [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | 95 | ci | 95 ci | 001 | 00 | cure | 001 001 | 001 001 001 | functional [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | need | functional | functional cure | high | living hiv | msm living hiv | msm living | living [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | functional | ci | functional cure | 95 | 95 ci | trial | 001 | 00 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hiv | cure | functional | ci | functional cure | 95 | 95 ci | trial | 001 | 00 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| MSM [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 356 | less than half | 42% ||| ||| STI ||| MSM ||| 90% | STI | the past year ||| ||| MSM [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] MSM ||| ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| MSM ||| Hong Kong's ||| ||| ||| ||| ||| 356 | less than half | 42% ||| ||| STI ||| MSM ||| 90% | STI | the past year ||| ||| MSM ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| MSM ||| Hong Kong's ||| ||| ||| ||| ||| 356 | less than half | 42% ||| ||| STI ||| MSM ||| 90% | STI | the past year ||| ||| MSM ||| ||| ||| [SUMMARY]"}}},{"rowIdx":3371,"cells":{"title":{"kind":"string","value":"The Impact of the COVID-19 Pandemic on an Israeli Acute Care Surgery Unit: Fewer Patients, More Disease."},"pmid":{"kind":"string","value":"33856956"},"background_abstract":{"kind":"string","value":"The COVID-19 pandemic has transformed and affected every aspect of health care. Like any catastrophic event, the stress on hospitals to maintain a certain level of function is immense. Acute surgical pathologies cannot be prevented or curtailed; therefore, it is important to understand patterns and outcomes during catastrophes in order to optimize care and organize the health care system."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In a single urban tertiary care center, a retrospective study examined the first complete lockdown period of Israel during the COVID-19 pandemic. This was compared to the same time period the previous year."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"During the pandemic, time to hospitalization was significantly decreased. There was also an overall reduction in surgical admissions yet with a higher percentage being hospitalized for further treatment (69.2% vs 23.5%). The patients admitted during this time had a higher APACHE-II score and Charlson comorbidity index score. During the pandemic, time to surgery was decreased, there were less laparoscopic procedures, and more RBC units were used per patient. There were no differences in overall complications, except when sub-analyzed for major complications (9.7% vs 6.3%). There was no significant difference in overall in-house mortality or morbidity. Length of hospitalization was significantly decreased in the elderly population during the pandemic."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"During the COVID-19 pandemic, despite a significantly less number of patients presenting to the hospital, there was a higher percentage of those admitted needing surgical intervention, and they were overall sicker than the previous year."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Aged","COVID-19","Pandemics","Israel","SARS-CoV-2","Retrospective Studies","Communicable Disease Control"],"string":"[\n \"Humans\",\n \"Aged\",\n \"COVID-19\",\n \"Pandemics\",\n \"Israel\",\n \"SARS-CoV-2\",\n \"Retrospective Studies\",\n \"Communicable Disease Control\"\n]"},"pmcid":{"kind":"string","value":"9629022"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6\nA perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11\nUnderstanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15\nThere is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"All patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.\nOutline of study process.\nDuring the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.\nStatistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\nOverall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\n Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.\nFor the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Patient Demographics and Clinical Characteristics","Treatment and Outcome Characteristics"],"string":"[\n \"Patient Demographics and Clinical Characteristics\",\n \"Treatment and Outcome Characteristics\"\n]"},"other_sections_texts":{"kind":"list like","value":["Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.","For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients."],"string":"[\n \"Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nDemographics, Intervention, and Diagnosis.\\nAbbreviations: ED, emergency department.\\nPatients Who Had Surgical Intervention.\\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\",\n \"For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nElderly Patients Sub-Analysis.\\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\\n10 Patients Originally Triaged as Potential COVID-19 Patients.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null],"string":"[\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Results","Patient Demographics and Clinical Characteristics","Treatment and Outcome Characteristics","Discussion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Results\",\n \"Patient Demographics and Clinical Characteristics\",\n \"Treatment and Outcome Characteristics\",\n \"Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["The COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6\nA perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11\nUnderstanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15\nThere is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic.","All patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.\nOutline of study process.\nDuring the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.\nStatistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant."," Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\nOverall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\n Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.\nFor the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.","Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.","For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.","During this period, we have shown that despite a significant decrease in the number of patients presenting to our ER, a majority of them presented with more severe disease when compared to the previous year. In general, the patients undergoing an operation were in worse condition than the patients without operation. Clearly, during this pandemic, or any other natural disaster or military campaign, there is limited ability to curtail the inevitable presentation of acute surgical pathologies or traumatic events. During challenging circumstances, hospitals must be prepared for and must be able to maintain emergent surgical services in order to avoid preventable deaths and mortality. The results of this study have shown the effect of the COVID-19 pandemic on our patient population suffering from acute general surgery illnesses.\nThere was no difference in the pathologies presenting between the two periods, which is an important statistical finding, yet must be taken in proper context. Depending on the type of catastrophe, the required mobilization of specific medical specialties and strain on the systems will be drastically different. During times of war or natural disasters, hospitals will require a larger ratio of orthopedic surgeons, general surgeons, surgical specialties, OR access, and ICU beds.14 In contrast, pathogen-derived epidemics/pandemics will require more specialists in the fields of internal medicine (ie, infectious disease, pulmonology, intensivist, and hematologist), ICU beds, and potential access to extracorporeal membrane oxygenation.15 As seen with our results, this primarily respiratory pandemic did not significantly change the acute surgical pathologies.\nIn regard to the younger patient population, there were several observations that were of interest yet not statistically significant. These patients appeared to present later in their course of disease, with a higher rate of complicated pathologies (ie, perforated appendix and gangrenous cholecystitis), postoperative complications, and blood transfusions. These younger patients were therefore more likely able to tolerate their symptoms at home for a longer period and therefore had a delay in diagnosis and treatment. In contrast, the older population, with their lower physiological reserve, appeared less able to delay medical treatment and came to the hospital regardless of fears and inconveniences caused by the pandemic restrictions.\nThe “fear” of presenting to the ER with severe illness has been documented from this current COVID-19 pandemic21 and from previously published literature during catastrophes. For example, during the SARS epidemic in 2002, Taiwan and Hong Kong showed a decrease in routine care not related to the severe acute respiratory syndrome.22 During this COVID-19 pandemic, one recent study reported the patterns of patients presenting to the ER of a busy level-1 trauma center in America. This group showed a global decrease in arrivals to the ER, along with a decreased proportion of patients presenting with abdominal pain when compared to other systemic complaints before the pandemic.23 This cannot be accurately compared to our data because we were only looking at acute surgical pathologies. Nevertheless, what was reported in this reference might also be a result of avoiding going to the hospital with abdominal pain during a pandemic that involves primarily respiratory symptoms. In Italy, toward the beginning of the pandemic, a series of children, without COVID-19-related illness, were shown to have presented late and in more critical condition, due to the fear of presenting to a hospital or lack of provisional care because of closure of health consults/centers.24 This corresponds with our overall results in general of sicker patients being admitted to the surgical ward.\nThe country where this study took place, Israel, also presents a unique confounding factor to these data. Our hospital is a public hospital within a socialized system where every citizen is born with universal health care coverage25; therefore, lacking health care is rarely a reason for not seeking medical attention.26 Compared to the rest of the world, our mortality has been comparatively low.27,28 Our “lockdown” measures and monitoring methods were considered aggressive, but the country also progressed in easing the regulations and opening its economy relatively fast. Nevertheless, the cooperation and coordination of our Ministry of Health, security sectors, and private industry played a pivotal role in preventing our hospital infrastructure from being completely overwhelmed. Our results showed a different demographic in terms of ethnicity presenting to the hospital during the COVID-19 pandemic. There was a significant increase in the ratio of Israeli-Jewish to Israeli-non-Jewish patients presenting to the ED and a smaller proportion of minorities, tourist, and foreign workers when compared to 2019. As mentioned above, the TASMC is located in the center of Tel Aviv, where 91% of the population is Jewish and 9% are non-Jewish. This difference might be explained by the lack of transport, hesitancy to travel distances, and fear of larger medical centers because of their treatment of COVID-19 patients.29 Hence, smaller local medical centers were potentially receiving more patients, regardless of appropriateness of level of care.\nWe found a low incidence of mis-triage of surgical patients into the COVID-19 isolation wing by our ER triage protocol. Out of the 209 patients admitted during this time, only 10 were initially placed in isolation. None of these 10 isolated patients had a positive COVID-19 test result. Despite being placed in triage, the average time to hospitalization, despite being statistically different, was only 30 minutes longer than the non-isolated group. When intervention (surgical and nonsurgical) was needed in the isolated group, there was no increase in time when compared to the non-isolated patients and, on average, even occurred more rapidly. With many surgical pathologies presenting with fever, it was difficult, especially at the beginning of the pandemic, not to over triage these patients and potentially delay intervention for surgical emergencies. This became especially relevant as evidence began to show that a possible presenting symptom of COVID-19 is fever with diarrhea.30 The significant reduction in time to hospitalization during the pandemic may be a confounding effect of both a decrease in volume to the ED and also sicker patients presenting to the ED, which would require quicker diagnosis and appropriate therapy. This decrease in hospitalization time happened despite a reduction in working staff.\nMultiple studies have established that this elderly population is at an increased risk to clinically significant COVID-19.31 Our elderly population (>70 years old) during the pandemic had slightly more comorbidities and had a greater 30-day mortality than the previous year. Despite their increased comorbidities, they did not present to the hospital sicker than their counterparts the previous year. This trend might demonstrate the opposite of the “fear” phenomenon mentioned above for younger patients that sicker, elder patients present more easily to the hospital driven by the distress that something is wrong. With the elderly patients who did not undergo surgery, we were able to significantly reduce their hospitalization time. We have emphasized in our department not to neglect surgical pathologies during this time in this vulnerable population.\nAn interesting finding from the patients undergoing surgery during the pandemic is that they had a higher APACHE score, more complications, more blood given, yet more cases were open (verses laparoscopic), and there was an attending present more often. This seemingly contradicting findings might be a true indicator of how severely ill/complicated the patients were when compared to the previous year. Yet, this finding would need to be compared to other large tertiary centers in order to gather a significant conclusion. There are additional limitations to this study, including the fact that it is coming from a single urban referral center. These data may not be representative of other smaller and/or rural centers. Other limitations include the short study time and not having a cohort of surgical patients who were COVID-19 positive. The short study time was chosen in order to describe the initial effect of the pandemic and the most constricting limitations imposed by the government during the initial lockdown. Therefore, the initial month of true, full lockdown was taken and compared to the previous year. Clearly, during different stages and evolution of the pandemic, societies and medical systems have changed/adapted; therefore, the time when this study was conducted needs to be taken into consideration. There was no (knowingly) COVID-19-positive patient in our cohort and therefore no comparison between the differences in surgical pathology progression between positive and negative surgical patients. As there is more understanding of the COVID-19 pathophysiology, this would be an important patient population to analyze.\nIn conclusion, we have shown that during this COVID-19 pandemic, the patients presenting were sicker than the patients during the same time frame in the previous year despite there being fewer patients needing surgical admission. We were able to decrease the time until hospitalization and the length of hospitalization on the highly vulnerable elderly population. These data support the need for advocating and educating the public to present to hospitals without delay if there are symptoms of a possible acute surgical illness. Clearly, there are difficult times that may force the population to stay home (natural disasters and war), but fear alone should not delay the presentation of a patient with abdominal pain to the hospital. With well-organized systems and cooperation between governing and health sectors, hospitalization can be streamlined and physician/hospital workforce exposure can be minimized, while not compromising the level of care and treatment needed for acute surgical pathologies. Through understanding the surgical needs of a population during a catastrophic event, the public health sector may more rapidly and precisely distribute needed personnel, essential resources, and allocate certain medical centers as referral points for surgical emergencies."],"string":"[\n \"The COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6\\nA perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11\\nUnderstanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15\\nThere is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic.\",\n \"All patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.\\nOutline of study process.\\nDuring the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.\\nStatistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant.\",\n \" Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nDemographics, Intervention, and Diagnosis.\\nAbbreviations: ED, emergency department.\\nPatients Who Had Surgical Intervention.\\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\\nOverall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nDemographics, Intervention, and Diagnosis.\\nAbbreviations: ED, emergency department.\\nPatients Who Had Surgical Intervention.\\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\\n Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nElderly Patients Sub-Analysis.\\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\\n10 Patients Originally Triaged as Potential COVID-19 Patients.\\nFor the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nElderly Patients Sub-Analysis.\\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\\n10 Patients Originally Triaged as Potential COVID-19 Patients.\",\n \"Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nDemographics, Intervention, and Diagnosis.\\nAbbreviations: ED, emergency department.\\nPatients Who Had Surgical Intervention.\\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\",\n \"For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nElderly Patients Sub-Analysis.\\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\\n10 Patients Originally Triaged as Potential COVID-19 Patients.\",\n \"During this period, we have shown that despite a significant decrease in the number of patients presenting to our ER, a majority of them presented with more severe disease when compared to the previous year. In general, the patients undergoing an operation were in worse condition than the patients without operation. Clearly, during this pandemic, or any other natural disaster or military campaign, there is limited ability to curtail the inevitable presentation of acute surgical pathologies or traumatic events. During challenging circumstances, hospitals must be prepared for and must be able to maintain emergent surgical services in order to avoid preventable deaths and mortality. The results of this study have shown the effect of the COVID-19 pandemic on our patient population suffering from acute general surgery illnesses.\\nThere was no difference in the pathologies presenting between the two periods, which is an important statistical finding, yet must be taken in proper context. Depending on the type of catastrophe, the required mobilization of specific medical specialties and strain on the systems will be drastically different. During times of war or natural disasters, hospitals will require a larger ratio of orthopedic surgeons, general surgeons, surgical specialties, OR access, and ICU beds.14 In contrast, pathogen-derived epidemics/pandemics will require more specialists in the fields of internal medicine (ie, infectious disease, pulmonology, intensivist, and hematologist), ICU beds, and potential access to extracorporeal membrane oxygenation.15 As seen with our results, this primarily respiratory pandemic did not significantly change the acute surgical pathologies.\\nIn regard to the younger patient population, there were several observations that were of interest yet not statistically significant. These patients appeared to present later in their course of disease, with a higher rate of complicated pathologies (ie, perforated appendix and gangrenous cholecystitis), postoperative complications, and blood transfusions. These younger patients were therefore more likely able to tolerate their symptoms at home for a longer period and therefore had a delay in diagnosis and treatment. In contrast, the older population, with their lower physiological reserve, appeared less able to delay medical treatment and came to the hospital regardless of fears and inconveniences caused by the pandemic restrictions.\\nThe “fear” of presenting to the ER with severe illness has been documented from this current COVID-19 pandemic21 and from previously published literature during catastrophes. For example, during the SARS epidemic in 2002, Taiwan and Hong Kong showed a decrease in routine care not related to the severe acute respiratory syndrome.22 During this COVID-19 pandemic, one recent study reported the patterns of patients presenting to the ER of a busy level-1 trauma center in America. This group showed a global decrease in arrivals to the ER, along with a decreased proportion of patients presenting with abdominal pain when compared to other systemic complaints before the pandemic.23 This cannot be accurately compared to our data because we were only looking at acute surgical pathologies. Nevertheless, what was reported in this reference might also be a result of avoiding going to the hospital with abdominal pain during a pandemic that involves primarily respiratory symptoms. In Italy, toward the beginning of the pandemic, a series of children, without COVID-19-related illness, were shown to have presented late and in more critical condition, due to the fear of presenting to a hospital or lack of provisional care because of closure of health consults/centers.24 This corresponds with our overall results in general of sicker patients being admitted to the surgical ward.\\nThe country where this study took place, Israel, also presents a unique confounding factor to these data. Our hospital is a public hospital within a socialized system where every citizen is born with universal health care coverage25; therefore, lacking health care is rarely a reason for not seeking medical attention.26 Compared to the rest of the world, our mortality has been comparatively low.27,28 Our “lockdown” measures and monitoring methods were considered aggressive, but the country also progressed in easing the regulations and opening its economy relatively fast. Nevertheless, the cooperation and coordination of our Ministry of Health, security sectors, and private industry played a pivotal role in preventing our hospital infrastructure from being completely overwhelmed. Our results showed a different demographic in terms of ethnicity presenting to the hospital during the COVID-19 pandemic. There was a significant increase in the ratio of Israeli-Jewish to Israeli-non-Jewish patients presenting to the ED and a smaller proportion of minorities, tourist, and foreign workers when compared to 2019. As mentioned above, the TASMC is located in the center of Tel Aviv, where 91% of the population is Jewish and 9% are non-Jewish. This difference might be explained by the lack of transport, hesitancy to travel distances, and fear of larger medical centers because of their treatment of COVID-19 patients.29 Hence, smaller local medical centers were potentially receiving more patients, regardless of appropriateness of level of care.\\nWe found a low incidence of mis-triage of surgical patients into the COVID-19 isolation wing by our ER triage protocol. Out of the 209 patients admitted during this time, only 10 were initially placed in isolation. None of these 10 isolated patients had a positive COVID-19 test result. Despite being placed in triage, the average time to hospitalization, despite being statistically different, was only 30 minutes longer than the non-isolated group. When intervention (surgical and nonsurgical) was needed in the isolated group, there was no increase in time when compared to the non-isolated patients and, on average, even occurred more rapidly. With many surgical pathologies presenting with fever, it was difficult, especially at the beginning of the pandemic, not to over triage these patients and potentially delay intervention for surgical emergencies. This became especially relevant as evidence began to show that a possible presenting symptom of COVID-19 is fever with diarrhea.30 The significant reduction in time to hospitalization during the pandemic may be a confounding effect of both a decrease in volume to the ED and also sicker patients presenting to the ED, which would require quicker diagnosis and appropriate therapy. This decrease in hospitalization time happened despite a reduction in working staff.\\nMultiple studies have established that this elderly population is at an increased risk to clinically significant COVID-19.31 Our elderly population (>70 years old) during the pandemic had slightly more comorbidities and had a greater 30-day mortality than the previous year. Despite their increased comorbidities, they did not present to the hospital sicker than their counterparts the previous year. This trend might demonstrate the opposite of the “fear” phenomenon mentioned above for younger patients that sicker, elder patients present more easily to the hospital driven by the distress that something is wrong. With the elderly patients who did not undergo surgery, we were able to significantly reduce their hospitalization time. We have emphasized in our department not to neglect surgical pathologies during this time in this vulnerable population.\\nAn interesting finding from the patients undergoing surgery during the pandemic is that they had a higher APACHE score, more complications, more blood given, yet more cases were open (verses laparoscopic), and there was an attending present more often. This seemingly contradicting findings might be a true indicator of how severely ill/complicated the patients were when compared to the previous year. Yet, this finding would need to be compared to other large tertiary centers in order to gather a significant conclusion. There are additional limitations to this study, including the fact that it is coming from a single urban referral center. These data may not be representative of other smaller and/or rural centers. Other limitations include the short study time and not having a cohort of surgical patients who were COVID-19 positive. The short study time was chosen in order to describe the initial effect of the pandemic and the most constricting limitations imposed by the government during the initial lockdown. Therefore, the initial month of true, full lockdown was taken and compared to the previous year. Clearly, during different stages and evolution of the pandemic, societies and medical systems have changed/adapted; therefore, the time when this study was conducted needs to be taken into consideration. There was no (knowingly) COVID-19-positive patient in our cohort and therefore no comparison between the differences in surgical pathology progression between positive and negative surgical patients. As there is more understanding of the COVID-19 pathophysiology, this would be an important patient population to analyze.\\nIn conclusion, we have shown that during this COVID-19 pandemic, the patients presenting were sicker than the patients during the same time frame in the previous year despite there being fewer patients needing surgical admission. We were able to decrease the time until hospitalization and the length of hospitalization on the highly vulnerable elderly population. These data support the need for advocating and educating the public to present to hospitals without delay if there are symptoms of a possible acute surgical illness. Clearly, there are difficult times that may force the population to stay home (natural disasters and war), but fear alone should not delay the presentation of a patient with abdominal pain to the hospital. With well-organized systems and cooperation between governing and health sectors, hospitalization can be streamlined and physician/hospital workforce exposure can be minimized, while not compromising the level of care and treatment needed for acute surgical pathologies. Through understanding the surgical needs of a population during a catastrophic event, the public health sector may more rapidly and precisely distribute needed personnel, essential resources, and allocate certain medical centers as referral points for surgical emergencies.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results",null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["COVID-19","acute care surgery","pandemic"],"string":"[\n \"COVID-19\",\n \"acute care surgery\",\n \"pandemic\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6\nA perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11\nUnderstanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15\nThere is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic.\n\nMethods:\nAll patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.\nOutline of study process.\nDuring the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.\nStatistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant.\n\nResults:\n Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\nOverall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\n Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.\nFor the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.\n\nPatient Demographics and Clinical Characteristics:\nOverall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\n\nTreatment and Outcome Characteristics:\nFor the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.\n\nDiscussion:\nDuring this period, we have shown that despite a significant decrease in the number of patients presenting to our ER, a majority of them presented with more severe disease when compared to the previous year. In general, the patients undergoing an operation were in worse condition than the patients without operation. Clearly, during this pandemic, or any other natural disaster or military campaign, there is limited ability to curtail the inevitable presentation of acute surgical pathologies or traumatic events. During challenging circumstances, hospitals must be prepared for and must be able to maintain emergent surgical services in order to avoid preventable deaths and mortality. The results of this study have shown the effect of the COVID-19 pandemic on our patient population suffering from acute general surgery illnesses.\nThere was no difference in the pathologies presenting between the two periods, which is an important statistical finding, yet must be taken in proper context. Depending on the type of catastrophe, the required mobilization of specific medical specialties and strain on the systems will be drastically different. During times of war or natural disasters, hospitals will require a larger ratio of orthopedic surgeons, general surgeons, surgical specialties, OR access, and ICU beds.14 In contrast, pathogen-derived epidemics/pandemics will require more specialists in the fields of internal medicine (ie, infectious disease, pulmonology, intensivist, and hematologist), ICU beds, and potential access to extracorporeal membrane oxygenation.15 As seen with our results, this primarily respiratory pandemic did not significantly change the acute surgical pathologies.\nIn regard to the younger patient population, there were several observations that were of interest yet not statistically significant. These patients appeared to present later in their course of disease, with a higher rate of complicated pathologies (ie, perforated appendix and gangrenous cholecystitis), postoperative complications, and blood transfusions. These younger patients were therefore more likely able to tolerate their symptoms at home for a longer period and therefore had a delay in diagnosis and treatment. In contrast, the older population, with their lower physiological reserve, appeared less able to delay medical treatment and came to the hospital regardless of fears and inconveniences caused by the pandemic restrictions.\nThe “fear” of presenting to the ER with severe illness has been documented from this current COVID-19 pandemic21 and from previously published literature during catastrophes. For example, during the SARS epidemic in 2002, Taiwan and Hong Kong showed a decrease in routine care not related to the severe acute respiratory syndrome.22 During this COVID-19 pandemic, one recent study reported the patterns of patients presenting to the ER of a busy level-1 trauma center in America. This group showed a global decrease in arrivals to the ER, along with a decreased proportion of patients presenting with abdominal pain when compared to other systemic complaints before the pandemic.23 This cannot be accurately compared to our data because we were only looking at acute surgical pathologies. Nevertheless, what was reported in this reference might also be a result of avoiding going to the hospital with abdominal pain during a pandemic that involves primarily respiratory symptoms. In Italy, toward the beginning of the pandemic, a series of children, without COVID-19-related illness, were shown to have presented late and in more critical condition, due to the fear of presenting to a hospital or lack of provisional care because of closure of health consults/centers.24 This corresponds with our overall results in general of sicker patients being admitted to the surgical ward.\nThe country where this study took place, Israel, also presents a unique confounding factor to these data. Our hospital is a public hospital within a socialized system where every citizen is born with universal health care coverage25; therefore, lacking health care is rarely a reason for not seeking medical attention.26 Compared to the rest of the world, our mortality has been comparatively low.27,28 Our “lockdown” measures and monitoring methods were considered aggressive, but the country also progressed in easing the regulations and opening its economy relatively fast. Nevertheless, the cooperation and coordination of our Ministry of Health, security sectors, and private industry played a pivotal role in preventing our hospital infrastructure from being completely overwhelmed. Our results showed a different demographic in terms of ethnicity presenting to the hospital during the COVID-19 pandemic. There was a significant increase in the ratio of Israeli-Jewish to Israeli-non-Jewish patients presenting to the ED and a smaller proportion of minorities, tourist, and foreign workers when compared to 2019. As mentioned above, the TASMC is located in the center of Tel Aviv, where 91% of the population is Jewish and 9% are non-Jewish. This difference might be explained by the lack of transport, hesitancy to travel distances, and fear of larger medical centers because of their treatment of COVID-19 patients.29 Hence, smaller local medical centers were potentially receiving more patients, regardless of appropriateness of level of care.\nWe found a low incidence of mis-triage of surgical patients into the COVID-19 isolation wing by our ER triage protocol. Out of the 209 patients admitted during this time, only 10 were initially placed in isolation. None of these 10 isolated patients had a positive COVID-19 test result. Despite being placed in triage, the average time to hospitalization, despite being statistically different, was only 30 minutes longer than the non-isolated group. When intervention (surgical and nonsurgical) was needed in the isolated group, there was no increase in time when compared to the non-isolated patients and, on average, even occurred more rapidly. With many surgical pathologies presenting with fever, it was difficult, especially at the beginning of the pandemic, not to over triage these patients and potentially delay intervention for surgical emergencies. This became especially relevant as evidence began to show that a possible presenting symptom of COVID-19 is fever with diarrhea.30 The significant reduction in time to hospitalization during the pandemic may be a confounding effect of both a decrease in volume to the ED and also sicker patients presenting to the ED, which would require quicker diagnosis and appropriate therapy. This decrease in hospitalization time happened despite a reduction in working staff.\nMultiple studies have established that this elderly population is at an increased risk to clinically significant COVID-19.31 Our elderly population (>70 years old) during the pandemic had slightly more comorbidities and had a greater 30-day mortality than the previous year. Despite their increased comorbidities, they did not present to the hospital sicker than their counterparts the previous year. This trend might demonstrate the opposite of the “fear” phenomenon mentioned above for younger patients that sicker, elder patients present more easily to the hospital driven by the distress that something is wrong. With the elderly patients who did not undergo surgery, we were able to significantly reduce their hospitalization time. We have emphasized in our department not to neglect surgical pathologies during this time in this vulnerable population.\nAn interesting finding from the patients undergoing surgery during the pandemic is that they had a higher APACHE score, more complications, more blood given, yet more cases were open (verses laparoscopic), and there was an attending present more often. This seemingly contradicting findings might be a true indicator of how severely ill/complicated the patients were when compared to the previous year. Yet, this finding would need to be compared to other large tertiary centers in order to gather a significant conclusion. There are additional limitations to this study, including the fact that it is coming from a single urban referral center. These data may not be representative of other smaller and/or rural centers. Other limitations include the short study time and not having a cohort of surgical patients who were COVID-19 positive. The short study time was chosen in order to describe the initial effect of the pandemic and the most constricting limitations imposed by the government during the initial lockdown. Therefore, the initial month of true, full lockdown was taken and compared to the previous year. Clearly, during different stages and evolution of the pandemic, societies and medical systems have changed/adapted; therefore, the time when this study was conducted needs to be taken into consideration. There was no (knowingly) COVID-19-positive patient in our cohort and therefore no comparison between the differences in surgical pathology progression between positive and negative surgical patients. As there is more understanding of the COVID-19 pathophysiology, this would be an important patient population to analyze.\nIn conclusion, we have shown that during this COVID-19 pandemic, the patients presenting were sicker than the patients during the same time frame in the previous year despite there being fewer patients needing surgical admission. We were able to decrease the time until hospitalization and the length of hospitalization on the highly vulnerable elderly population. These data support the need for advocating and educating the public to present to hospitals without delay if there are symptoms of a possible acute surgical illness. Clearly, there are difficult times that may force the population to stay home (natural disasters and war), but fear alone should not delay the presentation of a patient with abdominal pain to the hospital. With well-organized systems and cooperation between governing and health sectors, hospitalization can be streamlined and physician/hospital workforce exposure can be minimized, while not compromising the level of care and treatment needed for acute surgical pathologies. Through understanding the surgical needs of a population during a catastrophic event, the public health sector may more rapidly and precisely distribute needed personnel, essential resources, and allocate certain medical centers as referral points for surgical emergencies.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe COVID-19 pandemic has transformed and affected every aspect of health care. Like any catastrophic event, the stress on hospitals to maintain a certain level of function is immense. Acute surgical pathologies cannot be prevented or curtailed; therefore, it is important to understand patterns and outcomes during catastrophes in order to optimize care and organize the health care system.\n\nMethods:\nIn a single urban tertiary care center, a retrospective study examined the first complete lockdown period of Israel during the COVID-19 pandemic. This was compared to the same time period the previous year.\n\nResults:\nDuring the pandemic, time to hospitalization was significantly decreased. There was also an overall reduction in surgical admissions yet with a higher percentage being hospitalized for further treatment (69.2% vs 23.5%). The patients admitted during this time had a higher APACHE-II score and Charlson comorbidity index score. During the pandemic, time to surgery was decreased, there were less laparoscopic procedures, and more RBC units were used per patient. There were no differences in overall complications, except when sub-analyzed for major complications (9.7% vs 6.3%). There was no significant difference in overall in-house mortality or morbidity. Length of hospitalization was significantly decreased in the elderly population during the pandemic.\n\nConclusions:\nDuring the COVID-19 pandemic, despite a significantly less number of patients presenting to the hospital, there was a higher percentage of those admitted needing surgical intervention, and they were overall sicker than the previous year."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":7876,"string":"7,876"},"whole_article_abstract_length":{"kind":"number","value":290,"string":"290"},"other_sections_lengths":{"kind":"list like","value":[723,928],"string":"[\n 723,\n 928\n]"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["patients","19","covid","covid 19","vs","surgical","hours","time","10","surgery"],"string":"[\n \"patients\",\n \"19\",\n \"covid\",\n \"covid 19\",\n \"vs\",\n \"surgical\",\n \"hours\",\n \"time\",\n \"10\",\n \"surgery\"\n]"},"keybert_topics":{"kind":"list like","value":["surgical illnesses disaster","caused pandemic restrictions","intervention surgical emergencies","countries restricted surgical","hospitalization pandemic confounding"],"string":"[\n \"surgical illnesses disaster\",\n \"caused pandemic restrictions\",\n \"intervention surgical emergencies\",\n \"countries restricted surgical\",\n \"hospitalization pandemic confounding\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | acute care surgery | pandemic [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | acute care surgery | pandemic [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | acute care surgery | pandemic [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | acute care surgery | pandemic [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | COVID-19 | Pandemics | Israel | SARS-CoV-2 | Retrospective Studies | Communicable Disease Control [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | COVID-19 | Pandemics | Israel | SARS-CoV-2 | Retrospective Studies | Communicable Disease Control [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | COVID-19 | Pandemics | Israel | SARS-CoV-2 | Retrospective Studies | Communicable Disease Control [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | COVID-19 | Pandemics | Israel | SARS-CoV-2 | Retrospective Studies | Communicable Disease Control [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] surgical illnesses disaster | caused pandemic restrictions | intervention surgical emergencies | countries restricted surgical | hospitalization pandemic confounding [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] surgical illnesses disaster | caused pandemic restrictions | intervention surgical emergencies | countries restricted surgical | hospitalization pandemic confounding [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] surgical illnesses disaster | caused pandemic restrictions | intervention surgical emergencies | countries restricted surgical | hospitalization pandemic confounding [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] surgical illnesses disaster | caused pandemic restrictions | intervention surgical emergencies | countries restricted surgical | hospitalization pandemic confounding [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | 19 | covid | covid 19 | vs | surgical | hours | time | 10 | surgery [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | 19 | covid | covid 19 | vs | surgical | hours | time | 10 | surgery [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | 19 | covid | covid 19 | vs | surgical | hours | time | 10 | surgery [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | 19 | covid | covid 19 | vs | surgical | hours | time | 10 | surgery [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] countries | surgical | elective | care | hospitals | services | surgical services | surgeons | level | continued [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | study | covid | 19 | covid 19 | data | ed | outline | outline study | study process [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vs | hours | patients | table | 10 | 19 | quarantine | time | 34 | covid [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | vs | 19 | hours | covid 19 | covid | surgical | time | 10 | table [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] tertiary | first | Israel | COVID-19 ||| the previous year [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 69.2% | 23.5% ||| APACHE-II | Charlson ||| RBC ||| 9.7% | 6.3% ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| ||| ||| tertiary | first | Israel | COVID-19 ||| the previous year ||| ||| 69.2% | 23.5% ||| APACHE-II | Charlson ||| RBC ||| 9.7% | 6.3% ||| ||| ||| COVID-19 | the previous year [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3372,"cells":{"title":{"kind":"string","value":"A novel variable number of tandem repeat of the natriuretic peptide precursor B gene's 5'-flanking region is associated with essential hypertension among Japanese females."},"pmid":{"kind":"string","value":"17554401"},"background_abstract":{"kind":"string","value":"Brain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["5' Flanking Region","Adult","Asian People","Base Sequence","Body Mass Index","Female","Gene Frequency","Genotype","Humans","Hypertension","Japan","Linkage Disequilibrium","Logistic Models","Middle Aged","Minisatellite Repeats","Molecular Sequence Data","Natriuretic Peptide, Brain","Phenotype","Polymerase Chain Reaction","Polymorphism, Genetic","Polymorphism, Single-Stranded Conformational","Sequence Analysis, DNA"],"string":"[\n \"5' Flanking Region\",\n \"Adult\",\n \"Asian People\",\n \"Base Sequence\",\n \"Body Mass Index\",\n \"Female\",\n \"Gene Frequency\",\n \"Genotype\",\n \"Humans\",\n \"Hypertension\",\n \"Japan\",\n \"Linkage Disequilibrium\",\n \"Logistic Models\",\n \"Middle Aged\",\n \"Minisatellite Repeats\",\n \"Molecular Sequence Data\",\n \"Natriuretic Peptide, Brain\",\n \"Phenotype\",\n \"Polymerase Chain Reaction\",\n \"Polymorphism, Genetic\",\n \"Polymorphism, Single-Stranded Conformational\",\n \"Sequence Analysis, DNA\"\n]"},"pmcid":{"kind":"string","value":"1885554"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Natriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.\nBNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.\nThe aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH."},"methods_title":{"kind":"string","value":"2. Subjects and Methods"},"methods_text":{"kind":"string","value":" Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\nThe EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\n Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\nPlasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\n Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\nGenomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\n Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\nTwo oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\n Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\nGenotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\n Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\nData are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant."},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"We discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).\nThe association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).\nOn multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).\nThe clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).\nThe plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.\nAll subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.525). There was no association between the genotype and the BMI among the groups (Table 5)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Subjects","Biochemical analysis","Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP)","Sequencing analysis","Genotyping","Statistical analysis"],"string":"[\n \"Subjects\",\n \"Biochemical analysis\",\n \"Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP)\",\n \"Sequencing analysis\",\n \"Genotyping\",\n \"Statistical analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.","Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.","Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.","Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.","Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).","Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant."],"string":"[\n \"The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\",\n \"Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\",\n \"Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\",\n \"Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\",\n \"Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\",\n \"Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Subjects and Methods","Subjects","Biochemical analysis","Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP)","Sequencing analysis","Genotyping","Statistical analysis","3. Results","4. Discussion"],"string":"[\n \"1. Introduction\",\n \"2. Subjects and Methods\",\n \"Subjects\",\n \"Biochemical analysis\",\n \"Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP)\",\n \"Sequencing analysis\",\n \"Genotyping\",\n \"Statistical analysis\",\n \"3. Results\",\n \"4. Discussion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Natriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.\nBNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.\nThe aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH."," Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\nThe EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\n Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\nPlasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\n Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\nGenomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\n Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\nTwo oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\n Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\nGenotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\n Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\nData are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.","The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.","Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.","Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.","Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.","Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).","Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.","We discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).\nThe association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).\nOn multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).\nThe clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).\nThe plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.\nAll subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.525). There was no association between the genotype and the BMI among the groups (Table 5).","Ogawa et al. reported that the 5'-flanking region of the 1.9 kilo-base pairs in the NPPB gene has a high transcriptional activity. Using the deletion mutant model, the deletion of sequences between -1288 and -1095 reduced transcriptional activity to approximately 30%. These deleted sequences contain a characteristic CT-rich region (-1248 to -1191), followed by an Alu family sequence (-1190 to -934). Thus, these results are consistent with the hypothesis that Alu repeat sequences in the 5'-flanking regions have regulatory roles in NPPB expression 14. Therefore, we assessed the association between mutations or polymorphisms in the 5'-flanking region of NPPB and the presence of EH. In the present study, a novel VNTR was discovered, and a significant association between the VNTR and EH was found in female patients. The present study found that the number of patients with a 16 repeat allele of VNTR was lower in EH women than in NT women. It is well known that the plasma BNP level is higher in EH patients than in NT subjects, since an elevated blood pressure results in a high plasma BNP level, which is one of the protective factors for hypertension 4. We compared the plasma BNP levels of patients with and without the 16 repeat allele and found that there was no significant difference between the two groups. In fact, there were not enough subjects to allow the association studies to be done by gender. Given our results, these limitations should be addressed in future studies. It is possible that factors other than the NPPB gene may have affected the BNP levels, since many factors, including cardiac function and blood pressure, are known to affect human plasma BNP levels. Thus, it is possible that the plasma BNP level is not an accurate reflection of the function of the NPPB gene.\nIn the present study, the overall distribution of the VNTR genotype and the allele frequency were significantly different in females but not in males. Gender-specific susceptibility to EH is an interesting finding, but its importance is still unclear 11. Redfield et al. reported that BNP levels increased with age, and were higher in females than males among subjects with no known cardiovascular or detectable structural heart disease 18. Maffei et al. reported that hormone replacement therapy increased BNP levels in postmenopausal women 19. Although the absolute BNP value was different between these two studies, which used two different assays, the associations of the BNP levels with age and gender were consistent. Furthermore, the BNP level that had the optimal sensitivity and specificity for detecting systolic dysfunction in the overall population increased with age and was higher in women. This underscores the clinical relevance of the relationship of age, gender, and BNP. In both studies that used different assays, the effect of gender on BNP was substantial and independent of other factors 18. Unfortunately, we were not able to obtain samples to measure plasma BNP and ANP levels, due to the difficulty in obtaining written informed consent for blood examinations from subjects not receiving medications.\nIn the Japanese population, it has been reported that plasma BNP levels are positively associated with age, urinary salt excretion, higher blood pressure, a high R-wave voltage in the 12-lead ECG (Code 3-1 or 3-3), and female gender 20,21. On the other hand, Freitag et al., based on multivariate models adjusting for known risk factors, showed that elevated plasma BNP levels were associated with an increased risk of blood pressure progression in males but not in females. However, there was no significant trend of an increasing incidence of hypertension among BNP categories in either males or females. In a community-based sample, higher plasma BNP levels were found to be associated with an increased risk of BP progression in males, but not in females 22. Further studies are needed to resolve these conflicting results.\nSince there are many loci with a high degree of polymorphism in the number of tandemly repeated nucleotide sequence units, VNTR polymorphisms, also called minisatellites, were originally studied for linkage-mapping purposes. VNTRs have a highly polymorphic nature that makes them very useful as markers, both in linkage studies to map disease loci in families and in forensic applications. Recent reports indicate that some VNTR sequences may function as transcriptional or translational regulators, and that they may modify the function of a protein when the tandemly repeated region lies within the coding region of the gene 23. Although no clear effect on transcription has been shown, it has been reported that a VNTR in the second intron of the serotonin transporter gene is associated with susceptibility to major depression 24.\nWe previously determined the structural organization of human natriuretic peptide receptor genes 25-28 and identified an insertion/deletion mutation in the 5'-untranslated region of NPRA 12. The deletion encompasses eight nucleotides and alters the binding sites for the AP2 and zeste transcription factors. Transcriptional activity of the deletion allele was less than 30% that of the wild-type allele. The deletion allele was significantly more common in the EH group than in the NT group. These findings suggest that in Japanese individuals, this deletion in the NPRA gene reduces receptor activity and may confer increased susceptibility for the individual to develop EH or left ventricular hypertrophy (LVH). Animal models with a deletion of this gene develop disorders that resemble the symptoms of subjects with a deleted allele in the 5'-untranslated region of NPRA. We previously isolated a missense mutation of the NPRA gene 29 and a VNTR polymorphism upstream of the NPRC gene; this VNTR influences blood pressure levels in obesity-associated hypertension 30. Since the sampling of the above reports was different from the present experiment, it was impossible to analyze the relationship between systemic natriuretic peptide genes and EH.\nWang et al. reported that obese individuals have low circulating natriuretic peptide levels, which may contribute to their susceptibility to hypertension and hypertension-related disorders. The mechanisms linking obesity to hypertension have not been established, but sodium retention and excessive sympathetic tone are key contributors. Natriuretic peptides are important regulators of sodium homeostasis and neurohormonal activation; this raises the possibility that obese individuals have an impaired natriuretic peptide response 31. Therefore, we examined the relationship between the genotype and BMI and found no significant association.\nIn conclusion, a novel VNTR in the 5'-flanking region of the NPPB gene was discovered. This polymorphism was associated with EH in female subjects. However, this finding does not necessarily imply that there is a relationship between the NPPB gene and EH. Further studies of other polymorphisms are needed to determine whether there is an association between the NPPB gene and EH."],"string":"[\n \"Natriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.\\nBNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.\\nThe aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH.\",\n \" Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\\nThe EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\\n Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\\nPlasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\\n Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\\nGenomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\\n Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\\nTwo oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\\n Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\\nGenotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\\n Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\\nData are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\",\n \"The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\",\n \"Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\",\n \"Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\",\n \"Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\",\n \"Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\",\n \"Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\",\n \"We discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).\\nThe association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).\\nOn multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).\\nThe clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).\\nThe plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.\\nAll subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.525). There was no association between the genotype and the BMI among the groups (Table 5).\",\n \"Ogawa et al. reported that the 5'-flanking region of the 1.9 kilo-base pairs in the NPPB gene has a high transcriptional activity. Using the deletion mutant model, the deletion of sequences between -1288 and -1095 reduced transcriptional activity to approximately 30%. These deleted sequences contain a characteristic CT-rich region (-1248 to -1191), followed by an Alu family sequence (-1190 to -934). Thus, these results are consistent with the hypothesis that Alu repeat sequences in the 5'-flanking regions have regulatory roles in NPPB expression 14. Therefore, we assessed the association between mutations or polymorphisms in the 5'-flanking region of NPPB and the presence of EH. In the present study, a novel VNTR was discovered, and a significant association between the VNTR and EH was found in female patients. The present study found that the number of patients with a 16 repeat allele of VNTR was lower in EH women than in NT women. It is well known that the plasma BNP level is higher in EH patients than in NT subjects, since an elevated blood pressure results in a high plasma BNP level, which is one of the protective factors for hypertension 4. We compared the plasma BNP levels of patients with and without the 16 repeat allele and found that there was no significant difference between the two groups. In fact, there were not enough subjects to allow the association studies to be done by gender. Given our results, these limitations should be addressed in future studies. It is possible that factors other than the NPPB gene may have affected the BNP levels, since many factors, including cardiac function and blood pressure, are known to affect human plasma BNP levels. Thus, it is possible that the plasma BNP level is not an accurate reflection of the function of the NPPB gene.\\nIn the present study, the overall distribution of the VNTR genotype and the allele frequency were significantly different in females but not in males. Gender-specific susceptibility to EH is an interesting finding, but its importance is still unclear 11. Redfield et al. reported that BNP levels increased with age, and were higher in females than males among subjects with no known cardiovascular or detectable structural heart disease 18. Maffei et al. reported that hormone replacement therapy increased BNP levels in postmenopausal women 19. Although the absolute BNP value was different between these two studies, which used two different assays, the associations of the BNP levels with age and gender were consistent. Furthermore, the BNP level that had the optimal sensitivity and specificity for detecting systolic dysfunction in the overall population increased with age and was higher in women. This underscores the clinical relevance of the relationship of age, gender, and BNP. In both studies that used different assays, the effect of gender on BNP was substantial and independent of other factors 18. Unfortunately, we were not able to obtain samples to measure plasma BNP and ANP levels, due to the difficulty in obtaining written informed consent for blood examinations from subjects not receiving medications.\\nIn the Japanese population, it has been reported that plasma BNP levels are positively associated with age, urinary salt excretion, higher blood pressure, a high R-wave voltage in the 12-lead ECG (Code 3-1 or 3-3), and female gender 20,21. On the other hand, Freitag et al., based on multivariate models adjusting for known risk factors, showed that elevated plasma BNP levels were associated with an increased risk of blood pressure progression in males but not in females. However, there was no significant trend of an increasing incidence of hypertension among BNP categories in either males or females. In a community-based sample, higher plasma BNP levels were found to be associated with an increased risk of BP progression in males, but not in females 22. Further studies are needed to resolve these conflicting results.\\nSince there are many loci with a high degree of polymorphism in the number of tandemly repeated nucleotide sequence units, VNTR polymorphisms, also called minisatellites, were originally studied for linkage-mapping purposes. VNTRs have a highly polymorphic nature that makes them very useful as markers, both in linkage studies to map disease loci in families and in forensic applications. Recent reports indicate that some VNTR sequences may function as transcriptional or translational regulators, and that they may modify the function of a protein when the tandemly repeated region lies within the coding region of the gene 23. Although no clear effect on transcription has been shown, it has been reported that a VNTR in the second intron of the serotonin transporter gene is associated with susceptibility to major depression 24.\\nWe previously determined the structural organization of human natriuretic peptide receptor genes 25-28 and identified an insertion/deletion mutation in the 5'-untranslated region of NPRA 12. The deletion encompasses eight nucleotides and alters the binding sites for the AP2 and zeste transcription factors. Transcriptional activity of the deletion allele was less than 30% that of the wild-type allele. The deletion allele was significantly more common in the EH group than in the NT group. These findings suggest that in Japanese individuals, this deletion in the NPRA gene reduces receptor activity and may confer increased susceptibility for the individual to develop EH or left ventricular hypertrophy (LVH). Animal models with a deletion of this gene develop disorders that resemble the symptoms of subjects with a deleted allele in the 5'-untranslated region of NPRA. We previously isolated a missense mutation of the NPRA gene 29 and a VNTR polymorphism upstream of the NPRC gene; this VNTR influences blood pressure levels in obesity-associated hypertension 30. Since the sampling of the above reports was different from the present experiment, it was impossible to analyze the relationship between systemic natriuretic peptide genes and EH.\\nWang et al. reported that obese individuals have low circulating natriuretic peptide levels, which may contribute to their susceptibility to hypertension and hypertension-related disorders. The mechanisms linking obesity to hypertension have not been established, but sodium retention and excessive sympathetic tone are key contributors. Natriuretic peptides are important regulators of sodium homeostasis and neurohormonal activation; this raises the possibility that obese individuals have an impaired natriuretic peptide response 31. Therefore, we examined the relationship between the genotype and BMI and found no significant association.\\nIn conclusion, a novel VNTR in the 5'-flanking region of the NPPB gene was discovered. This polymorphism was associated with EH in female subjects. However, this finding does not necessarily imply that there is a relationship between the NPPB gene and EH. Further studies of other polymorphisms are needed to determine whether there is an association between the NPPB gene and EH.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Brain natriuretic peptide","essential hypertension","variable number of tandem repeat","association study"],"string":"[\n \"Brain natriuretic peptide\",\n \"essential hypertension\",\n \"variable number of tandem repeat\",\n \"association study\"\n]"},"whole_article_text":{"kind":"string","value":"1. Introduction:\nNatriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.\nBNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.\nThe aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH.\n\n2. Subjects and Methods:\n Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\nThe EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\n Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\nPlasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\n Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\nGenomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\n Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\nTwo oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\n Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\nGenotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\n Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\nData are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\n\nSubjects:\nThe EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\n\nBiochemical analysis:\nPlasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\n\nPolymerase chain reaction - single strand conformation polymorphism (PCR-SSCP):\nGenomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\n\nSequencing analysis:\nTwo oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\n\nGenotyping:\nGenotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\n\nStatistical analysis:\nData are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\n\n3. Results:\nWe discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).\nThe association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).\nOn multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).\nThe clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).\nThe plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.\nAll subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.525). There was no association between the genotype and the BMI among the groups (Table 5).\n\n4. Discussion:\nOgawa et al. reported that the 5'-flanking region of the 1.9 kilo-base pairs in the NPPB gene has a high transcriptional activity. Using the deletion mutant model, the deletion of sequences between -1288 and -1095 reduced transcriptional activity to approximately 30%. These deleted sequences contain a characteristic CT-rich region (-1248 to -1191), followed by an Alu family sequence (-1190 to -934). Thus, these results are consistent with the hypothesis that Alu repeat sequences in the 5'-flanking regions have regulatory roles in NPPB expression 14. Therefore, we assessed the association between mutations or polymorphisms in the 5'-flanking region of NPPB and the presence of EH. In the present study, a novel VNTR was discovered, and a significant association between the VNTR and EH was found in female patients. The present study found that the number of patients with a 16 repeat allele of VNTR was lower in EH women than in NT women. It is well known that the plasma BNP level is higher in EH patients than in NT subjects, since an elevated blood pressure results in a high plasma BNP level, which is one of the protective factors for hypertension 4. We compared the plasma BNP levels of patients with and without the 16 repeat allele and found that there was no significant difference between the two groups. In fact, there were not enough subjects to allow the association studies to be done by gender. Given our results, these limitations should be addressed in future studies. It is possible that factors other than the NPPB gene may have affected the BNP levels, since many factors, including cardiac function and blood pressure, are known to affect human plasma BNP levels. Thus, it is possible that the plasma BNP level is not an accurate reflection of the function of the NPPB gene.\nIn the present study, the overall distribution of the VNTR genotype and the allele frequency were significantly different in females but not in males. Gender-specific susceptibility to EH is an interesting finding, but its importance is still unclear 11. Redfield et al. reported that BNP levels increased with age, and were higher in females than males among subjects with no known cardiovascular or detectable structural heart disease 18. Maffei et al. reported that hormone replacement therapy increased BNP levels in postmenopausal women 19. Although the absolute BNP value was different between these two studies, which used two different assays, the associations of the BNP levels with age and gender were consistent. Furthermore, the BNP level that had the optimal sensitivity and specificity for detecting systolic dysfunction in the overall population increased with age and was higher in women. This underscores the clinical relevance of the relationship of age, gender, and BNP. In both studies that used different assays, the effect of gender on BNP was substantial and independent of other factors 18. Unfortunately, we were not able to obtain samples to measure plasma BNP and ANP levels, due to the difficulty in obtaining written informed consent for blood examinations from subjects not receiving medications.\nIn the Japanese population, it has been reported that plasma BNP levels are positively associated with age, urinary salt excretion, higher blood pressure, a high R-wave voltage in the 12-lead ECG (Code 3-1 or 3-3), and female gender 20,21. On the other hand, Freitag et al., based on multivariate models adjusting for known risk factors, showed that elevated plasma BNP levels were associated with an increased risk of blood pressure progression in males but not in females. However, there was no significant trend of an increasing incidence of hypertension among BNP categories in either males or females. In a community-based sample, higher plasma BNP levels were found to be associated with an increased risk of BP progression in males, but not in females 22. Further studies are needed to resolve these conflicting results.\nSince there are many loci with a high degree of polymorphism in the number of tandemly repeated nucleotide sequence units, VNTR polymorphisms, also called minisatellites, were originally studied for linkage-mapping purposes. VNTRs have a highly polymorphic nature that makes them very useful as markers, both in linkage studies to map disease loci in families and in forensic applications. Recent reports indicate that some VNTR sequences may function as transcriptional or translational regulators, and that they may modify the function of a protein when the tandemly repeated region lies within the coding region of the gene 23. Although no clear effect on transcription has been shown, it has been reported that a VNTR in the second intron of the serotonin transporter gene is associated with susceptibility to major depression 24.\nWe previously determined the structural organization of human natriuretic peptide receptor genes 25-28 and identified an insertion/deletion mutation in the 5'-untranslated region of NPRA 12. The deletion encompasses eight nucleotides and alters the binding sites for the AP2 and zeste transcription factors. Transcriptional activity of the deletion allele was less than 30% that of the wild-type allele. The deletion allele was significantly more common in the EH group than in the NT group. These findings suggest that in Japanese individuals, this deletion in the NPRA gene reduces receptor activity and may confer increased susceptibility for the individual to develop EH or left ventricular hypertrophy (LVH). Animal models with a deletion of this gene develop disorders that resemble the symptoms of subjects with a deleted allele in the 5'-untranslated region of NPRA. We previously isolated a missense mutation of the NPRA gene 29 and a VNTR polymorphism upstream of the NPRC gene; this VNTR influences blood pressure levels in obesity-associated hypertension 30. Since the sampling of the above reports was different from the present experiment, it was impossible to analyze the relationship between systemic natriuretic peptide genes and EH.\nWang et al. reported that obese individuals have low circulating natriuretic peptide levels, which may contribute to their susceptibility to hypertension and hypertension-related disorders. The mechanisms linking obesity to hypertension have not been established, but sodium retention and excessive sympathetic tone are key contributors. Natriuretic peptides are important regulators of sodium homeostasis and neurohormonal activation; this raises the possibility that obese individuals have an impaired natriuretic peptide response 31. Therefore, we examined the relationship between the genotype and BMI and found no significant association.\nIn conclusion, a novel VNTR in the 5'-flanking region of the NPPB gene was discovered. This polymorphism was associated with EH in female subjects. However, this finding does not necessarily imply that there is a relationship between the NPPB gene and EH. Further studies of other polymorphisms are needed to determine whether there is an association between the NPPB gene and EH.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nBrain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH.\n\nMethods:\nThe polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants.\n\nResults:\nA novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046).\n\nConclusions:\nThe 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":4569,"string":"4,569"},"whole_article_abstract_length":{"kind":"number","value":319,"string":"319"},"other_sections_lengths":{"kind":"list like","value":[203,107,232,109,93,102],"string":"[\n 203,\n 107,\n 232,\n 109,\n 93,\n 102\n]"},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["eh","bnp","subjects","nt","pcr","hypertension","plasma","levels","patients","gene"],"string":"[\n \"eh\",\n \"bnp\",\n \"subjects\",\n \"nt\",\n \"pcr\",\n \"hypertension\",\n \"plasma\",\n \"levels\",\n \"patients\",\n \"gene\"\n]"},"keybert_topics":{"kind":"list like","value":["natriuretic peptide genes","atrial natriuretic peptide","bnp acts cardiac","brain natriuretic peptide","peptide bnp type"],"string":"[\n \"natriuretic peptide genes\",\n \"atrial natriuretic peptide\",\n \"bnp acts cardiac\",\n \"brain natriuretic peptide\",\n \"peptide bnp type\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] bnp | natriuretic | natriuretic peptide | anp | peptide | brain | gene | eh | variants | mainly [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] pcr | min | subjects | eh | mmhg | hypertension | products | measured | bases | usa [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] repeat | allele | group | repeat allele | 16 | 16 repeat | 16 repeat allele | bmi | nt group | table [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] bnp | eh | pcr | min | nt | subjects | hypertension | repeat | products | plasma [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| BNP ||| 5'-flanking | NPPB [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5'-flanking ||| 4 | TTTC ||| 8 | 9 | 19 ||| 317 | 262 ||| 11 | 88.2% | 16 | second | 10.5% ||| Hardy-Weinberg ||| EH ||| 16 | 6.5% | 12.2% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| BNP ||| 5'-flanking | NPPB ||| 5'-flanking ||| 4 | TTTC ||| 8 | 9 | 19 ||| 317 | 262 ||| 11 | 88.2% | 16 | second | 10.5% ||| Hardy-Weinberg ||| EH ||| 16 | 6.5% | 12.2% ||| 16 | 5'-flanking | NPPB [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3373,"cells":{"title":{"kind":"string","value":"Vitamin D deficiency and visceral adipose tissue in early pregnant women."},"pmid":{"kind":"string","value":"34215200"},"background_abstract":{"kind":"string","value":"We aimed to assess the correlation between vitamin D serum level and visceral fat tissue during early pregnancy."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This cross-sectional study was performed in Pernambuco, Brazil. 190 low risk pregnant women (8-16 gestational weeks) were eligible. Visceral adipose tissue was measured by ultrasonography following the technique described by Armellini. The 25(OH) D in serum was determined through chemiluminescence. The Spearman correlation test was applied to evaluate the correlation between vitamin D serum level and VAT, considering p < 0.05 to be significant."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Vitamin D insufficiency was present in 129 (67.8 %) of subjects. Pregnant women with or without vitamin D deficiency did not differ in age, gestational age, nutritional status and visceral adipose tissue. No correlation between visceral adipose tissue and 25(OH) D was observed: - 0.057 (p = 0.435)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Maternal visceral adipose tissue and vitamin D serum level are not correlated during pregnancy."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Body Mass Index","Brazil","Cross-Sectional Studies","Female","Gestational Age","Humans","Intra-Abdominal Fat","Nutritional Status","Pregnancy","Pregnant Women","Prenatal Nutritional Physiological Phenomena","Vitamin D","Vitamin D Deficiency","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Body Mass Index\",\n \"Brazil\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Gestational Age\",\n \"Humans\",\n \"Intra-Abdominal Fat\",\n \"Nutritional Status\",\n \"Pregnancy\",\n \"Pregnant Women\",\n \"Prenatal Nutritional Physiological Phenomena\",\n \"Vitamin D\",\n \"Vitamin D Deficiency\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"8252319"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.\n\nTable 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343\nCharacteristics of pregnant women studied according to the vitamin D status\nVitamin D (30ng/mL)\n< 30 (n = 129)\nMedian ± SD (Average)\n≥ 30 (n = 61)\nMedian ± SD (Average)\n\nTable 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero\nSpearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index\n(1)Statistically different from zero"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings."},"other_sections_titles":{"kind":"list like","value":["Background","Methods","Statistical analyses"],"string":"[\n \"Background\",\n \"Methods\",\n \"Statistical analyses\"\n]"},"other_sections_texts":{"kind":"list like","value":["Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.","This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.","Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25."],"string":"[\n \"Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.\",\n \"This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\",\n \"Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null],"string":"[\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Statistical analyses","Results","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Statistical analyses\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.","This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.","Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.","190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.\n\nTable 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343\nCharacteristics of pregnant women studied according to the vitamin D status\nVitamin D (30ng/mL)\n< 30 (n = 129)\nMedian ± SD (Average)\n≥ 30 (n = 61)\nMedian ± SD (Average)\n\nTable 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero\nSpearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index\n(1)Statistically different from zero","In the present study we did not find association between serum VD values and visceral fat in pregnant women. For the best of our knowledge, it is the first time that this association is assessed during pregnancy. Some studies have described an inverse association between VDD and visceral fat in non-pregnancy populations [24, 14]. However, among the various physiological changes that occur during pregnancy, the redistribution of adipose tissue is one of them. Although not yet properly studied, it is admitted that an increase in visceral adipose tissue occurs, but the specific function of visceral adipose in pregnancy is still unknown. Thus, it seems likely that visceral adipose tissue has somewhat distinct functions in pregnancy and our findings cannot be compared with non-pregnant populations.\nRecently, Carreras-Badosa et al. found that maternal serum VD was inversely associated with visceral fat and in their offspring at the age of 5–6 years [25]. However, diet and lifestyle habits were not studied in both mothers and their children, and this, rather than maternal VD status could explain the relationship between lower maternal serum VD and offspring adiposity.\nAn increased risk of VDD has been described worldwide in obese individual. The underlying explanations are not clear. Receptors of VD are expressed in adipose tissue and 25(OH) D could modulate adipogenesis through VD receptors-dependent inhibition of specific components [26]. Other explanation is based on experimental findings that deficiency of VD can increases lipogenesis upregulating adipocyte calcium signaling and improving the secretion of parathyroid hormone [27]. However, the role of VD in adipogenesis or other functions of VAT during pregnancy is still unknown.\nPregnancy VDD has been described with a prevalence of 18–84 % [9]. In the present study, the prevalence of VDD was high (68 %). This value was similar to other Brazilian studies and higher than that described in countries such as Spain and United States [28]. Brazil is a tropical country with continental dimensions and abundant sunshine. The municipality of Petrolina is located at approximately − 9°, which favors the synthesis of VD. However, there is a paradox in countries with low solar incidence and with a lower frequency of VDD when compared with countries with higher solar incidence, certainly because of protection measures that prevent its synthesis and bioavailability [29].\nIn the multicenter HAPO study, increased maternal BMI was associated with lower maternal 25(OH) D levels [30]. However, BMI is not an accurate measure of fat tissue, especially during pregnancy. Besides, the deposition of adipose tissue preferably occurs in two distinct anatomical places, VAT and subcutaneous adipose tissue, and BMI does not differentiate them. We determine VAT through ultrasound and this method has been shown to be safe, effective, simple and reproducible [31].\nOur study has some limitations. At first, a cross-sectional design was performed and we could not determine the causal relationship between VD and VAT. Second, other possible important variables confounding the VD status, like sun light exposition, dietary habits and possible seasonal variations in VD, were not evaluated in our study. However, despite these limitations our study has strengths. The present study is the first to assess the correlation of VD with VAT thickness in pregnant women. A large sample was studied and specific methods to measure VD and VAT were used.","Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings."],"string":"[\n \"Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.\",\n \"This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\",\n \"Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\",\n \"190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.\\n\\nTable 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343\\nCharacteristics of pregnant women studied according to the vitamin D status\\nVitamin D (30ng/mL)\\n< 30 (n = 129)\\nMedian ± SD (Average)\\n≥ 30 (n = 61)\\nMedian ± SD (Average)\\n\\nTable 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero\\nSpearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index\\n(1)Statistically different from zero\",\n \"In the present study we did not find association between serum VD values and visceral fat in pregnant women. For the best of our knowledge, it is the first time that this association is assessed during pregnancy. Some studies have described an inverse association between VDD and visceral fat in non-pregnancy populations [24, 14]. However, among the various physiological changes that occur during pregnancy, the redistribution of adipose tissue is one of them. Although not yet properly studied, it is admitted that an increase in visceral adipose tissue occurs, but the specific function of visceral adipose in pregnancy is still unknown. Thus, it seems likely that visceral adipose tissue has somewhat distinct functions in pregnancy and our findings cannot be compared with non-pregnant populations.\\nRecently, Carreras-Badosa et al. found that maternal serum VD was inversely associated with visceral fat and in their offspring at the age of 5–6 years [25]. However, diet and lifestyle habits were not studied in both mothers and their children, and this, rather than maternal VD status could explain the relationship between lower maternal serum VD and offspring adiposity.\\nAn increased risk of VDD has been described worldwide in obese individual. The underlying explanations are not clear. Receptors of VD are expressed in adipose tissue and 25(OH) D could modulate adipogenesis through VD receptors-dependent inhibition of specific components [26]. Other explanation is based on experimental findings that deficiency of VD can increases lipogenesis upregulating adipocyte calcium signaling and improving the secretion of parathyroid hormone [27]. However, the role of VD in adipogenesis or other functions of VAT during pregnancy is still unknown.\\nPregnancy VDD has been described with a prevalence of 18–84 % [9]. In the present study, the prevalence of VDD was high (68 %). This value was similar to other Brazilian studies and higher than that described in countries such as Spain and United States [28]. Brazil is a tropical country with continental dimensions and abundant sunshine. The municipality of Petrolina is located at approximately − 9°, which favors the synthesis of VD. However, there is a paradox in countries with low solar incidence and with a lower frequency of VDD when compared with countries with higher solar incidence, certainly because of protection measures that prevent its synthesis and bioavailability [29].\\nIn the multicenter HAPO study, increased maternal BMI was associated with lower maternal 25(OH) D levels [30]. However, BMI is not an accurate measure of fat tissue, especially during pregnancy. Besides, the deposition of adipose tissue preferably occurs in two distinct anatomical places, VAT and subcutaneous adipose tissue, and BMI does not differentiate them. We determine VAT through ultrasound and this method has been shown to be safe, effective, simple and reproducible [31].\\nOur study has some limitations. At first, a cross-sectional design was performed and we could not determine the causal relationship between VD and VAT. Second, other possible important variables confounding the VD status, like sun light exposition, dietary habits and possible seasonal variations in VD, were not evaluated in our study. However, despite these limitations our study has strengths. The present study is the first to assess the correlation of VD with VAT thickness in pregnant women. A large sample was studied and specific methods to measure VD and VAT were used.\",\n \"Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,"results","discussion","conclusion"],"string":"[\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusion\"\n]"},"keywords":{"kind":"list like","value":["Visceral adipose tissue","Vitamin D","Pregnancy","Ultrasound","Obesity"],"string":"[\n \"Visceral adipose tissue\",\n \"Vitamin D\",\n \"Pregnancy\",\n \"Ultrasound\",\n \"Obesity\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nVitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.\n\nMethods:\nThis cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\n\nStatistical analyses:\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\n\nResults:\n190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.\n\nTable 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343\nCharacteristics of pregnant women studied according to the vitamin D status\nVitamin D (30ng/mL)\n< 30 (n = 129)\nMedian ± SD (Average)\n≥ 30 (n = 61)\nMedian ± SD (Average)\n\nTable 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero\nSpearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index\n(1)Statistically different from zero\n\nDiscussion:\nIn the present study we did not find association between serum VD values and visceral fat in pregnant women. For the best of our knowledge, it is the first time that this association is assessed during pregnancy. Some studies have described an inverse association between VDD and visceral fat in non-pregnancy populations [24, 14]. However, among the various physiological changes that occur during pregnancy, the redistribution of adipose tissue is one of them. Although not yet properly studied, it is admitted that an increase in visceral adipose tissue occurs, but the specific function of visceral adipose in pregnancy is still unknown. Thus, it seems likely that visceral adipose tissue has somewhat distinct functions in pregnancy and our findings cannot be compared with non-pregnant populations.\nRecently, Carreras-Badosa et al. found that maternal serum VD was inversely associated with visceral fat and in their offspring at the age of 5–6 years [25]. However, diet and lifestyle habits were not studied in both mothers and their children, and this, rather than maternal VD status could explain the relationship between lower maternal serum VD and offspring adiposity.\nAn increased risk of VDD has been described worldwide in obese individual. The underlying explanations are not clear. Receptors of VD are expressed in adipose tissue and 25(OH) D could modulate adipogenesis through VD receptors-dependent inhibition of specific components [26]. Other explanation is based on experimental findings that deficiency of VD can increases lipogenesis upregulating adipocyte calcium signaling and improving the secretion of parathyroid hormone [27]. However, the role of VD in adipogenesis or other functions of VAT during pregnancy is still unknown.\nPregnancy VDD has been described with a prevalence of 18–84 % [9]. In the present study, the prevalence of VDD was high (68 %). This value was similar to other Brazilian studies and higher than that described in countries such as Spain and United States [28]. Brazil is a tropical country with continental dimensions and abundant sunshine. The municipality of Petrolina is located at approximately − 9°, which favors the synthesis of VD. However, there is a paradox in countries with low solar incidence and with a lower frequency of VDD when compared with countries with higher solar incidence, certainly because of protection measures that prevent its synthesis and bioavailability [29].\nIn the multicenter HAPO study, increased maternal BMI was associated with lower maternal 25(OH) D levels [30]. However, BMI is not an accurate measure of fat tissue, especially during pregnancy. Besides, the deposition of adipose tissue preferably occurs in two distinct anatomical places, VAT and subcutaneous adipose tissue, and BMI does not differentiate them. We determine VAT through ultrasound and this method has been shown to be safe, effective, simple and reproducible [31].\nOur study has some limitations. At first, a cross-sectional design was performed and we could not determine the causal relationship between VD and VAT. Second, other possible important variables confounding the VD status, like sun light exposition, dietary habits and possible seasonal variations in VD, were not evaluated in our study. However, despite these limitations our study has strengths. The present study is the first to assess the correlation of VD with VAT thickness in pregnant women. A large sample was studied and specific methods to measure VD and VAT were used.\n\nConclusions:\nOur data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe aimed to assess the correlation between vitamin D serum level and visceral fat tissue during early pregnancy.\n\nMethods:\nThis cross-sectional study was performed in Pernambuco, Brazil. 190 low risk pregnant women (8-16 gestational weeks) were eligible. Visceral adipose tissue was measured by ultrasonography following the technique described by Armellini. The 25(OH) D in serum was determined through chemiluminescence. The Spearman correlation test was applied to evaluate the correlation between vitamin D serum level and VAT, considering p < 0.05 to be significant.\n\nResults:\nVitamin D insufficiency was present in 129 (67.8 %) of subjects. Pregnant women with or without vitamin D deficiency did not differ in age, gestational age, nutritional status and visceral adipose tissue. No correlation between visceral adipose tissue and 25(OH) D was observed: - 0.057 (p = 0.435).\n\nConclusions:\nMaternal visceral adipose tissue and vitamin D serum level are not correlated during pregnancy."},"background_conclusion_text":{"kind":"string","value":"Background:\nVitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.\n\nConclusions:\nOur data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWe aimed to assess the correlation between vitamin D serum level and visceral fat tissue during early pregnancy.\n\nMethods:\nThis cross-sectional study was performed in Pernambuco, Brazil. 190 low risk pregnant women (8-16 gestational weeks) were eligible. Visceral adipose tissue was measured by ultrasonography following the technique described by Armellini. The 25(OH) D in serum was determined through chemiluminescence. The Spearman correlation test was applied to evaluate the correlation between vitamin D serum level and VAT, considering p < 0.05 to be significant.\n\nResults:\nVitamin D insufficiency was present in 129 (67.8 %) of subjects. Pregnant women with or without vitamin D deficiency did not differ in age, gestational age, nutritional status and visceral adipose tissue. No correlation between visceral adipose tissue and 25(OH) D was observed: - 0.057 (p = 0.435).\n\nConclusions:\nMaternal visceral adipose tissue and vitamin D serum level are not correlated during pregnancy."},"whole_article_text_length":{"kind":"number","value":2629,"string":"2,629"},"whole_article_abstract_length":{"kind":"number","value":192,"string":"192"},"other_sections_lengths":{"kind":"list like","value":[443,712,188],"string":"[\n 443,\n 712,\n 188\n]"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["vd","vat","25","test","variables","correlation","women","pregnant","adipose","pregnant women"],"string":"[\n \"vd\",\n \"vat\",\n \"25\",\n \"test\",\n \"variables\",\n \"correlation\",\n \"women\",\n \"pregnant\",\n \"adipose\",\n \"pregnant women\"\n]"},"keybert_topics":{"kind":"list like","value":["vitamin deficiency vdd","adipose tissue vat","modulate adipogenesis vd","vitamin vd factors","bioavailability visceral adipose"],"string":"[\n \"vitamin deficiency vdd\",\n \"adipose tissue vat\",\n \"modulate adipogenesis vd\",\n \"vitamin vd factors\",\n \"bioavailability visceral adipose\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity 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[SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vdd | vat | obesity | vitamin | associated | women | diseases | insulin resistance | health [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 25 | 30 | 00 | 80 | 50 | oh | age | sd average | percentile | 75 [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | vd associated pregnancy study | associated pregnancy study assess | maternal visceral | maternal visceral adiposity | pregnant women studies | pregnant women studies needed | maternal visceral adiposity low | confirm findings | women studies needed confirm [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | test | variables | vat | 25 | numerical | numerical variables | pregnancy | women | vdd [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vd | test | variables | vat | 25 | numerical | numerical variables | pregnancy | women | vdd [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Vitamin D | 129 | 67.8 % ||| ||| 25(OH | 0.057 | 0.435 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Pernambuco | Brazil ||| 190 | 8 ||| Armellini ||| 25(OH ||| ||| Spearman | VAT | 0.05 ||| ||| Vitamin D | 129 | 67.8 % ||| ||| 25(OH | 0.057 | 0.435 ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Pernambuco | Brazil ||| 190 | 8 ||| Armellini ||| 25(OH ||| ||| Spearman | VAT | 0.05 ||| ||| Vitamin D | 129 | 67.8 % ||| ||| 25(OH | 0.057 | 0.435 ||| [SUMMARY]"}}},{"rowIdx":3374,"cells":{"title":{"kind":"string","value":"The Relationship Between Thyroid Antibodies and Vitamin D Level in Primary Hypothyroidism."},"pmid":{"kind":"string","value":"33424090"},"background_abstract":{"kind":"string","value":"Vitamin D deficiency is a global health problem. Its deficiency has been reported to be associated with different autoimmune diseases."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"A total number of 150 individuals were enrolled in this study. They were divided into the fallowing groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease. Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibodies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Serum levels of 25 (OH) vitamin D recorded a highly significant difference between the studies groups (20,76±6,31 ng/ml in group I vs. 24,37±9,05ng/ml in group II vs. 24,57±6,45ng/ml in group III, p<0,01). Moreover, there was a highly significant difference between patients with AITD and patients without AITD (20,76±6,31ng/ml vs. 24,37±9,05ng/ml, respectively; p<0,01). The concentration of anti-TPO and anti-TG antibodies were statistically significant higher in patients with vitamin D deficiency (p< 0,001). Serum TSH were significantly higher in group I (p< 0,001)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Significantly low levels of vitamin D were documented in patients with AITD that were related to the presence of anti-thyroid antibodies and higher level thyroid-stimulation hormone (TSH), suggesting the involvement of vitamin D in the pathogenesis of AITD and the advisability of supplementation."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Autoantibodies","Female","Healthy Volunteers","Humans","Hypothyroidism","Immunoglobulins, Thyroid-Stimulating","Male","Middle Aged","Vitamin D","Vitamin D Deficiency"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Autoantibodies\",\n \"Female\",\n \"Healthy Volunteers\",\n \"Humans\",\n \"Hypothyroidism\",\n \"Immunoglobulins, Thyroid-Stimulating\",\n \"Male\",\n \"Middle Aged\",\n \"Vitamin D\",\n \"Vitamin D Deficiency\"\n]"},"pmcid":{"kind":"string","value":"7780811"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8)."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"The study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.\nAnti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.\nThe statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05)."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","AIM","RESULTS","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"AIM\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"other_sections_texts":{"kind":"list like","value":["Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).","The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.","This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).","Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.","Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD."],"string":"[\n \"Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).\",\n \"The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.\",\n \"This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \\nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).\",\n \"Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.\",\n \"Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","AIM","METHODS","RESULTS","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"AIM\",\n \"METHODS\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).","The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.","The study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.\nAnti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.\nThe statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05).","This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).","Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.","Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD."],"string":"[\n \"Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).\",\n \"The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.\",\n \"The study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.\\nAnti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.\\nThe statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05).\",\n \"This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \\nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).\",\n \"Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.\",\n \"Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,"methods",null,null,null],"string":"[\n null,\n null,\n \"methods\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["autoimmune thyroid disease","vitamin D","thyroid autoantibodies","thyroid-stimulation hormone"],"string":"[\n \"autoimmune thyroid disease\",\n \"vitamin D\",\n \"thyroid autoantibodies\",\n \"thyroid-stimulation hormone\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nVitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).\n\nAIM:\nThe aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.\n\nMETHODS:\nThe study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.\nAnti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.\nThe statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05).\n\nRESULTS:\nThis study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).\n\nDISCUSSION:\nThyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.\n\nCONCLUSION:\nSignificantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nVitamin D deficiency is a global health problem. Its deficiency has been reported to be associated with different autoimmune diseases.\n\nMethods:\nA total number of 150 individuals were enrolled in this study. They were divided into the fallowing groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease. Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibodies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG).\n\nResults:\nSerum levels of 25 (OH) vitamin D recorded a highly significant difference between the studies groups (20,76±6,31 ng/ml in group I vs. 24,37±9,05ng/ml in group II vs. 24,57±6,45ng/ml in group III, p<0,01). Moreover, there was a highly significant difference between patients with AITD and patients without AITD (20,76±6,31ng/ml vs. 24,37±9,05ng/ml, respectively; p<0,01). The concentration of anti-TPO and anti-TG antibodies were statistically significant higher in patients with vitamin D deficiency (p< 0,001). Serum TSH were significantly higher in group I (p< 0,001).\n\nConclusions:\nSignificantly low levels of vitamin D were documented in patients with AITD that were related to the presence of anti-thyroid antibodies and higher level thyroid-stimulation hormone (TSH), suggesting the involvement of vitamin D in the pathogenesis of AITD and the advisability of supplementation."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nVitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).\n\nCONCLUSION:\nSignificantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nVitamin D deficiency is a global health problem. Its deficiency has been reported to be associated with different autoimmune diseases.\n\nMethods:\nA total number of 150 individuals were enrolled in this study. They were divided into the fallowing groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease. Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibodies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG).\n\nResults:\nSerum levels of 25 (OH) vitamin D recorded a highly significant difference between the studies groups (20,76±6,31 ng/ml in group I vs. 24,37±9,05ng/ml in group II vs. 24,57±6,45ng/ml in group III, p<0,01). Moreover, there was a highly significant difference between patients with AITD and patients without AITD (20,76±6,31ng/ml vs. 24,37±9,05ng/ml, respectively; p<0,01). The concentration of anti-TPO and anti-TG antibodies were statistically significant higher in patients with vitamin D deficiency (p< 0,001). Serum TSH were significantly higher in group I (p< 0,001).\n\nConclusions:\nSignificantly low levels of vitamin D were documented in patients with AITD that were related to the presence of anti-thyroid antibodies and higher level thyroid-stimulation hormone (TSH), suggesting the involvement of vitamin D in the pathogenesis of AITD and the advisability of supplementation."},"whole_article_text_length":{"kind":"number","value":2368,"string":"2,368"},"whole_article_abstract_length":{"kind":"number","value":311,"string":"311"},"other_sections_lengths":{"kind":"list like","value":[342,29,718,771,70],"string":"[\n 342,\n 29,\n 718,\n 771,\n 70\n]"},"num_sections":{"kind":"number","value":6,"string":"6"},"most_frequent_words":{"kind":"list like","value":["vitamin","aitd","patients","thyroid","ml","group","anti","25","patients aitd","oh"],"string":"[\n \"vitamin\",\n \"aitd\",\n \"patients\",\n \"thyroid\",\n \"ml\",\n \"group\",\n \"anti\",\n \"25\",\n \"patients aitd\",\n \"oh\"\n]"},"keybert_topics":{"kind":"list like","value":["specific autoantibodies vitamin","autoimmune thyroid diseases","diseases autoimmune thyroid","patients aitd vitamin","vitamin pathogenesis aitd"],"string":"[\n \"specific autoantibodies vitamin\",\n \"autoimmune thyroid diseases\",\n \"diseases autoimmune thyroid\",\n \"patients aitd vitamin\",\n \"vitamin pathogenesis aitd\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] autoimmune thyroid disease | vitamin D | thyroid autoantibodies | thyroid-stimulation 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[SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | aitd | patients | thyroid | ml | group | anti | 25 | patients aitd | oh [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | aitd | patients | thyroid | ml | group | anti | 25 | patients aitd | oh [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | aitd | patients | thyroid | ml | group | anti | 25 | patients aitd | oh [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | autoimmune | thyroid | genes | vdr | diseases | association | specific | autoimmune thyroid | autoimmune thyroid diseases [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] anti | thyroid | vitamin | included | 25 | group | tg | ml | anti tg | tpo anti [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | aitd | deficiency | antibodies abnormal thyroid functions | antibodies abnormal thyroid | antibodies abnormal | deficiency causal | functions studies required determine | functions studies | functions [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | thyroid | aitd | patients | anti | ml | group | 25 | patients aitd | oh [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vitamin | thyroid | aitd | patients | anti | ml | group | 25 | patients aitd | oh [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Vitamin ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 150 ||| 50 | AITD | II | 50 ||| 50 ||| 25 | TSH | anti-TG [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] AITD | TSH | AITD [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Vitamin ||| ||| 150 ||| 50 | AITD | II | 50 ||| 50 ||| 25 | TSH | anti-TG ||| 25 | 20,76±6,31 ng/ml | II | III ||| AITD | AITD | 20,76±6,31ng/ml ||| anti-TG ||| Serum TSH ||| AITD | TSH | AITD [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Vitamin ||| ||| 150 ||| 50 | AITD | II | 50 ||| 50 ||| 25 | TSH | anti-TG ||| 25 | 20,76±6,31 ng/ml | II | III ||| AITD | AITD | 20,76±6,31ng/ml ||| anti-TG ||| Serum TSH ||| AITD | TSH | AITD [SUMMARY]"}}},{"rowIdx":3375,"cells":{"title":{"kind":"string","value":"Activation of pro-oncogenic pathways in colorectal hyperplastic polyps."},"pmid":{"kind":"string","value":"24209454"},"background_abstract":{"kind":"string","value":"In contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Mean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adenoma","Adult","Aged","Aged, 80 and over","Colonic Polyps","Extracellular Signal-Regulated MAP Kinases","Female","Gastrins","Humans","Hyperplasia","Intestinal Mucosa","Male","Middle Aged","Oncogene Proteins","Protein Precursors","Retrospective Studies","Risk Factors","STAT3 Transcription Factor","Signal Transduction"],"string":"[\n \"Adenoma\",\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Colonic Polyps\",\n \"Extracellular Signal-Regulated MAP Kinases\",\n \"Female\",\n \"Gastrins\",\n \"Humans\",\n \"Hyperplasia\",\n \"Intestinal Mucosa\",\n \"Male\",\n \"Middle Aged\",\n \"Oncogene Proteins\",\n \"Protein Precursors\",\n \"Retrospective Studies\",\n \"Risk Factors\",\n \"STAT3 Transcription Factor\",\n \"Signal Transduction\"\n]"},"pmcid":{"kind":"string","value":"3829387"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\nWe examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\n Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\nFor immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\n Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].\nUnivariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34]."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\nClinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\n Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\nRepresentative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\n The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\nRepresentative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\n The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\nRepresentative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116)."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.\n Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images."},"other_sections_titles":{"kind":"list like","value":["Background","Patients and data collection","Immunohistochemistry","Statistical analysis","Clinical and histological characteristics","Expression of progastrin in normal mucosa and colonic neoplasms","The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression","The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression","Consent","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Patients and data collection\",\n \"Immunohistochemistry\",\n \"Statistical analysis\",\n \"Clinical and histological characteristics\",\n \"Expression of progastrin in normal mucosa and colonic neoplasms\",\n \"The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression\",\n \"The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression\",\n \"Consent\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.","We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.","For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.","Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].","Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.","Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.","Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.","Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).","Written informed consent was obtained from the patient for the publication of this report and any accompanying images.","The author(s) declare that they have no competing interests.","CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\n"],"string":"[\n \"Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\\n[3-5].\\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\\n[12].\\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.\",\n \"We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \\n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\\nClinical and histological features of hyperplastic polyps\\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\",\n \"For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\",\n \"Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\\n[34].\",\n \"Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \\n1. To take into account the heterogeneity of PG staining in HP observed in Figure \\n1, PG expression was analyzed in Table \\n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\",\n \"Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \\n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\",\n \"Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \\n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \\n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \\n2).\\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\\nSD: standard deviation.\\n(a)Cuzick test for trend across ordered groups.\\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\",\n \"Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3.\\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \\n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \\n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\",\n \"Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\",\n \"The author(s) declare that they have no competing interests.\",\n \"CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Patients and data collection","Immunohistochemistry","Statistical analysis","Results","Clinical and histological characteristics","Expression of progastrin in normal mucosa and colonic neoplasms","The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression","The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression","Discussion","Conclusion","Consent","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Patients and data collection\",\n \"Immunohistochemistry\",\n \"Statistical analysis\",\n \"Results\",\n \"Clinical and histological characteristics\",\n \"Expression of progastrin in normal mucosa and colonic neoplasms\",\n \"The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression\",\n \"The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression\",\n \"Discussion\",\n \"Conclusion\",\n \"Consent\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression."," Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\nWe examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\n Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\nFor immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\n Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].\nUnivariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].","We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.","For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.","Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34]."," Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\nClinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\n Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\nRepresentative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\n The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\nRepresentative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\n The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\nRepresentative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).","Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.","Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.","Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.","Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).","In the present study, we demonstrated a significant increase in the activation of the pro-oncogenic pathway, ERK1/2, in HP as compared to normal tissue. More interestingly, we showed a significant correlation between ERK pathway activation in HP and the expression of PG that is recognized as a growth factor for colonic epithelial cells. ERK activation was significantly higher in lesions with strong PG expression. Activation of this signaling pathway by PG has been previously reported in normal colonic epithelial cells from a transgenic mouse model overexpressing PG and has been linked to an increased risk of developing preneoplastic lesions in the colonic epithelium\n[20]. Therefore HP overexpressing PG that have a high activation of the ERK pathway might reflect less latent lesions.\nThe PG gene has been previously shown to be a target of two pro-oncogenic pathways frequently activated in colorectal cancer: APC/β-catenin and K-ras\n[35-37]. APC deletions or β-catenin mutations have not been reported in HP and we recently published that this pathway is not activated in HP with PG overexpression\n[12]. Therefore, it is unlikely that this pathway is involved in the expression of PG in these lesions. In contrast, KRAS mutations have been observed in thirty-seven percent of HP\n[5] and might lead to the increase in PG expression and ERK activation observed in the present study. However we cannot exclude an additional mechanism leading to PG expression in HP since in our study, nearly to sixty percent of HP presented an overexpression of PG or p-ERK. In a recent publication, Bongers et al.\n[38] have reported the activation of the EGFR pathway in seventy percent of HP from a small cohort of 27 samples. Interestingly, EGFR ligands have been shown to be potent regulators of the progastrin gene and an EGF response element has been identified on the progastrin promoter\n[39,40]. Therefore the EGFR pathway activated in HP might also contribute to PG overexpression and ERK activation independently of K-ras.\nProgastrin is clearly recognized as an autocrine growth factor for colorectal cancer cells and blocking PG expression has been shown to inhibit cellular growth in vitro and in vivo on tumor xenografts\n[23,41,42]. It is probable that an autocrine mechanism occurs in HP producing PG and leading to ERK activation. However a recent publication from Duckworth et al.\n[43] suggests that an indirect mechanism might be also proposed. These authors have shown that PG is capable to activate colonic fibroblasts leading to growth factors secretion that in turn stimulate colonic epithelial cells. These results therefore suggest that PG produced by HP might also activate the ERK pathway in colonic epithelial cells via a dialogue with the fibroblasts present in the stroma.\nThe identity of the receptor mediating the PG effects on colonic epithelial cells or fibroblasts remains an important point of debate. Several publications have shown that the receptor specific for the mature form of gastrin, the CCK-2 receptor, is not involved in the PG effect on fibroblasts or colon cancer cells\n[18,43,44]. In contrast the data from Jin et al.\n[45] suggest a role of this receptor in the proliferative effects of PG in vivo, although the nature of the interaction between PG and the CCK2 receptor in this study remains to be identified. Other studies have shown a role of ferric ions, Annexin A2 or glycosaminoglycans in the binding of PG to cell surface\n[20,46,47]. However, the cell surface protein that directly binds PG remained to be identified.\nIn contrast to what we observed for the ERK pathway, STAT3 activation was not significantly different between HP and normal colon. In addition we did not observe a significant correlation with PG expression. We previously demonstrated an association between STAT3 activation and PG in vivo, in transgenic mice overexpressing the prohormone\n[20]. STAT3 activation by PG might required high level of progastrin expression, as found in adenomas or adenocarcinomas.\nPreviously we demonstrated that PG expression in HP may predict occurrence of metachronous adenomas\n[12]. Including additional biomarkers might improve the specificity of such a test. P-ERK might be an interesting factor since this pro-oncogenic factor is overexpressed in a subset of PG positive HP.","HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.\n Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images.","Written informed consent was obtained from the patient for the publication of this report and any accompanying images.","The author(s) declare that they have no competing interests.","CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\n"],"string":"[\n \"Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\\n[3-5].\\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\\n[12].\\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.\",\n \" Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \\n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\\nClinical and histological features of hyperplastic polyps\\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\\nWe examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \\n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\\nClinical and histological features of hyperplastic polyps\\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\\n Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\\nFor immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\\n Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\\n[34].\\nUnivariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\\n[34].\",\n \"We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \\n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\\nClinical and histological features of hyperplastic polyps\\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\",\n \"For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\",\n \"Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\\n[34].\",\n \" Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \\n1. To take into account the heterogeneity of PG staining in HP observed in Figure \\n1, PG expression was analyzed in Table \\n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\\nClinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \\n1. To take into account the heterogeneity of PG staining in HP observed in Figure \\n1, PG expression was analyzed in Table \\n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\\n Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \\n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\\nRepresentative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \\n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\\n The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \\n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \\n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \\n2).\\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\\nSD: standard deviation.\\n(a)Cuzick test for trend across ordered groups.\\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\\nRepresentative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \\n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \\n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \\n2).\\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\\nSD: standard deviation.\\n(a)Cuzick test for trend across ordered groups.\\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\\n The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3.\\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \\n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \\n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\\nRepresentative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3.\\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \\n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \\n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\",\n \"Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \\n1. To take into account the heterogeneity of PG staining in HP observed in Figure \\n1, PG expression was analyzed in Table \\n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\",\n \"Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \\n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\",\n \"Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \\n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \\n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \\n2).\\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\\nSD: standard deviation.\\n(a)Cuzick test for trend across ordered groups.\\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\",\n \"Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \\n2 and\\n3.\\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \\n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \\n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\",\n \"In the present study, we demonstrated a significant increase in the activation of the pro-oncogenic pathway, ERK1/2, in HP as compared to normal tissue. More interestingly, we showed a significant correlation between ERK pathway activation in HP and the expression of PG that is recognized as a growth factor for colonic epithelial cells. ERK activation was significantly higher in lesions with strong PG expression. Activation of this signaling pathway by PG has been previously reported in normal colonic epithelial cells from a transgenic mouse model overexpressing PG and has been linked to an increased risk of developing preneoplastic lesions in the colonic epithelium\\n[20]. Therefore HP overexpressing PG that have a high activation of the ERK pathway might reflect less latent lesions.\\nThe PG gene has been previously shown to be a target of two pro-oncogenic pathways frequently activated in colorectal cancer: APC/β-catenin and K-ras\\n[35-37]. APC deletions or β-catenin mutations have not been reported in HP and we recently published that this pathway is not activated in HP with PG overexpression\\n[12]. Therefore, it is unlikely that this pathway is involved in the expression of PG in these lesions. In contrast, KRAS mutations have been observed in thirty-seven percent of HP\\n[5] and might lead to the increase in PG expression and ERK activation observed in the present study. However we cannot exclude an additional mechanism leading to PG expression in HP since in our study, nearly to sixty percent of HP presented an overexpression of PG or p-ERK. In a recent publication, Bongers et al.\\n[38] have reported the activation of the EGFR pathway in seventy percent of HP from a small cohort of 27 samples. Interestingly, EGFR ligands have been shown to be potent regulators of the progastrin gene and an EGF response element has been identified on the progastrin promoter\\n[39,40]. Therefore the EGFR pathway activated in HP might also contribute to PG overexpression and ERK activation independently of K-ras.\\nProgastrin is clearly recognized as an autocrine growth factor for colorectal cancer cells and blocking PG expression has been shown to inhibit cellular growth in vitro and in vivo on tumor xenografts\\n[23,41,42]. It is probable that an autocrine mechanism occurs in HP producing PG and leading to ERK activation. However a recent publication from Duckworth et al.\\n[43] suggests that an indirect mechanism might be also proposed. These authors have shown that PG is capable to activate colonic fibroblasts leading to growth factors secretion that in turn stimulate colonic epithelial cells. These results therefore suggest that PG produced by HP might also activate the ERK pathway in colonic epithelial cells via a dialogue with the fibroblasts present in the stroma.\\nThe identity of the receptor mediating the PG effects on colonic epithelial cells or fibroblasts remains an important point of debate. Several publications have shown that the receptor specific for the mature form of gastrin, the CCK-2 receptor, is not involved in the PG effect on fibroblasts or colon cancer cells\\n[18,43,44]. In contrast the data from Jin et al.\\n[45] suggest a role of this receptor in the proliferative effects of PG in vivo, although the nature of the interaction between PG and the CCK2 receptor in this study remains to be identified. Other studies have shown a role of ferric ions, Annexin A2 or glycosaminoglycans in the binding of PG to cell surface\\n[20,46,47]. However, the cell surface protein that directly binds PG remained to be identified.\\nIn contrast to what we observed for the ERK pathway, STAT3 activation was not significantly different between HP and normal colon. In addition we did not observe a significant correlation with PG expression. We previously demonstrated an association between STAT3 activation and PG in vivo, in transgenic mice overexpressing the prohormone\\n[20]. STAT3 activation by PG might required high level of progastrin expression, as found in adenomas or adenocarcinomas.\\nPreviously we demonstrated that PG expression in HP may predict occurrence of metachronous adenomas\\n[12]. Including additional biomarkers might improve the specificity of such a test. P-ERK might be an interesting factor since this pro-oncogenic factor is overexpressed in a subset of PG positive HP.\",\n \"HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.\\n Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images.\",\n \"Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\",\n \"The author(s) declare that they have no competing interests.\",\n \"CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,"results",null,null,null,null,"discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Hyperplastic polyps","Colorectal","Progastrin","ERK","STAT3","Pro-oncogenic pathways"],"string":"[\n \"Hyperplastic polyps\",\n \"Colorectal\",\n \"Progastrin\",\n \"ERK\",\n \"STAT3\",\n \"Pro-oncogenic pathways\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nAmong colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.\n\nMethods:\n Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\nWe examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\n Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\nFor immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\n Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].\nUnivariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].\n\nPatients and data collection:\nWe examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\n\nImmunohistochemistry:\nFor immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\n\nStatistical analysis:\nUnivariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].\n\nResults:\n Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\nClinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\n Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\nRepresentative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\n The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\nRepresentative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\n The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\nRepresentative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\n\nClinical and histological characteristics:\nClinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\n\nExpression of progastrin in normal mucosa and colonic neoplasms:\nRepresentative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\n\nThe ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression:\nRepresentative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\n\nThe STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression:\nRepresentative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\n\nDiscussion:\nIn the present study, we demonstrated a significant increase in the activation of the pro-oncogenic pathway, ERK1/2, in HP as compared to normal tissue. More interestingly, we showed a significant correlation between ERK pathway activation in HP and the expression of PG that is recognized as a growth factor for colonic epithelial cells. ERK activation was significantly higher in lesions with strong PG expression. Activation of this signaling pathway by PG has been previously reported in normal colonic epithelial cells from a transgenic mouse model overexpressing PG and has been linked to an increased risk of developing preneoplastic lesions in the colonic epithelium\n[20]. Therefore HP overexpressing PG that have a high activation of the ERK pathway might reflect less latent lesions.\nThe PG gene has been previously shown to be a target of two pro-oncogenic pathways frequently activated in colorectal cancer: APC/β-catenin and K-ras\n[35-37]. APC deletions or β-catenin mutations have not been reported in HP and we recently published that this pathway is not activated in HP with PG overexpression\n[12]. Therefore, it is unlikely that this pathway is involved in the expression of PG in these lesions. In contrast, KRAS mutations have been observed in thirty-seven percent of HP\n[5] and might lead to the increase in PG expression and ERK activation observed in the present study. However we cannot exclude an additional mechanism leading to PG expression in HP since in our study, nearly to sixty percent of HP presented an overexpression of PG or p-ERK. In a recent publication, Bongers et al.\n[38] have reported the activation of the EGFR pathway in seventy percent of HP from a small cohort of 27 samples. Interestingly, EGFR ligands have been shown to be potent regulators of the progastrin gene and an EGF response element has been identified on the progastrin promoter\n[39,40]. Therefore the EGFR pathway activated in HP might also contribute to PG overexpression and ERK activation independently of K-ras.\nProgastrin is clearly recognized as an autocrine growth factor for colorectal cancer cells and blocking PG expression has been shown to inhibit cellular growth in vitro and in vivo on tumor xenografts\n[23,41,42]. It is probable that an autocrine mechanism occurs in HP producing PG and leading to ERK activation. However a recent publication from Duckworth et al.\n[43] suggests that an indirect mechanism might be also proposed. These authors have shown that PG is capable to activate colonic fibroblasts leading to growth factors secretion that in turn stimulate colonic epithelial cells. These results therefore suggest that PG produced by HP might also activate the ERK pathway in colonic epithelial cells via a dialogue with the fibroblasts present in the stroma.\nThe identity of the receptor mediating the PG effects on colonic epithelial cells or fibroblasts remains an important point of debate. Several publications have shown that the receptor specific for the mature form of gastrin, the CCK-2 receptor, is not involved in the PG effect on fibroblasts or colon cancer cells\n[18,43,44]. In contrast the data from Jin et al.\n[45] suggest a role of this receptor in the proliferative effects of PG in vivo, although the nature of the interaction between PG and the CCK2 receptor in this study remains to be identified. Other studies have shown a role of ferric ions, Annexin A2 or glycosaminoglycans in the binding of PG to cell surface\n[20,46,47]. However, the cell surface protein that directly binds PG remained to be identified.\nIn contrast to what we observed for the ERK pathway, STAT3 activation was not significantly different between HP and normal colon. In addition we did not observe a significant correlation with PG expression. We previously demonstrated an association between STAT3 activation and PG in vivo, in transgenic mice overexpressing the prohormone\n[20]. STAT3 activation by PG might required high level of progastrin expression, as found in adenomas or adenocarcinomas.\nPreviously we demonstrated that PG expression in HP may predict occurrence of metachronous adenomas\n[12]. Including additional biomarkers might improve the specificity of such a test. P-ERK might be an interesting factor since this pro-oncogenic factor is overexpressed in a subset of PG positive HP.\n\nConclusion:\nHP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.\n Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images.\n\nConsent:\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images.\n\nCompeting interests:\nThe author(s) declare that they have no competing interests.\n\nAuthors’ contributions:\nCD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIn contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP.\n\nMethods:\nWe retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients.\n\nResults:\nMean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression.\n\nConclusions:\nHP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential."},"background_conclusion_text":{"kind":"string","value":"Background:\nAmong colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.\n\nConclusion:\nHP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.\n Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIn contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP.\n\nMethods:\nWe retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients.\n\nResults:\nMean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression.\n\nConclusions:\nHP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential."},"whole_article_text_length":{"kind":"number","value":8620,"string":"8,620"},"whole_article_abstract_length":{"kind":"number","value":293,"string":"293"},"other_sections_lengths":{"kind":"list like","value":[703,276,563,149,232,422,348,249,19,11,93,16],"string":"[\n 703,\n 276,\n 563,\n 149,\n 232,\n 422,\n 348,\n 249,\n 19,\n 11,\n 93,\n 16\n]"},"num_sections":{"kind":"number","value":16,"string":"16"},"most_frequent_words":{"kind":"list like","value":["expression","staining","hp","pg","high","cells","stat3","progastrin","normal","adenomas"],"string":"[\n \"expression\",\n \"staining\",\n \"hp\",\n \"pg\",\n \"high\",\n \"cells\",\n \"stat3\",\n \"progastrin\",\n \"normal\",\n \"adenomas\"\n]"},"keybert_topics":{"kind":"list like","value":["colonic neoplasms representative","serrated adenomas ssa","features colonic cancer","adenomatous polyposis hyperplastic","adenocarcinomas microsatellite instability"],"string":"[\n \"colonic neoplasms representative\",\n \"serrated adenomas ssa\",\n \"features colonic cancer\",\n \"adenomatous polyposis hyperplastic\",\n \"adenocarcinomas microsatellite instability\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] colorectal | hp | tsa | ssa | adenomas | serrated | pathways | gastrin | risk | pg [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] staining | expression | epithelial | epithelial cells | cells | 50 epithelial cells | 50 epithelial | defined | stat3 | test [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] staining | expression | grade | high | hp | py stat3 | py | stat3 | adenomas | normal [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] consent | accompanying images | patient publication report | informed consent obtained patient | informed consent obtained | informed consent | informed | consent obtained | consent obtained patient | consent obtained patient publication [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | cells | high | stat3 | grade | py stat3 | py [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] expression | staining | hp | pg | cells | high | stat3 | grade | py stat3 | py [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| HP ||| HP ||| ERK | STAT3 ||| HP [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ERK | STAT3 | HP | 48 [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] PG | 31% | 33% | HP | 3% | p=0.0021 | 7% ||| ERK ||| STAT3 | HP [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ERK [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| HP ||| HP ||| ERK | STAT3 ||| HP ||| ERK | STAT3 | HP | 48 ||| PG | 31% | 33% | HP | 3% | p=0.0021 | 7% ||| ERK ||| STAT3 | HP ||| ERK [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| HP ||| HP ||| ERK | STAT3 ||| HP ||| ERK | STAT3 | HP | 48 ||| PG | 31% | 33% | HP | 3% | p=0.0021 | 7% ||| ERK ||| STAT3 | HP ||| ERK [SUMMARY]"}}},{"rowIdx":3376,"cells":{"title":{"kind":"string","value":"Effect of the interleukin 10 polymorphisms on interleukin 10 production and visceral hypersensitivity in Chinese patients with diarrhea-predominant irritable bowel syndrome."},"pmid":{"kind":"string","value":"31205078"},"background_abstract":{"kind":"string","value":"Irritable bowel syndrome (IBS), a functional gastrointestinal disorder, is characterized by cytokine imbalance. Previously, decreased plasma interleukin 10 (IL-10) level was reported in patients with IBS, which may be due to genetic polymorphisms. However, there are no reports correlating the IL-10 polymorphisms with IL-10 production in patients with IBS. This study aimed to analyze the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with diarrhea-predominant IBS (IBS-D)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Two IL-10 single nucleotide polymorphisms (rs1800871 and rs1800896) were detected in 120 patients with IBS-D and 144 healthy controls (HC) using SNaPshot. IBS symptom severity score, Bristol scale, hospital anxiety, and depressive scale (HADS) were used to evaluate the clinical symptoms, as well as the psychological status and visceral sensitivity of the subjects. IL-10 levels in the plasma and peripheral blood mononuclear cell (PBMC) culture supernatant were measured using enzyme-linked immunosorbent assay, while those in ileal and colonic mucosal biopsies were measured using immunohistochemistry."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The frequency of rs1800896 C allele was significantly lower in the patients with IBS-D than that in the HC (odds ratio: 0.49, 95% confidence interval: 0.27-0.92, P = 0.0240). The IL-10 levels in the plasma (P = 0.0030) and PBMC culture supernatant (P = 0.0500) of the CT genotype subjects were significantly higher than those in the TT genotype subjects. The CT genotype subjects exhibited a higher pain threshold in the rectal distention test than the TT genotype subjects. Moreover, IL-10 rs1800871 GG genotype subjects showed an increase in the HADS score compared to other genotype subjects."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"IL-10 rs1800896 C allele is correlated with higher IL-10 levels in the plasma and the PBMC culture supernatant, which is associated with a higher pain threshold in the Chinese patients with IBS-D. This study provides an explicit relationship of IL-10 polymorphisms with IL-10 production, which might help in understanding the pathogenesis of IBS-D."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Aged","Diarrhea","Genotype","Humans","Interleukin-10","Irritable Bowel Syndrome","Middle Aged","Peripheral Blood Stem Cells","Polymorphism, Single Nucleotide","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Aged\",\n \"Diarrhea\",\n \"Genotype\",\n \"Humans\",\n \"Interleukin-10\",\n \"Irritable Bowel Syndrome\",\n \"Middle Aged\",\n \"Peripheral Blood Stem Cells\",\n \"Polymorphism, Single Nucleotide\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"6616227"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]\nIL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.\nTherefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\n Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\nThis study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\n Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\nDNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\n Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\nThe 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\n Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\nSystematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\n Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\nImmunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\n Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\nData were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\nA total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\n Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\nThe genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\n Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\nThe levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\n Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\nThe correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Ethical approval","Study design and participants recruitment","Cytokine gene polymorphisms evaluated through SNaPshot","Isolation and culture of peripheral blood mononuclear cells","Enzyme-linked immunosorbent assay","Immunohistochemistry staining","Statistical analysis","Clinical characteristics of the subjects","Genotyping of IL-10 gene polymorphisms","Measurements for the IL-10 level","Association of IL-10 polymorphisms and clinical symptoms","Acknowledgements","Funding","Conflicts of interest"],"string":"[\n \"Ethical approval\",\n \"Study design and participants recruitment\",\n \"Cytokine gene polymorphisms evaluated through SNaPshot\",\n \"Isolation and culture of peripheral blood mononuclear cells\",\n \"Enzyme-linked immunosorbent assay\",\n \"Immunohistochemistry staining\",\n \"Statistical analysis\",\n \"Clinical characteristics of the subjects\",\n \"Genotyping of IL-10 gene polymorphisms\",\n \"Measurements for the IL-10 level\",\n \"Association of IL-10 polymorphisms and clinical symptoms\",\n \"Acknowledgements\",\n \"Funding\",\n \"Conflicts of interest\"\n]"},"other_sections_texts":{"kind":"list like","value":["The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.","This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.","DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.","The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.","Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.","Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.","Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.","A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.","The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.","The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.","The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.","The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.","The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).","None."],"string":"[\n \"The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\",\n \"This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\",\n \"DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\",\n \"The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\",\n \"Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\",\n \"Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\",\n \"Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\",\n \"A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\\nClinical characteristics for patients with IBS-D and HC.\",\n \"The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\\nGenotyping of IL-10 gene polymorphisms.\\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\",\n \"The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\\nIL-10 levels in different genotype of IL-10 polymorphisms.\\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\",\n \"The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\",\n \"The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.\",\n \"The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).\",\n \"None.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Ethical approval","Study design and participants recruitment","Cytokine gene polymorphisms evaluated through SNaPshot","Isolation and culture of peripheral blood mononuclear cells","Enzyme-linked immunosorbent assay","Immunohistochemistry staining","Statistical analysis","Results","Clinical characteristics of the subjects","Genotyping of IL-10 gene polymorphisms","Measurements for the IL-10 level","Association of IL-10 polymorphisms and clinical symptoms","Discussion","Acknowledgements","Funding","Conflicts of interest"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Ethical approval\",\n \"Study design and participants recruitment\",\n \"Cytokine gene polymorphisms evaluated through SNaPshot\",\n \"Isolation and culture of peripheral blood mononuclear cells\",\n \"Enzyme-linked immunosorbent assay\",\n \"Immunohistochemistry staining\",\n \"Statistical analysis\",\n \"Results\",\n \"Clinical characteristics of the subjects\",\n \"Genotyping of IL-10 gene polymorphisms\",\n \"Measurements for the IL-10 level\",\n \"Association of IL-10 polymorphisms and clinical symptoms\",\n \"Discussion\",\n \"Acknowledgements\",\n \"Funding\",\n \"Conflicts of interest\"\n]"},"all_sections_texts":{"kind":"list like","value":["Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]\nIL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.\nTherefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D."," Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\n Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\nThis study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\n Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\nDNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\n Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\nThe 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\n Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\nSystematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\n Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\nImmunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\n Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\nData were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.","The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.","This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.","DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.","The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.","Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.","Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.","Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed."," Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\nA total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\n Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\nThe genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\n Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\nThe levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\n Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\nThe correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.","A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.","The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.","The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.","The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.","Patients with IBS-D present a particularly VH with a looser stool consistency, which may result by chronic systemic and mucosal inflammation. IL-10 plays an important role in the anti-inflammation response and is considered to be a potent suppressor of T lymphocytes or macrophages and their derived effector molecules, such as proinflammatory cytokines (IL-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemotactic protein 1, macrophage inflammatory protein 1α).[18] It is speculated that IL-10 gene polymorphisms might be involved in the IL-10 production.[19] Our study suggested that the carriers with IL-10 rs1800896 C allele have a lower risk for developing IBS-D, which may be associated with higher production of IL-10.\nThe gene encoding IL-10 is located on the human chromosome 1q31–1q32. The two polymorphisms analyzed in this study were −1082 A/G and −819 C/T, which are located in the gene promoter region. According to the single nucleotide polymorphism database (http://www.ncbi.nlm.nih.gov/SNP/), they are annotated as rs1800896 (T > C) and rs1800871 (A > G), which represents the polymorphism in the complementary strands. Qin et al[13] investigated that the IL-10 rs1800871 polymorphism was associated with a decreased risk for developing IBS in the eastern population. Moreover, other studies have confirmed that the C allele of IL-10 rs1800896 was strongly associated with the decreased risk for developing IBS in the western population.[7] However, there are studies which have reported no correlation between IL-10 polymorphisms and the risk for developing IBS.[12,20] In this study, the frequency of IL-10 rs1800896 C allele was significantly (OR: 0.49, 95% CI: 0.27–0.92, P = 0.0240) lower in the patients with IBS-D and was associated with a decreased risk for developing IBS-D. While the polymorphism rs1800871 presented no association with developing IBS, which investigated in patients with IBS-D and the healthy subjects.\nIL-10 was initially described as a T helper 2-type cytokine and was reported to be expressed in various cells of the adaptive immune system as well as the cells of the innate immune system. Patients with IBS exhibit cytokine imbalance characterized by decreased levels of IL-10 and increased levels of TNF-α.[1,7] The lower levels of IL-10 can be a predictor for IBS development.[21] The T allele of IL-10 rs1800896 has been reported to be associated with lower production of IL-10, while the C allele is associated with higher IL-10 production.[16,22] The C allele is associated with IL-10 production in the low stimulated cell culture.[23] In addition, there are very few studies that report a direct correlation between IL-10 polymorphisms and IL-10 production. Our data confirmed that the subjects carrying C allele of IL-10 rs1800896 exhibited a higher IL-10 concentration in the plasma and the PBMC culture supernatant. Additionally, we also demonstrated that the subjects with CT genotype were less sensitive in the rectal distention test than the subjects with TT genotype. Earlier studies demonstrated that IL-10 and other inflammatory cytokine concentration in the PBMC correlated with the severity of symptoms in IBS-D, including the intensity and frequency of painful events and motility-associated symptoms.[24] Some researchers hypothesize that IBS is on a spectrum with inflammation bowel disease (IBD), because they have largely overlapping pathophysiological mechanisms and clinical symptoms.[25,26] For example, the abdominal pain and diarrhea are predominant symptoms for patients with IBS or IBD, and post-inflammatory abnormalities are important in IBD and IBS. The subjects without C allele are more likely to develop IBS after intestinal infection.[3,27] Additionally, the polymorphisms of IL-10 gene can exert a protective effect on patients with IBD, ulcerative colitis or Crohn disease.[28,29]\nOur finding was intriguing as the IL-10 rs1800871 polymorphism exhibited a marginally positive correlation with the HADS score. The development of depressive disorder is considered to be associated with the activation of systemic inflammation as well.[30,31] It has been reported that the genotypes IL-10 rs1800871 and IL-10 rs1800896 decrease the risk for developing depression and are correlated with the Hamilton depression rating scale.[32] Globally, patients with IBS have a high comorbidity rate for depression or anxiety.[33] Our study supports the correlation between high comorbidity in IBS and mental disorder in an aspect of genetics polymorphism.\nTo the best of our knowledge, this study took the lead in studying the correlates of IL-10 gene polymorphisms and the expression level of IL-10. However, this study has some limitations. Only two polymorphisms for IL-10 were analyzed in this study. There may be other SNPs in IL-10 corresponding to rs1800896 and/or rs1800871, which may contribute to IL-10 expression. On the other hand, further studies are required for evaluating the transcriptional and epigenetic effects in IL-10 polymorphism.\nIn summary, the IL-10 rs1800896 polymorphism has a correlation with a higher concentration of IL-10 in both the plasma and the PBMC culture supernatant of Chinese patients with IBS-D. Additionally, this SNP is also associated with a higher visceral pain threshold. This study demonstrated a correlation between the IL-10 polymorphisms and IL-10 production, which might help in understanding the pathogenesis of IBS-D.","The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.","The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).","None."],"string":"[\n \"Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]\\nIL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.\\nTherefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D.\",\n \" Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\\n Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\\nThis study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\\n Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\\nDNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\\n Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\\nThe 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\\n Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\\nSystematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\\n Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\\nImmunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\\n Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\\nData were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\",\n \"The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\",\n \"This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\",\n \"DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\",\n \"The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\",\n \"Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\",\n \"Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\",\n \"Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\",\n \" Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\\nClinical characteristics for patients with IBS-D and HC.\\nA total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\\nClinical characteristics for patients with IBS-D and HC.\\n Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\\nGenotyping of IL-10 gene polymorphisms.\\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\\nThe genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\\nGenotyping of IL-10 gene polymorphisms.\\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\\n Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\\nIL-10 levels in different genotype of IL-10 polymorphisms.\\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\\nThe levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\\nIL-10 levels in different genotype of IL-10 polymorphisms.\\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\\n Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\\nThe correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\",\n \"A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\\nClinical characteristics for patients with IBS-D and HC.\",\n \"The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\\nGenotyping of IL-10 gene polymorphisms.\\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\",\n \"The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\\nIL-10 levels in different genotype of IL-10 polymorphisms.\\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\",\n \"The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\",\n \"Patients with IBS-D present a particularly VH with a looser stool consistency, which may result by chronic systemic and mucosal inflammation. IL-10 plays an important role in the anti-inflammation response and is considered to be a potent suppressor of T lymphocytes or macrophages and their derived effector molecules, such as proinflammatory cytokines (IL-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemotactic protein 1, macrophage inflammatory protein 1α).[18] It is speculated that IL-10 gene polymorphisms might be involved in the IL-10 production.[19] Our study suggested that the carriers with IL-10 rs1800896 C allele have a lower risk for developing IBS-D, which may be associated with higher production of IL-10.\\nThe gene encoding IL-10 is located on the human chromosome 1q31–1q32. The two polymorphisms analyzed in this study were −1082 A/G and −819 C/T, which are located in the gene promoter region. According to the single nucleotide polymorphism database (http://www.ncbi.nlm.nih.gov/SNP/), they are annotated as rs1800896 (T > C) and rs1800871 (A > G), which represents the polymorphism in the complementary strands. Qin et al[13] investigated that the IL-10 rs1800871 polymorphism was associated with a decreased risk for developing IBS in the eastern population. Moreover, other studies have confirmed that the C allele of IL-10 rs1800896 was strongly associated with the decreased risk for developing IBS in the western population.[7] However, there are studies which have reported no correlation between IL-10 polymorphisms and the risk for developing IBS.[12,20] In this study, the frequency of IL-10 rs1800896 C allele was significantly (OR: 0.49, 95% CI: 0.27–0.92, P = 0.0240) lower in the patients with IBS-D and was associated with a decreased risk for developing IBS-D. While the polymorphism rs1800871 presented no association with developing IBS, which investigated in patients with IBS-D and the healthy subjects.\\nIL-10 was initially described as a T helper 2-type cytokine and was reported to be expressed in various cells of the adaptive immune system as well as the cells of the innate immune system. Patients with IBS exhibit cytokine imbalance characterized by decreased levels of IL-10 and increased levels of TNF-α.[1,7] The lower levels of IL-10 can be a predictor for IBS development.[21] The T allele of IL-10 rs1800896 has been reported to be associated with lower production of IL-10, while the C allele is associated with higher IL-10 production.[16,22] The C allele is associated with IL-10 production in the low stimulated cell culture.[23] In addition, there are very few studies that report a direct correlation between IL-10 polymorphisms and IL-10 production. Our data confirmed that the subjects carrying C allele of IL-10 rs1800896 exhibited a higher IL-10 concentration in the plasma and the PBMC culture supernatant. Additionally, we also demonstrated that the subjects with CT genotype were less sensitive in the rectal distention test than the subjects with TT genotype. Earlier studies demonstrated that IL-10 and other inflammatory cytokine concentration in the PBMC correlated with the severity of symptoms in IBS-D, including the intensity and frequency of painful events and motility-associated symptoms.[24] Some researchers hypothesize that IBS is on a spectrum with inflammation bowel disease (IBD), because they have largely overlapping pathophysiological mechanisms and clinical symptoms.[25,26] For example, the abdominal pain and diarrhea are predominant symptoms for patients with IBS or IBD, and post-inflammatory abnormalities are important in IBD and IBS. The subjects without C allele are more likely to develop IBS after intestinal infection.[3,27] Additionally, the polymorphisms of IL-10 gene can exert a protective effect on patients with IBD, ulcerative colitis or Crohn disease.[28,29]\\nOur finding was intriguing as the IL-10 rs1800871 polymorphism exhibited a marginally positive correlation with the HADS score. The development of depressive disorder is considered to be associated with the activation of systemic inflammation as well.[30,31] It has been reported that the genotypes IL-10 rs1800871 and IL-10 rs1800896 decrease the risk for developing depression and are correlated with the Hamilton depression rating scale.[32] Globally, patients with IBS have a high comorbidity rate for depression or anxiety.[33] Our study supports the correlation between high comorbidity in IBS and mental disorder in an aspect of genetics polymorphism.\\nTo the best of our knowledge, this study took the lead in studying the correlates of IL-10 gene polymorphisms and the expression level of IL-10. However, this study has some limitations. Only two polymorphisms for IL-10 were analyzed in this study. There may be other SNPs in IL-10 corresponding to rs1800896 and/or rs1800871, which may contribute to IL-10 expression. On the other hand, further studies are required for evaluating the transcriptional and epigenetic effects in IL-10 polymorphism.\\nIn summary, the IL-10 rs1800896 polymorphism has a correlation with a higher concentration of IL-10 in both the plasma and the PBMC culture supernatant of Chinese patients with IBS-D. Additionally, this SNP is also associated with a higher visceral pain threshold. This study demonstrated a correlation between the IL-10 polymorphisms and IL-10 production, which might help in understanding the pathogenesis of IBS-D.\",\n \"The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.\",\n \"The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).\",\n \"None.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,null,"results",null,null,null,null,"discussion",null,null,null],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Irritable bowel syndrome","Interleukin 10","Polymorphisms","Visceral hypersensitivity","Depression"],"string":"[\n \"Irritable bowel syndrome\",\n \"Interleukin 10\",\n \"Polymorphisms\",\n \"Visceral hypersensitivity\",\n \"Depression\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nIrritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]\nIL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.\nTherefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D.\n\nMethods:\n Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\n Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\nThis study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\n Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\nDNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\n Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\nThe 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\n Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\nSystematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\n Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\nImmunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\n Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\nData were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\n\nEthical approval:\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\n\nStudy design and participants recruitment:\nThis study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\n\nCytokine gene polymorphisms evaluated through SNaPshot:\nDNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\n\nIsolation and culture of peripheral blood mononuclear cells:\nThe 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\n\nEnzyme-linked immunosorbent assay:\nSystematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\n\nImmunohistochemistry staining:\nImmunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\n\nStatistical analysis:\nData were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\n\nResults:\n Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\nA total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\n Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\nThe genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\n Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\nThe levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\n Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\nThe correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\n\nClinical characteristics of the subjects:\nA total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\n\nGenotyping of IL-10 gene polymorphisms:\nThe genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\n\nMeasurements for the IL-10 level:\nThe levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\n\nAssociation of IL-10 polymorphisms and clinical symptoms:\nThe correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\n\nDiscussion:\nPatients with IBS-D present a particularly VH with a looser stool consistency, which may result by chronic systemic and mucosal inflammation. IL-10 plays an important role in the anti-inflammation response and is considered to be a potent suppressor of T lymphocytes or macrophages and their derived effector molecules, such as proinflammatory cytokines (IL-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemotactic protein 1, macrophage inflammatory protein 1α).[18] It is speculated that IL-10 gene polymorphisms might be involved in the IL-10 production.[19] Our study suggested that the carriers with IL-10 rs1800896 C allele have a lower risk for developing IBS-D, which may be associated with higher production of IL-10.\nThe gene encoding IL-10 is located on the human chromosome 1q31–1q32. The two polymorphisms analyzed in this study were −1082 A/G and −819 C/T, which are located in the gene promoter region. According to the single nucleotide polymorphism database (http://www.ncbi.nlm.nih.gov/SNP/), they are annotated as rs1800896 (T > C) and rs1800871 (A > G), which represents the polymorphism in the complementary strands. Qin et al[13] investigated that the IL-10 rs1800871 polymorphism was associated with a decreased risk for developing IBS in the eastern population. Moreover, other studies have confirmed that the C allele of IL-10 rs1800896 was strongly associated with the decreased risk for developing IBS in the western population.[7] However, there are studies which have reported no correlation between IL-10 polymorphisms and the risk for developing IBS.[12,20] In this study, the frequency of IL-10 rs1800896 C allele was significantly (OR: 0.49, 95% CI: 0.27–0.92, P = 0.0240) lower in the patients with IBS-D and was associated with a decreased risk for developing IBS-D. While the polymorphism rs1800871 presented no association with developing IBS, which investigated in patients with IBS-D and the healthy subjects.\nIL-10 was initially described as a T helper 2-type cytokine and was reported to be expressed in various cells of the adaptive immune system as well as the cells of the innate immune system. Patients with IBS exhibit cytokine imbalance characterized by decreased levels of IL-10 and increased levels of TNF-α.[1,7] The lower levels of IL-10 can be a predictor for IBS development.[21] The T allele of IL-10 rs1800896 has been reported to be associated with lower production of IL-10, while the C allele is associated with higher IL-10 production.[16,22] The C allele is associated with IL-10 production in the low stimulated cell culture.[23] In addition, there are very few studies that report a direct correlation between IL-10 polymorphisms and IL-10 production. Our data confirmed that the subjects carrying C allele of IL-10 rs1800896 exhibited a higher IL-10 concentration in the plasma and the PBMC culture supernatant. Additionally, we also demonstrated that the subjects with CT genotype were less sensitive in the rectal distention test than the subjects with TT genotype. Earlier studies demonstrated that IL-10 and other inflammatory cytokine concentration in the PBMC correlated with the severity of symptoms in IBS-D, including the intensity and frequency of painful events and motility-associated symptoms.[24] Some researchers hypothesize that IBS is on a spectrum with inflammation bowel disease (IBD), because they have largely overlapping pathophysiological mechanisms and clinical symptoms.[25,26] For example, the abdominal pain and diarrhea are predominant symptoms for patients with IBS or IBD, and post-inflammatory abnormalities are important in IBD and IBS. The subjects without C allele are more likely to develop IBS after intestinal infection.[3,27] Additionally, the polymorphisms of IL-10 gene can exert a protective effect on patients with IBD, ulcerative colitis or Crohn disease.[28,29]\nOur finding was intriguing as the IL-10 rs1800871 polymorphism exhibited a marginally positive correlation with the HADS score. The development of depressive disorder is considered to be associated with the activation of systemic inflammation as well.[30,31] It has been reported that the genotypes IL-10 rs1800871 and IL-10 rs1800896 decrease the risk for developing depression and are correlated with the Hamilton depression rating scale.[32] Globally, patients with IBS have a high comorbidity rate for depression or anxiety.[33] Our study supports the correlation between high comorbidity in IBS and mental disorder in an aspect of genetics polymorphism.\nTo the best of our knowledge, this study took the lead in studying the correlates of IL-10 gene polymorphisms and the expression level of IL-10. However, this study has some limitations. Only two polymorphisms for IL-10 were analyzed in this study. There may be other SNPs in IL-10 corresponding to rs1800896 and/or rs1800871, which may contribute to IL-10 expression. On the other hand, further studies are required for evaluating the transcriptional and epigenetic effects in IL-10 polymorphism.\nIn summary, the IL-10 rs1800896 polymorphism has a correlation with a higher concentration of IL-10 in both the plasma and the PBMC culture supernatant of Chinese patients with IBS-D. Additionally, this SNP is also associated with a higher visceral pain threshold. This study demonstrated a correlation between the IL-10 polymorphisms and IL-10 production, which might help in understanding the pathogenesis of IBS-D.\n\nAcknowledgements:\nThe authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.\n\nFunding:\nThe studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).\n\nConflicts of interest:\nNone.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIrritable bowel syndrome (IBS), a functional gastrointestinal disorder, is characterized by cytokine imbalance. Previously, decreased plasma interleukin 10 (IL-10) level was reported in patients with IBS, which may be due to genetic polymorphisms. However, there are no reports correlating the IL-10 polymorphisms with IL-10 production in patients with IBS. This study aimed to analyze the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with diarrhea-predominant IBS (IBS-D).\n\nMethods:\nTwo IL-10 single nucleotide polymorphisms (rs1800871 and rs1800896) were detected in 120 patients with IBS-D and 144 healthy controls (HC) using SNaPshot. IBS symptom severity score, Bristol scale, hospital anxiety, and depressive scale (HADS) were used to evaluate the clinical symptoms, as well as the psychological status and visceral sensitivity of the subjects. IL-10 levels in the plasma and peripheral blood mononuclear cell (PBMC) culture supernatant were measured using enzyme-linked immunosorbent assay, while those in ileal and colonic mucosal biopsies were measured using immunohistochemistry.\n\nResults:\nThe frequency of rs1800896 C allele was significantly lower in the patients with IBS-D than that in the HC (odds ratio: 0.49, 95% confidence interval: 0.27-0.92, P = 0.0240). The IL-10 levels in the plasma (P = 0.0030) and PBMC culture supernatant (P = 0.0500) of the CT genotype subjects were significantly higher than those in the TT genotype subjects. The CT genotype subjects exhibited a higher pain threshold in the rectal distention test than the TT genotype subjects. Moreover, IL-10 rs1800871 GG genotype subjects showed an increase in the HADS score compared to other genotype subjects.\n\nConclusions:\nIL-10 rs1800896 C allele is correlated with higher IL-10 levels in the plasma and the PBMC culture supernatant, which is associated with a higher pain threshold in the Chinese patients with IBS-D. This study provides an explicit relationship of IL-10 polymorphisms with IL-10 production, which might help in understanding the pathogenesis of IBS-D."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":10183,"string":"10,183"},"whole_article_abstract_length":{"kind":"number","value":396,"string":"396"},"other_sections_lengths":{"kind":"list like","value":[40,297,629,122,274,175,179,287,243,365,218,56,40,2],"string":"[\n 40,\n 297,\n 629,\n 122,\n 274,\n 175,\n 179,\n 287,\n 243,\n 365,\n 218,\n 56,\n 40,\n 2\n]"},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["10","il","il 10","ibs","group","genotype","μl","hc","rs1800896","rs1800871"],"string":"[\n \"10\",\n \"il\",\n \"il 10\",\n \"ibs\",\n \"group\",\n \"genotype\",\n \"μl\",\n \"hc\",\n \"rs1800896\",\n \"rs1800871\"\n]"},"keybert_topics":{"kind":"list like","value":["ibs symptom severity","activation patients ibs","ibs pathophysiological mechanisms","ibs spectrum inflammation","ibs exhibit cytokine"],"string":"[\n \"ibs symptom severity\",\n \"activation patients ibs\",\n \"ibs pathophysiological mechanisms\",\n \"ibs spectrum inflammation\",\n \"ibs exhibit cytokine\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Diarrhea | Genotype | Humans | Interleukin-10 | Irritable Bowel Syndrome | Middle Aged | Peripheral Blood Stem Cells | Polymorphism, Single Nucleotide | Young 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[SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | 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[SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 10 | IBS ||| IL-10 | IBS ||| IL-10 | IL-10 | Chinese [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Two | IL-10 | rs1800871 | rs1800896 | 120 | 144 ||| Bristol | HADS ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] HC | 0.49 | 95% | 0.27 | 0.0240 ||| IL-10 | 0.0030 | 0.0500 | CT | TT ||| CT | TT ||| IL-10 | rs1800871 GG | HADS [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 10 | IBS ||| IL-10 | IBS ||| IL-10 | IL-10 | Chinese ||| Two | IL-10 | rs1800871 | rs1800896 | 120 | 144 ||| Bristol | HADS ||| ||| HC | 0.49 | 95% | 0.27 | 0.0240 ||| IL-10 | 0.0030 | 0.0500 | CT | TT ||| CT | TT ||| IL-10 | rs1800871 GG | HADS ||| Chinese | IL-10 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3377,"cells":{"title":{"kind":"string","value":"Prevalence and pattern of sexual assault in Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria."},"pmid":{"kind":"string","value":"28154687"},"background_abstract":{"kind":"string","value":"Sexual violence is an important public health problem of growing concern all over the world. This study was conducted to determine the prevalence and pattern of sexual assault managed in Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"It was a retrospective study that looked into cases of sexual assault admitted into the hospital between January 2010 and December 2014. Information on patients' biodata, and relevant details on the cases were extracted from the patients' case files and analyzed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Out of the 5317 gynecological admissions during the period under study, 45 (0.84%) were cases of sexual assault. Of these, only 34 case files were available for data extraction. The patients' ages ranged from 2 to 37 years (mean = 12.6 + 8.3). About two thirds (61.8%) of those affected were young children (aged 12 years and below). In majority of cases (70.6%) the assault was penetrative, and in most of the cases (91.2%) only a single assailant was involved. In close to two thirds of cases, the assailant was either an acquaintance (38.2%) or a family member (20.6%). Although law enforcement agents were informed in majority (58.8%) of cases, arrests were made in less than half (41.2%)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Although the prevalence of sexual assault in this study appears to be low, a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children's movement, and stringent laws should be enacted and enforced to curb this heinous act."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Age Distribution","Child","Child Abuse, Sexual","Child, Preschool","Crime Victims","Female","Hospitalization","Hospitals, University","Humans","Male","Nigeria","Prevalence","Rape","Retrospective Studies","Sex Offenses","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Age Distribution\",\n \"Child\",\n \"Child Abuse, Sexual\",\n \"Child, Preschool\",\n \"Crime Victims\",\n \"Female\",\n \"Hospitalization\",\n \"Hospitals, University\",\n \"Humans\",\n \"Male\",\n \"Nigeria\",\n \"Prevalence\",\n \"Rape\",\n \"Retrospective Studies\",\n \"Sex Offenses\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"5267856"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].\nIn the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"This was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\nThere were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\n Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\nMajority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\n Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\nThe most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\n Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\nThe time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\n Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\nNo weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\n Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\nIn majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\n Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents\nAlthough reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.\n What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\n What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half."},"other_sections_titles":{"kind":"list like","value":["Prevalence of sexual assault and socio-demographic profile of patients","Place and type of assault","Mode of presentation and type of injury sustained","Time interval between occurrence of assault and presentation","Use of weapon in assault","Profile of assailants","Involvement of law enforcement agents","What is known about this topic","What this study adds"],"string":"[\n \"Prevalence of sexual assault and socio-demographic profile of patients\",\n \"Place and type of assault\",\n \"Mode of presentation and type of injury sustained\",\n \"Time interval between occurrence of assault and presentation\",\n \"Use of weapon in assault\",\n \"Profile of assailants\",\n \"Involvement of law enforcement agents\",\n \"What is known about this topic\",\n \"What this study adds\"\n]"},"other_sections_texts":{"kind":"list like","value":["There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients","Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault","The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.","The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation","No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault","In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants","Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents","Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.","Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half."],"string":"[\n \"There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\\nSocio-demographic profile of patients\",\n \"Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\\nPlace and type of assault\",\n \"The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\",\n \"The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\\nMode of presentation and type of injury sustained\\nTime interval between occurrence of assault and presentation\",\n \"No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\\nUse of weapons in assault\",\n \"In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\\nProfile of assailants\",\n \"Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\\nInvolvement of law enforcement agents\",\n \"Sexual assault is prevalent across Nigeria, similar to the situation across the world;\\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\",\n \"Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\\nVictims were predominantly young children aged 12 years and below;\\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Results","Prevalence of sexual assault and socio-demographic profile of patients","Place and type of assault","Mode of presentation and type of injury sustained","Time interval between occurrence of assault and presentation","Use of weapon in assault","Profile of assailants","Involvement of law enforcement agents","Discussion","Conclusion","What is known about this topic","What this study adds"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Results\",\n \"Prevalence of sexual assault and socio-demographic profile of patients\",\n \"Place and type of assault\",\n \"Mode of presentation and type of injury sustained\",\n \"Time interval between occurrence of assault and presentation\",\n \"Use of weapon in assault\",\n \"Profile of assailants\",\n \"Involvement of law enforcement agents\",\n \"Discussion\",\n \"Conclusion\",\n \"What is known about this topic\",\n \"What this study adds\"\n]"},"all_sections_texts":{"kind":"list like","value":["Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].\nIn the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.","This was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital."," Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\nThere were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\n Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\nMajority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\n Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\nThe most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\n Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\nThe time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\n Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\nNo weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\n Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\nIn majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\n Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents\nAlthough reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents","There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients","Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault","The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.","The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation","No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault","In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants","Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents","Although the prevalence of sexual assault was relatively low (0.84%) in this study, it confirms the existence of the phenomenon in Sokoto, Nigeria. This figure probably represents the tip of the iceberg in terms of its burden in the study area, if the low prevalence of sexual assault reported in health facilities based studies [15, 18, 21] is juxtaposed with the very high prevalence reported in community based studies [14, 16, 17] across Nigeria. It is important to note that the prevalence of sexual assault recorded in this study is much higher than that obtained in a study in Zaria, also located in north western Nigeria that reported a prevalence of 0.06% [21]. While studies conducted in Lagos and Ile-lfe that reported prevalence of 0.76% [15] and 0.69% [8] respectively, compare well with the finding in this study, other health facilities based studies by Ashimi et al, in a rural tertiary health facility in north western Nigeria [11] and Daru et al, in Jos [28] reported higher prevalence of 3.0% and 5.6% respectively. These findings show that sexual assault is incontrovertibly a problem in Sokoto, just like other parts of Nigeria. About two-thirds (61.8%) of the victims in this study were young children aged 12 years and below. This is in agreement with the findings in studies by Bhattacharyya et al [29] and Lakew [30] in which teenagers accounted for majority of cases. The assault took place in either the victims or assailants homes in more than half of cases (54%), and in two-thirds of cases the assailants were either acquaintances (38.2%) or family members (20.6%). These findings are in concordance with the findings in studies conducted in Benin [31] and Osogbo [19]. This is not surprising considering the vulnerability of the age group involved. Most of the victims were children, who were possibly left in the care of these family members or neighbors in the absence of their parents.\nVaginal bleeding due to laceration was the commonest mode of presentation seen in this study (29.4%). This is similar to the finding in a study conducted in Birnin-Kudu, North-West Nigeria, where vaginal laceration constituted 24% of cases [11]. A possible explanation for these findings is that the two studies were conducted within the same geopolitical zone of Nigeria, perhaps with similar socio-cultural factors. However, in contrast to the finding in this study where only a fifth (20.5%) of victims presented with no demonstrable injury, majority (70.8%) of the cases in the Birni-Kudu study had no demonstrable injury. Majority of the victims in this study (61.7%) presented within 8 to 16 hours of the assault. This is in contrast to the findings from Lagos [15] and Benin City [31], where 64.5% and 47% respectively presented late. Only a few (14.7%) victims were assaulted with weapons in this study, this could be due to the fact that they were mostly young children, and were less likely to struggle with the assailants to warrant the use of weapons. Also, majority of the victims (91.2%) were assaulted by only one assailant, who was also known to them in majority of cases. This is similar to the finding in other studies [8, 32] that reported that majority of victims were assaulted by person(s) known to them. One of the most interesting findings of this study is the fact that majority of cases (58.8%) were reported to law enforcement agents, even though arrests were made in less than half (41.2%) of cases. This may not be unconnected with the fact that most of the victims were young children and their parents could have taken the decision to report the incident on their behalf, unlike adult victims that would have concealed the incident and suffer in silence due to fear of stigmatization, rejection by the society and safety concerns. This finding agrees with the findings in other studies [20, 21] that reported higher likelihood of arrests by law enforcement agents in juvenile assaults.","Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.\n What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\n What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.","Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.","Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half."],"string":"[\n \"Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].\\nIn the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.\",\n \"This was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital.\",\n \" Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\\nSocio-demographic profile of patients\\nThere were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\\nSocio-demographic profile of patients\\n Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\\nPlace and type of assault\\nMajority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\\nPlace and type of assault\\n Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\\nThe most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\\n Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\\nMode of presentation and type of injury sustained\\nTime interval between occurrence of assault and presentation\\nThe time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\\nMode of presentation and type of injury sustained\\nTime interval between occurrence of assault and presentation\\n Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\\nUse of weapons in assault\\nNo weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\\nUse of weapons in assault\\n Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\\nProfile of assailants\\nIn majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\\nProfile of assailants\\n Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\\nInvolvement of law enforcement agents\\nAlthough reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\\nInvolvement of law enforcement agents\",\n \"There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\\nSocio-demographic profile of patients\",\n \"Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\\nPlace and type of assault\",\n \"The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\",\n \"The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\\nMode of presentation and type of injury sustained\\nTime interval between occurrence of assault and presentation\",\n \"No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\\nUse of weapons in assault\",\n \"In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\\nProfile of assailants\",\n \"Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\\nInvolvement of law enforcement agents\",\n \"Although the prevalence of sexual assault was relatively low (0.84%) in this study, it confirms the existence of the phenomenon in Sokoto, Nigeria. This figure probably represents the tip of the iceberg in terms of its burden in the study area, if the low prevalence of sexual assault reported in health facilities based studies [15, 18, 21] is juxtaposed with the very high prevalence reported in community based studies [14, 16, 17] across Nigeria. It is important to note that the prevalence of sexual assault recorded in this study is much higher than that obtained in a study in Zaria, also located in north western Nigeria that reported a prevalence of 0.06% [21]. While studies conducted in Lagos and Ile-lfe that reported prevalence of 0.76% [15] and 0.69% [8] respectively, compare well with the finding in this study, other health facilities based studies by Ashimi et al, in a rural tertiary health facility in north western Nigeria [11] and Daru et al, in Jos [28] reported higher prevalence of 3.0% and 5.6% respectively. These findings show that sexual assault is incontrovertibly a problem in Sokoto, just like other parts of Nigeria. About two-thirds (61.8%) of the victims in this study were young children aged 12 years and below. This is in agreement with the findings in studies by Bhattacharyya et al [29] and Lakew [30] in which teenagers accounted for majority of cases. The assault took place in either the victims or assailants homes in more than half of cases (54%), and in two-thirds of cases the assailants were either acquaintances (38.2%) or family members (20.6%). These findings are in concordance with the findings in studies conducted in Benin [31] and Osogbo [19]. This is not surprising considering the vulnerability of the age group involved. Most of the victims were children, who were possibly left in the care of these family members or neighbors in the absence of their parents.\\nVaginal bleeding due to laceration was the commonest mode of presentation seen in this study (29.4%). This is similar to the finding in a study conducted in Birnin-Kudu, North-West Nigeria, where vaginal laceration constituted 24% of cases [11]. A possible explanation for these findings is that the two studies were conducted within the same geopolitical zone of Nigeria, perhaps with similar socio-cultural factors. However, in contrast to the finding in this study where only a fifth (20.5%) of victims presented with no demonstrable injury, majority (70.8%) of the cases in the Birni-Kudu study had no demonstrable injury. Majority of the victims in this study (61.7%) presented within 8 to 16 hours of the assault. This is in contrast to the findings from Lagos [15] and Benin City [31], where 64.5% and 47% respectively presented late. Only a few (14.7%) victims were assaulted with weapons in this study, this could be due to the fact that they were mostly young children, and were less likely to struggle with the assailants to warrant the use of weapons. Also, majority of the victims (91.2%) were assaulted by only one assailant, who was also known to them in majority of cases. This is similar to the finding in other studies [8, 32] that reported that majority of victims were assaulted by person(s) known to them. One of the most interesting findings of this study is the fact that majority of cases (58.8%) were reported to law enforcement agents, even though arrests were made in less than half (41.2%) of cases. This may not be unconnected with the fact that most of the victims were young children and their parents could have taken the decision to report the incident on their behalf, unlike adult victims that would have concealed the incident and suffer in silence due to fear of stigmatization, rejection by the society and safety concerns. This finding agrees with the findings in other studies [20, 21] that reported higher likelihood of arrests by law enforcement agents in juvenile assaults.\",\n \"Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.\\n What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;\\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\\n What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\\nVictims were predominantly young children aged 12 years and below;\\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\\nVictims were predominantly young children aged 12 years and below;\\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\",\n \"Sexual assault is prevalent across Nigeria, similar to the situation across the world;\\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\",\n \"Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\\nVictims were predominantly young children aged 12 years and below;\\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results",null,null,null,null,null,null,null,"discussion","conclusion",null,null],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\",\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Prevalence","pattern","sexual assault","UDUTH","Sokoto"],"string":"[\n \"Prevalence\",\n \"pattern\",\n \"sexual assault\",\n \"UDUTH\",\n \"Sokoto\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nSexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].\nIn the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.\n\nMethods:\nThis was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital.\n\nResults:\n Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\nThere were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\n Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\nMajority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\n Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\nThe most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\n Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\nThe time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\n Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\nNo weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\n Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\nIn majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\n Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents\nAlthough reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents\n\nPrevalence of sexual assault and socio-demographic profile of patients:\nThere were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\n\nPlace and type of assault:\nMajority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\n\nMode of presentation and type of injury sustained:\nThe most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\n\nTime interval between occurrence of assault and presentation:\nThe time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\n\nUse of weapon in assault:\nNo weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\n\nProfile of assailants:\nIn majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\n\nInvolvement of law enforcement agents:\nAlthough reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents\n\nDiscussion:\nAlthough the prevalence of sexual assault was relatively low (0.84%) in this study, it confirms the existence of the phenomenon in Sokoto, Nigeria. This figure probably represents the tip of the iceberg in terms of its burden in the study area, if the low prevalence of sexual assault reported in health facilities based studies [15, 18, 21] is juxtaposed with the very high prevalence reported in community based studies [14, 16, 17] across Nigeria. It is important to note that the prevalence of sexual assault recorded in this study is much higher than that obtained in a study in Zaria, also located in north western Nigeria that reported a prevalence of 0.06% [21]. While studies conducted in Lagos and Ile-lfe that reported prevalence of 0.76% [15] and 0.69% [8] respectively, compare well with the finding in this study, other health facilities based studies by Ashimi et al, in a rural tertiary health facility in north western Nigeria [11] and Daru et al, in Jos [28] reported higher prevalence of 3.0% and 5.6% respectively. These findings show that sexual assault is incontrovertibly a problem in Sokoto, just like other parts of Nigeria. About two-thirds (61.8%) of the victims in this study were young children aged 12 years and below. This is in agreement with the findings in studies by Bhattacharyya et al [29] and Lakew [30] in which teenagers accounted for majority of cases. The assault took place in either the victims or assailants homes in more than half of cases (54%), and in two-thirds of cases the assailants were either acquaintances (38.2%) or family members (20.6%). These findings are in concordance with the findings in studies conducted in Benin [31] and Osogbo [19]. This is not surprising considering the vulnerability of the age group involved. Most of the victims were children, who were possibly left in the care of these family members or neighbors in the absence of their parents.\nVaginal bleeding due to laceration was the commonest mode of presentation seen in this study (29.4%). This is similar to the finding in a study conducted in Birnin-Kudu, North-West Nigeria, where vaginal laceration constituted 24% of cases [11]. A possible explanation for these findings is that the two studies were conducted within the same geopolitical zone of Nigeria, perhaps with similar socio-cultural factors. However, in contrast to the finding in this study where only a fifth (20.5%) of victims presented with no demonstrable injury, majority (70.8%) of the cases in the Birni-Kudu study had no demonstrable injury. Majority of the victims in this study (61.7%) presented within 8 to 16 hours of the assault. This is in contrast to the findings from Lagos [15] and Benin City [31], where 64.5% and 47% respectively presented late. Only a few (14.7%) victims were assaulted with weapons in this study, this could be due to the fact that they were mostly young children, and were less likely to struggle with the assailants to warrant the use of weapons. Also, majority of the victims (91.2%) were assaulted by only one assailant, who was also known to them in majority of cases. This is similar to the finding in other studies [8, 32] that reported that majority of victims were assaulted by person(s) known to them. One of the most interesting findings of this study is the fact that majority of cases (58.8%) were reported to law enforcement agents, even though arrests were made in less than half (41.2%) of cases. This may not be unconnected with the fact that most of the victims were young children and their parents could have taken the decision to report the incident on their behalf, unlike adult victims that would have concealed the incident and suffer in silence due to fear of stigmatization, rejection by the society and safety concerns. This finding agrees with the findings in other studies [20, 21] that reported higher likelihood of arrests by law enforcement agents in juvenile assaults.\n\nConclusion:\nAlthough the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.\n What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\n What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\n\nWhat is known about this topic:\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\n\nWhat this study adds:\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSexual violence is an important public health problem of growing concern all over the world. This study was conducted to determine the prevalence and pattern of sexual assault managed in Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria.\n\nMethods:\nIt was a retrospective study that looked into cases of sexual assault admitted into the hospital between January 2010 and December 2014. Information on patients' biodata, and relevant details on the cases were extracted from the patients' case files and analyzed.\n\nResults:\nOut of the 5317 gynecological admissions during the period under study, 45 (0.84%) were cases of sexual assault. Of these, only 34 case files were available for data extraction. The patients' ages ranged from 2 to 37 years (mean = 12.6 + 8.3). About two thirds (61.8%) of those affected were young children (aged 12 years and below). In majority of cases (70.6%) the assault was penetrative, and in most of the cases (91.2%) only a single assailant was involved. In close to two thirds of cases, the assailant was either an acquaintance (38.2%) or a family member (20.6%). Although law enforcement agents were informed in majority (58.8%) of cases, arrests were made in less than half (41.2%).\n\nConclusions:\nAlthough the prevalence of sexual assault in this study appears to be low, a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children's movement, and stringent laws should be enacted and enforced to curb this heinous act."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nSexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].\nIn the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.\n\nConclusion:\nAlthough the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.\n What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\n What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSexual violence is an important public health problem of growing concern all over the world. This study was conducted to determine the prevalence and pattern of sexual assault managed in Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria.\n\nMethods:\nIt was a retrospective study that looked into cases of sexual assault admitted into the hospital between January 2010 and December 2014. Information on patients' biodata, and relevant details on the cases were extracted from the patients' case files and analyzed.\n\nResults:\nOut of the 5317 gynecological admissions during the period under study, 45 (0.84%) were cases of sexual assault. Of these, only 34 case files were available for data extraction. The patients' ages ranged from 2 to 37 years (mean = 12.6 + 8.3). About two thirds (61.8%) of those affected were young children (aged 12 years and below). In majority of cases (70.6%) the assault was penetrative, and in most of the cases (91.2%) only a single assailant was involved. In close to two thirds of cases, the assailant was either an acquaintance (38.2%) or a family member (20.6%). Although law enforcement agents were informed in majority (58.8%) of cases, arrests were made in less than half (41.2%).\n\nConclusions:\nAlthough the prevalence of sexual assault in this study appears to be low, a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children's movement, and stringent laws should be enacted and enforced to curb this heinous act."},"whole_article_text_length":{"kind":"number","value":4541,"string":"4,541"},"whole_article_abstract_length":{"kind":"number","value":316,"string":"316"},"other_sections_lengths":{"kind":"list like","value":[153,130,86,89,115,70,48,58,51],"string":"[\n 153,\n 130,\n 86,\n 89,\n 115,\n 70,\n 48,\n 58,\n 51\n]"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["assault","victims","cases","sexual","majority","years","sexual assault","study","aged","prevalence"],"string":"[\n \"assault\",\n \"victims\",\n \"cases\",\n \"sexual\",\n \"majority\",\n \"years\",\n \"sexual assault\",\n \"study\",\n \"aged\",\n \"prevalence\"\n]"},"keybert_topics":{"kind":"list like","value":["victim sexual assault","concerns sexual assault","sexual assault study","sexual abuse occurs","child sexual 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Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] sexual | reported | abuse | female | rape | likely | study | assault | sexual assault | sexual abuse [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | data | files patients | statistical | ethical | case files patients | case files | case | files | patients [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cases | victims | assault | 34 | presentation | table | years | patients | assailants | majority [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] nigeria | predominantly young | appears low | appears | predominantly young children | predominantly | sexual assault | sexual | children | sokoto nigeria [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cases | victims | assault | majority | sexual | years | 34 | sexual assault | aged | table [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cases | victims | assault | majority | sexual | years | 34 | sexual assault | aged | table [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] between January 2010 and December 2014 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5317 | 45 | 0.84% ||| only 34 ||| 2 to 37 years | 12.6 + 8.3 ||| About two thirds | 61.8% | 12 years ||| 70.6% | 91.2% ||| close to two thirds | 38.2% | 20.6% ||| 58.8% | less than half | 41.2% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria ||| between January 2010 and December 2014 ||| ||| 5317 | 45 | 0.84% ||| only 34 ||| 2 to 37 years | 12.6 + 8.3 ||| About two thirds | 61.8% | 12 years ||| 70.6% | 91.2% ||| close to two thirds | 38.2% | 20.6% ||| 58.8% | less than half | 41.2% ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria ||| between January 2010 and December 2014 ||| ||| 5317 | 45 | 0.84% ||| only 34 ||| 2 to 37 years | 12.6 + 8.3 ||| About two thirds | 61.8% | 12 years ||| 70.6% | 91.2% ||| close to two thirds | 38.2% | 20.6% ||| 58.8% | less than half | 41.2% ||| ||| [SUMMARY]"}}},{"rowIdx":3378,"cells":{"title":{"kind":"string","value":"Association between Epstein-Barr Virus Gene Polymorphism and Breast Cancer Risk among Egyptian Females."},"pmid":{"kind":"string","value":"35225477"},"background_abstract":{"kind":"string","value":"Epstein-Barr virus (EBV) has been implicated in the development of breast cancer (BC) since 1995. It is classified into A/B genotypes, C/D subtypes, and F/f variants according to variations in its genome."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Three hundred and sixty-two participants (142 BC patients and 220 controls) were enrolled in this study. All participants were screened for EBV infection by determination of viral-capsid-IgG antibodies in their sera. EBNA-1 gene was detected by PCR in tumor biopsies of seropositive patients and in peripheral blood mononuclear cells of controls. A/B genotyping of EBV was performed by nested-PCR targeting the EBNA-2 gene. C/D subtypes and F/f variants were identified by Restriction fragment length polymorphism at BamHI-I W1/I1 and BamHI-F regions of EBV genome, respectively."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Among 362 participants, 300(82.9%) were EBV-seropositive, including 120/142(84.5%) of the BC patients and 180/220(81.8 %) of the controls. EBNA-1 gene was positive in 54(45%) of seropositive BC patients and in 38(21.1%) of seropositive controls. There was a significant association of EBNA-1 gene with breast cancer (OR=3.05, 95%CI=1.84-5.07). Moreover, EBNA-1 gene positivity was significantly associated with the more aggressive tumors. Genotype-A and prototype-F were predominant among patients (90.4%, 100%, respectively) as well as among controls (91.7%, 100%, respectively) with no statistical significant association with BC risk. However, subtype-D was significantly more frequent in patients (95.6%) than in controls (64.7%) and was significantly associated with a higher BC risk as compared to subtype-C (OR=11.7, 95%CI=2.4-57.08). Subtype-D was significantly associated with higher grades tumors (100% among grade III), with progesteron receptor-negative tumors and with HER2-positive tumors (100% for each). The combined genotypes that significantly associated with BC risk were ADF (OR=4.9) and BDF (OR=5.5)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Subtype-D of EBV could be the only EBV type implicated in BC development among Egyptian females and associated more with poor prognosis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Breast Neoplasms","Case-Control Studies","Egypt","Epstein-Barr Virus Infections","Epstein-Barr Virus Nuclear Antigens","Female","Genotype","Herpesvirus 4, Human","Humans","Leukocytes, Mononuclear","Middle Aged","Polymerase Chain Reaction","Polymorphism, Restriction Fragment Length","Prognosis","Viral Proteins"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Breast Neoplasms\",\n \"Case-Control Studies\",\n \"Egypt\",\n \"Epstein-Barr Virus Infections\",\n \"Epstein-Barr Virus Nuclear Antigens\",\n \"Female\",\n \"Genotype\",\n \"Herpesvirus 4, Human\",\n \"Humans\",\n \"Leukocytes, Mononuclear\",\n \"Middle Aged\",\n \"Polymerase Chain Reaction\",\n \"Polymorphism, Restriction Fragment Length\",\n \"Prognosis\",\n \"Viral Proteins\"\n]"},"pmcid":{"kind":"string","value":"9272632"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).\nEBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).\nThe presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).\nEBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015). \nEBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007). \nDiagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).\nBreast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015). \nIn 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).\nTo date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"\nDetection of anti-VCA IgG of EBV by ELISA\n\nScreening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.\n\nDetection of EBV DNA by PCR \n\nEBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.\n\nGenotyping of EBV \n\n\nAnalysis of A/B types of EBV \n\nSuccessful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.\n\nAnalysis of C/D subtypes of EBV \n\nAmplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.\n\nAnalysis of F/f variants of EBV \n\nThe BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.\n\nCombined Genotypes of EBV\n\nA comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.\n\nAssociation between EBV DNA positivity and tumor characteristics\n\nAmong 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4. \n\nAssociation between EBV genotypes and tumor characteristics\n\nA/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.\nTarget Genes and Their Primer Sequence\nbp, base pair; F, forward; R, reverse.\nFrequency of EBV Infection in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer. \nFlowchart Indicating the Design and Results of the Present Study\nEBV Typing in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions\nAssociation between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients\n*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2 \nAssociation between Specific EBV Genotypes and Tumor Features\nIDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth"},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Author Contribution Statement"],"string":"[\n \"Author Contribution Statement\"\n]"},"other_sections_texts":{"kind":"list like","value":["Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript."],"string":"[\n \"Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and Methods","Results","Discussion","Author Contribution Statement"],"string":"[\n \"Introduction\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion\",\n \"Author Contribution Statement\"\n]"},"all_sections_texts":{"kind":"list like","value":["Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).\nEBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).\nThe presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).\nEBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015). \nEBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007). \nDiagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).\nBreast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015). \nIn 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).\nTo date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC.","\nStudy subjects \n\nThis case-control study included 362 female participants; 142 breast cancer (BC) patients and 220 healthy controls. All patients and controls were recruited from the Oncology Center, Faculty of Medicine, Mansoura University (OCMU) in Egypt through the period from January 2019 to December 2020. The ages of all patients and controls were matched (p >0.05). Patients’ ages ranged from 37 to 79 years old with a median age of 54 years. The diagnosis of BC in all enrolled patients was based on clinical examination, radiological findings, and histopathological examination of tissue biopsy. Control subjects were selected from women who attended OCMU for annual mammography as part of a breast checkup and we excluded those with a history of BC and those with fibroadenomas.\n\nSample collection\n\nFresh tumor tissue and blood specimen were obtained from BC patients, whereas only blood specimen was obtained from the control group. The tissue sample from each tumor was divided into two parts; one part was fixed in formaldehyde and delivered to OCMU’s pathology laboratory for histopathological examination, while the other part was stored at -80°C to be used for PCR. The blood specimen was obtained via venipuncture and divided into two tubes; a plain tube for separation of serum and an EDTA tube for separation of peripheral blood mononuclear cells (PBMCs), both of which were kept at –80°C until use.\n\nStudy design \n\nA Flowchart showing the workflow of this study is presented in Figure 1. All included BC patients and controls were screened for EBV infection by the detection of anti-viral capsid antigen (VCA) IgG antibodies in their sera. Then EBV seropositive subjects were investigated for the presence of EBV DNA by PCR amplification of the EBNA-1 gene; either in tumor biopsy of BC patients or in PBMCs of controls. Thereafter, A/B genotypes, C/D subtypes and F/f variants of EBV were identified in all EBV-DNA positive specimens by analysis of three regions in the EBV genome (EBNA-2 gene, BamHI-I W1/I1 and BamHI-F regions, respectively).\n\nHistopathological examination \n\nDiagnosis of BC and determination of its histological type was achieved in OCMU’s pathology laboratory by examination of hematoxylin-eosin (H-E) stained slides prepared from tumor biopsies. Only tumor biopsies with cancerous cells of more than 60% were included in the study. Grading of the tumors was performed according to the Scarff Bloom Richardson classification (Bloom and Richardson, 1957). Detection of hormonal receptor status [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)] of all included tumor biopsies was done by immunohistochemical staining of slides cut from tissue microarray prepared blocks as described previously (Ahmed and Yussif, 2016).\n\nDetection of EBV-viral capsid antigen (VCA) IgG antibodies\n\nScreening of all included BC patients and controls for EBV infection was performed by detection of anti-VCA IgG antibodies by ELISA (Bio-Rad Medical Diagnostics, GmbH, Germany) following the manufacturer guidelines.\n\nPolymerase chain reaction (PCR) amplification of EBNA-1 gene\n\nDetection of EBV DNA was performed by PCR amplification of EBNA-1 gene. DNA was extracted from all specimens with serologically confirmed EBV infection (120 tumor biopsies from BC patients and 180 PMNCs from controls) using the QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s tissue and blood protocols. Then EBV DNA was detected by PCR amplification of the EBNA-1 gene using specific primers listed in Table 1 according to the previously described PCR protocol (Hashimoto et al., 1995).\n\nDetection of A/B genotypes of EBV \n\nNested PCR that targets EBNA-2 gene was used for genotyping of EBV into genotype-A and genotype-B (Ayee et al., 2020). In the first round of PCR amplification, a common region of EBNA-2 was amplified using EBNA-2A and EBNA-2B as forward and reverse primers, respectively. A PCR reaction was performed in a total volume of 12.5ul and the final concentrations of each component were as follows; 1X One TaqR Quick-Load 2X master mix with standard buffer (Bio Labs, UK), 0.5µM from each of the forward and reverse primers, and 0.5µg/µL of the extracted DNA. Amplification was achieved in a thermal cycler under the following conditions; 2min at 94°C for initial denaturation, then followed by 35 cycles of amplification (each cycle consisted of 60s at 94°C, 90s at 52°C and 4min at 72°C), followed by a cycle of 10min at 72°C for final extension.\nIn the 2nd round of nested PCR, the reaction volume was 12.5ul and the components were the same as in the 1st round except that 0.5ul of the amplified product from the 1st round was used as a template and three primers were used; EBNA-2C as a forward primer (common to genotype-A and genotype-B), EBNA-2D and EBNA-2E as reverse primers specific for genotype-A and genotype-B, respectively. The cycling conditions were as follows: An initial cycle of heat denaturation for 2min at 94°C, 35 amplification cycles (30s at 94°C, 60s at 52°C, and 2 min at 72°C), and a final extension cycle for 10min at 72°C. The resultant amplicon was visualized by electrophoresis on an agarose gel with 2% ethidium bromide. Samples were identified by the presence of their respective bands (250 bp for genotype-A and 300 bp for genotype-B).\n\nDetection of C/D subtypes and F/f variants of EBV \n\nRestriction fragment length polymorphism (RFLP) was used for classification of the EBV into C/D subtypes and F/f variants according to the existence of an extra restriction enzyme site at the BamHI-I W1/I1 and BamHI-F regions of the EBV genome, respectively. PCR was performed, followed by RFLP as previously described (Henry et al., 2001).\nIn brief, the reaction conditions for PCR amplification of each region were similar except for the use of forward and reverse primers specific for each region. The total reaction volume was 25ul consisting of 22.0µl of PCR master mix (1x), 1µl of each forward and reverse primer (0.5μM), and 1µl of DNA template. PCR cycles included an initial step of denaturation at 94°C for 5min followed by 40 amplification cycles; denaturation at 94ºC for 30s, annealing at 55°C for 30s and extension at 72°C for 1min , thereafter a step of final extension for 10min at 72°C. After PCR, the enzymatic digestion of the amplified product was performed in a total reaction volume of 20ul that contained 10µl of the amplified products, 2µl of reaction buffer, 1.5ul of distilled water, and 1.5µl of BamHI endonucleases (10U/ul) (Promega, Madison). This mixture was incubated at 37ºC for 2hs then followed by agarose gel (2%) electrophoresis of enzyme-digested BamHI-I W1/I1 region and BamHI-F region for determination of C/D subtypes and F/f variants, respectively. The product size of BamHI W1/I1 region after enzyme digestion is either 206 bp with subtype-C or 139bp and 67bp with subtype-D. For the products of enzyme digested BamHI F region, the presence of one band (199 bp) identified an F-variant whereas the presence of two bands (128bp and 71bp) indicated an f-variant. \n\nStatistical analysis \n\nThe Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The qualitative data were expressed as frequencies and percentages. The differences in distribution of EBV types between tissue biopsies of BC patients and sera of the control group were compared by a univarite binary logistic regression test with the calculation of odds ratio (OR) and 95% confidence intervals (CIs). The difference between groups was considered statistically significant if the p-value was less than 0.05.","\nDetection of anti-VCA IgG of EBV by ELISA\n\nScreening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.\n\nDetection of EBV DNA by PCR \n\nEBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.\n\nGenotyping of EBV \n\n\nAnalysis of A/B types of EBV \n\nSuccessful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.\n\nAnalysis of C/D subtypes of EBV \n\nAmplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.\n\nAnalysis of F/f variants of EBV \n\nThe BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.\n\nCombined Genotypes of EBV\n\nA comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.\n\nAssociation between EBV DNA positivity and tumor characteristics\n\nAmong 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4. \n\nAssociation between EBV genotypes and tumor characteristics\n\nA/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.\nTarget Genes and Their Primer Sequence\nbp, base pair; F, forward; R, reverse.\nFrequency of EBV Infection in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer. \nFlowchart Indicating the Design and Results of the Present Study\nEBV Typing in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions\nAssociation between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients\n*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2 \nAssociation between Specific EBV Genotypes and Tumor Features\nIDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth","The presence of different polymorphisms in certain regions of the EBV genome resulted in the presence of different genotypes, subtypes, and variants of EBV (Ayadi et al., 2007). However, little is known about how EBV genetic diversity may affect EBV-associated disease (Palser et al., 2015). Moreover, up to date, there is a controversy whether these EBV variants are geographically related, or disease-associated. In 1995, the association of EBV with BC was reported but no study has been conducted in Egypt since then to investigate if there is a specific association of BC with certain types of EBV or if it is simply a geographical distribution. \nIn this study, the seroprevalence of EBV was 84.5% in BC patients and was 81.8% in the control group with no statistical significant difference. This is consistent with the high prevalence of EBV in the world. It is estimated that more than 90% of the world’s population is EBV-seropositive (Tzellos and Farrell, 2012). Smatti et al., (2017) reported that the EBV seroprevalence rate among healthy blood donors was 100% in Egyptians and Pakistanis, 98.3% in Indians, 96.8% in Syrians, and 97.8% in Qataris.\nAlthough this study detected a high seroprevalence of EBV among all study participants, the latent infection with EBV (as detected by molecular detection of the EBNA-1 gene) was significantly higher in BC tumor biopsies (45%) as compared to PMNCs of the control group (21.1%). This is in line with two previous Egyptian studies that detected EBV infection in 35.3% and 25% of breast carcinomas (Mohamed et al., 2007; Fawzy et al., 2008). Additionally, using PCR as a method of EBV detection, the prevalence of EBV infection among BC patients in various regions of the world was about 10–50% (Perkins et al., 2006; Lorenzetti et al., 2010). Other studies, on the other hand, failed to detect EBV among BC patients (Lespagnard et al., 1995; Kijima et al., 2001). This discrepancy in the results could be attributed to a variety of factors, such as geographical variation, differences in EBV infection detection methods, specimen type, use of different genes in the EBV genome for PCR, and the inclusion of different histological types of BC. However, studies that used biopsy specimens and used nested PCR as a method for detection showed less heterogeneity (Farahmand et al., 2019).\nIn this case-control study, EBV-DNA positivity increased the risk of BC development by about threefold (OR = 3.05). This is similar to two recent meta-analysis that reported that the risk of BC association with EBV infection is 3.84 and 4.74 folds higher as compared to controls (Bae and Kim, 2016; Farahmand et al., 2019). \nIn the current study, in an attempt to clarify if there is an association between BC and specific types of EBV in Egypt, we detected the distribution frequency of different EBV types obtained from tumor tissues of BC women and compared them with those obtained from PMNCs of healthy non-BC females.\nRegarding the distribution of A/B genotypes of EBV among the studied BC patients and healthy controls, we found that most of the EBV detected in both BC tumor biopsies and PBMNCs of the controls were of genotype-A (90.9% and 91.7%, respectively, p= 0.8). The high prevalence of genotype-A among the participants of this study is consistent with its geographical distribution. Genotype-A is the most prevalent genotype of EBV in the world, predominantly in the United States, France, Germany, North Africa, Turky, and Asia. Whereas, genotype of EBV predominates more in Central Africa and New Guinea (Ibrahim et al., 2010). In this study, the distribution difference of genotype-A and gentype-B was statistically insignificant between BC patients and the control group. As a result, it appears that neither genotype-A nor genotype-B has a preferential association with BC in Egypt. In the same context, the association of A/B genotypes of EBV with other malignant diseases such as NPC seems to be variable worldwide. Tamura et al (1993) reported no association of A/B genotypes of EBV with NPC in Japan, as they were predominant in patients as well as in general populations. Klemenc et al., (2006) and Ayadi et al., (2007), reported a high incidence of genotype-A in Slovenian and Tunisian patients with NPC, respectively. Ayee et al., (2020) identified genotype-B as the virulent genotype in Ghana and associated it with the likelihood of NPC development in Ghanaian patients.\nRegarding the polymorphism in the BamHI-I W1/I1 region. This study demonstrated that the frequency of subtype-D EBV is significantly higher in BC tumor biopsies (95.6%) as compared to controls (64.7%) (OR =11.7, 95%CI = 2.4-57.1, P =0.002). Similarly, Zhang et al., (2017) found a significant association between subtype-D and the risk of BC among Chinese populations (OR = 2.86), suggesting that subtype-D may play a role in the pathogenesis of BC. It is worth noting that the association of EBV subtype-D with other malignant diseases has previously been demonstrated; Abdel Hamid et al., (1992) discovered that subtype-D is detected more frequently in Egyptian NPC than subtype-C. Similarly, it was reported that EBV of subtype-D was associated with NPC in each of Slovenian patients (62.5%), Tunisian population (98%) and Northern China (32.2%) (Cui et al., 2011). Moreover, subtype-D was found to be associated not only with NPC but also with gastric carcinoma in Iranian population (90%), in Southern Tunisian population (100%), in Latin American population (85%) (Corvalan et al., 2006; Abdirad et al., 2007; BenAyed-Guerfali et al., 2011). \nThe genetic evidence that C/D subtypes of EBV could be related to the development of tumors is that some EBV genes, such as BamH1-A Rightward Frame-1 (BARF1) and Latent Membrane Protein-2A are present near the C/D locus. These genes are involved in transformation and immortalization during the tumorigenic process (Corvalan et al., 2006). However, the precise mechanisms need more exploration.\nRegarding the polymorphism at the BamHI-F region, we detected that EBV from all BC tumor biopsies and control PMNCs was of prototype-F. Therefore, the association of prototype-F with Egyptian BC is not likely. It was proposed that f-variant is only a geographic polymorphism because the EBV that was detected in Egyptian, Algerian, and Tunisian NPCs was of prototype-F, whereas the EBV that was detected in Asian NPCs was of variant-f (Cui et al., 2011). However, Zhou et al., (2001) reported that variant-f is found only in Chinese NPCs and not in Chinese HD patients. Therefore, the hypothesis that the f-variant is a geographically restricted polymorphism needs further investigation. \nIn the present study, ADF was the most prevalent combination of EBV genotypes among BC patients as well as among control subjects. Likewise, Klemenc et al., (2006) found that ADF is the most predominant combination of EBV genotypes in Slovenian patients with NPC as well as in the healthy population. Additionally, the risk of BC was significantly higher with each of ADF (OR=4.92) and BDF (OR=5.54) combinations and not significantly increased with BCF (P=0.217). Therefore, our findings could point to the implication of specific combinations of EBV genotypes in the development of BC in Egypt. Consequently, our results are not consistent with the hypothesis suggested by Khanim et al (1996) that EBV strains are geographically determined rather than disease-associated. \nAs regards the association between EBV and the characteristics of BC. EBV was detected more frequently in BC of larger size, higher stage, and higher grade. These findings are similar to that made previously by Murray et al., (2003) and Mohamed et al., (2007) who found that EBV is significantly associated with the more aggressive tumors. Other studies, however, debate our findings as they did not find any statistically significant association between EBV positivity and either grade, stage, or metastasis of the tumor (Richardson et al., 2015; Ahmed and Yussif, 2016; El-Naby et al., 2017).\nIn this study, there was no demonstrable significant association between EBV positivity and the steroid-hormone receptor. This agrees with many previous studies (Tiwawech et al., 2008; Ahmed and Yussif, 2016; Bae and Kim, 2016; Preciado et al., 2005). On the contrary, other investigators have shown that EBV is significantly associated with steroid hormone receptor-negative tumors (Bonnet et al., 1999; Murray et al., 2003; El-Naby et al., 2017). \nWe found that 75% of EBV-DNA positive BC biopsies are also reactive to HER2 expression. This is close to the result reported by Mohamed et al., (2007) who reported that 55.9% of DNA positive cases were HER2 positive. This finding correlates with the recognition that EBV could transform epithelial cells of the breast into malignancy via stimulating the HER2/HER3 signalling pathways (Szostek et al., 2009). Furthermore, our findings point to some extent that subtype-D of EBV could be the only EBV type that might be implicated in the development of BC with poor prognosis because it was the only type that showed more association with tumor of higher grade and with progesterone receptor-free tumors (p= 0.04). \nIn Conclusion, to the best of our knowledge, this is the first study in Egypt that provides baseline data on the prevalence of different EBV types in BC patients as well as in healthy controls. Overall, this study found that the D-subtype of EBV is BC-associated. Whereas, A/B genotypes and F/f variants of EBV are not associated with BC. Additionally, specific combinations of EBV genotypes (ADF and BDF) were BC- associated. Moreover, our findings indicated that EBV could have an impact on the histological criteria of BC and thus could have a role in prognosis and in optimizing the therapeutic strategies for BC. Nevertheless, our results are considered a platform for any future studies with a larger number of patients with BC in Egypt as well as in other parts of the world to investigate whether variations in the distribution of different types of EBV are geographically related or BC related. ","Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript."],"string":"[\n \"Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).\\nEBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).\\nThe presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).\\nEBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015). \\nEBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007). \\nDiagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).\\nBreast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015). \\nIn 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).\\nTo date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC.\",\n \"\\nStudy subjects \\n\\nThis case-control study included 362 female participants; 142 breast cancer (BC) patients and 220 healthy controls. All patients and controls were recruited from the Oncology Center, Faculty of Medicine, Mansoura University (OCMU) in Egypt through the period from January 2019 to December 2020. The ages of all patients and controls were matched (p >0.05). Patients’ ages ranged from 37 to 79 years old with a median age of 54 years. The diagnosis of BC in all enrolled patients was based on clinical examination, radiological findings, and histopathological examination of tissue biopsy. Control subjects were selected from women who attended OCMU for annual mammography as part of a breast checkup and we excluded those with a history of BC and those with fibroadenomas.\\n\\nSample collection\\n\\nFresh tumor tissue and blood specimen were obtained from BC patients, whereas only blood specimen was obtained from the control group. The tissue sample from each tumor was divided into two parts; one part was fixed in formaldehyde and delivered to OCMU’s pathology laboratory for histopathological examination, while the other part was stored at -80°C to be used for PCR. The blood specimen was obtained via venipuncture and divided into two tubes; a plain tube for separation of serum and an EDTA tube for separation of peripheral blood mononuclear cells (PBMCs), both of which were kept at –80°C until use.\\n\\nStudy design \\n\\nA Flowchart showing the workflow of this study is presented in Figure 1. All included BC patients and controls were screened for EBV infection by the detection of anti-viral capsid antigen (VCA) IgG antibodies in their sera. Then EBV seropositive subjects were investigated for the presence of EBV DNA by PCR amplification of the EBNA-1 gene; either in tumor biopsy of BC patients or in PBMCs of controls. Thereafter, A/B genotypes, C/D subtypes and F/f variants of EBV were identified in all EBV-DNA positive specimens by analysis of three regions in the EBV genome (EBNA-2 gene, BamHI-I W1/I1 and BamHI-F regions, respectively).\\n\\nHistopathological examination \\n\\nDiagnosis of BC and determination of its histological type was achieved in OCMU’s pathology laboratory by examination of hematoxylin-eosin (H-E) stained slides prepared from tumor biopsies. Only tumor biopsies with cancerous cells of more than 60% were included in the study. Grading of the tumors was performed according to the Scarff Bloom Richardson classification (Bloom and Richardson, 1957). Detection of hormonal receptor status [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)] of all included tumor biopsies was done by immunohistochemical staining of slides cut from tissue microarray prepared blocks as described previously (Ahmed and Yussif, 2016).\\n\\nDetection of EBV-viral capsid antigen (VCA) IgG antibodies\\n\\nScreening of all included BC patients and controls for EBV infection was performed by detection of anti-VCA IgG antibodies by ELISA (Bio-Rad Medical Diagnostics, GmbH, Germany) following the manufacturer guidelines.\\n\\nPolymerase chain reaction (PCR) amplification of EBNA-1 gene\\n\\nDetection of EBV DNA was performed by PCR amplification of EBNA-1 gene. DNA was extracted from all specimens with serologically confirmed EBV infection (120 tumor biopsies from BC patients and 180 PMNCs from controls) using the QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s tissue and blood protocols. Then EBV DNA was detected by PCR amplification of the EBNA-1 gene using specific primers listed in Table 1 according to the previously described PCR protocol (Hashimoto et al., 1995).\\n\\nDetection of A/B genotypes of EBV \\n\\nNested PCR that targets EBNA-2 gene was used for genotyping of EBV into genotype-A and genotype-B (Ayee et al., 2020). In the first round of PCR amplification, a common region of EBNA-2 was amplified using EBNA-2A and EBNA-2B as forward and reverse primers, respectively. A PCR reaction was performed in a total volume of 12.5ul and the final concentrations of each component were as follows; 1X One TaqR Quick-Load 2X master mix with standard buffer (Bio Labs, UK), 0.5µM from each of the forward and reverse primers, and 0.5µg/µL of the extracted DNA. Amplification was achieved in a thermal cycler under the following conditions; 2min at 94°C for initial denaturation, then followed by 35 cycles of amplification (each cycle consisted of 60s at 94°C, 90s at 52°C and 4min at 72°C), followed by a cycle of 10min at 72°C for final extension.\\nIn the 2nd round of nested PCR, the reaction volume was 12.5ul and the components were the same as in the 1st round except that 0.5ul of the amplified product from the 1st round was used as a template and three primers were used; EBNA-2C as a forward primer (common to genotype-A and genotype-B), EBNA-2D and EBNA-2E as reverse primers specific for genotype-A and genotype-B, respectively. The cycling conditions were as follows: An initial cycle of heat denaturation for 2min at 94°C, 35 amplification cycles (30s at 94°C, 60s at 52°C, and 2 min at 72°C), and a final extension cycle for 10min at 72°C. The resultant amplicon was visualized by electrophoresis on an agarose gel with 2% ethidium bromide. Samples were identified by the presence of their respective bands (250 bp for genotype-A and 300 bp for genotype-B).\\n\\nDetection of C/D subtypes and F/f variants of EBV \\n\\nRestriction fragment length polymorphism (RFLP) was used for classification of the EBV into C/D subtypes and F/f variants according to the existence of an extra restriction enzyme site at the BamHI-I W1/I1 and BamHI-F regions of the EBV genome, respectively. PCR was performed, followed by RFLP as previously described (Henry et al., 2001).\\nIn brief, the reaction conditions for PCR amplification of each region were similar except for the use of forward and reverse primers specific for each region. The total reaction volume was 25ul consisting of 22.0µl of PCR master mix (1x), 1µl of each forward and reverse primer (0.5μM), and 1µl of DNA template. PCR cycles included an initial step of denaturation at 94°C for 5min followed by 40 amplification cycles; denaturation at 94ºC for 30s, annealing at 55°C for 30s and extension at 72°C for 1min , thereafter a step of final extension for 10min at 72°C. After PCR, the enzymatic digestion of the amplified product was performed in a total reaction volume of 20ul that contained 10µl of the amplified products, 2µl of reaction buffer, 1.5ul of distilled water, and 1.5µl of BamHI endonucleases (10U/ul) (Promega, Madison). This mixture was incubated at 37ºC for 2hs then followed by agarose gel (2%) electrophoresis of enzyme-digested BamHI-I W1/I1 region and BamHI-F region for determination of C/D subtypes and F/f variants, respectively. The product size of BamHI W1/I1 region after enzyme digestion is either 206 bp with subtype-C or 139bp and 67bp with subtype-D. For the products of enzyme digested BamHI F region, the presence of one band (199 bp) identified an F-variant whereas the presence of two bands (128bp and 71bp) indicated an f-variant. \\n\\nStatistical analysis \\n\\nThe Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The qualitative data were expressed as frequencies and percentages. The differences in distribution of EBV types between tissue biopsies of BC patients and sera of the control group were compared by a univarite binary logistic regression test with the calculation of odds ratio (OR) and 95% confidence intervals (CIs). The difference between groups was considered statistically significant if the p-value was less than 0.05.\",\n \"\\nDetection of anti-VCA IgG of EBV by ELISA\\n\\nScreening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.\\n\\nDetection of EBV DNA by PCR \\n\\nEBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.\\n\\nGenotyping of EBV \\n\\n\\nAnalysis of A/B types of EBV \\n\\nSuccessful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.\\n\\nAnalysis of C/D subtypes of EBV \\n\\nAmplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.\\n\\nAnalysis of F/f variants of EBV \\n\\nThe BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.\\n\\nCombined Genotypes of EBV\\n\\nA comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.\\n\\nAssociation between EBV DNA positivity and tumor characteristics\\n\\nAmong 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4. \\n\\nAssociation between EBV genotypes and tumor characteristics\\n\\nA/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.\\nTarget Genes and Their Primer Sequence\\nbp, base pair; F, forward; R, reverse.\\nFrequency of EBV Infection in Breast Cancer Patients and Controls\\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer. \\nFlowchart Indicating the Design and Results of the Present Study\\nEBV Typing in Breast Cancer Patients and Controls\\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions\\nAssociation between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients\\n*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2 \\nAssociation between Specific EBV Genotypes and Tumor Features\\nIDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth\",\n \"The presence of different polymorphisms in certain regions of the EBV genome resulted in the presence of different genotypes, subtypes, and variants of EBV (Ayadi et al., 2007). However, little is known about how EBV genetic diversity may affect EBV-associated disease (Palser et al., 2015). Moreover, up to date, there is a controversy whether these EBV variants are geographically related, or disease-associated. In 1995, the association of EBV with BC was reported but no study has been conducted in Egypt since then to investigate if there is a specific association of BC with certain types of EBV or if it is simply a geographical distribution. \\nIn this study, the seroprevalence of EBV was 84.5% in BC patients and was 81.8% in the control group with no statistical significant difference. This is consistent with the high prevalence of EBV in the world. It is estimated that more than 90% of the world’s population is EBV-seropositive (Tzellos and Farrell, 2012). Smatti et al., (2017) reported that the EBV seroprevalence rate among healthy blood donors was 100% in Egyptians and Pakistanis, 98.3% in Indians, 96.8% in Syrians, and 97.8% in Qataris.\\nAlthough this study detected a high seroprevalence of EBV among all study participants, the latent infection with EBV (as detected by molecular detection of the EBNA-1 gene) was significantly higher in BC tumor biopsies (45%) as compared to PMNCs of the control group (21.1%). This is in line with two previous Egyptian studies that detected EBV infection in 35.3% and 25% of breast carcinomas (Mohamed et al., 2007; Fawzy et al., 2008). Additionally, using PCR as a method of EBV detection, the prevalence of EBV infection among BC patients in various regions of the world was about 10–50% (Perkins et al., 2006; Lorenzetti et al., 2010). Other studies, on the other hand, failed to detect EBV among BC patients (Lespagnard et al., 1995; Kijima et al., 2001). This discrepancy in the results could be attributed to a variety of factors, such as geographical variation, differences in EBV infection detection methods, specimen type, use of different genes in the EBV genome for PCR, and the inclusion of different histological types of BC. However, studies that used biopsy specimens and used nested PCR as a method for detection showed less heterogeneity (Farahmand et al., 2019).\\nIn this case-control study, EBV-DNA positivity increased the risk of BC development by about threefold (OR = 3.05). This is similar to two recent meta-analysis that reported that the risk of BC association with EBV infection is 3.84 and 4.74 folds higher as compared to controls (Bae and Kim, 2016; Farahmand et al., 2019). \\nIn the current study, in an attempt to clarify if there is an association between BC and specific types of EBV in Egypt, we detected the distribution frequency of different EBV types obtained from tumor tissues of BC women and compared them with those obtained from PMNCs of healthy non-BC females.\\nRegarding the distribution of A/B genotypes of EBV among the studied BC patients and healthy controls, we found that most of the EBV detected in both BC tumor biopsies and PBMNCs of the controls were of genotype-A (90.9% and 91.7%, respectively, p= 0.8). The high prevalence of genotype-A among the participants of this study is consistent with its geographical distribution. Genotype-A is the most prevalent genotype of EBV in the world, predominantly in the United States, France, Germany, North Africa, Turky, and Asia. Whereas, genotype of EBV predominates more in Central Africa and New Guinea (Ibrahim et al., 2010). In this study, the distribution difference of genotype-A and gentype-B was statistically insignificant between BC patients and the control group. As a result, it appears that neither genotype-A nor genotype-B has a preferential association with BC in Egypt. In the same context, the association of A/B genotypes of EBV with other malignant diseases such as NPC seems to be variable worldwide. Tamura et al (1993) reported no association of A/B genotypes of EBV with NPC in Japan, as they were predominant in patients as well as in general populations. Klemenc et al., (2006) and Ayadi et al., (2007), reported a high incidence of genotype-A in Slovenian and Tunisian patients with NPC, respectively. Ayee et al., (2020) identified genotype-B as the virulent genotype in Ghana and associated it with the likelihood of NPC development in Ghanaian patients.\\nRegarding the polymorphism in the BamHI-I W1/I1 region. This study demonstrated that the frequency of subtype-D EBV is significantly higher in BC tumor biopsies (95.6%) as compared to controls (64.7%) (OR =11.7, 95%CI = 2.4-57.1, P =0.002). Similarly, Zhang et al., (2017) found a significant association between subtype-D and the risk of BC among Chinese populations (OR = 2.86), suggesting that subtype-D may play a role in the pathogenesis of BC. It is worth noting that the association of EBV subtype-D with other malignant diseases has previously been demonstrated; Abdel Hamid et al., (1992) discovered that subtype-D is detected more frequently in Egyptian NPC than subtype-C. Similarly, it was reported that EBV of subtype-D was associated with NPC in each of Slovenian patients (62.5%), Tunisian population (98%) and Northern China (32.2%) (Cui et al., 2011). Moreover, subtype-D was found to be associated not only with NPC but also with gastric carcinoma in Iranian population (90%), in Southern Tunisian population (100%), in Latin American population (85%) (Corvalan et al., 2006; Abdirad et al., 2007; BenAyed-Guerfali et al., 2011). \\nThe genetic evidence that C/D subtypes of EBV could be related to the development of tumors is that some EBV genes, such as BamH1-A Rightward Frame-1 (BARF1) and Latent Membrane Protein-2A are present near the C/D locus. These genes are involved in transformation and immortalization during the tumorigenic process (Corvalan et al., 2006). However, the precise mechanisms need more exploration.\\nRegarding the polymorphism at the BamHI-F region, we detected that EBV from all BC tumor biopsies and control PMNCs was of prototype-F. Therefore, the association of prototype-F with Egyptian BC is not likely. It was proposed that f-variant is only a geographic polymorphism because the EBV that was detected in Egyptian, Algerian, and Tunisian NPCs was of prototype-F, whereas the EBV that was detected in Asian NPCs was of variant-f (Cui et al., 2011). However, Zhou et al., (2001) reported that variant-f is found only in Chinese NPCs and not in Chinese HD patients. Therefore, the hypothesis that the f-variant is a geographically restricted polymorphism needs further investigation. \\nIn the present study, ADF was the most prevalent combination of EBV genotypes among BC patients as well as among control subjects. Likewise, Klemenc et al., (2006) found that ADF is the most predominant combination of EBV genotypes in Slovenian patients with NPC as well as in the healthy population. Additionally, the risk of BC was significantly higher with each of ADF (OR=4.92) and BDF (OR=5.54) combinations and not significantly increased with BCF (P=0.217). Therefore, our findings could point to the implication of specific combinations of EBV genotypes in the development of BC in Egypt. Consequently, our results are not consistent with the hypothesis suggested by Khanim et al (1996) that EBV strains are geographically determined rather than disease-associated. \\nAs regards the association between EBV and the characteristics of BC. EBV was detected more frequently in BC of larger size, higher stage, and higher grade. These findings are similar to that made previously by Murray et al., (2003) and Mohamed et al., (2007) who found that EBV is significantly associated with the more aggressive tumors. Other studies, however, debate our findings as they did not find any statistically significant association between EBV positivity and either grade, stage, or metastasis of the tumor (Richardson et al., 2015; Ahmed and Yussif, 2016; El-Naby et al., 2017).\\nIn this study, there was no demonstrable significant association between EBV positivity and the steroid-hormone receptor. This agrees with many previous studies (Tiwawech et al., 2008; Ahmed and Yussif, 2016; Bae and Kim, 2016; Preciado et al., 2005). On the contrary, other investigators have shown that EBV is significantly associated with steroid hormone receptor-negative tumors (Bonnet et al., 1999; Murray et al., 2003; El-Naby et al., 2017). \\nWe found that 75% of EBV-DNA positive BC biopsies are also reactive to HER2 expression. This is close to the result reported by Mohamed et al., (2007) who reported that 55.9% of DNA positive cases were HER2 positive. This finding correlates with the recognition that EBV could transform epithelial cells of the breast into malignancy via stimulating the HER2/HER3 signalling pathways (Szostek et al., 2009). Furthermore, our findings point to some extent that subtype-D of EBV could be the only EBV type that might be implicated in the development of BC with poor prognosis because it was the only type that showed more association with tumor of higher grade and with progesterone receptor-free tumors (p= 0.04). \\nIn Conclusion, to the best of our knowledge, this is the first study in Egypt that provides baseline data on the prevalence of different EBV types in BC patients as well as in healthy controls. Overall, this study found that the D-subtype of EBV is BC-associated. Whereas, A/B genotypes and F/f variants of EBV are not associated with BC. Additionally, specific combinations of EBV genotypes (ADF and BDF) were BC- associated. Moreover, our findings indicated that EBV could have an impact on the histological criteria of BC and thus could have a role in prognosis and in optimizing the therapeutic strategies for BC. Nevertheless, our results are considered a platform for any future studies with a larger number of patients with BC in Egypt as well as in other parts of the world to investigate whether variations in the distribution of different types of EBV are geographically related or BC related. \",\n \"Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods","results","discussion",null],"string":"[\n \"intro\",\n \"materials|methods\",\n \"results\",\n \"discussion\",\n null\n]"},"keywords":{"kind":"list like","value":["Epstein","Barr virus","breast","cancer","RFLP","genotyping","Egypt"],"string":"[\n \"Epstein\",\n \"Barr virus\",\n \"breast\",\n \"cancer\",\n \"RFLP\",\n \"genotyping\",\n \"Egypt\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nEpstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).\nEBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).\nThe presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).\nEBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015). \nEBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007). \nDiagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).\nBreast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015). \nIn 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).\nTo date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC.\n\nMaterials and Methods:\n\nStudy subjects \n\nThis case-control study included 362 female participants; 142 breast cancer (BC) patients and 220 healthy controls. All patients and controls were recruited from the Oncology Center, Faculty of Medicine, Mansoura University (OCMU) in Egypt through the period from January 2019 to December 2020. The ages of all patients and controls were matched (p >0.05). Patients’ ages ranged from 37 to 79 years old with a median age of 54 years. The diagnosis of BC in all enrolled patients was based on clinical examination, radiological findings, and histopathological examination of tissue biopsy. Control subjects were selected from women who attended OCMU for annual mammography as part of a breast checkup and we excluded those with a history of BC and those with fibroadenomas.\n\nSample collection\n\nFresh tumor tissue and blood specimen were obtained from BC patients, whereas only blood specimen was obtained from the control group. The tissue sample from each tumor was divided into two parts; one part was fixed in formaldehyde and delivered to OCMU’s pathology laboratory for histopathological examination, while the other part was stored at -80°C to be used for PCR. The blood specimen was obtained via venipuncture and divided into two tubes; a plain tube for separation of serum and an EDTA tube for separation of peripheral blood mononuclear cells (PBMCs), both of which were kept at –80°C until use.\n\nStudy design \n\nA Flowchart showing the workflow of this study is presented in Figure 1. All included BC patients and controls were screened for EBV infection by the detection of anti-viral capsid antigen (VCA) IgG antibodies in their sera. Then EBV seropositive subjects were investigated for the presence of EBV DNA by PCR amplification of the EBNA-1 gene; either in tumor biopsy of BC patients or in PBMCs of controls. Thereafter, A/B genotypes, C/D subtypes and F/f variants of EBV were identified in all EBV-DNA positive specimens by analysis of three regions in the EBV genome (EBNA-2 gene, BamHI-I W1/I1 and BamHI-F regions, respectively).\n\nHistopathological examination \n\nDiagnosis of BC and determination of its histological type was achieved in OCMU’s pathology laboratory by examination of hematoxylin-eosin (H-E) stained slides prepared from tumor biopsies. Only tumor biopsies with cancerous cells of more than 60% were included in the study. Grading of the tumors was performed according to the Scarff Bloom Richardson classification (Bloom and Richardson, 1957). Detection of hormonal receptor status [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)] of all included tumor biopsies was done by immunohistochemical staining of slides cut from tissue microarray prepared blocks as described previously (Ahmed and Yussif, 2016).\n\nDetection of EBV-viral capsid antigen (VCA) IgG antibodies\n\nScreening of all included BC patients and controls for EBV infection was performed by detection of anti-VCA IgG antibodies by ELISA (Bio-Rad Medical Diagnostics, GmbH, Germany) following the manufacturer guidelines.\n\nPolymerase chain reaction (PCR) amplification of EBNA-1 gene\n\nDetection of EBV DNA was performed by PCR amplification of EBNA-1 gene. DNA was extracted from all specimens with serologically confirmed EBV infection (120 tumor biopsies from BC patients and 180 PMNCs from controls) using the QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s tissue and blood protocols. Then EBV DNA was detected by PCR amplification of the EBNA-1 gene using specific primers listed in Table 1 according to the previously described PCR protocol (Hashimoto et al., 1995).\n\nDetection of A/B genotypes of EBV \n\nNested PCR that targets EBNA-2 gene was used for genotyping of EBV into genotype-A and genotype-B (Ayee et al., 2020). In the first round of PCR amplification, a common region of EBNA-2 was amplified using EBNA-2A and EBNA-2B as forward and reverse primers, respectively. A PCR reaction was performed in a total volume of 12.5ul and the final concentrations of each component were as follows; 1X One TaqR Quick-Load 2X master mix with standard buffer (Bio Labs, UK), 0.5µM from each of the forward and reverse primers, and 0.5µg/µL of the extracted DNA. Amplification was achieved in a thermal cycler under the following conditions; 2min at 94°C for initial denaturation, then followed by 35 cycles of amplification (each cycle consisted of 60s at 94°C, 90s at 52°C and 4min at 72°C), followed by a cycle of 10min at 72°C for final extension.\nIn the 2nd round of nested PCR, the reaction volume was 12.5ul and the components were the same as in the 1st round except that 0.5ul of the amplified product from the 1st round was used as a template and three primers were used; EBNA-2C as a forward primer (common to genotype-A and genotype-B), EBNA-2D and EBNA-2E as reverse primers specific for genotype-A and genotype-B, respectively. The cycling conditions were as follows: An initial cycle of heat denaturation for 2min at 94°C, 35 amplification cycles (30s at 94°C, 60s at 52°C, and 2 min at 72°C), and a final extension cycle for 10min at 72°C. The resultant amplicon was visualized by electrophoresis on an agarose gel with 2% ethidium bromide. Samples were identified by the presence of their respective bands (250 bp for genotype-A and 300 bp for genotype-B).\n\nDetection of C/D subtypes and F/f variants of EBV \n\nRestriction fragment length polymorphism (RFLP) was used for classification of the EBV into C/D subtypes and F/f variants according to the existence of an extra restriction enzyme site at the BamHI-I W1/I1 and BamHI-F regions of the EBV genome, respectively. PCR was performed, followed by RFLP as previously described (Henry et al., 2001).\nIn brief, the reaction conditions for PCR amplification of each region were similar except for the use of forward and reverse primers specific for each region. The total reaction volume was 25ul consisting of 22.0µl of PCR master mix (1x), 1µl of each forward and reverse primer (0.5μM), and 1µl of DNA template. PCR cycles included an initial step of denaturation at 94°C for 5min followed by 40 amplification cycles; denaturation at 94ºC for 30s, annealing at 55°C for 30s and extension at 72°C for 1min , thereafter a step of final extension for 10min at 72°C. After PCR, the enzymatic digestion of the amplified product was performed in a total reaction volume of 20ul that contained 10µl of the amplified products, 2µl of reaction buffer, 1.5ul of distilled water, and 1.5µl of BamHI endonucleases (10U/ul) (Promega, Madison). This mixture was incubated at 37ºC for 2hs then followed by agarose gel (2%) electrophoresis of enzyme-digested BamHI-I W1/I1 region and BamHI-F region for determination of C/D subtypes and F/f variants, respectively. The product size of BamHI W1/I1 region after enzyme digestion is either 206 bp with subtype-C or 139bp and 67bp with subtype-D. For the products of enzyme digested BamHI F region, the presence of one band (199 bp) identified an F-variant whereas the presence of two bands (128bp and 71bp) indicated an f-variant. \n\nStatistical analysis \n\nThe Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The qualitative data were expressed as frequencies and percentages. The differences in distribution of EBV types between tissue biopsies of BC patients and sera of the control group were compared by a univarite binary logistic regression test with the calculation of odds ratio (OR) and 95% confidence intervals (CIs). The difference between groups was considered statistically significant if the p-value was less than 0.05.\n\nResults:\n\nDetection of anti-VCA IgG of EBV by ELISA\n\nScreening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.\n\nDetection of EBV DNA by PCR \n\nEBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.\n\nGenotyping of EBV \n\n\nAnalysis of A/B types of EBV \n\nSuccessful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.\n\nAnalysis of C/D subtypes of EBV \n\nAmplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.\n\nAnalysis of F/f variants of EBV \n\nThe BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.\n\nCombined Genotypes of EBV\n\nA comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.\n\nAssociation between EBV DNA positivity and tumor characteristics\n\nAmong 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4. \n\nAssociation between EBV genotypes and tumor characteristics\n\nA/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.\nTarget Genes and Their Primer Sequence\nbp, base pair; F, forward; R, reverse.\nFrequency of EBV Infection in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer. \nFlowchart Indicating the Design and Results of the Present Study\nEBV Typing in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions\nAssociation between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients\n*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2 \nAssociation between Specific EBV Genotypes and Tumor Features\nIDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth\n\nDiscussion:\nThe presence of different polymorphisms in certain regions of the EBV genome resulted in the presence of different genotypes, subtypes, and variants of EBV (Ayadi et al., 2007). However, little is known about how EBV genetic diversity may affect EBV-associated disease (Palser et al., 2015). Moreover, up to date, there is a controversy whether these EBV variants are geographically related, or disease-associated. In 1995, the association of EBV with BC was reported but no study has been conducted in Egypt since then to investigate if there is a specific association of BC with certain types of EBV or if it is simply a geographical distribution. \nIn this study, the seroprevalence of EBV was 84.5% in BC patients and was 81.8% in the control group with no statistical significant difference. This is consistent with the high prevalence of EBV in the world. It is estimated that more than 90% of the world’s population is EBV-seropositive (Tzellos and Farrell, 2012). Smatti et al., (2017) reported that the EBV seroprevalence rate among healthy blood donors was 100% in Egyptians and Pakistanis, 98.3% in Indians, 96.8% in Syrians, and 97.8% in Qataris.\nAlthough this study detected a high seroprevalence of EBV among all study participants, the latent infection with EBV (as detected by molecular detection of the EBNA-1 gene) was significantly higher in BC tumor biopsies (45%) as compared to PMNCs of the control group (21.1%). This is in line with two previous Egyptian studies that detected EBV infection in 35.3% and 25% of breast carcinomas (Mohamed et al., 2007; Fawzy et al., 2008). Additionally, using PCR as a method of EBV detection, the prevalence of EBV infection among BC patients in various regions of the world was about 10–50% (Perkins et al., 2006; Lorenzetti et al., 2010). Other studies, on the other hand, failed to detect EBV among BC patients (Lespagnard et al., 1995; Kijima et al., 2001). This discrepancy in the results could be attributed to a variety of factors, such as geographical variation, differences in EBV infection detection methods, specimen type, use of different genes in the EBV genome for PCR, and the inclusion of different histological types of BC. However, studies that used biopsy specimens and used nested PCR as a method for detection showed less heterogeneity (Farahmand et al., 2019).\nIn this case-control study, EBV-DNA positivity increased the risk of BC development by about threefold (OR = 3.05). This is similar to two recent meta-analysis that reported that the risk of BC association with EBV infection is 3.84 and 4.74 folds higher as compared to controls (Bae and Kim, 2016; Farahmand et al., 2019). \nIn the current study, in an attempt to clarify if there is an association between BC and specific types of EBV in Egypt, we detected the distribution frequency of different EBV types obtained from tumor tissues of BC women and compared them with those obtained from PMNCs of healthy non-BC females.\nRegarding the distribution of A/B genotypes of EBV among the studied BC patients and healthy controls, we found that most of the EBV detected in both BC tumor biopsies and PBMNCs of the controls were of genotype-A (90.9% and 91.7%, respectively, p= 0.8). The high prevalence of genotype-A among the participants of this study is consistent with its geographical distribution. Genotype-A is the most prevalent genotype of EBV in the world, predominantly in the United States, France, Germany, North Africa, Turky, and Asia. Whereas, genotype of EBV predominates more in Central Africa and New Guinea (Ibrahim et al., 2010). In this study, the distribution difference of genotype-A and gentype-B was statistically insignificant between BC patients and the control group. As a result, it appears that neither genotype-A nor genotype-B has a preferential association with BC in Egypt. In the same context, the association of A/B genotypes of EBV with other malignant diseases such as NPC seems to be variable worldwide. Tamura et al (1993) reported no association of A/B genotypes of EBV with NPC in Japan, as they were predominant in patients as well as in general populations. Klemenc et al., (2006) and Ayadi et al., (2007), reported a high incidence of genotype-A in Slovenian and Tunisian patients with NPC, respectively. Ayee et al., (2020) identified genotype-B as the virulent genotype in Ghana and associated it with the likelihood of NPC development in Ghanaian patients.\nRegarding the polymorphism in the BamHI-I W1/I1 region. This study demonstrated that the frequency of subtype-D EBV is significantly higher in BC tumor biopsies (95.6%) as compared to controls (64.7%) (OR =11.7, 95%CI = 2.4-57.1, P =0.002). Similarly, Zhang et al., (2017) found a significant association between subtype-D and the risk of BC among Chinese populations (OR = 2.86), suggesting that subtype-D may play a role in the pathogenesis of BC. It is worth noting that the association of EBV subtype-D with other malignant diseases has previously been demonstrated; Abdel Hamid et al., (1992) discovered that subtype-D is detected more frequently in Egyptian NPC than subtype-C. Similarly, it was reported that EBV of subtype-D was associated with NPC in each of Slovenian patients (62.5%), Tunisian population (98%) and Northern China (32.2%) (Cui et al., 2011). Moreover, subtype-D was found to be associated not only with NPC but also with gastric carcinoma in Iranian population (90%), in Southern Tunisian population (100%), in Latin American population (85%) (Corvalan et al., 2006; Abdirad et al., 2007; BenAyed-Guerfali et al., 2011). \nThe genetic evidence that C/D subtypes of EBV could be related to the development of tumors is that some EBV genes, such as BamH1-A Rightward Frame-1 (BARF1) and Latent Membrane Protein-2A are present near the C/D locus. These genes are involved in transformation and immortalization during the tumorigenic process (Corvalan et al., 2006). However, the precise mechanisms need more exploration.\nRegarding the polymorphism at the BamHI-F region, we detected that EBV from all BC tumor biopsies and control PMNCs was of prototype-F. Therefore, the association of prototype-F with Egyptian BC is not likely. It was proposed that f-variant is only a geographic polymorphism because the EBV that was detected in Egyptian, Algerian, and Tunisian NPCs was of prototype-F, whereas the EBV that was detected in Asian NPCs was of variant-f (Cui et al., 2011). However, Zhou et al., (2001) reported that variant-f is found only in Chinese NPCs and not in Chinese HD patients. Therefore, the hypothesis that the f-variant is a geographically restricted polymorphism needs further investigation. \nIn the present study, ADF was the most prevalent combination of EBV genotypes among BC patients as well as among control subjects. Likewise, Klemenc et al., (2006) found that ADF is the most predominant combination of EBV genotypes in Slovenian patients with NPC as well as in the healthy population. Additionally, the risk of BC was significantly higher with each of ADF (OR=4.92) and BDF (OR=5.54) combinations and not significantly increased with BCF (P=0.217). Therefore, our findings could point to the implication of specific combinations of EBV genotypes in the development of BC in Egypt. Consequently, our results are not consistent with the hypothesis suggested by Khanim et al (1996) that EBV strains are geographically determined rather than disease-associated. \nAs regards the association between EBV and the characteristics of BC. EBV was detected more frequently in BC of larger size, higher stage, and higher grade. These findings are similar to that made previously by Murray et al., (2003) and Mohamed et al., (2007) who found that EBV is significantly associated with the more aggressive tumors. Other studies, however, debate our findings as they did not find any statistically significant association between EBV positivity and either grade, stage, or metastasis of the tumor (Richardson et al., 2015; Ahmed and Yussif, 2016; El-Naby et al., 2017).\nIn this study, there was no demonstrable significant association between EBV positivity and the steroid-hormone receptor. This agrees with many previous studies (Tiwawech et al., 2008; Ahmed and Yussif, 2016; Bae and Kim, 2016; Preciado et al., 2005). On the contrary, other investigators have shown that EBV is significantly associated with steroid hormone receptor-negative tumors (Bonnet et al., 1999; Murray et al., 2003; El-Naby et al., 2017). \nWe found that 75% of EBV-DNA positive BC biopsies are also reactive to HER2 expression. This is close to the result reported by Mohamed et al., (2007) who reported that 55.9% of DNA positive cases were HER2 positive. This finding correlates with the recognition that EBV could transform epithelial cells of the breast into malignancy via stimulating the HER2/HER3 signalling pathways (Szostek et al., 2009). Furthermore, our findings point to some extent that subtype-D of EBV could be the only EBV type that might be implicated in the development of BC with poor prognosis because it was the only type that showed more association with tumor of higher grade and with progesterone receptor-free tumors (p= 0.04). \nIn Conclusion, to the best of our knowledge, this is the first study in Egypt that provides baseline data on the prevalence of different EBV types in BC patients as well as in healthy controls. Overall, this study found that the D-subtype of EBV is BC-associated. Whereas, A/B genotypes and F/f variants of EBV are not associated with BC. Additionally, specific combinations of EBV genotypes (ADF and BDF) were BC- associated. Moreover, our findings indicated that EBV could have an impact on the histological criteria of BC and thus could have a role in prognosis and in optimizing the therapeutic strategies for BC. Nevertheless, our results are considered a platform for any future studies with a larger number of patients with BC in Egypt as well as in other parts of the world to investigate whether variations in the distribution of different types of EBV are geographically related or BC related. \n\nAuthor Contribution Statement:\nMashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nEpstein-Barr virus (EBV) has been implicated in the development of breast cancer (BC) since 1995. It is classified into A/B genotypes, C/D subtypes, and F/f variants according to variations in its genome.\n\nMethods:\nThree hundred and sixty-two participants (142 BC patients and 220 controls) were enrolled in this study. All participants were screened for EBV infection by determination of viral-capsid-IgG antibodies in their sera. EBNA-1 gene was detected by PCR in tumor biopsies of seropositive patients and in peripheral blood mononuclear cells of controls. A/B genotyping of EBV was performed by nested-PCR targeting the EBNA-2 gene. C/D subtypes and F/f variants were identified by Restriction fragment length polymorphism at BamHI-I W1/I1 and BamHI-F regions of EBV genome, respectively.\n\nResults:\nAmong 362 participants, 300(82.9%) were EBV-seropositive, including 120/142(84.5%) of the BC patients and 180/220(81.8 %) of the controls. EBNA-1 gene was positive in 54(45%) of seropositive BC patients and in 38(21.1%) of seropositive controls. There was a significant association of EBNA-1 gene with breast cancer (OR=3.05, 95%CI=1.84-5.07). Moreover, EBNA-1 gene positivity was significantly associated with the more aggressive tumors. Genotype-A and prototype-F were predominant among patients (90.4%, 100%, respectively) as well as among controls (91.7%, 100%, respectively) with no statistical significant association with BC risk. However, subtype-D was significantly more frequent in patients (95.6%) than in controls (64.7%) and was significantly associated with a higher BC risk as compared to subtype-C (OR=11.7, 95%CI=2.4-57.08). Subtype-D was significantly associated with higher grades tumors (100% among grade III), with progesteron receptor-negative tumors and with HER2-positive tumors (100% for each). The combined genotypes that significantly associated with BC risk were ADF (OR=4.9) and BDF (OR=5.5).\n\nConclusions:\nSubtype-D of EBV could be the only EBV type implicated in BC development among Egyptian females and associated more with poor prognosis."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5997,"string":"5,997"},"whole_article_abstract_length":{"kind":"number","value":430,"string":"430"},"other_sections_lengths":{"kind":"list like","value":[124],"string":"[\n 124\n]"},"num_sections":{"kind":"number","value":5,"string":"5"},"most_frequent_words":{"kind":"list like","value":["ebv","bc","patients","genotype","bc patients","association","tumor","bamhi","study","controls"],"string":"[\n \"ebv\",\n \"bc\",\n \"patients\",\n \"genotype\",\n \"bc patients\",\n \"association\",\n \"tumor\",\n \"bamhi\",\n \"study\",\n \"controls\"\n]"},"keybert_topics":{"kind":"list like","value":["revealed genotype ebv","ebv genome identified","genes ebv genome","genotypes ebv studied","different genes ebv"],"string":"[\n \"revealed genotype ebv\",\n \"ebv genome identified\",\n \"genes ebv genome\",\n \"genotypes ebv studied\",\n \"different genes ebv\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein | Barr virus | breast | cancer | RFLP | genotyping | Egypt [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein | Barr virus | breast | cancer | RFLP | genotyping | Egypt [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein | Barr virus | breast | cancer | RFLP | genotyping | Egypt [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breast Neoplasms | Case-Control Studies | Egypt | Epstein-Barr Virus Infections | Epstein-Barr Virus Nuclear Antigens | Female | Genotype | Herpesvirus 4, Human | Humans | Leukocytes, Mononuclear | Middle Aged | Polymerase Chain Reaction | Polymorphism, Restriction Fragment Length | Prognosis | Viral Proteins [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breast Neoplasms | Case-Control Studies | Egypt | Epstein-Barr Virus Infections | Epstein-Barr Virus Nuclear Antigens | Female | Genotype | Herpesvirus 4, Human | Humans | Leukocytes, Mononuclear | Middle Aged | Polymerase Chain Reaction | Polymorphism, Restriction Fragment Length | Prognosis | Viral Proteins [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Breast Neoplasms | Case-Control Studies | Egypt | Epstein-Barr Virus Infections | Epstein-Barr Virus Nuclear Antigens | Female | Genotype | Herpesvirus 4, Human | Humans | Leukocytes, Mononuclear | Middle Aged | Polymerase Chain Reaction | Polymorphism, Restriction Fragment Length | Prognosis | Viral Proteins [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] revealed genotype ebv | ebv genome identified | genes ebv genome | genotypes ebv studied | different genes ebv [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] revealed genotype ebv | ebv genome identified | genes ebv genome | genotypes ebv studied | different genes ebv [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] revealed genotype ebv | ebv genome identified | genes ebv genome | genotypes ebv studied | different genes ebv [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | bc | patients | genotype | bc patients | association | tumor | bamhi | study | controls [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | bc | patients | genotype | bc patients | association | tumor | bamhi | study | controls [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | bc | patients | genotype | bc patients | association | tumor | bamhi | study | controls [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | bamhi | genotype | bc | antibodies | vca | women | igg | genotype genotype | different [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | bc | patients | bc patients | tumors | tumor | dna | ebv dna | vs | table [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ebv | bc | patients | genotype | bc patients | study | association | bamhi | tumor | pcr [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein-Barr | EBV | BC | 1995 ||| F/ [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 362 | 300(82.9% | EBV | 120/142(84.5% | BC | 180/220(81.8 % ||| 54(45% | BC | 38(21.1% ||| OR=3.05 | 95%CI=1.84-5.07 ||| ||| Genotype-A | 90.4% | 100% | 91.7% | 100% | BC ||| 95.6% | 64.7% | BC | OR=11.7 | 95%CI=2.4-57.08 ||| 100% | 100% ||| BC | BDF [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Epstein-Barr | EBV | BC | 1995 ||| F/ ||| Three hundred and sixty-two | 142 | BC | 220 ||| EBV ||| PCR ||| EBV ||| F/ | Restriction | EBV ||| ||| 362 | 300(82.9% | EBV | 120/142(84.5% | BC | 180/220(81.8 % ||| 54(45% | BC | 38(21.1% ||| OR=3.05 | 95%CI=1.84-5.07 ||| ||| Genotype-A | 90.4% | 100% | 91.7% | 100% | BC ||| 95.6% | 64.7% | BC | OR=11.7 | 95%CI=2.4-57.08 ||| 100% | 100% ||| BC | BDF ||| EBV | EBV | BC | Egyptian [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3379,"cells":{"title":{"kind":"string","value":"Evaluation of Three Multiplex Real-time Reverse Transcription PCR Assays for Simultaneous Detection of SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus in Nasopharyngeal Swabs."},"pmid":{"kind":"string","value":"34904407"},"background_abstract":{"kind":"string","value":"In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["COVID-19","Cross Reactions","Humans","Influenza A virus","Influenza B virus","Influenza, Human","Limit of Detection","Multiplex Polymerase Chain Reaction","Nasopharynx","Nucleocapsid Proteins","Polyproteins","RNA, Viral","Reagent Kits, Diagnostic","Republic of Korea","Respiratory Syncytial Virus Infections","Respiratory Syncytial Virus, Human","SARS-CoV-2","Viral Matrix Proteins","Viral Proteins"],"string":"[\n \"COVID-19\",\n \"Cross Reactions\",\n \"Humans\",\n \"Influenza A virus\",\n \"Influenza B virus\",\n \"Influenza, Human\",\n \"Limit of Detection\",\n \"Multiplex Polymerase Chain Reaction\",\n \"Nasopharynx\",\n \"Nucleocapsid Proteins\",\n \"Polyproteins\",\n \"RNA, Viral\",\n \"Reagent Kits, Diagnostic\",\n \"Republic of Korea\",\n \"Respiratory Syncytial Virus Infections\",\n \"Respiratory Syncytial Virus, Human\",\n \"SARS-CoV-2\",\n \"Viral Matrix Proteins\",\n \"Viral Proteins\"\n]"},"pmcid":{"kind":"string","value":"8668494"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10\nSARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).\nSince the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.\nMolecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS)."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nPowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nSpecimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nWe selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nSample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nA 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nAnalytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nThe LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nEvaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nReference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nEthics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\nThis study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262)."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Analytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nIn comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nClinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\nCompared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV","Specimen collection","Sample processing and PCR","Analytical performance: limit of detection (LoD) and cross-reactivity","Evaluation of the clinical performance of the three molecular diagnostic kits","Ethics statement","Analytical performance","Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV"],"string":"[\n \"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV\",\n \"Specimen collection\",\n \"Sample processing and PCR\",\n \"Analytical performance: limit of detection (LoD) and cross-reactivity\",\n \"Evaluation of the clinical performance of the three molecular diagnostic kits\",\n \"Ethics statement\",\n \"Analytical performance\",\n \"Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV\"\n]"},"other_sections_texts":{"kind":"list like","value":["PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.","We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.","A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.","The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.","Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).","This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).","In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.","Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples."],"string":"[\n \"PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\",\n \"We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\",\n \"A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\",\n \"The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\",\n \"Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\",\n \"This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\",\n \"In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\",\n \"Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV","Specimen collection","Sample processing and PCR","Analytical performance: limit of detection (LoD) and cross-reactivity","Evaluation of the clinical performance of the three molecular diagnostic kits","Ethics statement","RESULTS","Analytical performance","Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV\",\n \"Specimen collection\",\n \"Sample processing and PCR\",\n \"Analytical performance: limit of detection (LoD) and cross-reactivity\",\n \"Evaluation of the clinical performance of the three molecular diagnostic kits\",\n \"Ethics statement\",\n \"RESULTS\",\n \"Analytical performance\",\n \"Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10\nSARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).\nSince the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.\nMolecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS).","Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nPowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nSpecimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nWe selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nSample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nA 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nAnalytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nThe LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nEvaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nReference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nEthics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\nThis study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).","PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.","We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.","A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.","The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.","Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).","This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).","Analytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nIn comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nClinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\nCompared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.","In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.","Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.","In the evaluation of analytical sensitivity, the LoD is dependent on reference material kits, extraction methods, PCR machines, and other experimental conditions. Therefore, the LoDs reported in our study may not correspond with practical LoDs in the real world. In the evaluation of cross-reactivity of the three assays, all three kits did not present cross-reactivity with common respiratory viruses. Especially, for SARS-CoV-2, three assays showed no reactive with coronavirus 229E, OC43, and NL63.\nAll three kits had 100% NPA in SARS-CoV-2 results, but PPAs in SARS-CoV-2 results ranged from 92.8% to 95.9%; among 97 SARS-CoV-2 positive samples, 10 (10.3%) produced discrepant results among the three assays. All three kits had 100% PPA in influenza A/B results. In contrast to 100% NPA in influenza B results in all three kits, NPAs in influenza A results ranged from 99.7% to 100.0% due to two false influenza A-positive reactions. According to the LoD testing of AdvanSure and Allplex kits and RSV sequencing results, Allplex detected RSV more sensitively than AdvanSure.\nMolecular diagnostic kits for detecting SARS-CoV-2 have shifted from single SARS-CoV-2 detection to multi-detection of SARS-CoV-2 and other common respiratory pathogens. Nörz et al.23 adapted a laboratory-developed multiplex RT-PCR assay for the simultaneous detection of SARS-CoV-2 and influenza A/B on a fully automated high-throughput system and demonstrated analytical performance comparable with that of currently available commercial tests. Sensitivities of SARS-CoV-2, influenza A, and influenza B in the multiplex assay were 98.1%, 97.67%, and 100%, respectively.23 Xpert Xpress SARS-CoV-2/Flu-RSV (Xpert 4-in-1 assay) was launched with Emergency Use Authorization of the United States Food and Drug Administration in September 2020 and demonstrated a performance highly comparable with those of Xpert SARS-CoV-2 and Xpert Flu/RSV assays.24 PPAs and NPAs of SARS-CoV-2, influenza A, influenza B, and RSV in Xpert -in-1 assay were 98.48%/100%, 100%/100%, 100%/99.54%, and 100%/100%, respectively.24\nA case of co-infection of SARS-CoV-2 and other respiratory viruses was absent in the present study. SARS-CoV-2-positive specimens were collected between September 2020 and November 2020. In Korea, RSV and influenza outbreak seasons classically begin in autumn and winter, respectively.25 Although the collection period overlapped with RSV outbreak season, due to COVID-19-related quarantine and improved hygiene, transmission rates of influenza and RSV were lower.26 A US research team reported that 20.7% of specimens positive for SARS-CoV-2 were positive for one or more additional pathogens, compared with 26.7% negative for SARS-CoV-2.27 The most commonly co-infected pathogens were rhinovirus/enterovirus followed by RSV and non-SARS-CoV-2 Coronaviridae.27 A single-centered retrospective study was performed in Wuhan, China, between January 12 and February 21, 2020 during the initial COVID-19 outbreak.28 Unexpectedly, among COVID-19 patients, only 42.7% (131/307) of patients were singly positive for SARS-CoV-2; 57.3% (176/307) of COVID-19 patients were also positive for influenza viruses including influenza A (49.8%) and influenza B (7.5%).28 Another single-centered retrospective study conducted in Lukou District, China showed that 14.1% (11/78) of COVID-19 patients were co-infected with other respiratory pathogens including RSV (3/11, 36.3%) and influenza B (1/11, 9.1%).14 In contrast, a Korean research team demonstrated co-infection cases of SARS-CoV-2 and influenza virus or RSV were absent.29 Using the Allplex RV-Essential Assay (Seegene) detecting seven respiratory viruses including adenovirus, influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, RSV, and human rhinovirus, the co-infection rate in COVID-19 patients was 2.2%.29 The most frequently detected virus was adenovirus, followed by human rhinovirus and metapneumovirus.29 However, comprehensive PCR testing to detect respiratory viruses other than influenza virus and RSV was not performed in SARS-CoV-2-positive specimens in the present study.\nPowerChek, STANDARD M, and Allplex kits each specified different cycle conditions for rRT-PCR. PowerChek did not require any pre-amplification cycles during PCR. In the PCR protocol of STANDARD M, five pre-amplification cycles preceded the amplification cycles, and the time and temperature conditions of both cycles were the same. Allplex had three pre-amplification cycles with different time and temperature conditions from those of the amplification cycles. Volume of template RNA per test and the number of pre-amplification cycles and amplification cycles of the three multi-target kits were compared with corresponding single SARS-CoV-2 detection kits: PowerChek 2019-nCoV Real-time PCR (Kogene Biotech), STANDARD M nCoV Real-Time Detection (SD BioSensor), and Allplex 2019-nCoV (Seegene).17 KogeneBiotech and SD BioSensor had the same volume of template RNA per test and number of pre-amplification and amplification cycles in each pair of single- and multi-target assays. However, the multi-target assay of Allplex required 10 μL of template RNA compared with 8 μL in the single-target assay. Allplex single-target assay performed 45 amplification cycles without pre-amplification but the multi-target assay executed 3 pre-amplification and 42 amplification cycles. Allplex multi-target assay was thought to be designed to increase sensitivity than the single-target assay by increasing the volume of template RNA.\nThis study had some limitations. First, as previously mentioned, the evaluation of the LoD of the kits may not have reflected the practical analytical sensitivity because the measured LoD may have been affected by extraction method, type of PCR machine, etc. Second, for SARS-CoV-2, cross-reactivity test with other coronaviruses except 229E, OC43, and NL63 was not performed. Third, although the AdvanSure kit was treated as a comparator kit in this study, a true reference method was absent. Indeed, the AdvanSure kit was less sensitive for RSV compared with the Allplex kit. Finally, because multiplex PCR used to detect respiratory viruses other than influenza A/B and RSV was not performed in SARS-CoV-2-positive samples, co-infection rates of other respiratory viruses and SARS-CoV-2 could not be estimated.\nIn conclusion, the overall performance of these three multiplex rRT-PCR assays for concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. In the COVID-19 pandemic era, all three kits are suitable for the prompt differential diagnosis of COVID-19, influenza, and RSV infection in patients with respiratory symptoms."],"string":"[\n \"The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10\\nSARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).\\nSince the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.\\nMolecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS).\",\n \"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\\nPowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\\nSpecimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\\nWe selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\\nSample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\\nA 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\\nAnalytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\\nThe LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\\nEvaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\\nReference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\\nEthics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\\nThis study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\",\n \"PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\",\n \"We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\",\n \"A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\",\n \"The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\",\n \"Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\",\n \"This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\",\n \"Analytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\\nIn comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\\nClinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\\nCompared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\",\n \"In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\",\n \"Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\",\n \"In the evaluation of analytical sensitivity, the LoD is dependent on reference material kits, extraction methods, PCR machines, and other experimental conditions. Therefore, the LoDs reported in our study may not correspond with practical LoDs in the real world. In the evaluation of cross-reactivity of the three assays, all three kits did not present cross-reactivity with common respiratory viruses. Especially, for SARS-CoV-2, three assays showed no reactive with coronavirus 229E, OC43, and NL63.\\nAll three kits had 100% NPA in SARS-CoV-2 results, but PPAs in SARS-CoV-2 results ranged from 92.8% to 95.9%; among 97 SARS-CoV-2 positive samples, 10 (10.3%) produced discrepant results among the three assays. All three kits had 100% PPA in influenza A/B results. In contrast to 100% NPA in influenza B results in all three kits, NPAs in influenza A results ranged from 99.7% to 100.0% due to two false influenza A-positive reactions. According to the LoD testing of AdvanSure and Allplex kits and RSV sequencing results, Allplex detected RSV more sensitively than AdvanSure.\\nMolecular diagnostic kits for detecting SARS-CoV-2 have shifted from single SARS-CoV-2 detection to multi-detection of SARS-CoV-2 and other common respiratory pathogens. Nörz et al.23 adapted a laboratory-developed multiplex RT-PCR assay for the simultaneous detection of SARS-CoV-2 and influenza A/B on a fully automated high-throughput system and demonstrated analytical performance comparable with that of currently available commercial tests. Sensitivities of SARS-CoV-2, influenza A, and influenza B in the multiplex assay were 98.1%, 97.67%, and 100%, respectively.23 Xpert Xpress SARS-CoV-2/Flu-RSV (Xpert 4-in-1 assay) was launched with Emergency Use Authorization of the United States Food and Drug Administration in September 2020 and demonstrated a performance highly comparable with those of Xpert SARS-CoV-2 and Xpert Flu/RSV assays.24 PPAs and NPAs of SARS-CoV-2, influenza A, influenza B, and RSV in Xpert -in-1 assay were 98.48%/100%, 100%/100%, 100%/99.54%, and 100%/100%, respectively.24\\nA case of co-infection of SARS-CoV-2 and other respiratory viruses was absent in the present study. SARS-CoV-2-positive specimens were collected between September 2020 and November 2020. In Korea, RSV and influenza outbreak seasons classically begin in autumn and winter, respectively.25 Although the collection period overlapped with RSV outbreak season, due to COVID-19-related quarantine and improved hygiene, transmission rates of influenza and RSV were lower.26 A US research team reported that 20.7% of specimens positive for SARS-CoV-2 were positive for one or more additional pathogens, compared with 26.7% negative for SARS-CoV-2.27 The most commonly co-infected pathogens were rhinovirus/enterovirus followed by RSV and non-SARS-CoV-2 Coronaviridae.27 A single-centered retrospective study was performed in Wuhan, China, between January 12 and February 21, 2020 during the initial COVID-19 outbreak.28 Unexpectedly, among COVID-19 patients, only 42.7% (131/307) of patients were singly positive for SARS-CoV-2; 57.3% (176/307) of COVID-19 patients were also positive for influenza viruses including influenza A (49.8%) and influenza B (7.5%).28 Another single-centered retrospective study conducted in Lukou District, China showed that 14.1% (11/78) of COVID-19 patients were co-infected with other respiratory pathogens including RSV (3/11, 36.3%) and influenza B (1/11, 9.1%).14 In contrast, a Korean research team demonstrated co-infection cases of SARS-CoV-2 and influenza virus or RSV were absent.29 Using the Allplex RV-Essential Assay (Seegene) detecting seven respiratory viruses including adenovirus, influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, RSV, and human rhinovirus, the co-infection rate in COVID-19 patients was 2.2%.29 The most frequently detected virus was adenovirus, followed by human rhinovirus and metapneumovirus.29 However, comprehensive PCR testing to detect respiratory viruses other than influenza virus and RSV was not performed in SARS-CoV-2-positive specimens in the present study.\\nPowerChek, STANDARD M, and Allplex kits each specified different cycle conditions for rRT-PCR. PowerChek did not require any pre-amplification cycles during PCR. In the PCR protocol of STANDARD M, five pre-amplification cycles preceded the amplification cycles, and the time and temperature conditions of both cycles were the same. Allplex had three pre-amplification cycles with different time and temperature conditions from those of the amplification cycles. Volume of template RNA per test and the number of pre-amplification cycles and amplification cycles of the three multi-target kits were compared with corresponding single SARS-CoV-2 detection kits: PowerChek 2019-nCoV Real-time PCR (Kogene Biotech), STANDARD M nCoV Real-Time Detection (SD BioSensor), and Allplex 2019-nCoV (Seegene).17 KogeneBiotech and SD BioSensor had the same volume of template RNA per test and number of pre-amplification and amplification cycles in each pair of single- and multi-target assays. However, the multi-target assay of Allplex required 10 μL of template RNA compared with 8 μL in the single-target assay. Allplex single-target assay performed 45 amplification cycles without pre-amplification but the multi-target assay executed 3 pre-amplification and 42 amplification cycles. Allplex multi-target assay was thought to be designed to increase sensitivity than the single-target assay by increasing the volume of template RNA.\\nThis study had some limitations. First, as previously mentioned, the evaluation of the LoD of the kits may not have reflected the practical analytical sensitivity because the measured LoD may have been affected by extraction method, type of PCR machine, etc. Second, for SARS-CoV-2, cross-reactivity test with other coronaviruses except 229E, OC43, and NL63 was not performed. Third, although the AdvanSure kit was treated as a comparator kit in this study, a true reference method was absent. Indeed, the AdvanSure kit was less sensitive for RSV compared with the Allplex kit. Finally, because multiplex PCR used to detect respiratory viruses other than influenza A/B and RSV was not performed in SARS-CoV-2-positive samples, co-infection rates of other respiratory viruses and SARS-CoV-2 could not be estimated.\\nIn conclusion, the overall performance of these three multiplex rRT-PCR assays for concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. In the COVID-19 pandemic era, all three kits are suitable for the prompt differential diagnosis of COVID-19, influenza, and RSV infection in patients with respiratory symptoms.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,"results",null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["SARS-CoV-2","Influenza A Virus","Influenza B Virus","Respiratory Syncytial Viruses","Multiplex Polymerase Chain Reaction"],"string":"[\n \"SARS-CoV-2\",\n \"Influenza A Virus\",\n \"Influenza B Virus\",\n \"Respiratory Syncytial Viruses\",\n \"Multiplex Polymerase Chain Reaction\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nThe coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10\nSARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).\nSince the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.\nMolecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS).\n\nMETHODS:\nThree molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nPowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nSpecimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nWe selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nSample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nA 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nAnalytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nThe LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nEvaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nReference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nEthics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\nThis study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\n\nThree molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV:\nPowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\n\nSpecimen collection:\nWe selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\n\nSample processing and PCR:\nA 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\n\nAnalytical performance: limit of detection (LoD) and cross-reactivity:\nThe LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\n\nEvaluation of the clinical performance of the three molecular diagnostic kits:\nReference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\n\nEthics statement:\nThis study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\n\nRESULTS:\nAnalytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nIn comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nClinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\nCompared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\n\nAnalytical performance:\nIn comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\n\nClinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV:\nCompared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\n\nDISCUSSION:\nIn the evaluation of analytical sensitivity, the LoD is dependent on reference material kits, extraction methods, PCR machines, and other experimental conditions. Therefore, the LoDs reported in our study may not correspond with practical LoDs in the real world. In the evaluation of cross-reactivity of the three assays, all three kits did not present cross-reactivity with common respiratory viruses. Especially, for SARS-CoV-2, three assays showed no reactive with coronavirus 229E, OC43, and NL63.\nAll three kits had 100% NPA in SARS-CoV-2 results, but PPAs in SARS-CoV-2 results ranged from 92.8% to 95.9%; among 97 SARS-CoV-2 positive samples, 10 (10.3%) produced discrepant results among the three assays. All three kits had 100% PPA in influenza A/B results. In contrast to 100% NPA in influenza B results in all three kits, NPAs in influenza A results ranged from 99.7% to 100.0% due to two false influenza A-positive reactions. According to the LoD testing of AdvanSure and Allplex kits and RSV sequencing results, Allplex detected RSV more sensitively than AdvanSure.\nMolecular diagnostic kits for detecting SARS-CoV-2 have shifted from single SARS-CoV-2 detection to multi-detection of SARS-CoV-2 and other common respiratory pathogens. Nörz et al.23 adapted a laboratory-developed multiplex RT-PCR assay for the simultaneous detection of SARS-CoV-2 and influenza A/B on a fully automated high-throughput system and demonstrated analytical performance comparable with that of currently available commercial tests. Sensitivities of SARS-CoV-2, influenza A, and influenza B in the multiplex assay were 98.1%, 97.67%, and 100%, respectively.23 Xpert Xpress SARS-CoV-2/Flu-RSV (Xpert 4-in-1 assay) was launched with Emergency Use Authorization of the United States Food and Drug Administration in September 2020 and demonstrated a performance highly comparable with those of Xpert SARS-CoV-2 and Xpert Flu/RSV assays.24 PPAs and NPAs of SARS-CoV-2, influenza A, influenza B, and RSV in Xpert -in-1 assay were 98.48%/100%, 100%/100%, 100%/99.54%, and 100%/100%, respectively.24\nA case of co-infection of SARS-CoV-2 and other respiratory viruses was absent in the present study. SARS-CoV-2-positive specimens were collected between September 2020 and November 2020. In Korea, RSV and influenza outbreak seasons classically begin in autumn and winter, respectively.25 Although the collection period overlapped with RSV outbreak season, due to COVID-19-related quarantine and improved hygiene, transmission rates of influenza and RSV were lower.26 A US research team reported that 20.7% of specimens positive for SARS-CoV-2 were positive for one or more additional pathogens, compared with 26.7% negative for SARS-CoV-2.27 The most commonly co-infected pathogens were rhinovirus/enterovirus followed by RSV and non-SARS-CoV-2 Coronaviridae.27 A single-centered retrospective study was performed in Wuhan, China, between January 12 and February 21, 2020 during the initial COVID-19 outbreak.28 Unexpectedly, among COVID-19 patients, only 42.7% (131/307) of patients were singly positive for SARS-CoV-2; 57.3% (176/307) of COVID-19 patients were also positive for influenza viruses including influenza A (49.8%) and influenza B (7.5%).28 Another single-centered retrospective study conducted in Lukou District, China showed that 14.1% (11/78) of COVID-19 patients were co-infected with other respiratory pathogens including RSV (3/11, 36.3%) and influenza B (1/11, 9.1%).14 In contrast, a Korean research team demonstrated co-infection cases of SARS-CoV-2 and influenza virus or RSV were absent.29 Using the Allplex RV-Essential Assay (Seegene) detecting seven respiratory viruses including adenovirus, influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, RSV, and human rhinovirus, the co-infection rate in COVID-19 patients was 2.2%.29 The most frequently detected virus was adenovirus, followed by human rhinovirus and metapneumovirus.29 However, comprehensive PCR testing to detect respiratory viruses other than influenza virus and RSV was not performed in SARS-CoV-2-positive specimens in the present study.\nPowerChek, STANDARD M, and Allplex kits each specified different cycle conditions for rRT-PCR. PowerChek did not require any pre-amplification cycles during PCR. In the PCR protocol of STANDARD M, five pre-amplification cycles preceded the amplification cycles, and the time and temperature conditions of both cycles were the same. Allplex had three pre-amplification cycles with different time and temperature conditions from those of the amplification cycles. Volume of template RNA per test and the number of pre-amplification cycles and amplification cycles of the three multi-target kits were compared with corresponding single SARS-CoV-2 detection kits: PowerChek 2019-nCoV Real-time PCR (Kogene Biotech), STANDARD M nCoV Real-Time Detection (SD BioSensor), and Allplex 2019-nCoV (Seegene).17 KogeneBiotech and SD BioSensor had the same volume of template RNA per test and number of pre-amplification and amplification cycles in each pair of single- and multi-target assays. However, the multi-target assay of Allplex required 10 μL of template RNA compared with 8 μL in the single-target assay. Allplex single-target assay performed 45 amplification cycles without pre-amplification but the multi-target assay executed 3 pre-amplification and 42 amplification cycles. Allplex multi-target assay was thought to be designed to increase sensitivity than the single-target assay by increasing the volume of template RNA.\nThis study had some limitations. First, as previously mentioned, the evaluation of the LoD of the kits may not have reflected the practical analytical sensitivity because the measured LoD may have been affected by extraction method, type of PCR machine, etc. Second, for SARS-CoV-2, cross-reactivity test with other coronaviruses except 229E, OC43, and NL63 was not performed. Third, although the AdvanSure kit was treated as a comparator kit in this study, a true reference method was absent. Indeed, the AdvanSure kit was less sensitive for RSV compared with the Allplex kit. Finally, because multiplex PCR used to detect respiratory viruses other than influenza A/B and RSV was not performed in SARS-CoV-2-positive samples, co-infection rates of other respiratory viruses and SARS-CoV-2 could not be estimated.\nIn conclusion, the overall performance of these three multiplex rRT-PCR assays for concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. In the COVID-19 pandemic era, all three kits are suitable for the prompt differential diagnosis of COVID-19, influenza, and RSV infection in patients with respiratory symptoms.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIn the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits.\n\nMethods:\nA limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV.\n\nResults:\nExcept in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure.\n\nConclusions:\nThe overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":9168,"string":"9,168"},"whole_article_abstract_length":{"kind":"number","value":389,"string":"389"},"other_sections_lengths":{"kind":"list like","value":[444,347,141,292,258,36,220,607],"string":"[\n 444,\n 347,\n 141,\n 292,\n 258,\n 36,\n 220,\n 607\n]"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["cov","sars cov","sars","positive","influenza","rsv","kits","respiratory","samples","pcr"],"string":"[\n \"cov\",\n \"sars cov\",\n \"sars\",\n \"positive\",\n \"influenza\",\n \"rsv\",\n \"kits\",\n \"respiratory\",\n \"samples\",\n \"pcr\"\n]"},"keybert_topics":{"kind":"list like","value":["covid 19 pandemic","coronavirus disease","respiratory viruses influenza","age influenza virus","covid 19 disease"],"string":"[\n \"covid 19 pandemic\",\n \"coronavirus disease\",\n \"respiratory viruses influenza\",\n \"age influenza virus\",\n \"covid 19 disease\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] influenza | infection | cov | sars cov | sars | rsv | influenza rsv | sars cov influenza rsv | cov influenza rsv | cov influenza [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] positive | samples | sars cov | cov | sars | μl | results | kit | kits | pcr [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] positive | influenza | cov | sars cov | sars | rsv | table | npa | kits | respiratory [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | samples | respiratory | kit [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 | 2 | RSV ||| Three | transcription | Korea ||| PowerChek | Influenza | Multiplex Real-time | PCR Kit | PowerChek | KogeneBiotech | STANDARD | STANDARD M | SD BioSensor | Allplex | Allplex | Seegene ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Ninety-seven | 201 | 71 | 50 | 78 | RSV | 207 | the three assays ||| AdvanSure | AdvanSure | LG Life Sciences | RSV [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] three ||| three ||| three | 92% | ≥ 0.95 | RSV [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | COVID-19 | 2 | RSV ||| Three | transcription | Korea ||| PowerChek | Influenza | Multiplex Real-time | PCR Kit | PowerChek | KogeneBiotech | STANDARD | STANDARD M | SD BioSensor | Allplex | Allplex | Seegene ||| ||| ||| Ninety-seven | 201 | 71 | 50 | 78 | RSV | 207 | the three assays ||| AdvanSure | AdvanSure | LG Life Sciences | RSV ||| three ||| three ||| three | 92% | ≥ 0.95 | RSV ||| three | RSV ||| COVID-19 | influenza | RSV | COVID-19 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3380,"cells":{"title":{"kind":"string","value":"Regulation of pentraxin-3 by antioxidants."},"pmid":{"kind":"string","value":"19864306"},"background_abstract":{"kind":"string","value":"Pentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Human endothelial cells were cultured with lipopolysaccharide 2 microg ml(-1), peptidoglycan G 20 microg ml(-1), tumour necrosis factor (TNF) alpha 10 ng ml(-1), interleukin-1 (IL-1) beta 20 ng ml(-1), or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 microM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"PTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml(-1) compared with 6 (1-12) ng ml(-1), P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNFalpha plus IL-1beta. PTX3 concentrations were lower in cells co-treated with antioxidants (all P<0.05), associated with lower nuclear factor kappaB expression for NAC and trolox (P<0.05)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"PTX3 expression is down-regulated in vitro by antioxidants. Plasma levels of PTX3 are elevated in sepsis but seem to be unrelated to markers of oxidant stress or antioxidant capacity."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["APACHE","Adult","Aged","Aged, 80 and over","Antioxidants","Biomarkers","C-Reactive Protein","Cells, Cultured","Endothelium, Vascular","Female","Humans","Inflammation Mediators","Lipid Peroxides","Male","Middle Aged","Oxidative Stress","Pilot Projects","Sepsis","Serum Amyloid P-Component","Up-Regulation","Young Adult"],"string":"[\n \"APACHE\",\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Antioxidants\",\n \"Biomarkers\",\n \"C-Reactive Protein\",\n \"Cells, Cultured\",\n \"Endothelium, Vascular\",\n \"Female\",\n \"Humans\",\n \"Inflammation Mediators\",\n \"Lipid Peroxides\",\n \"Male\",\n \"Middle Aged\",\n \"Oxidative Stress\",\n \"Pilot Projects\",\n \"Sepsis\",\n \"Serum Amyloid P-Component\",\n \"Up-Regulation\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"2777941"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\nIn this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\n In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\nAll reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\n Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\nPTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\n Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\nNuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\n Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\nTotal antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\n Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\nFor the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Fifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.\n(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.\nCharacteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct\nTo assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.\nPlasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.\nWe also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.\nThe effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).\nNuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).\nThe effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Patient study","In vitro study","Pentraxin-3 and CRP measurement","Nuclear factor κB","Total antioxidant capacity and lipid peroxides","Data analysis","Funding"],"string":"[\n \"Patient study\",\n \"In vitro study\",\n \"Pentraxin-3 and CRP measurement\",\n \"Nuclear factor κB\",\n \"Total antioxidant capacity and lipid peroxides\",\n \"Data analysis\",\n \"Funding\"\n]"},"other_sections_texts":{"kind":"list like","value":["In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.","All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.","PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).","Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.","Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.","For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.","A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust."],"string":"[\n \"In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\",\n \"All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\",\n \"PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\",\n \"Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\",\n \"Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\",\n \"For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\",\n \"A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Methods","Patient study","In vitro study","Pentraxin-3 and CRP measurement","Nuclear factor κB","Total antioxidant capacity and lipid peroxides","Data analysis","Results","Discussion","Funding"],"string":"[\n \"Methods\",\n \"Patient study\",\n \"In vitro study\",\n \"Pentraxin-3 and CRP measurement\",\n \"Nuclear factor κB\",\n \"Total antioxidant capacity and lipid peroxides\",\n \"Data analysis\",\n \"Results\",\n \"Discussion\",\n \"Funding\"\n]"},"all_sections_texts":{"kind":"list like","value":[" Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\nIn this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\n In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\nAll reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\n Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\nPTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\n Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\nNuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\n Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\nTotal antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\n Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\nFor the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.","In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.","All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.","PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).","Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.","Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.","For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.","Fifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.\n(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.\nCharacteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct\nTo assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.\nPlasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.\nWe also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.\nThe effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).\nNuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).\nThe effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001).","We showed that PTX3 was up-regulated in human endothelial cells in response to bacterial cell proteins, heat-killed Candida cells, or cytokines, with the greatest response occurring with a combination of TNFα plus IL-1β. Endothelial PTX3 response to an inflammatory stimulus was significantly reduced by treatment with NAC, trolox, or idebenone, and was associated with lower nuclear NFκB expression in NAC and trolox-treated cells. Idebenone had no effect on NFκB expression. We also showed that circulating PTX3 concentrations are higher in patients with sepsis compared with healthy subjects and are higher in female patients than males. PTX3 was also related to APACHE II score quartile. In addition, total antioxidant capacity was low and lipid hydroperoxide levels were elevated in the patients with sepsis, indicating oxidative stress. However, PTX3 concentrations in patients with sepsis were not related to antioxidant capacity, despite the regulation of PTX3 expression by antioxidants in vitro.\nPTX3 is a member of the pentraxin superfamily, a family of proteins characterized by a multimeric, usually pentameric structure.12 CRP, the prototype of the pentraxin family, is an acute-phase protein produced in the liver in response to inflammatory signals such as IL-6. PTX3 is the prototype of the long pentraxin family and has some similarities with the short pentraxins but differs with respect to cellular source, and ligand recognition. PTX3 is produced in response by a variety of cell types, including macrophages and monocytes, dendritic cells, fibroblasts, epithelial, and endothelial cells, in response to inflammatory signals such as TNFα, IL-1β, microbial proteins including LPS, lipoarabinomannans, and PepG, but not IL-6.1–5\nWe found that exposure of endothelial cells to LPS, PepG, TNFα, or IL-1β resulted in PTX3 up-regulation, with an additive response when cells were treated with both TNFα and IL-1β, as previously reported.3–57 We also found that a ratio of between 1 and 10 heat-killed Candida cells to one endothelial cell resulted in an up-regulation of PTX3 expression by endothelial cells. Infection of mice with intra-cerebroventricular injection of live C. albicans was previously shown to result in up-regulation of PTX3 in the brain,19 but PTX3 expression by endothelial cells in response to C. albicans has not been described. Endothelial cells phagocytose live Candida cells, leading to secretion of cytokines including TNFα and IL-1β.20–23 The up-regulation of PTX3 expression found in our study may be related to TNFα and IL-1α produced as a result of phagocytosis of heat-killed Candida cells by the endothelial cells, although it has been reported that Candida cells killed with sodium periodate, although phagocytosed, are unable to elicit this cytokine response.2021 However, in another study, heat-killed Candida cells were able to stimulate monocytes to release pro-inflammatory cytokines,24 suggesting that the means of killing the cells may be relevant.\nPTX3 blood levels are usually <10 ng ml−1 in healthy subjects and increase rapidly during inflammation and infection, leading to investigation of the potential of PTX3 as an early biomarker for sepsis. In a heterogeneous group of critically ill patients ranging from those with no infection to those with septic shock, it was reported that PTX3 levels were higher in the more severely ill patients.25 We also found that PTX3 levels were related to severity of illness as assessed by APACHE II score. The APACHE scores were not presented in that report,25 making further comparisons difficult. PTX3 has also been found to be increased in patients with acute respiratory distress syndrome.26 In a recent study in children with meningococcal disease, high PTX3 and low CRP concentrations at admission discriminated between the presence and the absence of shock.27 PTX3 did not correlate with paediatric risk of mortality, whereas CRP correlated negatively. PTX3 blood levels increase more rapidly than CRP and only loose2526 or no27 correlations are found between circulating levels of PTX3 and CRP. PTX3 has also been shown to be up-regulated in patients with dengue virus infection.28\nMarked oxidative stress has been consistently reported in patients with sepsis8202930 and acts as a trigger for up-regulation of many inflammatory responses. The human PTX3 proximal promoter contains binding sites for several transcription factors, including activator protein-1 (AP-1), NFκB, and nuclear factor-IL-6. The NFκB proximal site is essential for induction by IL-1β and TNFα,67 and PTX3 was shown to be regulated by NFκB.31 NFκB is redox-sensitive32 and has been shown to be involved in regulation of inflammatory responses in patients with sepsis: we and others have found elevated activation of NFκB linked to increased mortality3334 and inhibitable by NAC treatment, resulting in down-regulation of inflammatory mediators.35 We therefore investigated whether antioxidants were able to down-regulate PTX3 expression by endothelial cells in vitro. We found that NAC, trolox, and idebenone treatment all resulted in lower PTX3 levels on exposure to an inflammatory stimulus. In the case of NAC and trolox, this was accompanied by lower nuclear expression of NFκB. In a study using macrophages, the antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced PTX3 expression in murine macrophages via an NFκB-dependent mechanism.31 However, there are other redox-sensitive transcription factors besides NFκB, such as AP-1,36 which may also be important for regulation of PTX3. Exogenous and endogenous antioxidants have been shown to be effective in blocking activation of NFκB either directly or indirectly, including NAC and trolox.3536 Although co-enzyme Q10 can inhibit NFκB,37 we can find no reports of the effect of idebenone on transcription, and so the mechanism of the down-regulation of PTX3 expression by idebenone remains unknown.\nOxidative stress can be defined as an imbalance between the production of reactive oxygen species and their removal by protective antioxidants. We measured lipid hydroperoxide levels and total antioxidant capacity in our patients with sepsis. Lipid peroxidation is the oxidative degradation of lipids and has been reported previously to be elevated in critically ill patients.8101230 Total antioxidant capacity reflects the ability of plasma to resist oxidative damage in vitro and has been widely used to assess the antioxidant status of patients with sepsis.8929 We found that lipid hydroperoxides were increased and total antioxidant capacity was decreased in our patients. The classical view of lipid peroxidation products is that they are essentially harmful, inducing and propagating chronic inflammatory reactions. However, it has been suggested that lipid peroxide breakdown products may promote cellular defence mechanisms,3839 such as induction of signalling pathways, resulting in up-regulation of anti-inflammatory mediators, and inhibition of signalling pathways which control pro-inflammatory mediator expression. Thus, the role of lipid hydroperoxides in inflammatory responses in sepsis is unclear. We found that neither lipid hydroperoxides nor antioxidant capacity was related to PTX3 levels, despite the effect of antioxidants on PTX3 expression in vitro. Although this was a small pilot study, there was a range of PTX values and oxidative stress was apparent, and so we would have expected some evidence of a relationship. These results may reflect the complex nature of the role of redox state involved in regulation of inflammatory pathways in vivo.\nOther factors also influence PTX3 levels. In a study of more than 2000 healthy Japanese subjects, plasma PTX3 levels were reported to be lower in men than in women, higher in older age groups, and inversely correlated with triglycerides.40 We also found that PTX3 levels in patients with sepsis were highest in the female patients, but the skewed age range of patients with sepsis did not allow assessment of any age. The previous study of critically ill patients25 did not report sex distribution. It remains unclear as to the importance of the contribution of these confounding factors.\nIn conclusion, we report that PTX3 expression in endothelial cells can be up-regulated by a variety of inflammatory mediators, including bacterial cell proteins, cytokines, and C. albicans and is down-regulated by NAC and trolox through an NFκB-mediated process. PTX3 levels are increased in patients with sepsis, and related to APACHE II score. Although we found evidence of oxidative stress in our patients, the regulation of PTX3 by antioxidants in vitro was not reflected clinically in this pilot study. Further work is needed to clarify regulatory mechanisms for PTX3 and its use as a biomarker in sepsis.","A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust."],"string":"[\n \" Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\\nIn this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\\n In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\\nAll reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\\n Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\\nPTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\\n Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\\nNuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\\n Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\\nTotal antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\\n Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\\nFor the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\",\n \"In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\",\n \"All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\",\n \"PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\",\n \"Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\",\n \"Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\",\n \"For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\",\n \"Fifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.\\n(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.\\nCharacteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct\\nTo assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.\\nPlasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.\\nWe also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.\\nThe effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).\\nNuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).\\nThe effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001).\",\n \"We showed that PTX3 was up-regulated in human endothelial cells in response to bacterial cell proteins, heat-killed Candida cells, or cytokines, with the greatest response occurring with a combination of TNFα plus IL-1β. Endothelial PTX3 response to an inflammatory stimulus was significantly reduced by treatment with NAC, trolox, or idebenone, and was associated with lower nuclear NFκB expression in NAC and trolox-treated cells. Idebenone had no effect on NFκB expression. We also showed that circulating PTX3 concentrations are higher in patients with sepsis compared with healthy subjects and are higher in female patients than males. PTX3 was also related to APACHE II score quartile. In addition, total antioxidant capacity was low and lipid hydroperoxide levels were elevated in the patients with sepsis, indicating oxidative stress. However, PTX3 concentrations in patients with sepsis were not related to antioxidant capacity, despite the regulation of PTX3 expression by antioxidants in vitro.\\nPTX3 is a member of the pentraxin superfamily, a family of proteins characterized by a multimeric, usually pentameric structure.12 CRP, the prototype of the pentraxin family, is an acute-phase protein produced in the liver in response to inflammatory signals such as IL-6. PTX3 is the prototype of the long pentraxin family and has some similarities with the short pentraxins but differs with respect to cellular source, and ligand recognition. PTX3 is produced in response by a variety of cell types, including macrophages and monocytes, dendritic cells, fibroblasts, epithelial, and endothelial cells, in response to inflammatory signals such as TNFα, IL-1β, microbial proteins including LPS, lipoarabinomannans, and PepG, but not IL-6.1–5\\nWe found that exposure of endothelial cells to LPS, PepG, TNFα, or IL-1β resulted in PTX3 up-regulation, with an additive response when cells were treated with both TNFα and IL-1β, as previously reported.3–57 We also found that a ratio of between 1 and 10 heat-killed Candida cells to one endothelial cell resulted in an up-regulation of PTX3 expression by endothelial cells. Infection of mice with intra-cerebroventricular injection of live C. albicans was previously shown to result in up-regulation of PTX3 in the brain,19 but PTX3 expression by endothelial cells in response to C. albicans has not been described. Endothelial cells phagocytose live Candida cells, leading to secretion of cytokines including TNFα and IL-1β.20–23 The up-regulation of PTX3 expression found in our study may be related to TNFα and IL-1α produced as a result of phagocytosis of heat-killed Candida cells by the endothelial cells, although it has been reported that Candida cells killed with sodium periodate, although phagocytosed, are unable to elicit this cytokine response.2021 However, in another study, heat-killed Candida cells were able to stimulate monocytes to release pro-inflammatory cytokines,24 suggesting that the means of killing the cells may be relevant.\\nPTX3 blood levels are usually <10 ng ml−1 in healthy subjects and increase rapidly during inflammation and infection, leading to investigation of the potential of PTX3 as an early biomarker for sepsis. In a heterogeneous group of critically ill patients ranging from those with no infection to those with septic shock, it was reported that PTX3 levels were higher in the more severely ill patients.25 We also found that PTX3 levels were related to severity of illness as assessed by APACHE II score. The APACHE scores were not presented in that report,25 making further comparisons difficult. PTX3 has also been found to be increased in patients with acute respiratory distress syndrome.26 In a recent study in children with meningococcal disease, high PTX3 and low CRP concentrations at admission discriminated between the presence and the absence of shock.27 PTX3 did not correlate with paediatric risk of mortality, whereas CRP correlated negatively. PTX3 blood levels increase more rapidly than CRP and only loose2526 or no27 correlations are found between circulating levels of PTX3 and CRP. PTX3 has also been shown to be up-regulated in patients with dengue virus infection.28\\nMarked oxidative stress has been consistently reported in patients with sepsis8202930 and acts as a trigger for up-regulation of many inflammatory responses. The human PTX3 proximal promoter contains binding sites for several transcription factors, including activator protein-1 (AP-1), NFκB, and nuclear factor-IL-6. The NFκB proximal site is essential for induction by IL-1β and TNFα,67 and PTX3 was shown to be regulated by NFκB.31 NFκB is redox-sensitive32 and has been shown to be involved in regulation of inflammatory responses in patients with sepsis: we and others have found elevated activation of NFκB linked to increased mortality3334 and inhibitable by NAC treatment, resulting in down-regulation of inflammatory mediators.35 We therefore investigated whether antioxidants were able to down-regulate PTX3 expression by endothelial cells in vitro. We found that NAC, trolox, and idebenone treatment all resulted in lower PTX3 levels on exposure to an inflammatory stimulus. In the case of NAC and trolox, this was accompanied by lower nuclear expression of NFκB. In a study using macrophages, the antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced PTX3 expression in murine macrophages via an NFκB-dependent mechanism.31 However, there are other redox-sensitive transcription factors besides NFκB, such as AP-1,36 which may also be important for regulation of PTX3. Exogenous and endogenous antioxidants have been shown to be effective in blocking activation of NFκB either directly or indirectly, including NAC and trolox.3536 Although co-enzyme Q10 can inhibit NFκB,37 we can find no reports of the effect of idebenone on transcription, and so the mechanism of the down-regulation of PTX3 expression by idebenone remains unknown.\\nOxidative stress can be defined as an imbalance between the production of reactive oxygen species and their removal by protective antioxidants. We measured lipid hydroperoxide levels and total antioxidant capacity in our patients with sepsis. Lipid peroxidation is the oxidative degradation of lipids and has been reported previously to be elevated in critically ill patients.8101230 Total antioxidant capacity reflects the ability of plasma to resist oxidative damage in vitro and has been widely used to assess the antioxidant status of patients with sepsis.8929 We found that lipid hydroperoxides were increased and total antioxidant capacity was decreased in our patients. The classical view of lipid peroxidation products is that they are essentially harmful, inducing and propagating chronic inflammatory reactions. However, it has been suggested that lipid peroxide breakdown products may promote cellular defence mechanisms,3839 such as induction of signalling pathways, resulting in up-regulation of anti-inflammatory mediators, and inhibition of signalling pathways which control pro-inflammatory mediator expression. Thus, the role of lipid hydroperoxides in inflammatory responses in sepsis is unclear. We found that neither lipid hydroperoxides nor antioxidant capacity was related to PTX3 levels, despite the effect of antioxidants on PTX3 expression in vitro. Although this was a small pilot study, there was a range of PTX values and oxidative stress was apparent, and so we would have expected some evidence of a relationship. These results may reflect the complex nature of the role of redox state involved in regulation of inflammatory pathways in vivo.\\nOther factors also influence PTX3 levels. In a study of more than 2000 healthy Japanese subjects, plasma PTX3 levels were reported to be lower in men than in women, higher in older age groups, and inversely correlated with triglycerides.40 We also found that PTX3 levels in patients with sepsis were highest in the female patients, but the skewed age range of patients with sepsis did not allow assessment of any age. The previous study of critically ill patients25 did not report sex distribution. It remains unclear as to the importance of the contribution of these confounding factors.\\nIn conclusion, we report that PTX3 expression in endothelial cells can be up-regulated by a variety of inflammatory mediators, including bacterial cell proteins, cytokines, and C. albicans and is down-regulated by NAC and trolox through an NFκB-mediated process. PTX3 levels are increased in patients with sepsis, and related to APACHE II score. Although we found evidence of oxidative stress in our patients, the regulation of PTX3 by antioxidants in vitro was not reflected clinically in this pilot study. Further work is needed to clarify regulatory mechanisms for PTX3 and its use as a biomarker in sepsis.\",\n \"A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["methods",null,null,null,null,null,null,"results","discussion",null],"string":"[\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n null\n]"},"keywords":{"kind":"list like","value":["immune response","infection, bacterial","infection, fungal","metabolism, protein, acute phase"],"string":"[\n \"immune response\",\n \"infection, bacterial\",\n \"infection, fungal\",\n \"metabolism, protein, acute phase\"\n]"},"whole_article_text":{"kind":"string","value":"Methods:\n Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\nIn this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\n In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\nAll reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\n Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\nPTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\n Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\nNuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\n Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\nTotal antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\n Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\nFor the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\n\nPatient study:\nIn this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\n\nIn vitro study:\nAll reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\n\nPentraxin-3 and CRP measurement:\nPTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\n\nNuclear factor κB:\nNuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\n\nTotal antioxidant capacity and lipid peroxides:\nTotal antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\n\nData analysis:\nFor the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\n\nResults:\nFifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.\n(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.\nCharacteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct\nTo assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.\nPlasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.\nWe also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.\nThe effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).\nNuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).\nThe effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001).\n\nDiscussion:\nWe showed that PTX3 was up-regulated in human endothelial cells in response to bacterial cell proteins, heat-killed Candida cells, or cytokines, with the greatest response occurring with a combination of TNFα plus IL-1β. Endothelial PTX3 response to an inflammatory stimulus was significantly reduced by treatment with NAC, trolox, or idebenone, and was associated with lower nuclear NFκB expression in NAC and trolox-treated cells. Idebenone had no effect on NFκB expression. We also showed that circulating PTX3 concentrations are higher in patients with sepsis compared with healthy subjects and are higher in female patients than males. PTX3 was also related to APACHE II score quartile. In addition, total antioxidant capacity was low and lipid hydroperoxide levels were elevated in the patients with sepsis, indicating oxidative stress. However, PTX3 concentrations in patients with sepsis were not related to antioxidant capacity, despite the regulation of PTX3 expression by antioxidants in vitro.\nPTX3 is a member of the pentraxin superfamily, a family of proteins characterized by a multimeric, usually pentameric structure.12 CRP, the prototype of the pentraxin family, is an acute-phase protein produced in the liver in response to inflammatory signals such as IL-6. PTX3 is the prototype of the long pentraxin family and has some similarities with the short pentraxins but differs with respect to cellular source, and ligand recognition. PTX3 is produced in response by a variety of cell types, including macrophages and monocytes, dendritic cells, fibroblasts, epithelial, and endothelial cells, in response to inflammatory signals such as TNFα, IL-1β, microbial proteins including LPS, lipoarabinomannans, and PepG, but not IL-6.1–5\nWe found that exposure of endothelial cells to LPS, PepG, TNFα, or IL-1β resulted in PTX3 up-regulation, with an additive response when cells were treated with both TNFα and IL-1β, as previously reported.3–57 We also found that a ratio of between 1 and 10 heat-killed Candida cells to one endothelial cell resulted in an up-regulation of PTX3 expression by endothelial cells. Infection of mice with intra-cerebroventricular injection of live C. albicans was previously shown to result in up-regulation of PTX3 in the brain,19 but PTX3 expression by endothelial cells in response to C. albicans has not been described. Endothelial cells phagocytose live Candida cells, leading to secretion of cytokines including TNFα and IL-1β.20–23 The up-regulation of PTX3 expression found in our study may be related to TNFα and IL-1α produced as a result of phagocytosis of heat-killed Candida cells by the endothelial cells, although it has been reported that Candida cells killed with sodium periodate, although phagocytosed, are unable to elicit this cytokine response.2021 However, in another study, heat-killed Candida cells were able to stimulate monocytes to release pro-inflammatory cytokines,24 suggesting that the means of killing the cells may be relevant.\nPTX3 blood levels are usually <10 ng ml−1 in healthy subjects and increase rapidly during inflammation and infection, leading to investigation of the potential of PTX3 as an early biomarker for sepsis. In a heterogeneous group of critically ill patients ranging from those with no infection to those with septic shock, it was reported that PTX3 levels were higher in the more severely ill patients.25 We also found that PTX3 levels were related to severity of illness as assessed by APACHE II score. The APACHE scores were not presented in that report,25 making further comparisons difficult. PTX3 has also been found to be increased in patients with acute respiratory distress syndrome.26 In a recent study in children with meningococcal disease, high PTX3 and low CRP concentrations at admission discriminated between the presence and the absence of shock.27 PTX3 did not correlate with paediatric risk of mortality, whereas CRP correlated negatively. PTX3 blood levels increase more rapidly than CRP and only loose2526 or no27 correlations are found between circulating levels of PTX3 and CRP. PTX3 has also been shown to be up-regulated in patients with dengue virus infection.28\nMarked oxidative stress has been consistently reported in patients with sepsis8202930 and acts as a trigger for up-regulation of many inflammatory responses. The human PTX3 proximal promoter contains binding sites for several transcription factors, including activator protein-1 (AP-1), NFκB, and nuclear factor-IL-6. The NFκB proximal site is essential for induction by IL-1β and TNFα,67 and PTX3 was shown to be regulated by NFκB.31 NFκB is redox-sensitive32 and has been shown to be involved in regulation of inflammatory responses in patients with sepsis: we and others have found elevated activation of NFκB linked to increased mortality3334 and inhibitable by NAC treatment, resulting in down-regulation of inflammatory mediators.35 We therefore investigated whether antioxidants were able to down-regulate PTX3 expression by endothelial cells in vitro. We found that NAC, trolox, and idebenone treatment all resulted in lower PTX3 levels on exposure to an inflammatory stimulus. In the case of NAC and trolox, this was accompanied by lower nuclear expression of NFκB. In a study using macrophages, the antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced PTX3 expression in murine macrophages via an NFκB-dependent mechanism.31 However, there are other redox-sensitive transcription factors besides NFκB, such as AP-1,36 which may also be important for regulation of PTX3. Exogenous and endogenous antioxidants have been shown to be effective in blocking activation of NFκB either directly or indirectly, including NAC and trolox.3536 Although co-enzyme Q10 can inhibit NFκB,37 we can find no reports of the effect of idebenone on transcription, and so the mechanism of the down-regulation of PTX3 expression by idebenone remains unknown.\nOxidative stress can be defined as an imbalance between the production of reactive oxygen species and their removal by protective antioxidants. We measured lipid hydroperoxide levels and total antioxidant capacity in our patients with sepsis. Lipid peroxidation is the oxidative degradation of lipids and has been reported previously to be elevated in critically ill patients.8101230 Total antioxidant capacity reflects the ability of plasma to resist oxidative damage in vitro and has been widely used to assess the antioxidant status of patients with sepsis.8929 We found that lipid hydroperoxides were increased and total antioxidant capacity was decreased in our patients. The classical view of lipid peroxidation products is that they are essentially harmful, inducing and propagating chronic inflammatory reactions. However, it has been suggested that lipid peroxide breakdown products may promote cellular defence mechanisms,3839 such as induction of signalling pathways, resulting in up-regulation of anti-inflammatory mediators, and inhibition of signalling pathways which control pro-inflammatory mediator expression. Thus, the role of lipid hydroperoxides in inflammatory responses in sepsis is unclear. We found that neither lipid hydroperoxides nor antioxidant capacity was related to PTX3 levels, despite the effect of antioxidants on PTX3 expression in vitro. Although this was a small pilot study, there was a range of PTX values and oxidative stress was apparent, and so we would have expected some evidence of a relationship. These results may reflect the complex nature of the role of redox state involved in regulation of inflammatory pathways in vivo.\nOther factors also influence PTX3 levels. In a study of more than 2000 healthy Japanese subjects, plasma PTX3 levels were reported to be lower in men than in women, higher in older age groups, and inversely correlated with triglycerides.40 We also found that PTX3 levels in patients with sepsis were highest in the female patients, but the skewed age range of patients with sepsis did not allow assessment of any age. The previous study of critically ill patients25 did not report sex distribution. It remains unclear as to the importance of the contribution of these confounding factors.\nIn conclusion, we report that PTX3 expression in endothelial cells can be up-regulated by a variety of inflammatory mediators, including bacterial cell proteins, cytokines, and C. albicans and is down-regulated by NAC and trolox through an NFκB-mediated process. PTX3 levels are increased in patients with sepsis, and related to APACHE II score. Although we found evidence of oxidative stress in our patients, the regulation of PTX3 by antioxidants in vitro was not reflected clinically in this pilot study. Further work is needed to clarify regulatory mechanisms for PTX3 and its use as a biomarker in sepsis.\n\nFunding:\nA.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis.\n\nMethods:\nHuman endothelial cells were cultured with lipopolysaccharide 2 microg ml(-1), peptidoglycan G 20 microg ml(-1), tumour necrosis factor (TNF) alpha 10 ng ml(-1), interleukin-1 (IL-1) beta 20 ng ml(-1), or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 microM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers.\n\nResults:\nPTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml(-1) compared with 6 (1-12) ng ml(-1), P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNFalpha plus IL-1beta. PTX3 concentrations were lower in cells co-treated with antioxidants (all P<0.05), associated with lower nuclear factor kappaB expression for NAC and trolox (P<0.05).\n\nConclusions:\nPTX3 expression is down-regulated in vitro by antioxidants. Plasma levels of PTX3 are elevated in sepsis but seem to be unrelated to markers of oxidant stress or antioxidant capacity."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":6611,"string":"6,611"},"whole_article_abstract_length":{"kind":"number","value":369,"string":"369"},"other_sections_lengths":{"kind":"list like","value":[237,441,195,319,131,93,40],"string":"[\n 237,\n 441,\n 195,\n 319,\n 131,\n 93,\n 40\n]"},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["ptx3","cells","patients","ml","plasma","il","sepsis","1β","endothelial","10"],"string":"[\n \"ptx3\",\n \"cells\",\n \"patients\",\n \"ml\",\n \"plasma\",\n \"il\",\n \"sepsis\",\n \"1β\",\n \"endothelial\",\n \"10\"\n]"},"keybert_topics":{"kind":"list like","value":["patients sepsis healthy","fulfilling criteria sepsis","use biomarker sepsis","elevated patients sepsis","sepsis taking statins"],"string":"[\n \"patients sepsis healthy\",\n \"fulfilling criteria sepsis\",\n \"use biomarker sepsis\",\n \"elevated patients sepsis\",\n \"sepsis taking statins\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"null"},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"null"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] immune response | infection, bacterial | infection, fungal | metabolism, protein, acute phase [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] immune response | infection, bacterial | infection, fungal | metabolism, protein, acute phase [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] immune response | infection, bacterial | infection, fungal | metabolism, protein, acute phase [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"null"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] APACHE | Adult | Aged | Aged, 80 and over | Antioxidants | Biomarkers | C-Reactive Protein | Cells, Cultured | Endothelium, Vascular | Female | Humans | Inflammation Mediators | Lipid Peroxides | Male | Middle Aged | Oxidative Stress | Pilot Projects | Sepsis | Serum Amyloid P-Component | Up-Regulation | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] APACHE | Adult | Aged | Aged, 80 and over | Antioxidants | Biomarkers | C-Reactive Protein | Cells, Cultured | Endothelium, Vascular | Female | Humans | Inflammation Mediators | Lipid Peroxides | Male | Middle Aged | Oxidative Stress | Pilot Projects | Sepsis | Serum Amyloid P-Component | Up-Regulation | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] APACHE | Adult | Aged | Aged, 80 and over | Antioxidants | Biomarkers | C-Reactive Protein | Cells, Cultured | Endothelium, Vascular | Female | Humans | Inflammation Mediators | Lipid Peroxides | Male | Middle Aged | Oxidative Stress | Pilot Projects | Sepsis | Serum Amyloid P-Component | Up-Regulation | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"null"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients sepsis healthy | fulfilling criteria sepsis | use biomarker sepsis | elevated patients sepsis | sepsis taking statins [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients sepsis healthy | fulfilling criteria sepsis | use biomarker sepsis | elevated patients sepsis | sepsis taking statins [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients sepsis healthy | fulfilling criteria sepsis | use biomarker sepsis | elevated patients sepsis | sepsis taking statins [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"null"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ptx3 | cells | patients | ml | plasma | il | sepsis | 1β | endothelial | 10 [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ptx3 | cells | patients | ml | plasma | il | sepsis | 1β | endothelial | 10 [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ptx3 | cells | patients | ml | plasma | il | sepsis | 1β | endothelial | 10 [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"null"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cells | incubated | ml | washed | assay | min | uk | plates | 37 | ptx3 [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] fig | cells | ptx3 | levels | patients sepsis | patients | ml | ptx3 levels | tnfα | sepsis [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ptx3 | cells | patients | ml | plasma | incubated | washed | 1β | sepsis | assay [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"null"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 2 | 20 | TNF | 10 ng ml(-1 | 20 ng | NAC | 25 mM | 100 | 1 | 15 | 11 [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 26 | 1 | 6 ||| 0.99 | 0.1-1.7 | mM | 2.2 | 1.3-3.3 | mM ||| 3.32 | 0.3-10.6 ||| ||| TNFalpha ||| kappaB | NAC [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| 2 | 20 | TNF | 10 ng ml(-1 | 20 ng | NAC | 25 mM | 100 | 1 | 15 | 11 ||| ||| 26 | 1 | 6 ||| 0.99 | 0.1-1.7 | mM | 2.2 | 1.3-3.3 | mM ||| 3.32 | 0.3-10.6 ||| ||| TNFalpha ||| kappaB | NAC ||| ||| Plasma [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3381,"cells":{"title":{"kind":"string","value":"Evaluation of Muscle Mass and Stiffness with Limb Ultrasound in COVID-19 Survivors."},"pmid":{"kind":"string","value":"35250860"},"background_abstract":{"kind":"string","value":"acute illnesses, like COVID-19, can act as a catabolic stimulus on muscles. So far, no study has evaluated muscle mass and quality through limb ultrasound in post-COVID-19 patients."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"cross sectional observational study, including patients seen one month after hospital discharge for SARS-CoV-2 pneumonia. The patients underwent a multidimensional evaluation. Moreover, we performed dominant medial gastrocnemius ultrasound (US) to characterize their muscle mass and quality."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"two hundred fifty-nine individuals (median age 67, 59.8% males) were included in the study. COVID-19 survivors with reduced muscle strength had a lower muscle US thickness (1.6 versus 1.73 cm, p =0.02) and a higher muscle stiffness (87 versus 76.3, p = 0.004) compared to patients with normal muscle strength. Also, patients with reduced Short Physical Performance Battery (SPPB) scores had a lower muscle US thickness (1.3 versus 1.71 cm, p = 0.01) and a higher muscle stiffness (104.9 versus 81.07, p = 0.04) compared to individuals with normal SPPB scores. The finding of increased muscle stiffness was also confirmed in patients with a pathological value (≥ 4) at the sarcopenia screening tool SARC-F (103.0 versus 79.55, p < 0.001). Muscle stiffness emerged as a significant predictor of probable sarcopenia (adjusted OR 1.02, 95% C.I. 1.002 - 1.04, p = 0.03). The optimal ultrasound cut-offs for probable sarcopenia were 1.51 cm for muscle thickness (p= 0.017) and 73.95 for muscle stiffness (p = 0.004)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"we described muscle ultrasound characteristics in post COVID-19 patients. Muscle ultrasound could be an innovative tool to assess muscle mass and quality in this population. Our preliminary findings need to be confirmed by future studies comparing muscle ultrasound with already validated techniques for measuring muscle mass and quality."},"conclusions_abstract_label":{"kind":"string","value":"DISCUSSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","COVID-19","Cross-Sectional Studies","Extremities","Female","Humans","Italy","Male","Middle Aged","Muscle Strength","Muscle, Skeletal","Muscular Diseases","Organ Size","SARS-CoV-2","Sarcopenia","Survivors","Ultrasonography"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"COVID-19\",\n \"Cross-Sectional Studies\",\n \"Extremities\",\n \"Female\",\n \"Humans\",\n \"Italy\",\n \"Male\",\n \"Middle Aged\",\n \"Muscle Strength\",\n \"Muscle, Skeletal\",\n \"Muscular Diseases\",\n \"Organ Size\",\n \"SARS-CoV-2\",\n \"Sarcopenia\",\n \"Survivors\",\n \"Ultrasonography\"\n]"},"pmcid":{"kind":"string","value":"8892603"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Two hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study. \nTable 1\n shows the main baseline characteristics of the study population. \nTable 2\n illustrates the main characteristics of their COVID-19 hospitalization. \nTable 3\n provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.\nBaseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).\nMain characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nMuscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\n**data on muscle stiffness were available for only 152 patients.\nSARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).\nThe comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in \nTables 1\n–\n3\n. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in \nFigure 1\n.\nNumber of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.\nThe patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04). \nFigure 2\n illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.\nComparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.\nOsteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).\nThe patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.\nThe patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).\n\n\nTable 4\n shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.\nSpearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.\nMNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nWe detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).\nPennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).\nAt the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.\nCut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in \nFigures 1S–3S\n."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Background","Material and Methods","Statistical Analyses"],"string":"[\n \"Background\",\n \"Material and Methods\",\n \"Statistical Analyses\"\n]"},"other_sections_texts":{"kind":"list like","value":["Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.","This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).","The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA)."],"string":"[\n \"Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.\",\n \"This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\",\n \"The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null],"string":"[\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Material and Methods","Statistical Analyses","Results","Discussion","Data Availability Statement","Ethics Statement","Author Contributions","Funding","Conflict of Interest","Publisher’s Note"],"string":"[\n \"Background\",\n \"Material and Methods\",\n \"Statistical Analyses\",\n \"Results\",\n \"Discussion\",\n \"Data Availability Statement\",\n \"Ethics Statement\",\n \"Author Contributions\",\n \"Funding\",\n \"Conflict of Interest\",\n \"Publisher’s Note\"\n]"},"all_sections_texts":{"kind":"list like","value":["Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.","This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).","The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).","Two hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study. \nTable 1\n shows the main baseline characteristics of the study population. \nTable 2\n illustrates the main characteristics of their COVID-19 hospitalization. \nTable 3\n provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.\nBaseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).\nMain characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nMuscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\n**data on muscle stiffness were available for only 152 patients.\nSARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).\nThe comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in \nTables 1\n–\n3\n. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in \nFigure 1\n.\nNumber of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.\nThe patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04). \nFigure 2\n illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.\nComparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.\nOsteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).\nThe patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.\nThe patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).\n\n\nTable 4\n shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.\nSpearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.\nMNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nWe detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).\nPennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).\nAt the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.\nCut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in \nFigures 1S–3S\n.","In this observational study, we found that one month after hospital discharge, COVID-19 survivors with reduced muscle function displayed low muscle mass and increased muscle stiffness at the ultrasound evaluation of the dominant medial gastrocnemius as compared with those with normal muscle function. The finding of increased muscle stiffness was confirmed in patients with a pathological SARC-F score too. Moreover, we detected a significant correlation between muscle ultrasound parameters and age, nutritional status and muscle performance. Finally, we detected an association between muscle stiffness and probable sarcopenia and with the ROC analyses we identified the cut-offs of the muscle ultrasound parameters, associated with probable sarcopenia.\nOur findings refer to preliminary data, collected one month after hospital discharge. The 3 and 6 months follow ups of the patients of this study are ongoing. Indeed, it is of paramount importance to continue follow up visits over time, because it has been reported that musculoskeletal symptoms can persist 3 and 6 months after hospitalization in COVID-19 survivors (45). Assessing whether these symptoms are underpinned by alteration of muscle function, mass and quality will allow the characterization of the COVID-19 disease on muscles, and the long-term effects of acute sarcopenia, that are presently unknown (46).\nOur results on the negative correlation between both muscle thickness and age, and pennation angle and age are in line with the typical architectural remodelling of ageing (47) characterized by decreased muscle size, and reduced pennation angles (48). Indeed, muscle thickness and pennation angle are characteristics of the muscle architecture, that are strongly related one to the other, as previously demonstrated by Kubo (49), and as confirmed in our study.\nDetecting changes in muscle architecture is of extreme importance, since these alterations have an impact on the mechanical behaviour of muscles (50). Our study confirmed the presence of a significant correlation between muscle ultrasound characteristics and measures of muscle function. Moreover, we found a significant correlation between muscle ultrasound aspects and nutritional status evaluated with the MNA-SF. Malnutrition, particularly when disease-associated, is known to be associated with alterations in body composition and reductions in fat free mass (51). Both the European Society of Clinical Nutrition and Metabolism (52) and the Global Leadership Initiative on Malnutrition (53) guidelines recommend the evaluation of fat free mass as a diagnostic criterion for malnutrition. Therefore, the association of a malnutrition screening tool with impaired measures of muscle mass and quality is not surprising. Muscle ultrasound is a simple and non-expensive tool to assess skeletal muscle characteristics, and could be a valuable instrument for the screening of malnutrition. Our finding is in line with the study by Mateos-Angulo et al. (54) that detected an association between MNA-SF and muscle thickness, measured with ultrasonography, in istitutionalized older adults.\nCompared to the study of Minetto et al. (55), the median values of muscle thickness were higher in our population (1.7, IQR 1.44 - 1.93 cm in the total sample of our study versus 1.42 ± 0.03 cm in men and 1.23 ± 0. 28 cm in women in the study of Minetto). However, Minetto et al. considered a small sample (44 people) of institutionalized pre-frail and frail older adults (mean age 79.2 ± 8.3 years) whereas our study included 259 community dwelling people with a median age of 67 years (IQR 56 – 75). Our data on muscle thickness are more in line with the findings of Kubo et al. (49) who detected a mean muscle thickness of 1.93 ± 0.27 cm in community dwelling men (mean age 69.5 ± 4.2 years) and of 1.77 ± 0.23 cm in community dwelling women (mean age 68.0 ± 5.3 years).\nDifferently from Kubo, the median values of the pennation angle, were higher in our sample (22.4° versus 16.5° in men and 15.6° in women). Anyway, the measurement of the pennation angle is strongly influenced by the pressure that the sonographer exerts on the muscle, and further data are needed to define normal and pathological values of this parameter in older people.\nOur study showed that muscle stiffness is augmented in post COVID-19 patients with reduced muscle function and pathological SARC-F score, as compared with those who had normal values of muscle function and SARC-F. Since we measured muscle stiffness with muscles in a resting condition, our finding refers to passive muscle stiffness. Passive muscle stiffness is an important characteristic, since it regulates the interactions between body and environment. When muscle stiffness is too elevated, the energy of the body-environment interactions can be transmitted to the tissues, causing an injury (56). For example, in people with an elevated muscle stiffness, there is a higher risk of muscle damage after eccentric exercise (57, 58).\nPassive muscle stiffness is influenced by collagen deposition, inflammation and swelling (59–61). Previous studies showed that the amount of collagen, of advanced glycation end-products and of collagen cross-linking in connective tissue, increase with ageing (62, 63). Indeed, we found a significant correlation between muscle stiffness and age. In addition to increasing muscle stiffness [as demonstrated in aged (64) and sarcopenic muscles (65)], the alterations of the extracellular matrix may also favour muscle mass decrease. The alterations in muscle extracellular matrix can alter the regenerative potential of the myogenic progenitor cells (66). However, the exact relation between muscle stiffness and aging has not been clearly elucidated so far. While some studies demonstrated higher muscle stiffness in older people (67–70), others detected opposite results (71). Our findings are in line with the first ones.\nIn this study we identified possible muscle ultrasound parameters cut-offs, for probable sarcopenia. Muscle ultrasound is a non-invasive, little expensive and low time-consuming technique. As such, it could potentially be considered an optimal screening test for probable sarcopenia. In our study, the muscle thickness cut off for probable sarcopenia had a high specificity (76%), but a low sensitivity (41%). It is known that highly specific screening tests unlikely yield false positive results (72). Therefore, people with a pathologic muscle thickness would likely have probable sarcopenia. On the contrary, the ultrasound cut-off for muscle stiffness had a high sensitivity (77%) but a low specificity (48%). Highly sensitive screening tests unlikely generate false negative outcomes (72). Thus, people with a normal muscle stiffness would not have probable sarcopenia.\nUnfortunately, the AUC of the ROC curves were < 0.7. These results indicate that muscle ultrasound has a low accuracy in detecting probable sarcopenia, compared to the gold standard hand grip test. Anyway, these results refer to a preliminary and reduced sample, and could be improved by future wider studies. Moreover, muscle ultrasound could be used as a complementary technique to hand grip test to assess the morphologic characteristics of skeletal muscle in patients with probable sarcopenia.\nOur study has the merit of having described for the first-time muscle mass and characteristics of post COVID-19 patients with the use of limb muscle ultrasound. Description of muscle ultrasound parameters of post COVID-19 patients with impaired muscle function and pathological SARC-F score is important, since no accepted definition of muscle quality exists so far. Characterizing the changes of muscle architecture through a non-invasive and easy to use tool as echography would provide information to better define muscle quality. Finally, we identified possible cut-off values of the muscle ultrasound parameters suggestive of the risk of sarcopenia in post COVID-19 patients. It could be speculated that muscle ultrasonography may detect subjects slowly recovering from COVID-19, and with potentially negative long-term sequelae.\nSome limitations of this study deserve to be mentioned: the relatively limited sample size, the fact that some patients with dementia (in the absence of their care-givers) could have improperly answered to some questions of the SARC-F, the missing information on muscle stiffness for 107 patients, and the dependency on the ability of the operator for the evaluation of muscle mass and quality. Due to the lack of measures of muscle mass/function prior to SARS-CoV-2 infection or during hospitalization, we could not specifically address the impact of COVID-19 on skeletal muscle. However, the main aim of our study was to characterize muscle mass and quality by muscle ultrasound in a population prone to skeletal muscle impairment (19, 20), and to assess the association of ultrasound parameters with established tools for the assessment of the risk of sarcopenia. Further studies are needed to assess whether our findings can be generalized to patient populations other than COVID-19 survivors. Finally, an important limit is that we did not compare ultrasound muscle characteristics against reference methods for measuring fat free mass, such as dual-energy X-ray absorptiometry, CT or MRI. In the future, wider, multicenter studies will help better define the role of ultrasound for the evaluation of muscle quantity and quality, and correlate these data to relevant clinical outcomes.\nIn conclusion, we showed that muscle ultrasound parameters have a significant correlation with age, nutritional status and muscle performance in COVID-19 survivors. Although our findings need to be confirmed by studies comparing muscle ultrasound against validated techniques for measuring muscle mass and quality, our study suggests, for the first time, that muscle ultrasound could be an innovative tool to assess muscle mass and quality in COVID-19 survivors.","The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.","The studies involving human participants were reviewed and approved by San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). The patients/participants provided their written informed consent to participate in this study.","All authors made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted.","This study was financially supported by Ministero della Salute, Italy, and by COVID-19 donations.","The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.","All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."],"string":"[\n \"Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.\",\n \"This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\",\n \"The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\",\n \"Two hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study. \\nTable 1\\n shows the main baseline characteristics of the study population. \\nTable 2\\n illustrates the main characteristics of their COVID-19 hospitalization. \\nTable 3\\n provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.\\nBaseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.\\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).\\nMain characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\\nMuscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\\n**data on muscle stiffness were available for only 152 patients.\\nSARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).\\nThe comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in \\nTables 1\\n–\\n3\\n. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in \\nFigure 1\\n.\\nNumber of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.\\nThe patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04). \\nFigure 2\\n illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.\\nComparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.\\nOsteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).\\nThe patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.\\nThe patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).\\n\\n\\nTable 4\\n shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.\\nSpearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.\\nMNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\\nWe detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).\\nPennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).\\nAt the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.\\nCut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in \\nFigures 1S–3S\\n.\",\n \"In this observational study, we found that one month after hospital discharge, COVID-19 survivors with reduced muscle function displayed low muscle mass and increased muscle stiffness at the ultrasound evaluation of the dominant medial gastrocnemius as compared with those with normal muscle function. The finding of increased muscle stiffness was confirmed in patients with a pathological SARC-F score too. Moreover, we detected a significant correlation between muscle ultrasound parameters and age, nutritional status and muscle performance. Finally, we detected an association between muscle stiffness and probable sarcopenia and with the ROC analyses we identified the cut-offs of the muscle ultrasound parameters, associated with probable sarcopenia.\\nOur findings refer to preliminary data, collected one month after hospital discharge. The 3 and 6 months follow ups of the patients of this study are ongoing. Indeed, it is of paramount importance to continue follow up visits over time, because it has been reported that musculoskeletal symptoms can persist 3 and 6 months after hospitalization in COVID-19 survivors (45). Assessing whether these symptoms are underpinned by alteration of muscle function, mass and quality will allow the characterization of the COVID-19 disease on muscles, and the long-term effects of acute sarcopenia, that are presently unknown (46).\\nOur results on the negative correlation between both muscle thickness and age, and pennation angle and age are in line with the typical architectural remodelling of ageing (47) characterized by decreased muscle size, and reduced pennation angles (48). Indeed, muscle thickness and pennation angle are characteristics of the muscle architecture, that are strongly related one to the other, as previously demonstrated by Kubo (49), and as confirmed in our study.\\nDetecting changes in muscle architecture is of extreme importance, since these alterations have an impact on the mechanical behaviour of muscles (50). Our study confirmed the presence of a significant correlation between muscle ultrasound characteristics and measures of muscle function. Moreover, we found a significant correlation between muscle ultrasound aspects and nutritional status evaluated with the MNA-SF. Malnutrition, particularly when disease-associated, is known to be associated with alterations in body composition and reductions in fat free mass (51). Both the European Society of Clinical Nutrition and Metabolism (52) and the Global Leadership Initiative on Malnutrition (53) guidelines recommend the evaluation of fat free mass as a diagnostic criterion for malnutrition. Therefore, the association of a malnutrition screening tool with impaired measures of muscle mass and quality is not surprising. Muscle ultrasound is a simple and non-expensive tool to assess skeletal muscle characteristics, and could be a valuable instrument for the screening of malnutrition. Our finding is in line with the study by Mateos-Angulo et al. (54) that detected an association between MNA-SF and muscle thickness, measured with ultrasonography, in istitutionalized older adults.\\nCompared to the study of Minetto et al. (55), the median values of muscle thickness were higher in our population (1.7, IQR 1.44 - 1.93 cm in the total sample of our study versus 1.42 ± 0.03 cm in men and 1.23 ± 0. 28 cm in women in the study of Minetto). However, Minetto et al. considered a small sample (44 people) of institutionalized pre-frail and frail older adults (mean age 79.2 ± 8.3 years) whereas our study included 259 community dwelling people with a median age of 67 years (IQR 56 – 75). Our data on muscle thickness are more in line with the findings of Kubo et al. (49) who detected a mean muscle thickness of 1.93 ± 0.27 cm in community dwelling men (mean age 69.5 ± 4.2 years) and of 1.77 ± 0.23 cm in community dwelling women (mean age 68.0 ± 5.3 years).\\nDifferently from Kubo, the median values of the pennation angle, were higher in our sample (22.4° versus 16.5° in men and 15.6° in women). Anyway, the measurement of the pennation angle is strongly influenced by the pressure that the sonographer exerts on the muscle, and further data are needed to define normal and pathological values of this parameter in older people.\\nOur study showed that muscle stiffness is augmented in post COVID-19 patients with reduced muscle function and pathological SARC-F score, as compared with those who had normal values of muscle function and SARC-F. Since we measured muscle stiffness with muscles in a resting condition, our finding refers to passive muscle stiffness. Passive muscle stiffness is an important characteristic, since it regulates the interactions between body and environment. When muscle stiffness is too elevated, the energy of the body-environment interactions can be transmitted to the tissues, causing an injury (56). For example, in people with an elevated muscle stiffness, there is a higher risk of muscle damage after eccentric exercise (57, 58).\\nPassive muscle stiffness is influenced by collagen deposition, inflammation and swelling (59–61). Previous studies showed that the amount of collagen, of advanced glycation end-products and of collagen cross-linking in connective tissue, increase with ageing (62, 63). Indeed, we found a significant correlation between muscle stiffness and age. In addition to increasing muscle stiffness [as demonstrated in aged (64) and sarcopenic muscles (65)], the alterations of the extracellular matrix may also favour muscle mass decrease. The alterations in muscle extracellular matrix can alter the regenerative potential of the myogenic progenitor cells (66). However, the exact relation between muscle stiffness and aging has not been clearly elucidated so far. While some studies demonstrated higher muscle stiffness in older people (67–70), others detected opposite results (71). Our findings are in line with the first ones.\\nIn this study we identified possible muscle ultrasound parameters cut-offs, for probable sarcopenia. Muscle ultrasound is a non-invasive, little expensive and low time-consuming technique. As such, it could potentially be considered an optimal screening test for probable sarcopenia. In our study, the muscle thickness cut off for probable sarcopenia had a high specificity (76%), but a low sensitivity (41%). It is known that highly specific screening tests unlikely yield false positive results (72). Therefore, people with a pathologic muscle thickness would likely have probable sarcopenia. On the contrary, the ultrasound cut-off for muscle stiffness had a high sensitivity (77%) but a low specificity (48%). Highly sensitive screening tests unlikely generate false negative outcomes (72). Thus, people with a normal muscle stiffness would not have probable sarcopenia.\\nUnfortunately, the AUC of the ROC curves were < 0.7. These results indicate that muscle ultrasound has a low accuracy in detecting probable sarcopenia, compared to the gold standard hand grip test. Anyway, these results refer to a preliminary and reduced sample, and could be improved by future wider studies. Moreover, muscle ultrasound could be used as a complementary technique to hand grip test to assess the morphologic characteristics of skeletal muscle in patients with probable sarcopenia.\\nOur study has the merit of having described for the first-time muscle mass and characteristics of post COVID-19 patients with the use of limb muscle ultrasound. Description of muscle ultrasound parameters of post COVID-19 patients with impaired muscle function and pathological SARC-F score is important, since no accepted definition of muscle quality exists so far. Characterizing the changes of muscle architecture through a non-invasive and easy to use tool as echography would provide information to better define muscle quality. Finally, we identified possible cut-off values of the muscle ultrasound parameters suggestive of the risk of sarcopenia in post COVID-19 patients. It could be speculated that muscle ultrasonography may detect subjects slowly recovering from COVID-19, and with potentially negative long-term sequelae.\\nSome limitations of this study deserve to be mentioned: the relatively limited sample size, the fact that some patients with dementia (in the absence of their care-givers) could have improperly answered to some questions of the SARC-F, the missing information on muscle stiffness for 107 patients, and the dependency on the ability of the operator for the evaluation of muscle mass and quality. Due to the lack of measures of muscle mass/function prior to SARS-CoV-2 infection or during hospitalization, we could not specifically address the impact of COVID-19 on skeletal muscle. However, the main aim of our study was to characterize muscle mass and quality by muscle ultrasound in a population prone to skeletal muscle impairment (19, 20), and to assess the association of ultrasound parameters with established tools for the assessment of the risk of sarcopenia. Further studies are needed to assess whether our findings can be generalized to patient populations other than COVID-19 survivors. Finally, an important limit is that we did not compare ultrasound muscle characteristics against reference methods for measuring fat free mass, such as dual-energy X-ray absorptiometry, CT or MRI. In the future, wider, multicenter studies will help better define the role of ultrasound for the evaluation of muscle quantity and quality, and correlate these data to relevant clinical outcomes.\\nIn conclusion, we showed that muscle ultrasound parameters have a significant correlation with age, nutritional status and muscle performance in COVID-19 survivors. Although our findings need to be confirmed by studies comparing muscle ultrasound against validated techniques for measuring muscle mass and quality, our study suggests, for the first time, that muscle ultrasound could be an innovative tool to assess muscle mass and quality in COVID-19 survivors.\",\n \"The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\",\n \"The studies involving human participants were reviewed and approved by San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). The patients/participants provided their written informed consent to participate in this study.\",\n \"All authors made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted.\",\n \"This study was financially supported by Ministero della Salute, Italy, and by COVID-19 donations.\",\n \"The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\",\n \"All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,"results","discussion","data-availability","ethics-statement","author-contributions","funding-information","COI-statement","disclaimer"],"string":"[\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"data-availability\",\n \"ethics-statement\",\n \"author-contributions\",\n \"funding-information\",\n \"COI-statement\",\n \"disclaimer\"\n]"},"keywords":{"kind":"list like","value":["muscle","ultrasound","sarcopenia","COVID - 19","muscle mass","muscle quality"],"string":"[\n \"muscle\",\n \"ultrasound\",\n \"sarcopenia\",\n \"COVID - 19\",\n \"muscle mass\",\n \"muscle quality\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nSarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.\n\nMaterial and Methods:\nThis was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\n\nStatistical Analyses:\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\n\nResults:\nTwo hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study. \nTable 1\n shows the main baseline characteristics of the study population. \nTable 2\n illustrates the main characteristics of their COVID-19 hospitalization. \nTable 3\n provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.\nBaseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).\nMain characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nMuscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\n**data on muscle stiffness were available for only 152 patients.\nSARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).\nThe comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in \nTables 1\n–\n3\n. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in \nFigure 1\n.\nNumber of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.\nThe patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04). \nFigure 2\n illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.\nComparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.\nOsteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).\nThe patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.\nThe patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).\n\n\nTable 4\n shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.\nSpearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.\nMNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nWe detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).\nPennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).\nAt the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.\nCut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in \nFigures 1S–3S\n.\n\nDiscussion:\nIn this observational study, we found that one month after hospital discharge, COVID-19 survivors with reduced muscle function displayed low muscle mass and increased muscle stiffness at the ultrasound evaluation of the dominant medial gastrocnemius as compared with those with normal muscle function. The finding of increased muscle stiffness was confirmed in patients with a pathological SARC-F score too. Moreover, we detected a significant correlation between muscle ultrasound parameters and age, nutritional status and muscle performance. Finally, we detected an association between muscle stiffness and probable sarcopenia and with the ROC analyses we identified the cut-offs of the muscle ultrasound parameters, associated with probable sarcopenia.\nOur findings refer to preliminary data, collected one month after hospital discharge. The 3 and 6 months follow ups of the patients of this study are ongoing. Indeed, it is of paramount importance to continue follow up visits over time, because it has been reported that musculoskeletal symptoms can persist 3 and 6 months after hospitalization in COVID-19 survivors (45). Assessing whether these symptoms are underpinned by alteration of muscle function, mass and quality will allow the characterization of the COVID-19 disease on muscles, and the long-term effects of acute sarcopenia, that are presently unknown (46).\nOur results on the negative correlation between both muscle thickness and age, and pennation angle and age are in line with the typical architectural remodelling of ageing (47) characterized by decreased muscle size, and reduced pennation angles (48). Indeed, muscle thickness and pennation angle are characteristics of the muscle architecture, that are strongly related one to the other, as previously demonstrated by Kubo (49), and as confirmed in our study.\nDetecting changes in muscle architecture is of extreme importance, since these alterations have an impact on the mechanical behaviour of muscles (50). Our study confirmed the presence of a significant correlation between muscle ultrasound characteristics and measures of muscle function. Moreover, we found a significant correlation between muscle ultrasound aspects and nutritional status evaluated with the MNA-SF. Malnutrition, particularly when disease-associated, is known to be associated with alterations in body composition and reductions in fat free mass (51). Both the European Society of Clinical Nutrition and Metabolism (52) and the Global Leadership Initiative on Malnutrition (53) guidelines recommend the evaluation of fat free mass as a diagnostic criterion for malnutrition. Therefore, the association of a malnutrition screening tool with impaired measures of muscle mass and quality is not surprising. Muscle ultrasound is a simple and non-expensive tool to assess skeletal muscle characteristics, and could be a valuable instrument for the screening of malnutrition. Our finding is in line with the study by Mateos-Angulo et al. (54) that detected an association between MNA-SF and muscle thickness, measured with ultrasonography, in istitutionalized older adults.\nCompared to the study of Minetto et al. (55), the median values of muscle thickness were higher in our population (1.7, IQR 1.44 - 1.93 cm in the total sample of our study versus 1.42 ± 0.03 cm in men and 1.23 ± 0. 28 cm in women in the study of Minetto). However, Minetto et al. considered a small sample (44 people) of institutionalized pre-frail and frail older adults (mean age 79.2 ± 8.3 years) whereas our study included 259 community dwelling people with a median age of 67 years (IQR 56 – 75). Our data on muscle thickness are more in line with the findings of Kubo et al. (49) who detected a mean muscle thickness of 1.93 ± 0.27 cm in community dwelling men (mean age 69.5 ± 4.2 years) and of 1.77 ± 0.23 cm in community dwelling women (mean age 68.0 ± 5.3 years).\nDifferently from Kubo, the median values of the pennation angle, were higher in our sample (22.4° versus 16.5° in men and 15.6° in women). Anyway, the measurement of the pennation angle is strongly influenced by the pressure that the sonographer exerts on the muscle, and further data are needed to define normal and pathological values of this parameter in older people.\nOur study showed that muscle stiffness is augmented in post COVID-19 patients with reduced muscle function and pathological SARC-F score, as compared with those who had normal values of muscle function and SARC-F. Since we measured muscle stiffness with muscles in a resting condition, our finding refers to passive muscle stiffness. Passive muscle stiffness is an important characteristic, since it regulates the interactions between body and environment. When muscle stiffness is too elevated, the energy of the body-environment interactions can be transmitted to the tissues, causing an injury (56). For example, in people with an elevated muscle stiffness, there is a higher risk of muscle damage after eccentric exercise (57, 58).\nPassive muscle stiffness is influenced by collagen deposition, inflammation and swelling (59–61). Previous studies showed that the amount of collagen, of advanced glycation end-products and of collagen cross-linking in connective tissue, increase with ageing (62, 63). Indeed, we found a significant correlation between muscle stiffness and age. In addition to increasing muscle stiffness [as demonstrated in aged (64) and sarcopenic muscles (65)], the alterations of the extracellular matrix may also favour muscle mass decrease. The alterations in muscle extracellular matrix can alter the regenerative potential of the myogenic progenitor cells (66). However, the exact relation between muscle stiffness and aging has not been clearly elucidated so far. While some studies demonstrated higher muscle stiffness in older people (67–70), others detected opposite results (71). Our findings are in line with the first ones.\nIn this study we identified possible muscle ultrasound parameters cut-offs, for probable sarcopenia. Muscle ultrasound is a non-invasive, little expensive and low time-consuming technique. As such, it could potentially be considered an optimal screening test for probable sarcopenia. In our study, the muscle thickness cut off for probable sarcopenia had a high specificity (76%), but a low sensitivity (41%). It is known that highly specific screening tests unlikely yield false positive results (72). Therefore, people with a pathologic muscle thickness would likely have probable sarcopenia. On the contrary, the ultrasound cut-off for muscle stiffness had a high sensitivity (77%) but a low specificity (48%). Highly sensitive screening tests unlikely generate false negative outcomes (72). Thus, people with a normal muscle stiffness would not have probable sarcopenia.\nUnfortunately, the AUC of the ROC curves were < 0.7. These results indicate that muscle ultrasound has a low accuracy in detecting probable sarcopenia, compared to the gold standard hand grip test. Anyway, these results refer to a preliminary and reduced sample, and could be improved by future wider studies. Moreover, muscle ultrasound could be used as a complementary technique to hand grip test to assess the morphologic characteristics of skeletal muscle in patients with probable sarcopenia.\nOur study has the merit of having described for the first-time muscle mass and characteristics of post COVID-19 patients with the use of limb muscle ultrasound. Description of muscle ultrasound parameters of post COVID-19 patients with impaired muscle function and pathological SARC-F score is important, since no accepted definition of muscle quality exists so far. Characterizing the changes of muscle architecture through a non-invasive and easy to use tool as echography would provide information to better define muscle quality. Finally, we identified possible cut-off values of the muscle ultrasound parameters suggestive of the risk of sarcopenia in post COVID-19 patients. It could be speculated that muscle ultrasonography may detect subjects slowly recovering from COVID-19, and with potentially negative long-term sequelae.\nSome limitations of this study deserve to be mentioned: the relatively limited sample size, the fact that some patients with dementia (in the absence of their care-givers) could have improperly answered to some questions of the SARC-F, the missing information on muscle stiffness for 107 patients, and the dependency on the ability of the operator for the evaluation of muscle mass and quality. Due to the lack of measures of muscle mass/function prior to SARS-CoV-2 infection or during hospitalization, we could not specifically address the impact of COVID-19 on skeletal muscle. However, the main aim of our study was to characterize muscle mass and quality by muscle ultrasound in a population prone to skeletal muscle impairment (19, 20), and to assess the association of ultrasound parameters with established tools for the assessment of the risk of sarcopenia. Further studies are needed to assess whether our findings can be generalized to patient populations other than COVID-19 survivors. Finally, an important limit is that we did not compare ultrasound muscle characteristics against reference methods for measuring fat free mass, such as dual-energy X-ray absorptiometry, CT or MRI. In the future, wider, multicenter studies will help better define the role of ultrasound for the evaluation of muscle quantity and quality, and correlate these data to relevant clinical outcomes.\nIn conclusion, we showed that muscle ultrasound parameters have a significant correlation with age, nutritional status and muscle performance in COVID-19 survivors. Although our findings need to be confirmed by studies comparing muscle ultrasound against validated techniques for measuring muscle mass and quality, our study suggests, for the first time, that muscle ultrasound could be an innovative tool to assess muscle mass and quality in COVID-19 survivors.\n\nData Availability Statement:\nThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\n\nEthics Statement:\nThe studies involving human participants were reviewed and approved by San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). The patients/participants provided their written informed consent to participate in this study.\n\nAuthor Contributions:\nAll authors made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted.\n\nFunding:\nThis study was financially supported by Ministero della Salute, Italy, and by COVID-19 donations.\n\nConflict of Interest:\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\n\nPublisher’s Note:\nAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nacute illnesses, like COVID-19, can act as a catabolic stimulus on muscles. So far, no study has evaluated muscle mass and quality through limb ultrasound in post-COVID-19 patients.\n\nMethods:\ncross sectional observational study, including patients seen one month after hospital discharge for SARS-CoV-2 pneumonia. The patients underwent a multidimensional evaluation. Moreover, we performed dominant medial gastrocnemius ultrasound (US) to characterize their muscle mass and quality.\n\nResults:\ntwo hundred fifty-nine individuals (median age 67, 59.8% males) were included in the study. COVID-19 survivors with reduced muscle strength had a lower muscle US thickness (1.6 versus 1.73 cm, p =0.02) and a higher muscle stiffness (87 versus 76.3, p = 0.004) compared to patients with normal muscle strength. Also, patients with reduced Short Physical Performance Battery (SPPB) scores had a lower muscle US thickness (1.3 versus 1.71 cm, p = 0.01) and a higher muscle stiffness (104.9 versus 81.07, p = 0.04) compared to individuals with normal SPPB scores. The finding of increased muscle stiffness was also confirmed in patients with a pathological value (≥ 4) at the sarcopenia screening tool SARC-F (103.0 versus 79.55, p < 0.001). Muscle stiffness emerged as a significant predictor of probable sarcopenia (adjusted OR 1.02, 95% C.I. 1.002 - 1.04, p = 0.03). The optimal ultrasound cut-offs for probable sarcopenia were 1.51 cm for muscle thickness (p= 0.017) and 73.95 for muscle stiffness (p = 0.004).\n\nConclusions:\nwe described muscle ultrasound characteristics in post COVID-19 patients. Muscle ultrasound could be an innovative tool to assess muscle mass and quality in this population. Our preliminary findings need to be confirmed by future studies comparing muscle ultrasound with already validated techniques for measuring muscle mass and quality."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":7069,"string":"7,069"},"whole_article_abstract_length":{"kind":"number","value":356,"string":"356"},"other_sections_lengths":{"kind":"list like","value":[681,1969,567],"string":"[\n 681,\n 1969,\n 567\n]"},"num_sections":{"kind":"number","value":11,"string":"11"},"most_frequent_words":{"kind":"list like","value":["muscle","sarcopenia","ultrasound","strength","patients","stiffness","hospital","study","muscle stiffness","muscle ultrasound"],"string":"[\n \"muscle\",\n \"sarcopenia\",\n \"ultrasound\",\n \"strength\",\n \"patients\",\n \"stiffness\",\n \"hospital\",\n \"study\",\n \"muscle stiffness\",\n \"muscle ultrasound\"\n]"},"keybert_topics":{"kind":"list like","value":["sarcopenic muscles","sarcopenia muscle stiffness","muscle quality assessing","muscle quality determined","sarcopenia reduced muscle"],"string":"[\n \"sarcopenic muscles\",\n \"sarcopenia muscle stiffness\",\n \"muscle quality assessing\",\n \"muscle quality determined\",\n \"sarcopenia reduced muscle\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | ultrasound | sarcopenia | COVID - 19 | muscle mass | muscle quality [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | ultrasound | sarcopenia | COVID - 19 | muscle mass | muscle quality [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | ultrasound | sarcopenia | COVID - 19 | muscle mass | muscle quality [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | COVID-19 | Cross-Sectional Studies | Extremities | Female | Humans | Italy | Male | Middle Aged | Muscle Strength | Muscle, Skeletal | Muscular Diseases | Organ Size | SARS-CoV-2 | Sarcopenia | Survivors | Ultrasonography [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | COVID-19 | Cross-Sectional Studies | Extremities | Female | Humans | Italy | Male | Middle Aged | Muscle Strength | Muscle, Skeletal | Muscular Diseases | Organ Size | SARS-CoV-2 | Sarcopenia | Survivors | Ultrasonography [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | COVID-19 | Cross-Sectional Studies | Extremities | Female | Humans | Italy | Male | Middle Aged | Muscle Strength | Muscle, Skeletal | Muscular Diseases | Organ Size | SARS-CoV-2 | Sarcopenia | Survivors | Ultrasonography [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] sarcopenic muscles | sarcopenia muscle stiffness | muscle quality assessing | muscle quality determined | sarcopenia reduced muscle [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] sarcopenic muscles | sarcopenia muscle stiffness | muscle quality assessing | muscle quality determined | sarcopenia reduced muscle [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] sarcopenic muscles | sarcopenia muscle stiffness | muscle quality assessing | muscle quality determined | sarcopenia reduced muscle [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | sarcopenia | ultrasound | strength | patients | stiffness | hospital | study | muscle stiffness | muscle ultrasound [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | sarcopenia | ultrasound | strength | patients | stiffness | hospital | study | muscle stiffness | muscle ultrasound [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | sarcopenia | ultrasound | strength | patients | stiffness | hospital | study | muscle stiffness | muscle ultrasound [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | sarcopenia | muscle mass | quality | mass | acute | muscle quality | acute sarcopenia | ultrasound | developing [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | 001 | 95 | strength | normal | 04 | sarcopenia | patients | 01 | performance [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] muscle | sarcopenia | strength | ultrasound | article | authors | study | patients | hospital | 19 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] two hundred fifty-nine | age 67 | 59.8% ||| COVID-19 | US | 1.6 | 1.73 cm | 0.02 | 87 | 76.3 | 0.004 ||| Short Physical Performance Battery | SPPB | US | 1.3 | 1.71 cm | 0.01 | 104.9 | 81.07 | 0.04 | SPPB ||| ≥ | 4 | 103.0 | 79.55 | p < 0.001 ||| 1.02 | 95% | C.I. | 1.002 - 1.04 | 0.03 ||| 1.51 cm | 0.017 | 73.95 | 0.004 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| ||| one month ||| ||| US ||| two hundred fifty-nine | age 67 | 59.8% ||| COVID-19 | US | 1.6 | 1.73 cm | 0.02 | 87 | 76.3 | 0.004 ||| Short Physical Performance Battery | SPPB | US | 1.3 | 1.71 cm | 0.01 | 104.9 | 81.07 | 0.04 | SPPB ||| ≥ | 4 | 103.0 | 79.55 | p < 0.001 ||| 1.02 | 95% | C.I. | 1.002 - 1.04 | 0.03 ||| 1.51 cm | 0.017 | 73.95 | 0.004 ||| COVID-19 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3382,"cells":{"title":{"kind":"string","value":"Medical residents reflect on their prejudices toward poverty: a photovoice training project."},"pmid":{"kind":"string","value":"25551370"},"background_abstract":{"kind":"string","value":"Clinicians face challenges in delivering care to socioeconomically disadvantaged patients. While both the public and academic sectors recognize the importance of addressing social inequities in healthcare, there is room for improvement in the training of family physicians, who report being ill-equipped to provide care that is responsive to the living conditions of these patients. This study explored: (i) residents' perceptions and experience in relation to providing care for socioeconomically disadvantaged patients, and (ii) how participating in a photovoice study helped them uncover and examine some of their prejudices and assumptions about poverty."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We conducted a participatory photovoice study. Participants were four family medicine residents, two medical supervisors, and two researchers. Residents attended six photovoice meetings at which they discussed photos they had taken. In collaboration with the researchers, the participants defined the research questions, took photos, and participated in data analysis and results dissemination. Meetings were recorded and transcribed for analysis, which consisted of coding, peer debriefing, thematic analysis, and interpretation."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The medical residents uncovered and examined their own prejudices and misconceptions about poverty. They reported feeling unprepared to provide care to socioeconomically disadvantaged patients. Supported by medical supervisors and researchers, the residents underwent a three-phase reflexive process of: (1) engaging reflexively, (2) break(ing) through, and (3) taking action. The results indicated that medical residents subsequently felt encouraged to adopt a care approach that helped them overcome the social distance between themselves and their socioeconomically disadvantaged patients."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Attitude of Health Personnel","Canada","Family Practice","Humans","Internship and Residency","Poverty","Prejudice","Students, Medical"],"string":"[\n \"Attitude of Health Personnel\",\n \"Canada\",\n \"Family Practice\",\n \"Humans\",\n \"Internship and Residency\",\n \"Poverty\",\n \"Prejudice\",\n \"Students, Medical\"\n]"},"pmcid":{"kind":"string","value":"4323214"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].\nClinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].\nPhysicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].\nHence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].\nPhotovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].\nThis participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.\n Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\nThe process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\n Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nZigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\n Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\nBeing reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."},"other_sections_titles":{"kind":"list like","value":["Study design","Recruitment and participants","Design and procedure","Data analysis","Phase1: engaging reflexively with poverty","Phase 2: break(ing)through","Phase 3: taking action"],"string":"[\n \"Study design\",\n \"Recruitment and participants\",\n \"Design and procedure\",\n \"Data analysis\",\n \"Phase1: engaging reflexively with poverty\",\n \"Phase 2: break(ing)through\",\n \"Phase 3: taking action\"\n]"},"other_sections_texts":{"kind":"list like","value":["This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].","The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.","We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.","All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.","The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.","Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.","Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents."],"string":"[\n \"This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\",\n \"The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\",\n \"We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\",\n \"All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\",\n \"The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\",\n \"Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\",\n \"Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study design","Recruitment and participants","Design and procedure","Data analysis","Results","Phase1: engaging reflexively with poverty","Phase 2: break(ing)through","Phase 3: taking action","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design\",\n \"Recruitment and participants\",\n \"Design and procedure\",\n \"Data analysis\",\n \"Results\",\n \"Phase1: engaging reflexively with poverty\",\n \"Phase 2: break(ing)through\",\n \"Phase 3: taking action\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].\nClinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].\nPhysicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].\nHence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].\nPhotovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].\nThis participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty."," Study design This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\nThis study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\n Recruitment and participants The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\nThe first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\n Design and procedure We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\nWe conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\n Data analysis All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\nAll meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.","This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].","The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.","We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.","All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.","The medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.\n Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\nThe process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\n Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nZigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\n Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\nBeing reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.","The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.","Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.","Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.","This study explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons. It also examined how residents’ involvement in a photovoice project helped them uncover their prejudices and assumptions concerning poverty. In this project, residents underwent a three-phase learning process of: (1) engaging reflexively; (2) break(ing)through; and (3) taking action. Sharing experiences involved lengthy conversations in which participants responded to photos presented by others, made connections with other participants’ comments, and highlighted differences and similarities. In fact, most “individual” photos became “group” photos as they were appropriated by each of the participants, who were stimulated by the photos to reflect on their own concerns.\nAs in other photovoice projects, this research generated a reflexive process that led to sustained critical consciousness and constituted “transformative learning” [27,28]. As noted by Sandars [29], the medical residents reported undergoing transformations in their perceptions of themselves and of the world triggered by a process of introspection during which feelings of powerlessness, sadness, shame, and anger emerged.\nWe believe the medical residents’ experience with the photovoice project encouraged them to take poverty into serious consideration in their practice. The entire photovoice project, encompassing three phases, was a learning process for all participants. Residents’ concrete actions in direct response to the project could be seen as evidence that it was an effective teaching tool. As reported by Carlson, Engebretson, and Chamberlain [17], photovoice can be a means for expanding social consciousness and triggering social change, because its use fosters both reflection and action.\nWe acknowledge that the research was limited by the small number of participants. Small sample size in photovoice research initiatives is not uncommon because of the time requirements and the nature of the participants’ involvement. For example, in their oft-cited article on African-American mothers, Killion & Wang [30] had five participants. Another limitation of our study relates to the fact that the medical supervisors fulfilled multiple roles with the medical residents. A posteriori we considered it to be an asset for our data collection, because the supervisors contributed valuable input to the photovoice meetings. However, we observed that power relations could arise among the participants because the supervisors were involved in the residents’ evaluations.\nBy involving both medical residents and supervisors in this photovoice study, we made one of the first attempts in a medical academic milieu to involve healthcare professionals in identifying ways to improve physicians’ competencies in delivering care to socioeconomically disadvantaged patients. The study and its outcomes also generated momentum for changes to the family medicine curriculum at a teaching university. Thus, to improve the knowledge and skills of future primary care doctors and incorporate a “social competence” component into residency programs, we recommend developing an experiential learning approach by giving residents opportunities to be involved in underserved community activities and in reflexive discussion with medical supervisors.\nIn conclusion, photovoice appears to be a promising and innovative teaching approach in medical education, especially for medical residents. More specifically, our participatory research project helped future physicians become aware of their prejudices and motivated them to acquire skills for delivering healthcare to socioeconomically disadvantaged patients. Photovoice can be an effective tool for raising health professionals’ awareness of socioeconomic realities of their patients.","This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."],"string":"[\n \"Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].\\nClinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].\\nPhysicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].\\nHence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].\\nPhotovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].\\nThis participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty.\",\n \" Study design This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\\nThis study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\\n Recruitment and participants The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\\nThe first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\\n Design and procedure We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\\nWe conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\\n Data analysis All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\\nAll meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\",\n \"This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\",\n \"The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\",\n \"We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\",\n \"All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\",\n \"The medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.\\n Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\\nThe process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\\n Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\\nZigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\\n Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\\nBeing reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\",\n \"The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\",\n \"Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\",\n \"Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\",\n \"This study explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons. It also examined how residents’ involvement in a photovoice project helped them uncover their prejudices and assumptions concerning poverty. In this project, residents underwent a three-phase learning process of: (1) engaging reflexively; (2) break(ing)through; and (3) taking action. Sharing experiences involved lengthy conversations in which participants responded to photos presented by others, made connections with other participants’ comments, and highlighted differences and similarities. In fact, most “individual” photos became “group” photos as they were appropriated by each of the participants, who were stimulated by the photos to reflect on their own concerns.\\nAs in other photovoice projects, this research generated a reflexive process that led to sustained critical consciousness and constituted “transformative learning” [27,28]. As noted by Sandars [29], the medical residents reported undergoing transformations in their perceptions of themselves and of the world triggered by a process of introspection during which feelings of powerlessness, sadness, shame, and anger emerged.\\nWe believe the medical residents’ experience with the photovoice project encouraged them to take poverty into serious consideration in their practice. The entire photovoice project, encompassing three phases, was a learning process for all participants. Residents’ concrete actions in direct response to the project could be seen as evidence that it was an effective teaching tool. As reported by Carlson, Engebretson, and Chamberlain [17], photovoice can be a means for expanding social consciousness and triggering social change, because its use fosters both reflection and action.\\nWe acknowledge that the research was limited by the small number of participants. Small sample size in photovoice research initiatives is not uncommon because of the time requirements and the nature of the participants’ involvement. For example, in their oft-cited article on African-American mothers, Killion & Wang [30] had five participants. Another limitation of our study relates to the fact that the medical supervisors fulfilled multiple roles with the medical residents. A posteriori we considered it to be an asset for our data collection, because the supervisors contributed valuable input to the photovoice meetings. However, we observed that power relations could arise among the participants because the supervisors were involved in the residents’ evaluations.\\nBy involving both medical residents and supervisors in this photovoice study, we made one of the first attempts in a medical academic milieu to involve healthcare professionals in identifying ways to improve physicians’ competencies in delivering care to socioeconomically disadvantaged patients. The study and its outcomes also generated momentum for changes to the family medicine curriculum at a teaching university. Thus, to improve the knowledge and skills of future primary care doctors and incorporate a “social competence” component into residency programs, we recommend developing an experiential learning approach by giving residents opportunities to be involved in underserved community activities and in reflexive discussion with medical supervisors.\\nIn conclusion, photovoice appears to be a promising and innovative teaching approach in medical education, especially for medical residents. More specifically, our participatory research project helped future physicians become aware of their prejudices and motivated them to acquire skills for delivering healthcare to socioeconomically disadvantaged patients. Photovoice can be an effective tool for raising health professionals’ awareness of socioeconomic realities of their patients.\",\n \"This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,"results",null,null,null,"discussion","conclusions"],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]"},"keywords":{"kind":"list like","value":["Healthcare disparities","Poverty","Participatory action research","Photovoice","Residents","Education","Healthcare professionals","Medicine"],"string":"[\n \"Healthcare disparities\",\n \"Poverty\",\n \"Participatory action research\",\n \"Photovoice\",\n \"Residents\",\n \"Education\",\n \"Healthcare professionals\",\n \"Medicine\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nPoverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].\nClinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].\nPhysicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].\nHence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].\nPhotovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].\nThis participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty.\n\nMethods:\n Study design This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\nThis study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\n Recruitment and participants The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\nThe first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\n Design and procedure We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\nWe conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\n Data analysis All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\nAll meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\n\nStudy design:\nThis study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\n\nRecruitment and participants:\nThe first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\n\nDesign and procedure:\nWe conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\n\nData analysis:\nAll meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\n\nResults:\nThe medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.\n Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\nThe process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\n Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nZigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\n Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\nBeing reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\n\nPhase1: engaging reflexively with poverty:\nThe process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\n\nPhase 2: break(ing)through:\nZigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\n\nPhase 3: taking action:\nBeing reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\n\nDiscussion:\nThis study explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons. It also examined how residents’ involvement in a photovoice project helped them uncover their prejudices and assumptions concerning poverty. In this project, residents underwent a three-phase learning process of: (1) engaging reflexively; (2) break(ing)through; and (3) taking action. Sharing experiences involved lengthy conversations in which participants responded to photos presented by others, made connections with other participants’ comments, and highlighted differences and similarities. In fact, most “individual” photos became “group” photos as they were appropriated by each of the participants, who were stimulated by the photos to reflect on their own concerns.\nAs in other photovoice projects, this research generated a reflexive process that led to sustained critical consciousness and constituted “transformative learning” [27,28]. As noted by Sandars [29], the medical residents reported undergoing transformations in their perceptions of themselves and of the world triggered by a process of introspection during which feelings of powerlessness, sadness, shame, and anger emerged.\nWe believe the medical residents’ experience with the photovoice project encouraged them to take poverty into serious consideration in their practice. The entire photovoice project, encompassing three phases, was a learning process for all participants. Residents’ concrete actions in direct response to the project could be seen as evidence that it was an effective teaching tool. As reported by Carlson, Engebretson, and Chamberlain [17], photovoice can be a means for expanding social consciousness and triggering social change, because its use fosters both reflection and action.\nWe acknowledge that the research was limited by the small number of participants. Small sample size in photovoice research initiatives is not uncommon because of the time requirements and the nature of the participants’ involvement. For example, in their oft-cited article on African-American mothers, Killion & Wang [30] had five participants. Another limitation of our study relates to the fact that the medical supervisors fulfilled multiple roles with the medical residents. A posteriori we considered it to be an asset for our data collection, because the supervisors contributed valuable input to the photovoice meetings. However, we observed that power relations could arise among the participants because the supervisors were involved in the residents’ evaluations.\nBy involving both medical residents and supervisors in this photovoice study, we made one of the first attempts in a medical academic milieu to involve healthcare professionals in identifying ways to improve physicians’ competencies in delivering care to socioeconomically disadvantaged patients. The study and its outcomes also generated momentum for changes to the family medicine curriculum at a teaching university. Thus, to improve the knowledge and skills of future primary care doctors and incorporate a “social competence” component into residency programs, we recommend developing an experiential learning approach by giving residents opportunities to be involved in underserved community activities and in reflexive discussion with medical supervisors.\nIn conclusion, photovoice appears to be a promising and innovative teaching approach in medical education, especially for medical residents. More specifically, our participatory research project helped future physicians become aware of their prejudices and motivated them to acquire skills for delivering healthcare to socioeconomically disadvantaged patients. Photovoice can be an effective tool for raising health professionals’ awareness of socioeconomic realities of their patients.\n\nConclusions:\nThis study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nClinicians face challenges in delivering care to socioeconomically disadvantaged patients. While both the public and academic sectors recognize the importance of addressing social inequities in healthcare, there is room for improvement in the training of family physicians, who report being ill-equipped to provide care that is responsive to the living conditions of these patients. This study explored: (i) residents' perceptions and experience in relation to providing care for socioeconomically disadvantaged patients, and (ii) how participating in a photovoice study helped them uncover and examine some of their prejudices and assumptions about poverty.\n\nMethods:\nWe conducted a participatory photovoice study. Participants were four family medicine residents, two medical supervisors, and two researchers. Residents attended six photovoice meetings at which they discussed photos they had taken. In collaboration with the researchers, the participants defined the research questions, took photos, and participated in data analysis and results dissemination. Meetings were recorded and transcribed for analysis, which consisted of coding, peer debriefing, thematic analysis, and interpretation.\n\nResults:\nThe medical residents uncovered and examined their own prejudices and misconceptions about poverty. They reported feeling unprepared to provide care to socioeconomically disadvantaged patients. Supported by medical supervisors and researchers, the residents underwent a three-phase reflexive process of: (1) engaging reflexively, (2) break(ing) through, and (3) taking action. The results indicated that medical residents subsequently felt encouraged to adopt a care approach that helped them overcome the social distance between themselves and their socioeconomically disadvantaged patients.\n\nConclusions:\nThis study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."},"background_conclusion_text":{"kind":"string","value":"Background:\nPoverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].\nClinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].\nPhysicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].\nHence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].\nPhotovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].\nThis participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty.\n\nConclusions:\nThis study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nClinicians face challenges in delivering care to socioeconomically disadvantaged patients. While both the public and academic sectors recognize the importance of addressing social inequities in healthcare, there is room for improvement in the training of family physicians, who report being ill-equipped to provide care that is responsive to the living conditions of these patients. This study explored: (i) residents' perceptions and experience in relation to providing care for socioeconomically disadvantaged patients, and (ii) how participating in a photovoice study helped them uncover and examine some of their prejudices and assumptions about poverty.\n\nMethods:\nWe conducted a participatory photovoice study. Participants were four family medicine residents, two medical supervisors, and two researchers. Residents attended six photovoice meetings at which they discussed photos they had taken. In collaboration with the researchers, the participants defined the research questions, took photos, and participated in data analysis and results dissemination. Meetings were recorded and transcribed for analysis, which consisted of coding, peer debriefing, thematic analysis, and interpretation.\n\nResults:\nThe medical residents uncovered and examined their own prejudices and misconceptions about poverty. They reported feeling unprepared to provide care to socioeconomically disadvantaged patients. Supported by medical supervisors and researchers, the residents underwent a three-phase reflexive process of: (1) engaging reflexively, (2) break(ing) through, and (3) taking action. The results indicated that medical residents subsequently felt encouraged to adopt a care approach that helped them overcome the social distance between themselves and their socioeconomically disadvantaged patients.\n\nConclusions:\nThis study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."},"whole_article_text_length":{"kind":"number","value":10155,"string":"10,155"},"whole_article_abstract_length":{"kind":"number","value":355,"string":"355"},"other_sections_lengths":{"kind":"list like","value":[162,237,598,199,704,762,167],"string":"[\n 162,\n 237,\n 598,\n 199,\n 704,\n 762,\n 167\n]"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["residents","poverty","medical","photovoice","participants","medical residents","research","photos","project","researchers"],"string":"[\n \"residents\",\n \"poverty\",\n \"medical\",\n \"photovoice\",\n \"participants\",\n \"medical residents\",\n \"research\",\n \"photos\",\n \"project\",\n \"researchers\"\n]"},"keybert_topics":{"kind":"list like","value":["disadvantaged patients 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Clinicians ||| ||| ||| ||| four | two | two ||| six ||| ||| ||| ||| ||| ||| three | 1 | 2 | 3 ||| ||| ||| clinicians [SUMMARY]"}}},{"rowIdx":3383,"cells":{"title":{"kind":"string","value":"TH1/TH2 Cytokine profile in relapsing-remitting multiple sclerosis patients treated with Glatiramer acetate or Natalizumab."},"pmid":{"kind":"string","value":"22989378"},"background_abstract":{"kind":"string","value":"The balance between T helper cells Th2- and Th1-related cytokines plays a key role in multiple sclerosis (MS). A shift from a Th1 towards a Th2 cytokine profile could have a beneficial effect on the clinical course of the disease. The objective of this study was to assess Th2/Th1 cytokine profile in relapsing-remitting MS (RRMS) patients receiving an immunosuppressive treatment with natalizumab (NAT), or an immunomodulatory treatment with glatiramer acetate (GA) after one year of treatment."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This was an observational cross-sectional study. All consecutive patients diagnosed with RRMS who had received GA or NAT for 12 months were included in the study. We determined serum levels of Th1 and Th2 cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) by flow cytometry. Th2/Th1 bias was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2 cytokines and proinflammatory INF-γ or TNF-α Th1 cytokines."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated. RRMS patients treated with NAT showed significantly higher levels of IL-6 (p < 0.05), MCP-1 (p < 0.01), and GM-CSF (p < 0.05) compared to GA patients after one year of treatment. A trend for increasing of IL-12p70, IL-1b, TNF- α and IFN- γ levels was also found in patients receiving NAT compared to GA patients. IL-4/IFN-γ, IFN-γ/TNF-α and IL-10/IFN-γ ratios as markers of Th2/Th1 ratio were significantly elevated in GA patients compared to those receiving NAT (p < 0.05)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In conclusion, our findings suggest that GA promotes a superior Th2-biased anti-inflammatory response as compared with NAT in the systemic circulation of RRMS patients. Future studies with larger cohorts will determine whether this immune Th2 shift in GA patients is associated with a beneficial effect on disease outcome."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adjuvants, Immunologic","Adult","Antibodies, Monoclonal, Humanized","Cytokines","Glatiramer Acetate","Humans","Male","Multiple Sclerosis","Natalizumab","Peptides","Secondary Prevention","Th1-Th2 Balance","Treatment Outcome"],"string":"[\n \"Adjuvants, Immunologic\",\n \"Adult\",\n \"Antibodies, Monoclonal, Humanized\",\n \"Cytokines\",\n \"Glatiramer Acetate\",\n \"Humans\",\n \"Male\",\n \"Multiple Sclerosis\",\n \"Natalizumab\",\n \"Peptides\",\n \"Secondary Prevention\",\n \"Th1-Th2 Balance\",\n \"Treatment Outcome\"\n]"},"pmcid":{"kind":"string","value":"3517482"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"The present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.\n Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\nAll consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\n Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\nBriefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\n Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\nMedian serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\nA total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\n Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\nOverall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA."},"other_sections_titles":{"kind":"list like","value":["Background","Patients","Cytokine analysis","Statistical analysis","Patient characteristics","Th1/Th2 cytokine profile","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Patients\",\n \"Cytokine analysis\",\n \"Statistical analysis\",\n \"Patient characteristics\",\n \"Th1/Th2 cytokine profile\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.","All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.","Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.","Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.","A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.","Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.","E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.","All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\n"],"string":"[\n \"Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.\",\n \"All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\",\n \"Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\",\n \"Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\",\n \"A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\",\n \"Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\\n\\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\\n\\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\",\n \"E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.\",\n \"All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Patients","Cytokine analysis","Statistical analysis","Results","Patient characteristics","Th1/Th2 cytokine profile","Discussion","Conclusion","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Patients\",\n \"Cytokine analysis\",\n \"Statistical analysis\",\n \"Results\",\n \"Patient characteristics\",\n \"Th1/Th2 cytokine profile\",\n \"Discussion\",\n \"Conclusion\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.","The present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.\n Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\nAll consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\n Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\nBriefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\n Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\nMedian serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.","All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.","Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.","Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented."," Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\nA total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\n Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\nOverall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.","A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.","Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.","The results of the present study suggest a Th2/Th1 balance shift in favour of a Th2 cytokine profile on GA-treated patients, while NAT causes a predominant Th1-biased response. This superior anti-inflammatory shift of GA seems to be mainly mediated by raising IL-4 and IL-10 levels which could lead to a down-regulation of Th1 cytokine secretion. Accordingly, the enhancement of circulating IL-4 and IL-10 and the subsequent detrimental effect on IFN-γ and TNF-α seen in GA-treated RRMS patients may play a protective role from inflammatory response that could affect the clinical course of disease in these patients. In this regard, a stabilization of disability score was found in both NAT and GA patients after one year of treatment. The potential clinical implications of immune response in GA-treated patients have been previously assessed [30-32]. Indeed, Valenzuela et al. [31] reported an increased IL-4/IFN-γ ratio to be associated with a favourable clinical outcome in a study of 36 RRMS patients treated with GA. However, the available findings suggesting the potential association between specific cytokine patterns and clinical response to GA were controversial primarily due to the short follow-up period. A recent study with a longer follow-up period of 3 years has demonstrated that IL-2 + IFN-γ/IL-10 + IL-4 ratio was significantly elevated in those patients with RRMS that suffered from relapses and progressing brain atrophy, suggesting that a specific pattern of Th2/Th1 cytokines may predict clinical response to GA therapy [33]. Moreover, this study suggests that the quotient IL-4 + IL-10/IL-2 + IFN-γ could be a promising parameter to identify patients associated with a highly beneficial response to GA therapy. However, although follow-up data over 3 years were available in this study, the sample size was relatively small to draw firm conclusions. Consequently, further studies including larger cohorts of patients will be required to validate that clinical and immune response correlate in patients treated with GA. Additionally, the mechanisms underlying the relation between cytokine response and clinical outcome in GA treated patients remain as a matter of debate.\nThe results of the present study show that patients treated with NAT exhibit higher levels of circulating proinflammatory cytokines and chemokines than those treated with GA. These findings are in agreement with previous studies where NAT treatment has been associated with an increased expression of proinflammatory cytokines in peripheral blood mononuclear cells [34,35]. Accordingly, an increase in activated leukocytes producing proinflammatory cytokines has been found in peripheral blood of NAT-treated patients [34,36]. Although it is not clear, these findings could probably be due to the inhibition of transmigration of lymphocytes into CNS, resulting in sequestration of activated T cells in the peripheral circulation [36]. The prolonged T-cell activation could result in decreased local immunosurveillance, reactivation of latent viral infections or opportunistic CNS infections, as evidenced by the rare but severe occurrence of progressive multifocal leukoencephalopathy caused by JC virus in NAT-treated patients [37]. Recent evidence has suggested that NAT seem to exert its beneficial effect without affecting regulatory T cell function [28]. Interestingly, NAT therapy has been associated with an increase in some pro-inflammatory and anti-inflammatory cytokines within the first 2 months of therapy, whereas relevant cytokines for MS such as IL-2, IL-7, or IL-1β have been found to be increased after one year of treatment, suggesting different immunological mechanisms [28]. The changes in the Th1/Th2 paradigm do not appear to be applicable to explain the beneficial effect of NAT. The increase of circulating Th1 cytokines could be related to a “rebound” effect that led to the development of new and enlarging T2 lesions previously seen in cohorts of patients discontinuing NAT due to safety issues related to this therapy, particularly regarding PML [38]. This finding has led to the concern that cessation of NAT might promote a worsening of MS disease by increasing inflammatory activity. The most likely explanation is that short exposure to NAT (e.g., 2 infusions) results in blockade of migration and accumulation of activated lymphocytes in the periphery that retain their capacity to cause CNS disease [39].\nThe main limitations of this study include the small sample size and a one-point measurement of cytokine patterns. However, despite these limitations, our findings are potentially interesting given that to our knowledge, this is the first study to compare the Th1/Th2 bias between GA and NAT treated RRMS patients.","In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA.","E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.","All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\n"],"string":"[\n \"Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.\",\n \"The present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.\\n Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\\nAll consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\\n Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\\nBriefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\\n Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\\nMedian serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\",\n \"All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\",\n \"Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\",\n \"Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\",\n \" Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\\nA total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\\n Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\\n\\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\\n\\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\\nOverall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\\n\\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\\n\\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\",\n \"A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\",\n \"Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\\n\\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\\n\\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\",\n \"The results of the present study suggest a Th2/Th1 balance shift in favour of a Th2 cytokine profile on GA-treated patients, while NAT causes a predominant Th1-biased response. This superior anti-inflammatory shift of GA seems to be mainly mediated by raising IL-4 and IL-10 levels which could lead to a down-regulation of Th1 cytokine secretion. Accordingly, the enhancement of circulating IL-4 and IL-10 and the subsequent detrimental effect on IFN-γ and TNF-α seen in GA-treated RRMS patients may play a protective role from inflammatory response that could affect the clinical course of disease in these patients. In this regard, a stabilization of disability score was found in both NAT and GA patients after one year of treatment. The potential clinical implications of immune response in GA-treated patients have been previously assessed [30-32]. Indeed, Valenzuela et al. [31] reported an increased IL-4/IFN-γ ratio to be associated with a favourable clinical outcome in a study of 36 RRMS patients treated with GA. However, the available findings suggesting the potential association between specific cytokine patterns and clinical response to GA were controversial primarily due to the short follow-up period. A recent study with a longer follow-up period of 3 years has demonstrated that IL-2 + IFN-γ/IL-10 + IL-4 ratio was significantly elevated in those patients with RRMS that suffered from relapses and progressing brain atrophy, suggesting that a specific pattern of Th2/Th1 cytokines may predict clinical response to GA therapy [33]. Moreover, this study suggests that the quotient IL-4 + IL-10/IL-2 + IFN-γ could be a promising parameter to identify patients associated with a highly beneficial response to GA therapy. However, although follow-up data over 3 years were available in this study, the sample size was relatively small to draw firm conclusions. Consequently, further studies including larger cohorts of patients will be required to validate that clinical and immune response correlate in patients treated with GA. Additionally, the mechanisms underlying the relation between cytokine response and clinical outcome in GA treated patients remain as a matter of debate.\\nThe results of the present study show that patients treated with NAT exhibit higher levels of circulating proinflammatory cytokines and chemokines than those treated with GA. These findings are in agreement with previous studies where NAT treatment has been associated with an increased expression of proinflammatory cytokines in peripheral blood mononuclear cells [34,35]. Accordingly, an increase in activated leukocytes producing proinflammatory cytokines has been found in peripheral blood of NAT-treated patients [34,36]. Although it is not clear, these findings could probably be due to the inhibition of transmigration of lymphocytes into CNS, resulting in sequestration of activated T cells in the peripheral circulation [36]. The prolonged T-cell activation could result in decreased local immunosurveillance, reactivation of latent viral infections or opportunistic CNS infections, as evidenced by the rare but severe occurrence of progressive multifocal leukoencephalopathy caused by JC virus in NAT-treated patients [37]. Recent evidence has suggested that NAT seem to exert its beneficial effect without affecting regulatory T cell function [28]. Interestingly, NAT therapy has been associated with an increase in some pro-inflammatory and anti-inflammatory cytokines within the first 2 months of therapy, whereas relevant cytokines for MS such as IL-2, IL-7, or IL-1β have been found to be increased after one year of treatment, suggesting different immunological mechanisms [28]. The changes in the Th1/Th2 paradigm do not appear to be applicable to explain the beneficial effect of NAT. The increase of circulating Th1 cytokines could be related to a “rebound” effect that led to the development of new and enlarging T2 lesions previously seen in cohorts of patients discontinuing NAT due to safety issues related to this therapy, particularly regarding PML [38]. This finding has led to the concern that cessation of NAT might promote a worsening of MS disease by increasing inflammatory activity. The most likely explanation is that short exposure to NAT (e.g., 2 infusions) results in blockade of migration and accumulation of activated lymphocytes in the periphery that retain their capacity to cause CNS disease [39].\\nThe main limitations of this study include the small sample size and a one-point measurement of cytokine patterns. However, despite these limitations, our findings are potentially interesting given that to our knowledge, this is the first study to compare the Th1/Th2 bias between GA and NAT treated RRMS patients.\",\n \"In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA.\",\n \"E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.\",\n \"All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,"results",null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Multiple sclerosis","Th2","Th1","Cytokines","Glatiramer acetate","Natalizumab"],"string":"[\n \"Multiple sclerosis\",\n \"Th2\",\n \"Th1\",\n \"Cytokines\",\n \"Glatiramer acetate\",\n \"Natalizumab\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nMultiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.\n\nMethods:\nThe present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.\n Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\nAll consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\n Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\nBriefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\n Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\nMedian serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\n\nPatients:\nAll consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\n\nCytokine analysis:\nBriefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\n\nStatistical analysis:\nMedian serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\n\nResults:\n Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\nA total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\n Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\nOverall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\n\nPatient characteristics:\nA total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\n\nTh1/Th2 cytokine profile:\nOverall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\n\nDiscussion:\nThe results of the present study suggest a Th2/Th1 balance shift in favour of a Th2 cytokine profile on GA-treated patients, while NAT causes a predominant Th1-biased response. This superior anti-inflammatory shift of GA seems to be mainly mediated by raising IL-4 and IL-10 levels which could lead to a down-regulation of Th1 cytokine secretion. Accordingly, the enhancement of circulating IL-4 and IL-10 and the subsequent detrimental effect on IFN-γ and TNF-α seen in GA-treated RRMS patients may play a protective role from inflammatory response that could affect the clinical course of disease in these patients. In this regard, a stabilization of disability score was found in both NAT and GA patients after one year of treatment. The potential clinical implications of immune response in GA-treated patients have been previously assessed [30-32]. Indeed, Valenzuela et al. [31] reported an increased IL-4/IFN-γ ratio to be associated with a favourable clinical outcome in a study of 36 RRMS patients treated with GA. However, the available findings suggesting the potential association between specific cytokine patterns and clinical response to GA were controversial primarily due to the short follow-up period. A recent study with a longer follow-up period of 3 years has demonstrated that IL-2 + IFN-γ/IL-10 + IL-4 ratio was significantly elevated in those patients with RRMS that suffered from relapses and progressing brain atrophy, suggesting that a specific pattern of Th2/Th1 cytokines may predict clinical response to GA therapy [33]. Moreover, this study suggests that the quotient IL-4 + IL-10/IL-2 + IFN-γ could be a promising parameter to identify patients associated with a highly beneficial response to GA therapy. However, although follow-up data over 3 years were available in this study, the sample size was relatively small to draw firm conclusions. Consequently, further studies including larger cohorts of patients will be required to validate that clinical and immune response correlate in patients treated with GA. Additionally, the mechanisms underlying the relation between cytokine response and clinical outcome in GA treated patients remain as a matter of debate.\nThe results of the present study show that patients treated with NAT exhibit higher levels of circulating proinflammatory cytokines and chemokines than those treated with GA. These findings are in agreement with previous studies where NAT treatment has been associated with an increased expression of proinflammatory cytokines in peripheral blood mononuclear cells [34,35]. Accordingly, an increase in activated leukocytes producing proinflammatory cytokines has been found in peripheral blood of NAT-treated patients [34,36]. Although it is not clear, these findings could probably be due to the inhibition of transmigration of lymphocytes into CNS, resulting in sequestration of activated T cells in the peripheral circulation [36]. The prolonged T-cell activation could result in decreased local immunosurveillance, reactivation of latent viral infections or opportunistic CNS infections, as evidenced by the rare but severe occurrence of progressive multifocal leukoencephalopathy caused by JC virus in NAT-treated patients [37]. Recent evidence has suggested that NAT seem to exert its beneficial effect without affecting regulatory T cell function [28]. Interestingly, NAT therapy has been associated with an increase in some pro-inflammatory and anti-inflammatory cytokines within the first 2 months of therapy, whereas relevant cytokines for MS such as IL-2, IL-7, or IL-1β have been found to be increased after one year of treatment, suggesting different immunological mechanisms [28]. The changes in the Th1/Th2 paradigm do not appear to be applicable to explain the beneficial effect of NAT. The increase of circulating Th1 cytokines could be related to a “rebound” effect that led to the development of new and enlarging T2 lesions previously seen in cohorts of patients discontinuing NAT due to safety issues related to this therapy, particularly regarding PML [38]. This finding has led to the concern that cessation of NAT might promote a worsening of MS disease by increasing inflammatory activity. The most likely explanation is that short exposure to NAT (e.g., 2 infusions) results in blockade of migration and accumulation of activated lymphocytes in the periphery that retain their capacity to cause CNS disease [39].\nThe main limitations of this study include the small sample size and a one-point measurement of cytokine patterns. However, despite these limitations, our findings are potentially interesting given that to our knowledge, this is the first study to compare the Th1/Th2 bias between GA and NAT treated RRMS patients.\n\nConclusion:\nIn summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA.\n\nCompeting interests:\nE. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.\n\nAuthors’ contributions:\nAll authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe balance between T helper cells Th2- and Th1-related cytokines plays a key role in multiple sclerosis (MS). A shift from a Th1 towards a Th2 cytokine profile could have a beneficial effect on the clinical course of the disease. The objective of this study was to assess Th2/Th1 cytokine profile in relapsing-remitting MS (RRMS) patients receiving an immunosuppressive treatment with natalizumab (NAT), or an immunomodulatory treatment with glatiramer acetate (GA) after one year of treatment.\n\nMethods:\nThis was an observational cross-sectional study. All consecutive patients diagnosed with RRMS who had received GA or NAT for 12 months were included in the study. We determined serum levels of Th1 and Th2 cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) by flow cytometry. Th2/Th1 bias was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2 cytokines and proinflammatory INF-γ or TNF-α Th1 cytokines.\n\nResults:\nEleven patients under treatment with NAT and 12 patients treated with GA were evaluated. RRMS patients treated with NAT showed significantly higher levels of IL-6 (p < 0.05), MCP-1 (p < 0.01), and GM-CSF (p < 0.05) compared to GA patients after one year of treatment. A trend for increasing of IL-12p70, IL-1b, TNF- α and IFN- γ levels was also found in patients receiving NAT compared to GA patients. IL-4/IFN-γ, IFN-γ/TNF-α and IL-10/IFN-γ ratios as markers of Th2/Th1 ratio were significantly elevated in GA patients compared to those receiving NAT (p < 0.05).\n\nConclusions:\nIn conclusion, our findings suggest that GA promotes a superior Th2-biased anti-inflammatory response as compared with NAT in the systemic circulation of RRMS patients. Future studies with larger cohorts will determine whether this immune Th2 shift in GA patients is associated with a beneficial effect on disease outcome."},"background_conclusion_text":{"kind":"string","value":"Background:\nMultiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.\n\nConclusion:\nIn summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe balance between T helper cells Th2- and Th1-related cytokines plays a key role in multiple sclerosis (MS). A shift from a Th1 towards a Th2 cytokine profile could have a beneficial effect on the clinical course of the disease. The objective of this study was to assess Th2/Th1 cytokine profile in relapsing-remitting MS (RRMS) patients receiving an immunosuppressive treatment with natalizumab (NAT), or an immunomodulatory treatment with glatiramer acetate (GA) after one year of treatment.\n\nMethods:\nThis was an observational cross-sectional study. All consecutive patients diagnosed with RRMS who had received GA or NAT for 12 months were included in the study. We determined serum levels of Th1 and Th2 cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) by flow cytometry. Th2/Th1 bias was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2 cytokines and proinflammatory INF-γ or TNF-α Th1 cytokines.\n\nResults:\nEleven patients under treatment with NAT and 12 patients treated with GA were evaluated. RRMS patients treated with NAT showed significantly higher levels of IL-6 (p < 0.05), MCP-1 (p < 0.01), and GM-CSF (p < 0.05) compared to GA patients after one year of treatment. A trend for increasing of IL-12p70, IL-1b, TNF- α and IFN- γ levels was also found in patients receiving NAT compared to GA patients. IL-4/IFN-γ, IFN-γ/TNF-α and IL-10/IFN-γ ratios as markers of Th2/Th1 ratio were significantly elevated in GA patients compared to those receiving NAT (p < 0.05).\n\nConclusions:\nIn conclusion, our findings suggest that GA promotes a superior Th2-biased anti-inflammatory response as compared with NAT in the systemic circulation of RRMS patients. Future studies with larger cohorts will determine whether this immune Th2 shift in GA patients is associated with a beneficial effect on disease outcome."},"whole_article_text_length":{"kind":"number","value":5026,"string":"5,026"},"whole_article_abstract_length":{"kind":"number","value":427,"string":"427"},"other_sections_lengths":{"kind":"list like","value":[453,107,171,117,206,495,93,83,16],"string":"[\n 453,\n 107,\n 171,\n 117,\n 206,\n 495,\n 93,\n 83,\n 16\n]"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["patients","il","ga","nat","th2","treated","th1","cytokines","il il","ratio"],"string":"[\n \"patients\",\n \"il\",\n \"ga\",\n \"nat\",\n \"th2\",\n \"treated\",\n \"th1\",\n \"cytokines\",\n \"il il\",\n \"ratio\"\n]"},"keybert_topics":{"kind":"list like","value":["th2 related cytokines","cytokines ms","relevant cytokines ms","th1 cytokines interferon","inflammation demyielination cytokines"],"string":"[\n \"th2 related cytokines\",\n \"cytokines ms\",\n \"relevant cytokines ms\",\n \"th1 cytokines interferon\",\n \"inflammation demyielination cytokines\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cytokines | th2 | th1 cytokines | ms | th1 | disease | inflammation | cns | inflammatory | treatment [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] il | il il | patients | il il il | usa | protein | test | ratio | il il il il | th1 [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] nat | il | patients | ga | 05 | pg | higher | respectively | treated | mean [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] effect | larger | patients | beneficial effect | shift | beneficial | response | th2 | ga | systemic [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] il | patients | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] il | patients | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| RRMS | NAT | one year [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| RRMS | NAT | 12 months ||| IL-1b | IL-5 | IL-6 | IL-10 | IL-12p70 | monocyte ||| ||| GM-CSF ||| Th2 | IL-5 | IL-6 | IL-10 | INF | TNF [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] NAT | 12 ||| RRMS | NAT | IL-6 | 0.05 | MCP-1 | 0.01 | GM-CSF | 0.05 | one year ||| IL-12p70 | IL-1b | NAT ||| IFN | IFN | IL-10 | IFN | GA | NAT | 0.05 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] NAT | RRMS ||| GA [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| RRMS | NAT | one year ||| ||| RRMS | NAT | 12 months ||| IL-1b | IL-5 | IL-6 | IL-10 | IL-12p70 | monocyte ||| ||| GM-CSF ||| Th2 | IL-5 | IL-6 | IL-10 | INF | TNF ||| ||| NAT | 12 ||| RRMS | NAT | IL-6 | 0.05 | MCP-1 | 0.01 | GM-CSF | 0.05 | one year ||| IL-12p70 | IL-1b | NAT ||| IFN | IFN | IL-10 | IFN | GA | NAT | 0.05 ||| NAT | RRMS ||| GA [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| RRMS | NAT | one year ||| ||| RRMS | NAT | 12 months ||| IL-1b | IL-5 | IL-6 | IL-10 | IL-12p70 | monocyte ||| ||| GM-CSF ||| Th2 | IL-5 | IL-6 | IL-10 | INF | TNF ||| ||| NAT | 12 ||| RRMS | NAT | IL-6 | 0.05 | MCP-1 | 0.01 | GM-CSF | 0.05 | one year ||| IL-12p70 | IL-1b | NAT ||| IFN | IFN | IL-10 | IFN | GA | NAT | 0.05 ||| NAT | RRMS ||| GA [SUMMARY]"}}},{"rowIdx":3384,"cells":{"title":{"kind":"string","value":"PURIFICATION AND FRACTIONAL ANALYSIS OF METHANOLIC EXTRACT OF "},"pmid":{"kind":"string","value":"28480428"},"background_abstract":{"kind":"string","value":"Wedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components."},"methods_abstract_label":{"kind":"string","value":"MATERIALS AND METHODS"},"results_abstract":{"kind":"string","value":"Out of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Antineoplastic Agents, Phytogenic","Apoptosis","Cell Line, Tumor","Chromatography, Thin Layer","Humans","Megakaryocytes","Methanol","Methylene Chloride","Plant Extracts","Wedelia"],"string":"[\n \"Antineoplastic Agents, Phytogenic\",\n \"Apoptosis\",\n \"Cell Line, Tumor\",\n \"Chromatography, Thin Layer\",\n \"Humans\",\n \"Megakaryocytes\",\n \"Methanol\",\n \"Methylene Chloride\",\n \"Plant Extracts\",\n \"Wedelia\"\n]"},"pmcid":{"kind":"string","value":"5412222"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.\nLeukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.\nAsteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.\nA brief explanation of the entire process done in the study"},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Thin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.\nThin layer chromatogram showing the separation of methanolic extract into two bands under UV light.\nThymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.\nThe reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.\nFrom the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.\nDNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.\nlane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.\nSilica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.\nThymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.\nThe graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.\nNuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.\nNuclear staining of MEG01 cell with negative, positive control and fraction 3.\nFlow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.\nDetection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).\nHPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.\nHPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis."},"other_sections_titles":{"kind":"list like","value":["Collection of plant and obtaining crude extracts of Wedelia trilobata","Thin layer chromatography (TLC)","Activity analysis of the fractions obtained from TLC","Activity analysis of the fractions obtained from Silica gel chromatography"],"string":"[\n \"Collection of plant and obtaining crude extracts of Wedelia trilobata\",\n \"Thin layer chromatography (TLC)\",\n \"Activity analysis of the fractions obtained from TLC\",\n \"Activity analysis of the fractions obtained from Silica gel chromatography\"\n]"},"other_sections_texts":{"kind":"list like","value":["The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).","This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.","Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.","Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016)."],"string":"[\n \"The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\",\n \"This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\",\n \"Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\",\n \"Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null],"string":"[\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Collection of plant and obtaining crude extracts of Wedelia trilobata","Thin layer chromatography (TLC)","Activity analysis of the fractions obtained from TLC","Activity analysis of the fractions obtained from Silica gel chromatography","Results","Discussion","Conclusion"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Collection of plant and obtaining crude extracts of Wedelia trilobata\",\n \"Thin layer chromatography (TLC)\",\n \"Activity analysis of the fractions obtained from TLC\",\n \"Activity analysis of the fractions obtained from Silica gel chromatography\",\n \"Results\",\n \"Discussion\",\n \"Conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.\nLeukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.\nAsteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.\nA brief explanation of the entire process done in the study"," Collection of plant and obtaining crude extracts of Wedelia trilobata The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\nThe characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\n Thin layer chromatography (TLC) This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\nThis is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\n Activity analysis of the fractions obtained from TLC Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\nCell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\n Activity analysis of the fractions obtained from Silica gel chromatography Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\nThymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).","The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).","This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.","Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.","Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).","Thin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.\nThin layer chromatogram showing the separation of methanolic extract into two bands under UV light.\nThymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.\nThe reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.\nFrom the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.\nDNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.\nlane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.\nSilica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.\nThymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.\nThe graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.\nNuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.\nNuclear staining of MEG01 cell with negative, positive control and fraction 3.\nFlow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.\nDetection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).\nHPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.\nHPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes.","TLC: The initial separation of methanolic extract through TLC (Sasidharan et al. 2011) showed two prominent bands with methanol and dichloromethane solvent mixture taken at the ratio 6:4 whereas, the separation was improper with other solvent mixtures and ratios tried. Among the two bands obtained, the second band still retains certain color mixture which may indicate the presence of different compositions. Before going to the further purification, the characteristic analysis of both the bands was assessed for anti-proliferation and apoptosis activity.\nThymidine uptake assay: The thymidine uptake assay (Giridharan et al. 2002; Belakavadi et al. 2005) to check antiproliferation activity in MEG01 cells was performed twice, first was done on the bands obtained from TLC and analyzed that the second band showed the reduction in proliferation of MEG01 cells in comparison to the first band indicating that band 2 posses more active compounds in it. Second on the fraction obtained through silica gel column chromatography and in result the fractions 2, 3 and 4 showed the anti-proliferation activity among which the fraction 3 was more prominent. This gave the further lead to concentrate on fraction 3 for analysis.\nDNA fragmentation assay: The apoptosis activity of TLC separated bands when checked with the DNA degradation assay (Park et al. 2013) showed that, the band 2 possess the prominent bioactive compound having both antiproliferation as well as the apoptosis activity. This made the band 2 a clear selection for further purification.\nNuclear staining assay: The apoptosis activity was analyzed for fraction 3 in comparison with the activity of 2-Amino-N-quinolin-8-yl-benzenesulfonamide (Sigma Aldrich), taken as positive control. Both the positive control and fraction 3 produced orange fluorescent with green fluorescent in background due to apoptotic activity indicating that, the ruptured nucleus released apoptotic bodies containing DNA binds to EtBr and produced orange fluorescent (Srinivas et al, 2003).\nFlow cytometry analysis: Further analysis through flow cytometry using Ca2+ dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide; the fraction 3 was confirmed that it has more apoptotic activity. In the figure 7 the presence of cells in lower left square indicates the apoptotic cells and this clearly indicates the apoptotic activity by fraction 3 (Donal et al, 2009).\nHPLC: The HPLC analysis of fraction 3 produced a single peak at a retention time 3.65 min. The single peak and the early elution indicate the presence of pure and polar compound (Panawan et al, 2015; Ji-Yeon et al, 2016).","The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis."],"string":"[\n \"Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.\\nLeukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.\\nAsteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.\\nA brief explanation of the entire process done in the study\",\n \" Collection of plant and obtaining crude extracts of Wedelia trilobata The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\\nThe characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\\n Thin layer chromatography (TLC) This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\\nThis is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\\n Activity analysis of the fractions obtained from TLC Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\\nCell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\\n Activity analysis of the fractions obtained from Silica gel chromatography Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\\nThymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\",\n \"The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\",\n \"This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\",\n \"Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\",\n \"Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\",\n \"Thin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.\\nThin layer chromatogram showing the separation of methanolic extract into two bands under UV light.\\nThymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.\\nThe reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.\\nFrom the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.\\nDNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.\\nlane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.\\nSilica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.\\nThymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.\\nThe graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.\\nNuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.\\nNuclear staining of MEG01 cell with negative, positive control and fraction 3.\\nFlow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.\\nDetection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).\\nHPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.\\nHPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes.\",\n \"TLC: The initial separation of methanolic extract through TLC (Sasidharan et al. 2011) showed two prominent bands with methanol and dichloromethane solvent mixture taken at the ratio 6:4 whereas, the separation was improper with other solvent mixtures and ratios tried. Among the two bands obtained, the second band still retains certain color mixture which may indicate the presence of different compositions. Before going to the further purification, the characteristic analysis of both the bands was assessed for anti-proliferation and apoptosis activity.\\nThymidine uptake assay: The thymidine uptake assay (Giridharan et al. 2002; Belakavadi et al. 2005) to check antiproliferation activity in MEG01 cells was performed twice, first was done on the bands obtained from TLC and analyzed that the second band showed the reduction in proliferation of MEG01 cells in comparison to the first band indicating that band 2 posses more active compounds in it. Second on the fraction obtained through silica gel column chromatography and in result the fractions 2, 3 and 4 showed the anti-proliferation activity among which the fraction 3 was more prominent. This gave the further lead to concentrate on fraction 3 for analysis.\\nDNA fragmentation assay: The apoptosis activity of TLC separated bands when checked with the DNA degradation assay (Park et al. 2013) showed that, the band 2 possess the prominent bioactive compound having both antiproliferation as well as the apoptosis activity. This made the band 2 a clear selection for further purification.\\nNuclear staining assay: The apoptosis activity was analyzed for fraction 3 in comparison with the activity of 2-Amino-N-quinolin-8-yl-benzenesulfonamide (Sigma Aldrich), taken as positive control. Both the positive control and fraction 3 produced orange fluorescent with green fluorescent in background due to apoptotic activity indicating that, the ruptured nucleus released apoptotic bodies containing DNA binds to EtBr and produced orange fluorescent (Srinivas et al, 2003).\\nFlow cytometry analysis: Further analysis through flow cytometry using Ca2+ dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide; the fraction 3 was confirmed that it has more apoptotic activity. In the figure 7 the presence of cells in lower left square indicates the apoptotic cells and this clearly indicates the apoptotic activity by fraction 3 (Donal et al, 2009).\\nHPLC: The HPLC analysis of fraction 3 produced a single peak at a retention time 3.65 min. The single peak and the early elution indicate the presence of pure and polar compound (Panawan et al, 2015; Ji-Yeon et al, 2016).\",\n \"The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,null,"results","discussion:","conclusion"],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion:\",\n \"conclusion\"\n]"},"keywords":{"kind":"list like","value":["\nWedelia trilobata\n","MEG-01","Antiproliferation","Apoptosis"],"string":"[\n \"\\nWedelia trilobata\\n\",\n \"MEG-01\",\n \"Antiproliferation\",\n \"Apoptosis\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nApproach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.\nLeukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.\nAsteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.\nA brief explanation of the entire process done in the study\n\nMaterials and methods:\n Collection of plant and obtaining crude extracts of Wedelia trilobata The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\nThe characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\n Thin layer chromatography (TLC) This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\nThis is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\n Activity analysis of the fractions obtained from TLC Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\nCell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\n Activity analysis of the fractions obtained from Silica gel chromatography Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\nThymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\n\nCollection of plant and obtaining crude extracts of Wedelia trilobata:\nThe characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\n\nThin layer chromatography (TLC):\nThis is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\n\nActivity analysis of the fractions obtained from TLC:\nCell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\n\nActivity analysis of the fractions obtained from Silica gel chromatography:\nThymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\n\nResults:\nThin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.\nThin layer chromatogram showing the separation of methanolic extract into two bands under UV light.\nThymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.\nThe reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.\nFrom the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.\nDNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.\nlane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.\nSilica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.\nThymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.\nThe graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.\nNuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.\nNuclear staining of MEG01 cell with negative, positive control and fraction 3.\nFlow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.\nDetection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).\nHPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.\nHPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes.\n\nDiscussion:\nTLC: The initial separation of methanolic extract through TLC (Sasidharan et al. 2011) showed two prominent bands with methanol and dichloromethane solvent mixture taken at the ratio 6:4 whereas, the separation was improper with other solvent mixtures and ratios tried. Among the two bands obtained, the second band still retains certain color mixture which may indicate the presence of different compositions. Before going to the further purification, the characteristic analysis of both the bands was assessed for anti-proliferation and apoptosis activity.\nThymidine uptake assay: The thymidine uptake assay (Giridharan et al. 2002; Belakavadi et al. 2005) to check antiproliferation activity in MEG01 cells was performed twice, first was done on the bands obtained from TLC and analyzed that the second band showed the reduction in proliferation of MEG01 cells in comparison to the first band indicating that band 2 posses more active compounds in it. Second on the fraction obtained through silica gel column chromatography and in result the fractions 2, 3 and 4 showed the anti-proliferation activity among which the fraction 3 was more prominent. This gave the further lead to concentrate on fraction 3 for analysis.\nDNA fragmentation assay: The apoptosis activity of TLC separated bands when checked with the DNA degradation assay (Park et al. 2013) showed that, the band 2 possess the prominent bioactive compound having both antiproliferation as well as the apoptosis activity. This made the band 2 a clear selection for further purification.\nNuclear staining assay: The apoptosis activity was analyzed for fraction 3 in comparison with the activity of 2-Amino-N-quinolin-8-yl-benzenesulfonamide (Sigma Aldrich), taken as positive control. Both the positive control and fraction 3 produced orange fluorescent with green fluorescent in background due to apoptotic activity indicating that, the ruptured nucleus released apoptotic bodies containing DNA binds to EtBr and produced orange fluorescent (Srinivas et al, 2003).\nFlow cytometry analysis: Further analysis through flow cytometry using Ca2+ dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide; the fraction 3 was confirmed that it has more apoptotic activity. In the figure 7 the presence of cells in lower left square indicates the apoptotic cells and this clearly indicates the apoptotic activity by fraction 3 (Donal et al, 2009).\nHPLC: The HPLC analysis of fraction 3 produced a single peak at a retention time 3.65 min. The single peak and the early elution indicate the presence of pure and polar compound (Panawan et al, 2015; Ji-Yeon et al, 2016).\n\nConclusion:\nThe phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity.\n\nMethods:\nThe methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components.\n\nResults:\nOut of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC.\n\nConclusions:\nThe single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nApproach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.\nLeukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.\nAsteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.\nA brief explanation of the entire process done in the study\n\nConclusion:\nThe phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity.\n\nMethods:\nThe methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components.\n\nResults:\nOut of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC.\n\nConclusions:\nThe single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis."},"whole_article_text_length":{"kind":"number","value":4738,"string":"4,738"},"whole_article_abstract_length":{"kind":"number","value":284,"string":"284"},"other_sections_lengths":{"kind":"list like","value":[36,174,316,488],"string":"[\n 36,\n 174,\n 316,\n 488\n]"},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["cells","activity","tlc","column","fractions","assay","obtained","fraction","apoptotic","chromatography"],"string":"[\n \"cells\",\n \"activity\",\n \"tlc\",\n \"column\",\n \"fractions\",\n \"assay\",\n \"obtained\",\n \"fraction\",\n \"apoptotic\",\n \"chromatography\"\n]"},"keybert_topics":{"kind":"list like","value":["relying plant extracts","plant extracts healthcare","bioactive compounds plant","extracts like diterpenes","disease bioactive compounds"],"string":"[\n \"relying plant extracts\",\n \"plant extracts healthcare\",\n \"bioactive compounds plant\",\n \"extracts like diterpenes\",\n \"disease bioactive compounds\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] \nWedelia trilobata\n | MEG-01 | Antiproliferation | Apoptosis [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] \nWedelia trilobata\n | MEG-01 | Antiproliferation | Apoptosis [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] \nWedelia trilobata\n | MEG-01 | Antiproliferation | Apoptosis [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] \nWedelia trilobata\n | MEG-01 | Antiproliferation | Apoptosis [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] \nWedelia trilobata\n | MEG-01 | Antiproliferation | Apoptosis [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia 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Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds 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| fraction | cells | indicates | meg01 | lane | annexin | activity [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] apoptotic anti | study | anti | revealed | single peak hplc | study emphasis | phytoconstituent purification | phytoconstituent purification analytical | phytoconstituent purification analytical study | extract wt [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cells | activity | plant | fraction | assay | tlc | column | apoptotic | dna | band [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cells | activity | plant | fraction | assay | tlc | column | apoptotic | dna | band [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Wedelia | Ayurveda | Siddha | MEG- 01 ||| WT [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 6:4 | two | TLC | second ||| second | five | 3rd | HPLC [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] HPLC [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Wedelia | Ayurveda | Siddha | MEG- 01 ||| WT ||| WT ||| TLC ||| HPLC ||| 6:4 | two | TLC | second ||| second | five | 3rd | HPLC ||| HPLC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Wedelia | Ayurveda | Siddha | MEG- 01 ||| WT ||| WT ||| TLC ||| HPLC ||| 6:4 | two | TLC | second ||| second | five | 3rd | HPLC ||| HPLC [SUMMARY]"}}},{"rowIdx":3385,"cells":{"title":{"kind":"string","value":"Melatonin mitigates the adverse effect of hypoxia during myocardial differentiation in mouse embryonic stem cells."},"pmid":{"kind":"string","value":"34313039"},"background_abstract":{"kind":"string","value":"Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Biomarkers","Cell Differentiation","Embryonic Stem Cells","Gene Expression Regulation","Heart","Hypoxia","Melatonin","Myocytes, Cardiac","Oxygen"],"string":"[\n \"Animals\",\n \"Biomarkers\",\n \"Cell Differentiation\",\n \"Embryonic Stem Cells\",\n \"Gene Expression Regulation\",\n \"Heart\",\n \"Hypoxia\",\n \"Melatonin\",\n \"Myocytes, Cardiac\",\n \"Oxygen\"\n]"},"pmcid":{"kind":"string","value":"8318788"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Abnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.\nOxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].\nEmbryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].\nThe pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].\nPrevious studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Hypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nTo determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nEffect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nThe effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nEffect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nMelatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Mouse embryonic stem cell (mESC) culture","Differentiation into cardiomyocytes","Hypoxia induction and melatonin and/or luzindole treatment","Assessment of beating ratio in mESCs-derived cardiomyocytes","Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR)","Western blotting analysis","Statistical analyses","Hypoxia-induced inhibition on myocardial differentiation","Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia","Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation"],"string":"[\n \"Mouse embryonic stem cell (mESC) culture\",\n \"Differentiation into cardiomyocytes\",\n \"Hypoxia induction and melatonin and/or luzindole treatment\",\n \"Assessment of beating ratio in mESCs-derived cardiomyocytes\",\n \"Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR)\",\n \"Western blotting analysis\",\n \"Statistical analyses\",\n \"Hypoxia-induced inhibition on myocardial differentiation\",\n \"Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia\",\n \"Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation\"\n]"},"other_sections_texts":{"kind":"list like","value":["mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].","The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.","Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.","Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.","Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.","Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].","Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.","To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).","The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.","Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase."],"string":"[\n \"mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\",\n \"The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\",\n \"Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\",\n \"Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\",\n \"Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\",\n \"Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\",\n \"Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\",\n \"To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\\n*p < 0.05 versus Normoxia (Control).\\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control).\",\n \"The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\",\n \"Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Mouse embryonic stem cell (mESC) culture","Differentiation into cardiomyocytes","Hypoxia induction and melatonin and/or luzindole treatment","Assessment of beating ratio in mESCs-derived cardiomyocytes","Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR)","Western blotting analysis","Statistical analyses","RESULTS","Hypoxia-induced inhibition on myocardial differentiation","Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia","Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Mouse embryonic stem cell (mESC) culture\",\n \"Differentiation into cardiomyocytes\",\n \"Hypoxia induction and melatonin and/or luzindole treatment\",\n \"Assessment of beating ratio in mESCs-derived cardiomyocytes\",\n \"Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR)\",\n \"Western blotting analysis\",\n \"Statistical analyses\",\n \"RESULTS\",\n \"Hypoxia-induced inhibition on myocardial differentiation\",\n \"Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia\",\n \"Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Abnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.\nOxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].\nEmbryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].\nThe pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].\nPrevious studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation.","Mouse embryonic stem cell (mESC) culture mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\nmESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\nDifferentiation into cardiomyocytes The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\nThe mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\nHypoxia induction and melatonin and/or luzindole treatment Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\nDifferentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\nAssessment of beating ratio in mESCs-derived cardiomyocytes Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\nContraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\nTotal RNA extraction and real-time quantitative polymerase chain reaction (qPCR) Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\nTotal RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\nWestern blotting analysis Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\nProteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\nStatistical analyses Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\nSignificant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.","mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].","The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.","Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.","Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.","Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.","Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].","Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.","Hypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nTo determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nEffect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nThe effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nEffect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nMelatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.","To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).","The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.","Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.","In the present study, it was observed that differentiation into cardiomyocytes is impaired under hypoxic conditions. In addition, it was observed that the differentiation of hypoxia-treated cardiomyocytes was recovered by treatment with melatonin. Our results suggest that supraphysiological melatonin treatment can counteract the adverse effect of hypoxia during the myocardial differentiation of mESCs.\nmRNA expression of Mtnr1a was decreased under hypoxic conditions. It was determined that the adverse effect of hypoxia was altered by melatonin pretreatment, which affected the mRNA expressions of melatonin receptors during myocardial differentiation of mESCs.\nMelatonin is reported to have a neuroprotective effect by relieving brain edema and improving nerve function [2526]. Albeit physiological concentration of melatonin has been known as between 30 to 400 pM in normal plasma; however, supraphysiological dose of melatonin treatment has been reported to hold anti-tumor activity in head and neck squamous cell carcinoma cell lines [2728]. In addition, Kudová et al. [29] reported that 100 mM promoted myocardial differentiation., while physiological dose of melatonin did not show a protective role against hypoxia. In the present study, three different concentrations (50–100 mM) of melatonin were set. We performed MTT assay and confirmed that melatonin per se did not affect cell viability in every concentration. In hypoxia-treated mESCs, treatment with melatonin 500 μM stabilized the cells' beating ratio. Beating ratio of stem cell-derived cardiomyocyte has been known to be sensitive to environmental stress during differentiation [30]. Our results suggest that 500 µM of melatonin treatment hold a protective effect against hypoxic stress during differentiation. Melatonin significantly reduced protein expression of HIF-1α; the mRNA expression of the melatonin receptors was restored in a concentration-dependent way. These results indicated that melatonin alleviates hypoxia-induced inhibition on cardiomyocytes differentiation.\nThe mechanism associated with the protective action of melatonin on hypoxia-induced effects in mESCs-derived cardiomyocytes was investigated. Previous studies have shown that activation of the ERK/AKT pathway is involved with melatonin-mediated anti-proliferative effects [3132]. In the present study, hypoxia increased the expression of p-ERK in cardiomyocytes during differentiation, while melatonin suppressed the expression of p-ERK in hypoxia-treated cardiomyocytes in a concentration-dependent manner. These results provide that melatonin can inhibit phosphorylation of ERK, which is consistent with the findings in previous studies; PI3K and AKT regulate various cellular events and are involved in cellular survival and apoptosis [3334]. In the present study, hypoxia reduced the protein expression of p-AKT and PI3K, while melatonin increased those expressions in hypoxia-treated cardiomyocytes in a concentration-dependent manner, in contrast to the results presented in other studies. Alteration of expression of Bcl-2 family members associated with apoptosis was also identified. Bcl-2 expression was decreased in hypoxia-treated cardiomyocytes during differentiation but was increased by melatonin pretreatment. In contrast, the expression of Bax increased in hypoxia-treated cardiomyocytes but decreased with melatonin pretreatment. Taken together, the results show that melatonin, acting via the AKT and PI3K pathway, can mitigate hypoxia-related effects on the myocardial differentiation of mESCs.\nROS are generated as a byproduct during cellular metabolism and involve in cellular signalling and homeostasis. When ROS significant increase in the cytoplasm, it leads to oxidative stress [3536]. Melatonin reduces oxidative stress by directly promoting the ROS neutralizers and by increasing the expression of antioxidant enzymes [3738]. The expression level of Cu/Zn-SOD, Mn-SOD, and catalase in differentiating cardiomyocytes was decreased under hypoxic conditions, but they were increased by pretreatment of melatonin before the induction of hypoxia.\nThis study demonstrates that melatonin can partially mitigate the adverse effect of hypoxia in hypoxia-treated cardiomyocytes during differentiation by regulating apoptosis and by reducing and oxidative stress of the cells via the actions of the AKT and PI3K pathway. Previous evidence suggests that melatonin hold protective roles in vitro and clinically. Kudová et al. [29] reported that melatonin promoted myocardial differentiation in HIF-1α deficient mESCs by stabilizing HIF-2α. Albeit Kudová et al. [29] did not implement a physical hypoxia environment, their observation and our result both concluded that melatonin exerted a protect effect during myocardial differentiation. In addition, in ischemic heart disease, melatonin can prevent cardiac malfunctions related to ischemic heart disease and myocardial cell death through its antioxidant and anti-inflammatory properties [39]. Overexpression of melatonin receptor 2 can attenuate the hypoxia derived cell injury via the Notch1/HES1/RORa signalling [40]. Furthermore, Guerra-Librero et al. [28] reported that high-dose of melatonin treatment exerted anticancer activity in neck and head squamous cell carcinoma cell line, suggesting the potential possibility for melatonin as clinical use. Our results can support that melatonin hold a regulatory function against hypoxia also in differentiation stage. For a reason, findings from the present study may provide helpful information for understanding the mechanism of melatonin as a mitigator of hypoxic damage."],"string":"[\n \"Abnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.\\nOxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].\\nEmbryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].\\nThe pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].\\nPrevious studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation.\",\n \"Mouse embryonic stem cell (mESC) culture mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\\nmESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\\nDifferentiation into cardiomyocytes The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\\nThe mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\\nHypoxia induction and melatonin and/or luzindole treatment Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\\nDifferentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\\nAssessment of beating ratio in mESCs-derived cardiomyocytes Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\\nContraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\\nTotal RNA extraction and real-time quantitative polymerase chain reaction (qPCR) Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\\nTotal RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\\nWestern blotting analysis Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\\nProteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\\nStatistical analyses Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\\nSignificant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\",\n \"mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\",\n \"The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\",\n \"Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\",\n \"Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\",\n \"Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\",\n \"Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\",\n \"Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\",\n \"Hypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\\n*p < 0.05 versus Normoxia (Control).\\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control).\\nTo determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\\n*p < 0.05 versus Normoxia (Control).\\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control).\\nEffect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\\nThe effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\\nEffect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\nMelatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\",\n \"To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\\n*p < 0.05 versus Normoxia (Control).\\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control).\",\n \"The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\n*p < 0.05 versus Normoxia (Control). #\\np < 0.05 versus Hypoxia.\",\n \"Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\",\n \"In the present study, it was observed that differentiation into cardiomyocytes is impaired under hypoxic conditions. In addition, it was observed that the differentiation of hypoxia-treated cardiomyocytes was recovered by treatment with melatonin. Our results suggest that supraphysiological melatonin treatment can counteract the adverse effect of hypoxia during the myocardial differentiation of mESCs.\\nmRNA expression of Mtnr1a was decreased under hypoxic conditions. It was determined that the adverse effect of hypoxia was altered by melatonin pretreatment, which affected the mRNA expressions of melatonin receptors during myocardial differentiation of mESCs.\\nMelatonin is reported to have a neuroprotective effect by relieving brain edema and improving nerve function [2526]. Albeit physiological concentration of melatonin has been known as between 30 to 400 pM in normal plasma; however, supraphysiological dose of melatonin treatment has been reported to hold anti-tumor activity in head and neck squamous cell carcinoma cell lines [2728]. In addition, Kudová et al. [29] reported that 100 mM promoted myocardial differentiation., while physiological dose of melatonin did not show a protective role against hypoxia. In the present study, three different concentrations (50–100 mM) of melatonin were set. We performed MTT assay and confirmed that melatonin per se did not affect cell viability in every concentration. In hypoxia-treated mESCs, treatment with melatonin 500 μM stabilized the cells' beating ratio. Beating ratio of stem cell-derived cardiomyocyte has been known to be sensitive to environmental stress during differentiation [30]. Our results suggest that 500 µM of melatonin treatment hold a protective effect against hypoxic stress during differentiation. Melatonin significantly reduced protein expression of HIF-1α; the mRNA expression of the melatonin receptors was restored in a concentration-dependent way. These results indicated that melatonin alleviates hypoxia-induced inhibition on cardiomyocytes differentiation.\\nThe mechanism associated with the protective action of melatonin on hypoxia-induced effects in mESCs-derived cardiomyocytes was investigated. Previous studies have shown that activation of the ERK/AKT pathway is involved with melatonin-mediated anti-proliferative effects [3132]. In the present study, hypoxia increased the expression of p-ERK in cardiomyocytes during differentiation, while melatonin suppressed the expression of p-ERK in hypoxia-treated cardiomyocytes in a concentration-dependent manner. These results provide that melatonin can inhibit phosphorylation of ERK, which is consistent with the findings in previous studies; PI3K and AKT regulate various cellular events and are involved in cellular survival and apoptosis [3334]. In the present study, hypoxia reduced the protein expression of p-AKT and PI3K, while melatonin increased those expressions in hypoxia-treated cardiomyocytes in a concentration-dependent manner, in contrast to the results presented in other studies. Alteration of expression of Bcl-2 family members associated with apoptosis was also identified. Bcl-2 expression was decreased in hypoxia-treated cardiomyocytes during differentiation but was increased by melatonin pretreatment. In contrast, the expression of Bax increased in hypoxia-treated cardiomyocytes but decreased with melatonin pretreatment. Taken together, the results show that melatonin, acting via the AKT and PI3K pathway, can mitigate hypoxia-related effects on the myocardial differentiation of mESCs.\\nROS are generated as a byproduct during cellular metabolism and involve in cellular signalling and homeostasis. When ROS significant increase in the cytoplasm, it leads to oxidative stress [3536]. Melatonin reduces oxidative stress by directly promoting the ROS neutralizers and by increasing the expression of antioxidant enzymes [3738]. The expression level of Cu/Zn-SOD, Mn-SOD, and catalase in differentiating cardiomyocytes was decreased under hypoxic conditions, but they were increased by pretreatment of melatonin before the induction of hypoxia.\\nThis study demonstrates that melatonin can partially mitigate the adverse effect of hypoxia in hypoxia-treated cardiomyocytes during differentiation by regulating apoptosis and by reducing and oxidative stress of the cells via the actions of the AKT and PI3K pathway. Previous evidence suggests that melatonin hold protective roles in vitro and clinically. Kudová et al. [29] reported that melatonin promoted myocardial differentiation in HIF-1α deficient mESCs by stabilizing HIF-2α. Albeit Kudová et al. [29] did not implement a physical hypoxia environment, their observation and our result both concluded that melatonin exerted a protect effect during myocardial differentiation. In addition, in ischemic heart disease, melatonin can prevent cardiac malfunctions related to ischemic heart disease and myocardial cell death through its antioxidant and anti-inflammatory properties [39]. Overexpression of melatonin receptor 2 can attenuate the hypoxia derived cell injury via the Notch1/HES1/RORa signalling [40]. Furthermore, Guerra-Librero et al. [28] reported that high-dose of melatonin treatment exerted anticancer activity in neck and head squamous cell carcinoma cell line, suggesting the potential possibility for melatonin as clinical use. Our results can support that melatonin hold a regulatory function against hypoxia also in differentiation stage. For a reason, findings from the present study may provide helpful information for understanding the mechanism of melatonin as a mitigator of hypoxic damage.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Melatonin","hypoxia","cardiomyocytes","mESCs","Apoptosis"],"string":"[\n \"Melatonin\",\n \"hypoxia\",\n \"cardiomyocytes\",\n \"mESCs\",\n \"Apoptosis\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nAbnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.\nOxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].\nEmbryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].\nThe pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].\nPrevious studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation.\n\nMATERIALS AND METHODS:\nMouse embryonic stem cell (mESC) culture mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\nmESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\nDifferentiation into cardiomyocytes The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\nThe mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\nHypoxia induction and melatonin and/or luzindole treatment Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\nDifferentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\nAssessment of beating ratio in mESCs-derived cardiomyocytes Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\nContraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\nTotal RNA extraction and real-time quantitative polymerase chain reaction (qPCR) Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\nTotal RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\nWestern blotting analysis Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\nProteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\nStatistical analyses Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\nSignificant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\n\nMouse embryonic stem cell (mESC) culture:\nmESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\n\nDifferentiation into cardiomyocytes:\nThe mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\n\nHypoxia induction and melatonin and/or luzindole treatment:\nDifferentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\n\nAssessment of beating ratio in mESCs-derived cardiomyocytes:\nContraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\n\nTotal RNA extraction and real-time quantitative polymerase chain reaction (qPCR):\nTotal RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\n\nWestern blotting analysis:\nProteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\n\nStatistical analyses:\nSignificant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\n\nRESULTS:\nHypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nTo determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nEffect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nThe effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nEffect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nMelatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n\nHypoxia-induced inhibition on myocardial differentiation:\nTo determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\n\nEffect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia:\nThe effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\n\nEffect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation:\nMelatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n\nDISCUSSION:\nIn the present study, it was observed that differentiation into cardiomyocytes is impaired under hypoxic conditions. In addition, it was observed that the differentiation of hypoxia-treated cardiomyocytes was recovered by treatment with melatonin. Our results suggest that supraphysiological melatonin treatment can counteract the adverse effect of hypoxia during the myocardial differentiation of mESCs.\nmRNA expression of Mtnr1a was decreased under hypoxic conditions. It was determined that the adverse effect of hypoxia was altered by melatonin pretreatment, which affected the mRNA expressions of melatonin receptors during myocardial differentiation of mESCs.\nMelatonin is reported to have a neuroprotective effect by relieving brain edema and improving nerve function [2526]. Albeit physiological concentration of melatonin has been known as between 30 to 400 pM in normal plasma; however, supraphysiological dose of melatonin treatment has been reported to hold anti-tumor activity in head and neck squamous cell carcinoma cell lines [2728]. In addition, Kudová et al. [29] reported that 100 mM promoted myocardial differentiation., while physiological dose of melatonin did not show a protective role against hypoxia. In the present study, three different concentrations (50–100 mM) of melatonin were set. We performed MTT assay and confirmed that melatonin per se did not affect cell viability in every concentration. In hypoxia-treated mESCs, treatment with melatonin 500 μM stabilized the cells' beating ratio. Beating ratio of stem cell-derived cardiomyocyte has been known to be sensitive to environmental stress during differentiation [30]. Our results suggest that 500 µM of melatonin treatment hold a protective effect against hypoxic stress during differentiation. Melatonin significantly reduced protein expression of HIF-1α; the mRNA expression of the melatonin receptors was restored in a concentration-dependent way. These results indicated that melatonin alleviates hypoxia-induced inhibition on cardiomyocytes differentiation.\nThe mechanism associated with the protective action of melatonin on hypoxia-induced effects in mESCs-derived cardiomyocytes was investigated. Previous studies have shown that activation of the ERK/AKT pathway is involved with melatonin-mediated anti-proliferative effects [3132]. In the present study, hypoxia increased the expression of p-ERK in cardiomyocytes during differentiation, while melatonin suppressed the expression of p-ERK in hypoxia-treated cardiomyocytes in a concentration-dependent manner. These results provide that melatonin can inhibit phosphorylation of ERK, which is consistent with the findings in previous studies; PI3K and AKT regulate various cellular events and are involved in cellular survival and apoptosis [3334]. In the present study, hypoxia reduced the protein expression of p-AKT and PI3K, while melatonin increased those expressions in hypoxia-treated cardiomyocytes in a concentration-dependent manner, in contrast to the results presented in other studies. Alteration of expression of Bcl-2 family members associated with apoptosis was also identified. Bcl-2 expression was decreased in hypoxia-treated cardiomyocytes during differentiation but was increased by melatonin pretreatment. In contrast, the expression of Bax increased in hypoxia-treated cardiomyocytes but decreased with melatonin pretreatment. Taken together, the results show that melatonin, acting via the AKT and PI3K pathway, can mitigate hypoxia-related effects on the myocardial differentiation of mESCs.\nROS are generated as a byproduct during cellular metabolism and involve in cellular signalling and homeostasis. When ROS significant increase in the cytoplasm, it leads to oxidative stress [3536]. Melatonin reduces oxidative stress by directly promoting the ROS neutralizers and by increasing the expression of antioxidant enzymes [3738]. The expression level of Cu/Zn-SOD, Mn-SOD, and catalase in differentiating cardiomyocytes was decreased under hypoxic conditions, but they were increased by pretreatment of melatonin before the induction of hypoxia.\nThis study demonstrates that melatonin can partially mitigate the adverse effect of hypoxia in hypoxia-treated cardiomyocytes during differentiation by regulating apoptosis and by reducing and oxidative stress of the cells via the actions of the AKT and PI3K pathway. Previous evidence suggests that melatonin hold protective roles in vitro and clinically. Kudová et al. [29] reported that melatonin promoted myocardial differentiation in HIF-1α deficient mESCs by stabilizing HIF-2α. Albeit Kudová et al. [29] did not implement a physical hypoxia environment, their observation and our result both concluded that melatonin exerted a protect effect during myocardial differentiation. In addition, in ischemic heart disease, melatonin can prevent cardiac malfunctions related to ischemic heart disease and myocardial cell death through its antioxidant and anti-inflammatory properties [39]. Overexpression of melatonin receptor 2 can attenuate the hypoxia derived cell injury via the Notch1/HES1/RORa signalling [40]. Furthermore, Guerra-Librero et al. [28] reported that high-dose of melatonin treatment exerted anticancer activity in neck and head squamous cell carcinoma cell line, suggesting the potential possibility for melatonin as clinical use. Our results can support that melatonin hold a regulatory function against hypoxia also in differentiation stage. For a reason, findings from the present study may provide helpful information for understanding the mechanism of melatonin as a mitigator of hypoxic damage.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase.\n\nMethods:\nMouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated.\n\nResults:\nUnder hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects.\n\nConclusions:\nThis study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":7977,"string":"7,977"},"whole_article_abstract_length":{"kind":"number","value":318,"string":"318"},"other_sections_lengths":{"kind":"list like","value":[59,163,95,43,97,323,64,326,416,534],"string":"[\n 59,\n 163,\n 95,\n 43,\n 97,\n 323,\n 64,\n 326,\n 416,\n 534\n]"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list 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[SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Tbx20 ||| mESCs ||| ||| p-ERK | Bcl-2 | Bax | 3 | 2 | Bcl-2 | Cu/Zn-SOD | Mn-SOD ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Melatonin ||| mESCs ||| ||| ||| ||| Tbx20 ||| mESCs ||| ||| p-ERK | Bcl-2 | Bax | 3 | 2 | Bcl-2 | Cu/Zn-SOD | Mn-SOD ||| ||| the p-AKT | PI3K [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3386,"cells":{"title":{"kind":"string","value":"Multi-contrast atherosclerosis characterization (MATCH) of carotid plaque with a single 5-min scan: technical development and clinical feasibility."},"pmid":{"kind":"string","value":"25184808"},"background_abstract":{"kind":"string","value":"Multi-contrast weighted imaging is a commonly used cardiovascular magnetic resonance (CMR) protocol for characterization of carotid plaque composition. However, this approach is limited in several aspects including low slice resolution, long scan time, image mis-registration, and complex image interpretation. In this work, a 3D CMR technique, named Multi-contrast Atherosclerosis Characterization (MATCH), was developed to mitigate the above limitations."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"MATCH employs a 3D spoiled segmented fast low angle shot readout to acquire data with three different contrast weightings in an interleaved fashion. The inherently co-registered image sets, hyper T1-weighting, gray blood, and T2-weighting, are used to detect intra-plaque hemorrhage (IPH), calcification (CA), lipid-rich necrotic core (LRNC), and loose-matrix (LM). The MATCH sequence was optimized by computer simulations and testing on four healthy volunteers and then evaluated in a pilot study of six patients with carotid plaque, using the conventional multi-contrast protocol as a reference."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"On MATCH images, the major plaque components were easy to identify. Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation. Based on Cohen's kappa tests, moderate to excellent agreement in the image-based or artery-based component detection between the two protocols was obtained for LRNC, IPH, CA, and LM, respectively. Compared with the conventional multi-contrast protocol, the MATCH protocol yield significantly higher signal contrast ratio for IPH (3.1±1.3 vs. 0.4±0.3, p<0.001) and CA (1.6±1.5 vs. 0.7±0.6, p=0.012) with respect to the vessel wall."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for characterization of carotid plaque composition. Our pilot clinical study suggests that the MATCH-based protocol may outperform the conventional multi-contrast protocol in several respects. With further technical improvements and large-scale clinical validation, MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Algorithms","Carotid Arteries","Carotid Stenosis","Computer Simulation","Contrast Media","Feasibility Studies","Fibrosis","Hemorrhage","Humans","Image Interpretation, Computer-Assisted","Imaging, Three-Dimensional","Magnetic Resonance Angiography","Male","Middle Aged","Models, Cardiovascular","Necrosis","Pilot Projects","Plaque, Atherosclerotic","Predictive Value of Tests","Reproducibility of Results","Signal-To-Noise Ratio","Vascular Calcification"],"string":"[\n \"Aged\",\n \"Algorithms\",\n \"Carotid Arteries\",\n \"Carotid Stenosis\",\n \"Computer Simulation\",\n \"Contrast Media\",\n \"Feasibility Studies\",\n \"Fibrosis\",\n \"Hemorrhage\",\n \"Humans\",\n \"Image Interpretation, Computer-Assisted\",\n \"Imaging, Three-Dimensional\",\n \"Magnetic Resonance Angiography\",\n \"Male\",\n \"Middle Aged\",\n \"Models, Cardiovascular\",\n \"Necrosis\",\n \"Pilot Projects\",\n \"Plaque, Atherosclerotic\",\n \"Predictive Value of Tests\",\n \"Reproducibility of Results\",\n \"Signal-To-Noise Ratio\",\n \"Vascular Calcification\"\n]"},"pmcid":{"kind":"string","value":"4222690"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\nThe proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\n Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\nA hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\n Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\nSix male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\n Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\nAll image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\nFor the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\n Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\nThe two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup."},"other_sections_titles":{"kind":"list like","value":["Background","Sequence design","Optimization","Patient studies","Image analysis","Computer simulations and in vivo verification","Patient studies","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Sequence design\",\n \"Optimization\",\n \"Patient studies\",\n \"Image analysis\",\n \"Computer simulations and in vivo verification\",\n \"Patient studies\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"other_sections_texts":{"kind":"list like","value":["Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.","The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.","A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.","Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.","All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.","For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.","The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.","The authors declare that they have no competing interests.","ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.\",\n \"The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\",\n \"A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\\nRelevant imaging parameters for the sequences used\\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\",\n \"Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\\nArtery-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\",\n \"All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\",\n \"For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\",\n \"The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\\nSlice-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\",\n \"The authors declare that they have no competing interests.\",\n \"ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Sequence design","Optimization","Patient studies","Image analysis","Results","Computer simulations and in vivo verification","Patient studies","Discussion","Conclusions","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Sequence design\",\n \"Optimization\",\n \"Patient studies\",\n \"Image analysis\",\n \"Results\",\n \"Computer simulations and in vivo verification\",\n \"Patient studies\",\n \"Discussion\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference."," Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\nThe proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\n Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\nA hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\n Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\nSix male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\n Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\nAll image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.","The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.","A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.","Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.","All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations."," Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\nFor the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\n Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\nThe two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.","For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.","The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.","To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for adequate characterization of carotid plaques. Compared with the conventional multi-contrast protocol, the presented MATCH CMR protocol features substantially shortened scan time and simplified image interpretation. The pilot study on patients demonstrated that the MATCH protocol is in good agreement with the conventional protocol in the detection of IPH, CA, LM, and LRNC as well as differentiation of IPH age.\nTo enable a short scan time and expedited identification of these components, the MATCH protocol included a minimum of three contrast weightings. The first contrast weighting, hyper T1-w, adopted a nonselective inversion preparation that has been employed in the MPRAGE method and proven to outperform several other T1-w sequences for the detection of IPH [29]. Such an approach, however, may not adequately suppress the blood signals while achieving good contrast between the IPH and vessel wall. Although a phase-sensitive (PS) reconstruction method could be used to address the issue [25],[39], an FSD black-blood preparation module was used instead to eliminate the need of post-processing and avoid phase manipulation-related errors. With such a combination of magnetization preparations, computer simulations revealed an IPH-wall signal contrast of 0.173 that is substantially higher than that theoretically available from regular MPRAGE imaging [39]. In our pilot study, 7 images with IPH-like signal characteristics were observed by the MATCH protocol only, suggesting its higher sensitivity to IPH than the conventional multi-contrast protocol that included T1-w TSE instead of MPRAGE and thus could be of limited performance according to Ota et al. [29].\nThe second contrast weighting, gray blood, was designed specifically for the detection of CA, especially the superficial calcified nodules. Due to hypointense signals and juxtaluminal location, superficial calcified nodules are often poorly visualized on the black-blood images and likely mistaken as the lumen or wall surface ulceration. On the other hand, the shadowing effect from the bright lumen on the TOF images may limit the discrimination of this constituent from others. In contrast, gray blood contrast makes the lumen slightly brighter than calcification while maintaining an adequate contrast between the lumen and the vessel wall. In contrast to the original implementation by Koktzoglou I. [28], gray blood contrast in this work was acquired following relatively long blood T1-recovery, resulting in a slightly brighter lumen. Nevertheless, the advantages associated with the gray blood contrast were preserved and have been clearly demonstrated in the patient studies.\nThe third contrast weighting, T2-w, was chosen to provide overall plaque morphology, detect LM and LRNC, and, when combined with the hyper T1-w contrast, differentiate acute and recent hemorrhage. With the optimized parameters, the T2-w MATCH images were nearly identical to the conventional T2-w TSE images with respect to overall image contrast. Therefore, concordant composition analyses were obtained with both characterization protocols in our study.\nAn interleaved acquisition of the three contrast weightings is one of noteworthy features in MATCH. This may significantly reduce the likelihood of mis-registration between multiple contrasts and potentially avoid substantial data exclusion in patient studies [11],[14]. Easier identification of co-existent components and better appreciation of their spatial relations were perceived by the reviewer when using MATCH. As an important ingredient for the interleaved acquisition strategy, a low-flip-angle segmented FLASH readout employed in this work allowed for a large amount of k-space lines to be acquired and three-phase signal sampling per TR with a very minimal interruption of T1-recovery.\nLong scan times commonly associated with 3D imaging for plaque characterization is a limitation because some patients, particularly the elderly in whom carotid atherosclerosis is common, may not tolerate the long procedure. In a recent multi-center trial using the conventional plaque characterization protocol, patient movement-induced motion artifact accounted for 46% of failed cases (15% of all) [40]. Thus, the proposed MATCH protocol was developed so that imaging could be completed within a clinically acceptable scan time, i.e. 5 minutes. The use of fast FLASH readout, interleaved image acquisition, and parallel imaging has contributed to the achievement of this goal. As a trade-off, however, high slice resolution (e.g. 1 mm) or even isotropic spatial resolution was not attempted in this work. Therefore, the reduced partial volume effect by 3D imaging was essentially not demonstrated herein. Nevertheless, the MATCH sequence has the potential to yield higher spatial resolution images if advanced fast imaging methods such as compressed sensing can be integrated into it.\nDue to the interleaved acquisition manner and relatively long scan times, MATCH could be more prone to motion than the conventional sequences. Random motion events, such as bulk motion or swallowing, would corrupt all three MATCH data sets and necessitate a repeat scan. In contrast, the conventional protocol may still have part of scans providing valid information. Although no evident motion artifacts were observed in MATCH with the limited patient size in this work, this shortcoming merits a systematic investigation and further technical improvement such as motion self-gating [41].\nIt should be acknowledged that MATCH is not capable of identifying all important features in carotid atherosclerotic plaques. Thinning/rupture of the fibrous cap has been shown to have the highest hazard ratio compared with IPH and LRNC and therefore serves as one of important imaging marker for plaque vulnerability [42]. However, the current non-contrast MRI techniques have been shown to be poor in reproducibility of identifying fibrous cap [43]. Hence, MATCH, as a non-contrast technique, was not designed in this first development work to characterize the fibrous status. Further innovation is desirable to incorporate the ability into MATCH. Alternatively, contrast-enhanced MRI that has proven good reproducibility of fibrous cap status assessment [44] may be combined with MATCH.\nThere are several limitations in this study. First, plaque histology, the gold standard for plaque characterization, was not available in the pilot study. The capability of MATCH in detecting each of histological components was evaluated through a comparison with the conventional multi-contrast protocol. Despite good agreement in image-based composition analysis, there was some discrepancy between the two protocols. Histological verification is needed to determine the accuracy of composition analysis. Second, the patient sample size is rather small. This preliminary work presents technical aspects and the feasibility of the new sequence. While the pilot data have been encouraging, a large-scale investigation is warranted to further elucidate its clinical utility. Third, the conventional multi-contrast protocol that served as a reference could be further refined. For example, the TOF sequence should have used the same slice thickness and in-plane resolution as in other scans; MPRAGE [29] and contrast-enhanced T1-w TSE [45]-[47] might be included for an improved detection of IPH and lipid core, respectively, although this could be unsuitable for patients who cannot tolerate longer imaging protocol or have renal insufficiency. Last, some potential technical advantages of the MATCH sequence over the conventional protocol await more stringent validation. For example, higher slice resolution was not demonstrated due to the consideration of scan time and patient tolerance; more simplified image interpretation was only subjectively reported by the image reader but not quantitatively validated by a comparison of interpretation times. Further investigations with focus on these aspects are highly necessary.","A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup.","The authors declare that they have no competing interests.","ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.\",\n \" Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\\nThe proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\\n Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\\nRelevant imaging parameters for the sequences used\\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\\nA hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\\nRelevant imaging parameters for the sequences used\\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\\n Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\\nArtery-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\\nSix male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\\nArtery-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\\n Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\\nAll image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\",\n \"The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\",\n \"A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\\nRelevant imaging parameters for the sequences used\\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\",\n \"Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\\nArtery-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\",\n \"All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\",\n \" Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\\nFor the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\\n Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\\nSlice-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\\nThe two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\\nSlice-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\",\n \"For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\",\n \"The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\\nSlice-based composition analyses: MATCH vs. Conventional protocol\\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\",\n \"To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for adequate characterization of carotid plaques. Compared with the conventional multi-contrast protocol, the presented MATCH CMR protocol features substantially shortened scan time and simplified image interpretation. The pilot study on patients demonstrated that the MATCH protocol is in good agreement with the conventional protocol in the detection of IPH, CA, LM, and LRNC as well as differentiation of IPH age.\\nTo enable a short scan time and expedited identification of these components, the MATCH protocol included a minimum of three contrast weightings. The first contrast weighting, hyper T1-w, adopted a nonselective inversion preparation that has been employed in the MPRAGE method and proven to outperform several other T1-w sequences for the detection of IPH [29]. Such an approach, however, may not adequately suppress the blood signals while achieving good contrast between the IPH and vessel wall. Although a phase-sensitive (PS) reconstruction method could be used to address the issue [25],[39], an FSD black-blood preparation module was used instead to eliminate the need of post-processing and avoid phase manipulation-related errors. With such a combination of magnetization preparations, computer simulations revealed an IPH-wall signal contrast of 0.173 that is substantially higher than that theoretically available from regular MPRAGE imaging [39]. In our pilot study, 7 images with IPH-like signal characteristics were observed by the MATCH protocol only, suggesting its higher sensitivity to IPH than the conventional multi-contrast protocol that included T1-w TSE instead of MPRAGE and thus could be of limited performance according to Ota et al. [29].\\nThe second contrast weighting, gray blood, was designed specifically for the detection of CA, especially the superficial calcified nodules. Due to hypointense signals and juxtaluminal location, superficial calcified nodules are often poorly visualized on the black-blood images and likely mistaken as the lumen or wall surface ulceration. On the other hand, the shadowing effect from the bright lumen on the TOF images may limit the discrimination of this constituent from others. In contrast, gray blood contrast makes the lumen slightly brighter than calcification while maintaining an adequate contrast between the lumen and the vessel wall. In contrast to the original implementation by Koktzoglou I. [28], gray blood contrast in this work was acquired following relatively long blood T1-recovery, resulting in a slightly brighter lumen. Nevertheless, the advantages associated with the gray blood contrast were preserved and have been clearly demonstrated in the patient studies.\\nThe third contrast weighting, T2-w, was chosen to provide overall plaque morphology, detect LM and LRNC, and, when combined with the hyper T1-w contrast, differentiate acute and recent hemorrhage. With the optimized parameters, the T2-w MATCH images were nearly identical to the conventional T2-w TSE images with respect to overall image contrast. Therefore, concordant composition analyses were obtained with both characterization protocols in our study.\\nAn interleaved acquisition of the three contrast weightings is one of noteworthy features in MATCH. This may significantly reduce the likelihood of mis-registration between multiple contrasts and potentially avoid substantial data exclusion in patient studies [11],[14]. Easier identification of co-existent components and better appreciation of their spatial relations were perceived by the reviewer when using MATCH. As an important ingredient for the interleaved acquisition strategy, a low-flip-angle segmented FLASH readout employed in this work allowed for a large amount of k-space lines to be acquired and three-phase signal sampling per TR with a very minimal interruption of T1-recovery.\\nLong scan times commonly associated with 3D imaging for plaque characterization is a limitation because some patients, particularly the elderly in whom carotid atherosclerosis is common, may not tolerate the long procedure. In a recent multi-center trial using the conventional plaque characterization protocol, patient movement-induced motion artifact accounted for 46% of failed cases (15% of all) [40]. Thus, the proposed MATCH protocol was developed so that imaging could be completed within a clinically acceptable scan time, i.e. 5 minutes. The use of fast FLASH readout, interleaved image acquisition, and parallel imaging has contributed to the achievement of this goal. As a trade-off, however, high slice resolution (e.g. 1 mm) or even isotropic spatial resolution was not attempted in this work. Therefore, the reduced partial volume effect by 3D imaging was essentially not demonstrated herein. Nevertheless, the MATCH sequence has the potential to yield higher spatial resolution images if advanced fast imaging methods such as compressed sensing can be integrated into it.\\nDue to the interleaved acquisition manner and relatively long scan times, MATCH could be more prone to motion than the conventional sequences. Random motion events, such as bulk motion or swallowing, would corrupt all three MATCH data sets and necessitate a repeat scan. In contrast, the conventional protocol may still have part of scans providing valid information. Although no evident motion artifacts were observed in MATCH with the limited patient size in this work, this shortcoming merits a systematic investigation and further technical improvement such as motion self-gating [41].\\nIt should be acknowledged that MATCH is not capable of identifying all important features in carotid atherosclerotic plaques. Thinning/rupture of the fibrous cap has been shown to have the highest hazard ratio compared with IPH and LRNC and therefore serves as one of important imaging marker for plaque vulnerability [42]. However, the current non-contrast MRI techniques have been shown to be poor in reproducibility of identifying fibrous cap [43]. Hence, MATCH, as a non-contrast technique, was not designed in this first development work to characterize the fibrous status. Further innovation is desirable to incorporate the ability into MATCH. Alternatively, contrast-enhanced MRI that has proven good reproducibility of fibrous cap status assessment [44] may be combined with MATCH.\\nThere are several limitations in this study. First, plaque histology, the gold standard for plaque characterization, was not available in the pilot study. The capability of MATCH in detecting each of histological components was evaluated through a comparison with the conventional multi-contrast protocol. Despite good agreement in image-based composition analysis, there was some discrepancy between the two protocols. Histological verification is needed to determine the accuracy of composition analysis. Second, the patient sample size is rather small. This preliminary work presents technical aspects and the feasibility of the new sequence. While the pilot data have been encouraging, a large-scale investigation is warranted to further elucidate its clinical utility. Third, the conventional multi-contrast protocol that served as a reference could be further refined. For example, the TOF sequence should have used the same slice thickness and in-plane resolution as in other scans; MPRAGE [29] and contrast-enhanced T1-w TSE [45]-[47] might be included for an improved detection of IPH and lipid core, respectively, although this could be unsuitable for patients who cannot tolerate longer imaging protocol or have renal insufficiency. Last, some potential technical advantages of the MATCH sequence over the conventional protocol await more stringent validation. For example, higher slice resolution was not demonstrated due to the consideration of scan time and patient tolerance; more simplified image interpretation was only subjectively reported by the image reader but not quantitatively validated by a comparison of interpretation times. Further investigations with focus on these aspects are highly necessary.\",\n \"A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup.\",\n \"The authors declare that they have no competing interests.\",\n \"ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results",null,null,"discussion","conclusions",null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Carotid plaque","Atherosclerosis","Multi-contrast","Composition characterization","Magnetic resonance"],"string":"[\n \"Carotid plaque\",\n \"Atherosclerosis\",\n \"Multi-contrast\",\n \"Composition characterization\",\n \"Magnetic resonance\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nDisrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.\n\nMethods:\n Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\nThe proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\n Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\nA hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\n Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\nSix male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\n Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\nAll image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\n\nSequence design:\nThe proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\n\nOptimization:\nA hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\n\nPatient studies:\nSix male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\n\nImage analysis:\nAll image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\n\nResults:\n Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\nFor the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\n Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\nThe two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\n\nComputer simulations and in vivo verification:\nFor the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\n\nPatient studies:\nThe two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\n\nDiscussion:\nTo the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for adequate characterization of carotid plaques. Compared with the conventional multi-contrast protocol, the presented MATCH CMR protocol features substantially shortened scan time and simplified image interpretation. The pilot study on patients demonstrated that the MATCH protocol is in good agreement with the conventional protocol in the detection of IPH, CA, LM, and LRNC as well as differentiation of IPH age.\nTo enable a short scan time and expedited identification of these components, the MATCH protocol included a minimum of three contrast weightings. The first contrast weighting, hyper T1-w, adopted a nonselective inversion preparation that has been employed in the MPRAGE method and proven to outperform several other T1-w sequences for the detection of IPH [29]. Such an approach, however, may not adequately suppress the blood signals while achieving good contrast between the IPH and vessel wall. Although a phase-sensitive (PS) reconstruction method could be used to address the issue [25],[39], an FSD black-blood preparation module was used instead to eliminate the need of post-processing and avoid phase manipulation-related errors. With such a combination of magnetization preparations, computer simulations revealed an IPH-wall signal contrast of 0.173 that is substantially higher than that theoretically available from regular MPRAGE imaging [39]. In our pilot study, 7 images with IPH-like signal characteristics were observed by the MATCH protocol only, suggesting its higher sensitivity to IPH than the conventional multi-contrast protocol that included T1-w TSE instead of MPRAGE and thus could be of limited performance according to Ota et al. [29].\nThe second contrast weighting, gray blood, was designed specifically for the detection of CA, especially the superficial calcified nodules. Due to hypointense signals and juxtaluminal location, superficial calcified nodules are often poorly visualized on the black-blood images and likely mistaken as the lumen or wall surface ulceration. On the other hand, the shadowing effect from the bright lumen on the TOF images may limit the discrimination of this constituent from others. In contrast, gray blood contrast makes the lumen slightly brighter than calcification while maintaining an adequate contrast between the lumen and the vessel wall. In contrast to the original implementation by Koktzoglou I. [28], gray blood contrast in this work was acquired following relatively long blood T1-recovery, resulting in a slightly brighter lumen. Nevertheless, the advantages associated with the gray blood contrast were preserved and have been clearly demonstrated in the patient studies.\nThe third contrast weighting, T2-w, was chosen to provide overall plaque morphology, detect LM and LRNC, and, when combined with the hyper T1-w contrast, differentiate acute and recent hemorrhage. With the optimized parameters, the T2-w MATCH images were nearly identical to the conventional T2-w TSE images with respect to overall image contrast. Therefore, concordant composition analyses were obtained with both characterization protocols in our study.\nAn interleaved acquisition of the three contrast weightings is one of noteworthy features in MATCH. This may significantly reduce the likelihood of mis-registration between multiple contrasts and potentially avoid substantial data exclusion in patient studies [11],[14]. Easier identification of co-existent components and better appreciation of their spatial relations were perceived by the reviewer when using MATCH. As an important ingredient for the interleaved acquisition strategy, a low-flip-angle segmented FLASH readout employed in this work allowed for a large amount of k-space lines to be acquired and three-phase signal sampling per TR with a very minimal interruption of T1-recovery.\nLong scan times commonly associated with 3D imaging for plaque characterization is a limitation because some patients, particularly the elderly in whom carotid atherosclerosis is common, may not tolerate the long procedure. In a recent multi-center trial using the conventional plaque characterization protocol, patient movement-induced motion artifact accounted for 46% of failed cases (15% of all) [40]. Thus, the proposed MATCH protocol was developed so that imaging could be completed within a clinically acceptable scan time, i.e. 5 minutes. The use of fast FLASH readout, interleaved image acquisition, and parallel imaging has contributed to the achievement of this goal. As a trade-off, however, high slice resolution (e.g. 1 mm) or even isotropic spatial resolution was not attempted in this work. Therefore, the reduced partial volume effect by 3D imaging was essentially not demonstrated herein. Nevertheless, the MATCH sequence has the potential to yield higher spatial resolution images if advanced fast imaging methods such as compressed sensing can be integrated into it.\nDue to the interleaved acquisition manner and relatively long scan times, MATCH could be more prone to motion than the conventional sequences. Random motion events, such as bulk motion or swallowing, would corrupt all three MATCH data sets and necessitate a repeat scan. In contrast, the conventional protocol may still have part of scans providing valid information. Although no evident motion artifacts were observed in MATCH with the limited patient size in this work, this shortcoming merits a systematic investigation and further technical improvement such as motion self-gating [41].\nIt should be acknowledged that MATCH is not capable of identifying all important features in carotid atherosclerotic plaques. Thinning/rupture of the fibrous cap has been shown to have the highest hazard ratio compared with IPH and LRNC and therefore serves as one of important imaging marker for plaque vulnerability [42]. However, the current non-contrast MRI techniques have been shown to be poor in reproducibility of identifying fibrous cap [43]. Hence, MATCH, as a non-contrast technique, was not designed in this first development work to characterize the fibrous status. Further innovation is desirable to incorporate the ability into MATCH. Alternatively, contrast-enhanced MRI that has proven good reproducibility of fibrous cap status assessment [44] may be combined with MATCH.\nThere are several limitations in this study. First, plaque histology, the gold standard for plaque characterization, was not available in the pilot study. The capability of MATCH in detecting each of histological components was evaluated through a comparison with the conventional multi-contrast protocol. Despite good agreement in image-based composition analysis, there was some discrepancy between the two protocols. Histological verification is needed to determine the accuracy of composition analysis. Second, the patient sample size is rather small. This preliminary work presents technical aspects and the feasibility of the new sequence. While the pilot data have been encouraging, a large-scale investigation is warranted to further elucidate its clinical utility. Third, the conventional multi-contrast protocol that served as a reference could be further refined. For example, the TOF sequence should have used the same slice thickness and in-plane resolution as in other scans; MPRAGE [29] and contrast-enhanced T1-w TSE [45]-[47] might be included for an improved detection of IPH and lipid core, respectively, although this could be unsuitable for patients who cannot tolerate longer imaging protocol or have renal insufficiency. Last, some potential technical advantages of the MATCH sequence over the conventional protocol await more stringent validation. For example, higher slice resolution was not demonstrated due to the consideration of scan time and patient tolerance; more simplified image interpretation was only subjectively reported by the image reader but not quantitatively validated by a comparison of interpretation times. Further investigations with focus on these aspects are highly necessary.\n\nConclusions:\nA 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMulti-contrast weighted imaging is a commonly used cardiovascular magnetic resonance (CMR) protocol for characterization of carotid plaque composition. However, this approach is limited in several aspects including low slice resolution, long scan time, image mis-registration, and complex image interpretation. In this work, a 3D CMR technique, named Multi-contrast Atherosclerosis Characterization (MATCH), was developed to mitigate the above limitations.\n\nMethods:\nMATCH employs a 3D spoiled segmented fast low angle shot readout to acquire data with three different contrast weightings in an interleaved fashion. The inherently co-registered image sets, hyper T1-weighting, gray blood, and T2-weighting, are used to detect intra-plaque hemorrhage (IPH), calcification (CA), lipid-rich necrotic core (LRNC), and loose-matrix (LM). The MATCH sequence was optimized by computer simulations and testing on four healthy volunteers and then evaluated in a pilot study of six patients with carotid plaque, using the conventional multi-contrast protocol as a reference.\n\nResults:\nOn MATCH images, the major plaque components were easy to identify. Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation. Based on Cohen's kappa tests, moderate to excellent agreement in the image-based or artery-based component detection between the two protocols was obtained for LRNC, IPH, CA, and LM, respectively. Compared with the conventional multi-contrast protocol, the MATCH protocol yield significantly higher signal contrast ratio for IPH (3.1±1.3 vs. 0.4±0.3, p<0.001) and CA (1.6±1.5 vs. 0.7±0.6, p=0.012) with respect to the vessel wall.\n\nConclusions:\nTo the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for characterization of carotid plaque composition. Our pilot clinical study suggests that the MATCH-based protocol may outperform the conventional multi-contrast protocol in several respects. With further technical improvements and large-scale clinical validation, MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup."},"background_conclusion_text":{"kind":"string","value":"Background:\nDisrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.\n\nConclusions:\nA 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMulti-contrast weighted imaging is a commonly used cardiovascular magnetic resonance (CMR) protocol for characterization of carotid plaque composition. However, this approach is limited in several aspects including low slice resolution, long scan time, image mis-registration, and complex image interpretation. In this work, a 3D CMR technique, named Multi-contrast Atherosclerosis Characterization (MATCH), was developed to mitigate the above limitations.\n\nMethods:\nMATCH employs a 3D spoiled segmented fast low angle shot readout to acquire data with three different contrast weightings in an interleaved fashion. The inherently co-registered image sets, hyper T1-weighting, gray blood, and T2-weighting, are used to detect intra-plaque hemorrhage (IPH), calcification (CA), lipid-rich necrotic core (LRNC), and loose-matrix (LM). The MATCH sequence was optimized by computer simulations and testing on four healthy volunteers and then evaluated in a pilot study of six patients with carotid plaque, using the conventional multi-contrast protocol as a reference.\n\nResults:\nOn MATCH images, the major plaque components were easy to identify. Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation. Based on Cohen's kappa tests, moderate to excellent agreement in the image-based or artery-based component detection between the two protocols was obtained for LRNC, IPH, CA, and LM, respectively. Compared with the conventional multi-contrast protocol, the MATCH protocol yield significantly higher signal contrast ratio for IPH (3.1±1.3 vs. 0.4±0.3, p<0.001) and CA (1.6±1.5 vs. 0.7±0.6, p=0.012) with respect to the vessel wall.\n\nConclusions:\nTo the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for characterization of carotid plaque composition. Our pilot clinical study suggests that the MATCH-based protocol may outperform the conventional multi-contrast protocol in several respects. With further technical improvements and large-scale clinical validation, MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup."},"whole_article_text_length":{"kind":"number","value":14891,"string":"14,891"},"whole_article_abstract_length":{"kind":"number","value":433,"string":"433"},"other_sections_lengths":{"kind":"list like","value":[829,565,546,254,885,459,1357,10,159],"string":"[\n 829,\n 565,\n 546,\n 254,\n 885,\n 459,\n 1357,\n 10,\n 159\n]"},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["match","images","iph","contrast","t1","wall","hyper","t2","protocol","blood"],"string":"[\n \"match\",\n \"images\",\n \"iph\",\n \"contrast\",\n \"t1\",\n \"wall\",\n \"hyper\",\n \"t2\",\n \"protocol\",\n \"blood\"\n]"},"keybert_topics":{"kind":"list like","value":["assessment plaque stability","carotid plaque imaging","plaque mr characterization","application cmr plaque","cmr plaque characterization"],"string":"[\n \"assessment plaque stability\",\n \"carotid plaque imaging\",\n \"plaque mr characterization\",\n \"application cmr plaque\",\n \"cmr plaque characterization\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | 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Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization 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| iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] plaque | contrast | technique | multi | multi contrast | cmr | contrasts | weighted | carotid | multiple [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ms | wall | images | iph | match | t1 | blood | t2 | vessel | vessel wall [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] images | match | hyper | iph | figure | intense | hyper intense | t1 | protocol | tse [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] carotid plaque | carotid | plaque | match | technique | cmr | clinical | multi contrast | multi | validation particular [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] match | images | iph | contrast | wall | t1 | ms | protocol | hyper | t2 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] match | images | iph | contrast | wall | t1 | ms | protocol | hyper | t2 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] CMR ||| ||| CMR | Multi [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] three ||| T1 | T2 | IPH | CA | LRNC ||| four | six [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| three ||| Cohen | two | LRNC | IPH | CA ||| IPH | 3.1±1.3 | 0.4±0.3 | CA | 1.6±1.5 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] first | CMR ||| ||| CMR [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CMR ||| ||| CMR | Multi ||| three ||| T1 | T2 | IPH | CA | LRNC ||| four | six ||| ||| three ||| Cohen | two | LRNC | IPH | CA ||| IPH | 3.1±1.3 | 0.4±0.3 | CA | 1.6±1.5 ||| first | CMR ||| ||| CMR [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] CMR ||| ||| CMR | Multi ||| three ||| T1 | T2 | IPH | CA | LRNC ||| four | six ||| ||| three ||| Cohen | two | LRNC | IPH | CA ||| IPH | 3.1±1.3 | 0.4±0.3 | CA | 1.6±1.5 ||| first | CMR ||| ||| CMR [SUMMARY]"}}},{"rowIdx":3387,"cells":{"title":{"kind":"string","value":"Factors Associated with Health Literacy, Self-Efficacy, Social Support, and Oral Health Care Behaviors Among Elderly in Northern Border Community Thailand."},"pmid":{"kind":"string","value":"34326634"},"background_abstract":{"kind":"string","value":"Oral health problems among elderly people are an important public health issues worldwide. Oral healthcare is essential to the health and well-being of elders and is one of the key indicators determining their quality of life. This research aimed to study oral health literacy, self-efficacy, social support, and demographic characteristic factors associated with the oral health care behaviors of elderly people living in the rural areas of northern Thailand."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"This research was a cross-sectional study that recruited 406 elderly participants using convenience and snowball samplings. Participants' names were obtained from the registration list of the Java Health Center Information System (JHCIS) program, where they received a health service between 2018 and 2020. Data were obtained through face-to-face interviews with participants, while they were waiting to receive a health service or through a phone interview. Linear regression was analyzed to determine the factors associated with oral healthcare behaviors."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The majority of participants (85%) had inadequate functional health literacy, 52% had moderate self-efficacy toward oral health behaviors, 91.9% had moderate social support, and 53% admitted to moderate oral health behaviors. The results from the model show that self-efficacy, social support, and oral health literacy are positively associated with oral health care behaviors among the elderly (p-value < 0.05). The multiple regression model can account for 47.2% of the variance in oral health care behaviors."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Improving oral health care behaviors among elderly people should be considered by health care providers and those who provide social support. Self-esteem, communication skills among service providers and service receivers, and self-management of oral healthcare should receive special attention. Moreover, social support and relevant agencies can help promote oral healthcare by collaborating with other healthcare providers for better oral health outcomes among elderly people."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","Cross-Sectional Studies","Delivery of Health Care","Female","Health Literacy","Humans","Male","Middle Aged","Quality of Life","Self Efficacy","Social Support","Thailand"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"Cross-Sectional Studies\",\n \"Delivery of Health Care\",\n \"Female\",\n \"Health Literacy\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Quality of Life\",\n \"Self Efficacy\",\n \"Social Support\",\n \"Thailand\"\n]"},"pmcid":{"kind":"string","value":"8314679"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3\nA Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15\nHealth literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information."},"other_sections_titles":{"kind":"list like","value":["Methodology","Statistical Analysis","Results","Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly","Discussion","Conclusion"],"string":"[\n \"Methodology\",\n \"Statistical Analysis\",\n \"Results\",\n \"Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly\",\n \"Discussion\",\n \"Conclusion\"\n]"},"other_sections_texts":{"kind":"list like","value":["This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.","Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.","According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\n\nDemographic Characteristics of Elderly (N=406)\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\n\nOral Health Characteristics of Elderly (N=406)\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\n\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).","Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).","The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.","In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information."],"string":"[\n \"This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\",\n \"Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\",\n \"According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\\n\\nDemographic Characteristics of Elderly (N=406)\\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\\n\\nOral Health Characteristics of Elderly (N=406)\\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\\n\\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\\n\\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\\nNote: *Significant at the 0.05 level (2-tailed).\\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\\n\\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\\nNote: *Significant at the 0.05 level (2-tailed).\",\n \"Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\\n\\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\\nNote: *Significant at the 0.05 level (2-tailed).\",\n \"The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.\",\n \"In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methodology","Statistical Analysis","Results","Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly","Discussion","Conclusion"],"string":"[\n \"Introduction\",\n \"Methodology\",\n \"Statistical Analysis\",\n \"Results\",\n \"Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly\",\n \"Discussion\",\n \"Conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3\nA Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15\nHealth literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.","This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.","Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.","According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\n\nDemographic Characteristics of Elderly (N=406)\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\n\nOral Health Characteristics of Elderly (N=406)\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\n\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).","Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).","The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.","In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information."],"string":"[\n \"Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3\\nA Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15\\nHealth literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.\",\n \"This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\",\n \"Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\",\n \"According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\\n\\nDemographic Characteristics of Elderly (N=406)\\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\\n\\nOral Health Characteristics of Elderly (N=406)\\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\\n\\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\\n\\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\\nNote: *Significant at the 0.05 level (2-tailed).\\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\\n\\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\\nNote: *Significant at the 0.05 level (2-tailed).\",\n \"Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\\n\\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\\nNote: *Significant at the 0.05 level (2-tailed).\",\n \"The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.\",\n \"In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null,null],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["health literacy","self-efficacy","social support","oral health","elderly"],"string":"[\n \"health literacy\",\n \"self-efficacy\",\n \"social support\",\n \"oral health\",\n \"elderly\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nOral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3\nA Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15\nHealth literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.\n\nMethodology:\nThis research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\n\nStatistical Analysis:\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\n\nResults:\nAccording to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\n\nDemographic Characteristics of Elderly (N=406)\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\n\nOral Health Characteristics of Elderly (N=406)\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\n\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\n\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly:\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\n\nDiscussion:\nThe study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.\n\nConclusion:\nIn conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nOral health problems among elderly people are an important public health issues worldwide. Oral healthcare is essential to the health and well-being of elders and is one of the key indicators determining their quality of life. This research aimed to study oral health literacy, self-efficacy, social support, and demographic characteristic factors associated with the oral health care behaviors of elderly people living in the rural areas of northern Thailand.\n\nMethods:\nThis research was a cross-sectional study that recruited 406 elderly participants using convenience and snowball samplings. Participants' names were obtained from the registration list of the Java Health Center Information System (JHCIS) program, where they received a health service between 2018 and 2020. Data were obtained through face-to-face interviews with participants, while they were waiting to receive a health service or through a phone interview. Linear regression was analyzed to determine the factors associated with oral healthcare behaviors.\n\nResults:\nThe majority of participants (85%) had inadequate functional health literacy, 52% had moderate self-efficacy toward oral health behaviors, 91.9% had moderate social support, and 53% admitted to moderate oral health behaviors. The results from the model show that self-efficacy, social support, and oral health literacy are positively associated with oral health care behaviors among the elderly (p-value < 0.05). The multiple regression model can account for 47.2% of the variance in oral health care behaviors.\n\nConclusions:\nImproving oral health care behaviors among elderly people should be considered by health care providers and those who provide social support. Self-esteem, communication skills among service providers and service receivers, and self-management of oral healthcare should receive special attention. Moreover, social support and relevant agencies can help promote oral healthcare by collaborating with other healthcare providers for better oral health outcomes among elderly people."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nOral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3\nA Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15\nHealth literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.\n\nConclusion:\nIn conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nOral health problems among elderly people are an important public health issues worldwide. Oral healthcare is essential to the health and well-being of elders and is one of the key indicators determining their quality of life. This research aimed to study oral health literacy, self-efficacy, social support, and demographic characteristic factors associated with the oral health care behaviors of elderly people living in the rural areas of northern Thailand.\n\nMethods:\nThis research was a cross-sectional study that recruited 406 elderly participants using convenience and snowball samplings. Participants' names were obtained from the registration list of the Java Health Center Information System (JHCIS) program, where they received a health service between 2018 and 2020. Data were obtained through face-to-face interviews with participants, while they were waiting to receive a health service or through a phone interview. Linear regression was analyzed to determine the factors associated with oral healthcare behaviors.\n\nResults:\nThe majority of participants (85%) had inadequate functional health literacy, 52% had moderate self-efficacy toward oral health behaviors, 91.9% had moderate social support, and 53% admitted to moderate oral health behaviors. The results from the model show that self-efficacy, social support, and oral health literacy are positively associated with oral health care behaviors among the elderly (p-value < 0.05). The multiple regression model can account for 47.2% of the variance in oral health care behaviors.\n\nConclusions:\nImproving oral health care behaviors among elderly people should be considered by health care providers and those who provide social support. Self-esteem, communication skills among service providers and service receivers, and self-management of oral healthcare should receive special attention. Moreover, social support and relevant agencies can help promote oral healthcare by collaborating with other healthcare providers for better oral health outcomes among elderly people."},"whole_article_text_length":{"kind":"number","value":6606,"string":"6,606"},"whole_article_abstract_length":{"kind":"number","value":359,"string":"359"},"other_sections_lengths":{"kind":"list like","value":[1688,120,1480,237,1898,254],"string":"[\n 1688,\n 120,\n 1480,\n 237,\n 1898,\n 254\n]"},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["health","oral","oral health","elderly","behaviors","literacy","health literacy","healthcare","self","study"],"string":"[\n \"health\",\n \"oral\",\n \"oral health\",\n \"elderly\",\n \"behaviors\",\n \"literacy\",\n \"health literacy\",\n \"healthcare\",\n \"self\",\n \"study\"\n]"},"keybert_topics":{"kind":"list like","value":["improve elderly oral","loss teeth elderly","teeth elderly report","oral 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[SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] health literacy | self-efficacy | social support | oral health | elderly [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] health literacy | self-efficacy | social support | oral health | elderly [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] health literacy | self-efficacy | social support | oral health | elderly [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Cross-Sectional Studies | Delivery of Health Care | Female | Health Literacy | Humans | Male | Middle Aged | Quality of Life | Self Efficacy | Social Support | Thailand 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[SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Thailand [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Thailand ||| 406 ||| the Java Health Center Information System | between 2018 and 2020 ||| ||| Linear ||| ||| 85% | 52% | 91.9% | 53% ||| ||| 47.2% ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Thailand ||| 406 ||| the Java Health Center Information System | between 2018 and 2020 ||| ||| Linear ||| ||| 85% | 52% | 91.9% | 53% ||| ||| 47.2% ||| ||| ||| [SUMMARY]"}}},{"rowIdx":3388,"cells":{"title":{"kind":"string","value":"Comprehension and Practice Patterns of Korean Urologists for Retractile and Gliding Testes."},"pmid":{"kind":"string","value":"35347906"},"background_abstract":{"kind":"string","value":"It is quite difficult to distinguish retractile testis from gliding testis, which requires different treatment planning in the clinic setting. We evaluated practice patterns of urologists in Korea regarding the diagnosis and management of retractile and gliding testes."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We mailed or e-mailed self-completion questionnaires consisting of 20 items to 106 urologists practicing in Korean hospitals concerning the diagnosis and treatment of cryptorchidism. We collected and analyzed the responses statistically."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Responses were received from 62 urologists. The response rate was 58.5%. Thirty-seven urologists (59.7%) actually felt they had difficulty in distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than for pediatric urologists (40.0%) (P = 0.006). In cases of infant retractile testis, only five urologists (8.1%) said that they would perform orchiopexy immediately, with 54 (87.1%) urologists saying they would do follow-up. In cases of preschool-age children with retractile testis, 17 urologists (27.4%) said that they would perform orchiopexy immediately with 41 (66.1%) urologists saying they would do follow-up. In cases of infant gliding testis, 37 urologists (59.7%) said that they would perform orchiopexy immediately with 24 (38.7%) urologists saying they would do a follow-up."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"More than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing a practical guideline."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Asian People","Child","Child, Preschool","Comprehension","Cryptorchidism","Humans","Infant","Male","Urologists"],"string":"[\n \"Asian People\",\n \"Child\",\n \"Child, Preschool\",\n \"Comprehension\",\n \"Cryptorchidism\",\n \"Humans\",\n \"Infant\",\n \"Male\",\n \"Urologists\"\n]"},"pmcid":{"kind":"string","value":"8960942"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Recently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.\nThe normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4\nIn prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9\nRetractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"We conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.\nThe questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.\nWe received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.\nThirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.\nStatistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nWe performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nEthics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\nThe present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"\nFig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.\nThirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).\nGT = gliding testis, RT = retractile testis.\nMost of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).\nCase scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nOf the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nCase scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nOf the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nCase scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nOf the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nSummary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\nScrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Statistical analysis","Ethics statement","Case scenario of an infant with retractile testis","Case scenario of a 6-year-old child with retractile testis","Case scenario of an infant with a gliding testis","Summary"],"string":"[\n \"Statistical analysis\",\n \"Ethics statement\",\n \"Case scenario of an infant with retractile testis\",\n \"Case scenario of a 6-year-old child with retractile testis\",\n \"Case scenario of an infant with a gliding testis\",\n \"Summary\"\n]"},"other_sections_texts":{"kind":"list like","value":["We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.","The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.","Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).","Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).","Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).","Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied."],"string":"[\n \"We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\",\n \"The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\",\n \"Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\\nValues are presented as number (%).\\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\\nValues are presented as number (%).\",\n \"Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\",\n \"Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\",\n \"Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Statistical analysis","Ethics statement","RESULTS","Case scenario of an infant with retractile testis","Case scenario of a 6-year-old child with retractile testis","Case scenario of an infant with a gliding testis","Summary","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Statistical analysis\",\n \"Ethics statement\",\n \"RESULTS\",\n \"Case scenario of an infant with retractile testis\",\n \"Case scenario of a 6-year-old child with retractile testis\",\n \"Case scenario of an infant with a gliding testis\",\n \"Summary\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["Recently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.\nThe normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4\nIn prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9\nRetractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey.","We conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.\nThe questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.\nWe received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.\nThirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.\nStatistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nWe performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nEthics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\nThe present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.","We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.","The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.","\nFig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.\nThirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).\nGT = gliding testis, RT = retractile testis.\nMost of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).\nCase scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nOf the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nCase scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nOf the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nCase scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nOf the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nSummary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\nScrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.","Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).","Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).","Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).","Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.","Although cryptorchidism is one of the most common diseases in pediatric urology, there have been no systematic investigations into the recognition and treatment of unstable high scrotal testis, such retractile or gliding testes.\nGliding testis should be clearly distinguished from retractile testis. There are many tips on how to distinguish gliding from retractile testis. Van Essen10 described that having a patient take a squatting position on physical examination is essential in distinguishing gliding from retractile testis. Wyllie11 stated that retractile testis could be brought into a low, stable scrotal position and that traction on the cord structures is not painful. In contrast, gliding testis can only be brought into a high unstable scrotal position, and further traction on cord structures is painful.11 Ferro et al.12 defines retractile testis as a phenomenon in which a testis can manipulated into the scrotum and remain in position without tension. A gliding testis is defined as when it can be manipulated into the upper scrotum, but then it retracts when released.12 However, both are two different and distinct non-scrotal entities, and each definition is so clear, but there are many cases where it is often difficult to apply it in practice.\nIn the beginning, both conditions were thought to be identical, and a gliding testis was therefore also called a “pathological retractile testis”.1314 Many studies about retractile testis could have some bias because they include some cases of gliding testis in their studies. Often it was concluded that the course of retracted testis was similar to that of cryptorchidism. It should be realized that this can create vague fears about retractile testis. However, most urologists believe that a retractile testis is a physiological variant of a fully descended testis, and therefore active treatment and long-term follow-up are usually not indicated. In contrast to a retractile testis, a gliding testis belongs to the spectrum of undescended testis. Therefore, it should be noted that retractile testis do not need surgery but indicates careful physical examinations and periodic follow-ups. However, in this study, 59.7% of urologists felt they had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than pediatric urologists (40.0%).\nThe treatment for retractile testis remains controversial, whereas treatment methods for undescended testis have been well established through many studies.151617 The most typical indication for orchidopexy is when a retractile testis has any decrease in testicular volume and when it ascends because of increased spermatic cord tension. This makes it impossible to reposition the testis into its correct anatomic location. In this study, treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. They recommended follow-up or orchiopexy at a similar rate.\nA retractile testis is attributed to an overactive cremasteric reflex or to alterations within the contractile properties of the cremasteric muscle.18 It is especially common in boys around the school starting age (5–6 years).19 Some authors have suggested that retractile testis may not be as innocuous as has been widely believed.20 In addition, recent evidence suggests that a retractile testis may evolve into an acquired-undescended testis.8\nIt has been reported that retractile testis is accompanied by histological changes. Abnormalities on semen analysis have been found during follow-up when patients with retractile testis had become adults.2122\nIn this study, most of the urologists (98.4%) agreed that cryptorchidism could affect infertility and testicular function in the future. Fifty urologists (80.6%) agreed that these adverse effects were also true with gliding testis, and selected surgical treatment. The distribution of treatment decisions shows no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108). Meanwhile, 13 urologists (21.0%) agreed that retractile testis lead to adverse effects, including 31.2% of non-pediatric urologists and 10% of pediatric urologists. Regarding infant with retractile testis, treatment distribution varied among groups (P = 0.030). These differences show the result of a more conservative treatment trend among pediatric urologists than in non-pediatric urologists. To overcome such discrepancies in clinical practice, it is necessary to establish an appropriate guideline for the proper management of retractile testis.\nLa Scala and Ein7 reported that 150 boys with retractile testis needed periodic follow-up. In their series, only 22.7% of the patients required orchidopexy. They recommend that patients with retractile testis be attentively followed annually. They suggested that most patients with retractile testis can expect a spontaneously favorable resolution without surgical treatment, even after 14 years of age. In this study, the follow-up period in retractile and gliding testes is not clear. Half of the urologists wanted to follow-up before school age or until 2 years of age. Some urologists (12.5–24.1%) wanted to follow-up after puberty. Therefore, we need further evaluation about what is the proper follow-up period for retractile and gliding testes.\nIn this study, there are some limitations. This is a survey study of urologists. As a result of raising questions about the treatment trends of these urologists, we do not know if this occurs in the real clinical setting. However, we tried to elicit answers that are as realistic as possible, with scenarios for patients seen in the actual clinical field. Second, the number of respondents is small. We think it is likely that the urologists who seldom treat pediatric patients did not respond. However, we tried to include all pediatric urologists in the Korean Society of Pediatric Urology, and most members of our society participated in this survey study.\nMoreover, we found some differences in practice patterns between pediatric urologists and non-pediatric urologists. Overall, non-pediatric urologists found it challenging to distinguish retractile testis from gliding testis. Compared to pediatric urologists, they had a higher tendency to agree with the statement that retractile testis could cause infertility and testicular dysfunction in the future and chose to perform orchiopexy as early as the time of the first detection. To overcome these differences, adequate education about retractile and gliding testes is essential. The results of this study are expected to be utilized as basic data for establishing a practical guideline for the diagnosis and treatment of retractile testis in Korea.\nIn conclusion, more than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. Among urologists, non-pediatric urologists had more difficulty than pediatric urologists, and this disparity was more pronounced in children compared to infants. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing consistent management plans. The results of this study are expected to be used as basic data for making a practical guideline for the diagnosis and treatment of retractile testis in Korea."],"string":"[\n \"Recently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.\\nThe normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4\\nIn prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9\\nRetractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey.\",\n \"We conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.\\nThe questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.\\nWe received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.\\nThirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.\\nStatistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\\nWe performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\\nEthics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\\nThe present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\",\n \"We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\",\n \"The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\",\n \"\\nFig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.\\nThirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).\\nGT = gliding testis, RT = retractile testis.\\nMost of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).\\nCase scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\\nValues are presented as number (%).\\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\\nValues are presented as number (%).\\nOf the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\\nValues are presented as number (%).\\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\\nValues are presented as number (%).\\nCase scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\\nOf the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\\nCase scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\\nOf the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\\nSummary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\\nScrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\",\n \"Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\\nValues are presented as number (%).\\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\\nValues are presented as number (%).\",\n \"Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\",\n \"Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\",\n \"Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\",\n \"Although cryptorchidism is one of the most common diseases in pediatric urology, there have been no systematic investigations into the recognition and treatment of unstable high scrotal testis, such retractile or gliding testes.\\nGliding testis should be clearly distinguished from retractile testis. There are many tips on how to distinguish gliding from retractile testis. Van Essen10 described that having a patient take a squatting position on physical examination is essential in distinguishing gliding from retractile testis. Wyllie11 stated that retractile testis could be brought into a low, stable scrotal position and that traction on the cord structures is not painful. In contrast, gliding testis can only be brought into a high unstable scrotal position, and further traction on cord structures is painful.11 Ferro et al.12 defines retractile testis as a phenomenon in which a testis can manipulated into the scrotum and remain in position without tension. A gliding testis is defined as when it can be manipulated into the upper scrotum, but then it retracts when released.12 However, both are two different and distinct non-scrotal entities, and each definition is so clear, but there are many cases where it is often difficult to apply it in practice.\\nIn the beginning, both conditions were thought to be identical, and a gliding testis was therefore also called a “pathological retractile testis”.1314 Many studies about retractile testis could have some bias because they include some cases of gliding testis in their studies. Often it was concluded that the course of retracted testis was similar to that of cryptorchidism. It should be realized that this can create vague fears about retractile testis. However, most urologists believe that a retractile testis is a physiological variant of a fully descended testis, and therefore active treatment and long-term follow-up are usually not indicated. In contrast to a retractile testis, a gliding testis belongs to the spectrum of undescended testis. Therefore, it should be noted that retractile testis do not need surgery but indicates careful physical examinations and periodic follow-ups. However, in this study, 59.7% of urologists felt they had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than pediatric urologists (40.0%).\\nThe treatment for retractile testis remains controversial, whereas treatment methods for undescended testis have been well established through many studies.151617 The most typical indication for orchidopexy is when a retractile testis has any decrease in testicular volume and when it ascends because of increased spermatic cord tension. This makes it impossible to reposition the testis into its correct anatomic location. In this study, treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. They recommended follow-up or orchiopexy at a similar rate.\\nA retractile testis is attributed to an overactive cremasteric reflex or to alterations within the contractile properties of the cremasteric muscle.18 It is especially common in boys around the school starting age (5–6 years).19 Some authors have suggested that retractile testis may not be as innocuous as has been widely believed.20 In addition, recent evidence suggests that a retractile testis may evolve into an acquired-undescended testis.8\\nIt has been reported that retractile testis is accompanied by histological changes. Abnormalities on semen analysis have been found during follow-up when patients with retractile testis had become adults.2122\\nIn this study, most of the urologists (98.4%) agreed that cryptorchidism could affect infertility and testicular function in the future. Fifty urologists (80.6%) agreed that these adverse effects were also true with gliding testis, and selected surgical treatment. The distribution of treatment decisions shows no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108). Meanwhile, 13 urologists (21.0%) agreed that retractile testis lead to adverse effects, including 31.2% of non-pediatric urologists and 10% of pediatric urologists. Regarding infant with retractile testis, treatment distribution varied among groups (P = 0.030). These differences show the result of a more conservative treatment trend among pediatric urologists than in non-pediatric urologists. To overcome such discrepancies in clinical practice, it is necessary to establish an appropriate guideline for the proper management of retractile testis.\\nLa Scala and Ein7 reported that 150 boys with retractile testis needed periodic follow-up. In their series, only 22.7% of the patients required orchidopexy. They recommend that patients with retractile testis be attentively followed annually. They suggested that most patients with retractile testis can expect a spontaneously favorable resolution without surgical treatment, even after 14 years of age. In this study, the follow-up period in retractile and gliding testes is not clear. Half of the urologists wanted to follow-up before school age or until 2 years of age. Some urologists (12.5–24.1%) wanted to follow-up after puberty. Therefore, we need further evaluation about what is the proper follow-up period for retractile and gliding testes.\\nIn this study, there are some limitations. This is a survey study of urologists. As a result of raising questions about the treatment trends of these urologists, we do not know if this occurs in the real clinical setting. However, we tried to elicit answers that are as realistic as possible, with scenarios for patients seen in the actual clinical field. Second, the number of respondents is small. We think it is likely that the urologists who seldom treat pediatric patients did not respond. However, we tried to include all pediatric urologists in the Korean Society of Pediatric Urology, and most members of our society participated in this survey study.\\nMoreover, we found some differences in practice patterns between pediatric urologists and non-pediatric urologists. Overall, non-pediatric urologists found it challenging to distinguish retractile testis from gliding testis. Compared to pediatric urologists, they had a higher tendency to agree with the statement that retractile testis could cause infertility and testicular dysfunction in the future and chose to perform orchiopexy as early as the time of the first detection. To overcome these differences, adequate education about retractile and gliding testes is essential. The results of this study are expected to be utilized as basic data for establishing a practical guideline for the diagnosis and treatment of retractile testis in Korea.\\nIn conclusion, more than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. Among urologists, non-pediatric urologists had more difficulty than pediatric urologists, and this disparity was more pronounced in children compared to infants. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing consistent management plans. The results of this study are expected to be used as basic data for making a practical guideline for the diagnosis and treatment of retractile testis in Korea.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,"results",null,null,null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["Cryptorchidism","Child","Testis","Practice Patterns, Physicians","Surveys and Questionnaires","Practice Guideline"],"string":"[\n \"Cryptorchidism\",\n \"Child\",\n \"Testis\",\n \"Practice Patterns, Physicians\",\n \"Surveys and Questionnaires\",\n \"Practice Guideline\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nRecently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.\nThe normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4\nIn prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9\nRetractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey.\n\nMETHODS:\nWe conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.\nThe questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.\nWe received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.\nThirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.\nStatistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nWe performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nEthics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\nThe present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\n\nStatistical analysis:\nWe performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\n\nEthics statement:\nThe present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\n\nRESULTS:\n\nFig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.\nThirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).\nGT = gliding testis, RT = retractile testis.\nMost of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).\nCase scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nOf the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nCase scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nOf the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nCase scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nOf the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nSummary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\nScrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\n\nCase scenario of an infant with retractile testis:\nOf the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\n\nCase scenario of a 6-year-old child with retractile testis:\nOf the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\n\nCase scenario of an infant with a gliding testis:\nOf the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\n\nSummary:\nScrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\n\nDISCUSSION:\nAlthough cryptorchidism is one of the most common diseases in pediatric urology, there have been no systematic investigations into the recognition and treatment of unstable high scrotal testis, such retractile or gliding testes.\nGliding testis should be clearly distinguished from retractile testis. There are many tips on how to distinguish gliding from retractile testis. Van Essen10 described that having a patient take a squatting position on physical examination is essential in distinguishing gliding from retractile testis. Wyllie11 stated that retractile testis could be brought into a low, stable scrotal position and that traction on the cord structures is not painful. In contrast, gliding testis can only be brought into a high unstable scrotal position, and further traction on cord structures is painful.11 Ferro et al.12 defines retractile testis as a phenomenon in which a testis can manipulated into the scrotum and remain in position without tension. A gliding testis is defined as when it can be manipulated into the upper scrotum, but then it retracts when released.12 However, both are two different and distinct non-scrotal entities, and each definition is so clear, but there are many cases where it is often difficult to apply it in practice.\nIn the beginning, both conditions were thought to be identical, and a gliding testis was therefore also called a “pathological retractile testis”.1314 Many studies about retractile testis could have some bias because they include some cases of gliding testis in their studies. Often it was concluded that the course of retracted testis was similar to that of cryptorchidism. It should be realized that this can create vague fears about retractile testis. However, most urologists believe that a retractile testis is a physiological variant of a fully descended testis, and therefore active treatment and long-term follow-up are usually not indicated. In contrast to a retractile testis, a gliding testis belongs to the spectrum of undescended testis. Therefore, it should be noted that retractile testis do not need surgery but indicates careful physical examinations and periodic follow-ups. However, in this study, 59.7% of urologists felt they had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than pediatric urologists (40.0%).\nThe treatment for retractile testis remains controversial, whereas treatment methods for undescended testis have been well established through many studies.151617 The most typical indication for orchidopexy is when a retractile testis has any decrease in testicular volume and when it ascends because of increased spermatic cord tension. This makes it impossible to reposition the testis into its correct anatomic location. In this study, treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. They recommended follow-up or orchiopexy at a similar rate.\nA retractile testis is attributed to an overactive cremasteric reflex or to alterations within the contractile properties of the cremasteric muscle.18 It is especially common in boys around the school starting age (5–6 years).19 Some authors have suggested that retractile testis may not be as innocuous as has been widely believed.20 In addition, recent evidence suggests that a retractile testis may evolve into an acquired-undescended testis.8\nIt has been reported that retractile testis is accompanied by histological changes. Abnormalities on semen analysis have been found during follow-up when patients with retractile testis had become adults.2122\nIn this study, most of the urologists (98.4%) agreed that cryptorchidism could affect infertility and testicular function in the future. Fifty urologists (80.6%) agreed that these adverse effects were also true with gliding testis, and selected surgical treatment. The distribution of treatment decisions shows no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108). Meanwhile, 13 urologists (21.0%) agreed that retractile testis lead to adverse effects, including 31.2% of non-pediatric urologists and 10% of pediatric urologists. Regarding infant with retractile testis, treatment distribution varied among groups (P = 0.030). These differences show the result of a more conservative treatment trend among pediatric urologists than in non-pediatric urologists. To overcome such discrepancies in clinical practice, it is necessary to establish an appropriate guideline for the proper management of retractile testis.\nLa Scala and Ein7 reported that 150 boys with retractile testis needed periodic follow-up. In their series, only 22.7% of the patients required orchidopexy. They recommend that patients with retractile testis be attentively followed annually. They suggested that most patients with retractile testis can expect a spontaneously favorable resolution without surgical treatment, even after 14 years of age. In this study, the follow-up period in retractile and gliding testes is not clear. Half of the urologists wanted to follow-up before school age or until 2 years of age. Some urologists (12.5–24.1%) wanted to follow-up after puberty. Therefore, we need further evaluation about what is the proper follow-up period for retractile and gliding testes.\nIn this study, there are some limitations. This is a survey study of urologists. As a result of raising questions about the treatment trends of these urologists, we do not know if this occurs in the real clinical setting. However, we tried to elicit answers that are as realistic as possible, with scenarios for patients seen in the actual clinical field. Second, the number of respondents is small. We think it is likely that the urologists who seldom treat pediatric patients did not respond. However, we tried to include all pediatric urologists in the Korean Society of Pediatric Urology, and most members of our society participated in this survey study.\nMoreover, we found some differences in practice patterns between pediatric urologists and non-pediatric urologists. Overall, non-pediatric urologists found it challenging to distinguish retractile testis from gliding testis. Compared to pediatric urologists, they had a higher tendency to agree with the statement that retractile testis could cause infertility and testicular dysfunction in the future and chose to perform orchiopexy as early as the time of the first detection. To overcome these differences, adequate education about retractile and gliding testes is essential. The results of this study are expected to be utilized as basic data for establishing a practical guideline for the diagnosis and treatment of retractile testis in Korea.\nIn conclusion, more than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. Among urologists, non-pediatric urologists had more difficulty than pediatric urologists, and this disparity was more pronounced in children compared to infants. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing consistent management plans. The results of this study are expected to be used as basic data for making a practical guideline for the diagnosis and treatment of retractile testis in Korea.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIt is quite difficult to distinguish retractile testis from gliding testis, which requires different treatment planning in the clinic setting. We evaluated practice patterns of urologists in Korea regarding the diagnosis and management of retractile and gliding testes.\n\nMethods:\nWe mailed or e-mailed self-completion questionnaires consisting of 20 items to 106 urologists practicing in Korean hospitals concerning the diagnosis and treatment of cryptorchidism. We collected and analyzed the responses statistically.\n\nResults:\nResponses were received from 62 urologists. The response rate was 58.5%. Thirty-seven urologists (59.7%) actually felt they had difficulty in distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than for pediatric urologists (40.0%) (P = 0.006). In cases of infant retractile testis, only five urologists (8.1%) said that they would perform orchiopexy immediately, with 54 (87.1%) urologists saying they would do follow-up. In cases of preschool-age children with retractile testis, 17 urologists (27.4%) said that they would perform orchiopexy immediately with 41 (66.1%) urologists saying they would do follow-up. In cases of infant gliding testis, 37 urologists (59.7%) said that they would perform orchiopexy immediately with 24 (38.7%) urologists saying they would do a follow-up.\n\nConclusions:\nMore than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing a practical guideline."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":4576,"string":"4,576"},"whole_article_abstract_length":{"kind":"number","value":334,"string":"334"},"other_sections_lengths":{"kind":"list like","value":[56,42,190,126,152,177],"string":"[\n 56,\n 42,\n 190,\n 126,\n 152,\n 177\n]"},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["testis","urologists","retractile","retractile testis","pediatric","gliding","pediatric urologists","treatment","non","follow"],"string":"[\n \"testis\",\n \"urologists\",\n \"retractile\",\n \"retractile testis\",\n \"pediatric\",\n \"gliding\",\n \"pediatric urologists\",\n \"treatment\",\n \"non\",\n \"follow\"\n]"},"keybert_topics":{"kind":"list like","value":["acquired undescended testis","testicles cryptorchidism undescended","high scrotal testis","scrotal testis retract","retractile testis scrotal"],"string":"[\n \"acquired undescended testis\",\n \"testicles cryptorchidism undescended\",\n \"high scrotal testis\",\n \"scrotal testis retract\",\n \"retractile testis scrotal\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] testis | retractile | undescended | retractile testis | scrotal | stable | undescended testis | cryptorchidism | gliding | testis retractile testis [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hospital | urologist | pediatric | patients | test | ibm | groups | 05 | pediatric patients | questionnaire [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] urologists | testis | pediatric | retractile | retractile testis | fig | gliding | pediatric urologists | non pediatric urologists | non pediatric [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] urologists | testis | retractile | pediatric | retractile testis | gliding | follow | pediatric urologists | non pediatric | said [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Korea [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 20 | 106 | Korean ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 62 ||| 58.5% ||| Thirty-seven | 59.7% ||| 78.1% | 40.0% ||| 0.006 ||| only five | 8.1% | 54 | 87.1% ||| 17 | 27.4% | 41 | 66.1% ||| 37 | 59.7% | 24 | 38.7% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Korea ||| 20 | 106 | Korean ||| ||| ||| 62 ||| 58.5% ||| Thirty-seven | 59.7% ||| 78.1% | 40.0% ||| 0.006 ||| only five | 8.1% | 54 | 87.1% ||| 17 | 27.4% | 41 | 66.1% ||| 37 | 59.7% | 24 | 38.7% ||| More than half | 59.7% | Korean ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3389,"cells":{"title":{"kind":"string","value":"Comparison of perceived quality amongst migrant and local patients using primary health care delivered by community health centres in Shenzhen, China."},"pmid":{"kind":"string","value":"24779564"},"background_abstract":{"kind":"string","value":"Providing good quality primary health care to all inhabitants is one of the Chinese Government's health care objectives. However, information is scarce regarding the difference in quality of primary health care delivered to migrants and local residents respectively. This study aimed to compare patients' perceptions of quality of primary health care between migrants and local patients, and their willingness to use and recommend primary health care to others."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A cross-sectional survey was conducted. 787 patients in total were chosen from four randomly drawn Community Health Centers (CHCs) for interviews."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Local residents scored higher than migrants in terms of their satisfaction with types of drugs available (3.62 vs. 3.45, p=0.035), attitude of health workers (4.41 vs. 4.14, p=0.042) and waiting time (4.30 vs. 3.86, p<0.001). Even though there was no significant difference in overall satisfaction between local residents and migrants (4.16 vs. 3.91, p=0.159), migrants were more likely to utilize primary health care as the first choice for their usual health problems (94.1% vs. 87.1%, p=0.032), while local residents were more inclined to recommend Traditional Chinese Medicine to others (65.6% vs. 56.6%, p=0.026)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Quality of primary health care given to migrants is less satisfactory than to local residents in terms of attitude of health workers and waiting time. Our study suggests quality of care could be improved through extending opening hours of CHCs and strengthening professional ethics education. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals' recommendations for traditional Chinese medicine were possibly because of cultural differences."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Attitude of Health Personnel","China","Community Health Centers","Cross-Sectional Studies","Female","Health Services Accessibility","Humans","Interviews as Topic","Male","Primary Health Care","Quality of Health Care","Transients and Migrants","Waiting Lists"],"string":"[\n \"Attitude of Health Personnel\",\n \"China\",\n \"Community Health Centers\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Health Services Accessibility\",\n \"Humans\",\n \"Interviews as Topic\",\n \"Male\",\n \"Primary Health Care\",\n \"Quality of Health Care\",\n \"Transients and Migrants\",\n \"Waiting Lists\"\n]"},"pmcid":{"kind":"string","value":"4012177"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\nThis cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\n Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\nIn this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\n Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\nWe compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\n Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\nEthical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"In total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).\nSocio-economic and demographic characteristics of respondents\nThe mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).\nThe mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status\n†Adjusted for demographic and socio-economic characteristics of the respondents.\nCompared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).\nThe willingness to use by the respondents, and to recommend to others by registration type\n†Adjusted for demographic and socio-economic characteristics of the respondents."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences."},"other_sections_titles":{"kind":"list like","value":["Background","Study design, settings and data collection process","Key measures","Statistical analysis","Ethical approval","Major findings","Strengths and limitations","Comparisons with existing literature","Implications for research and practice","Competing interests","Authors’ contribution","Pre-publication history"],"string":"[\n \"Background\",\n \"Study design, settings and data collection process\",\n \"Key measures\",\n \"Statistical analysis\",\n \"Ethical approval\",\n \"Major findings\",\n \"Strengths and limitations\",\n \"Comparisons with existing literature\",\n \"Implications for research and practice\",\n \"Competing interests\",\n \"Authors’ contribution\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.","This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.","In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.","We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).","Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).","The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.","A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.","Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].","Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.","The authors declare that they have no competing interests.","XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\n"],"string":"[\n \"China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.\",\n \"This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\",\n \"In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\\nQuestions to measure patient satisfaction\\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\\nQuestions on patients’ willingness to use and recommend primary health care to others\\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\",\n \"We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\",\n \"Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\",\n \"The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\",\n \"A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\",\n \"Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\",\n \"Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\",\n \"The authors declare that they have no competing interests.\",\n \"XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study design, settings and data collection process","Key measures","Statistical analysis","Ethical approval","Results","Discussion","Major findings","Strengths and limitations","Comparisons with existing literature","Implications for research and practice","Conclusions","Competing interests","Authors’ contribution","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design, settings and data collection process\",\n \"Key measures\",\n \"Statistical analysis\",\n \"Ethical approval\",\n \"Results\",\n \"Discussion\",\n \"Major findings\",\n \"Strengths and limitations\",\n \"Comparisons with existing literature\",\n \"Implications for research and practice\",\n \"Conclusions\",\n \"Competing interests\",\n \"Authors’ contribution\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others."," Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\nThis cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\n Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\nIn this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\n Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\nWe compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\n Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\nEthical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).","This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.","In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.","We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).","Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).","In total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).\nSocio-economic and demographic characteristics of respondents\nThe mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).\nThe mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status\n†Adjusted for demographic and socio-economic characteristics of the respondents.\nCompared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).\nThe willingness to use by the respondents, and to recommend to others by registration type\n†Adjusted for demographic and socio-economic characteristics of the respondents."," Major findings The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\nThe findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\n Strengths and limitations A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\nA particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\n Comparisons with existing literature Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\nUnlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\n Implications for research and practice Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\nLocal residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.","The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.","A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.","Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].","Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.","In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences.","The authors declare that they have no competing interests.","XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\n"],"string":"[\n \"China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.\",\n \" Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\\nThis cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\\n Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\\nQuestions to measure patient satisfaction\\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\\nQuestions on patients’ willingness to use and recommend primary health care to others\\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\\nIn this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\\nQuestions to measure patient satisfaction\\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\\nQuestions on patients’ willingness to use and recommend primary health care to others\\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\\n Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\\nWe compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\\n Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\\nEthical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\",\n \"This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\",\n \"In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\\nQuestions to measure patient satisfaction\\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\\nQuestions on patients’ willingness to use and recommend primary health care to others\\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\",\n \"We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\",\n \"Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\",\n \"In total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).\\nSocio-economic and demographic characteristics of respondents\\nThe mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).\\nThe mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status\\n†Adjusted for demographic and socio-economic characteristics of the respondents.\\nCompared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).\\nThe willingness to use by the respondents, and to recommend to others by registration type\\n†Adjusted for demographic and socio-economic characteristics of the respondents.\",\n \" Major findings The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\\nThe findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\\n Strengths and limitations A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\\nA particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\\n Comparisons with existing literature Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\\nUnlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\\n Implications for research and practice Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\\nLocal residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\",\n \"The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\",\n \"A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\",\n \"Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\",\n \"Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\",\n \"In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences.\",\n \"The authors declare that they have no competing interests.\",\n \"XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results","discussion",null,null,null,null,"conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n null,\n null,\n null,\n null,\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Patient satisfaction","Quality of care","Primary health care","Migration"],"string":"[\n \"Patient satisfaction\",\n \"Quality of care\",\n \"Primary health care\",\n \"Migration\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nChina’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.\n\nMethods:\n Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\nThis cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\n Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\nIn this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\n Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\nWe compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\n Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\nEthical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\n\nStudy design, settings and data collection process:\nThis cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\n\nKey measures:\nIn this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\n\nStatistical analysis:\nWe compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\n\nEthical approval:\nEthical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\n\nResults:\nIn total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).\nSocio-economic and demographic characteristics of respondents\nThe mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).\nThe mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status\n†Adjusted for demographic and socio-economic characteristics of the respondents.\nCompared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).\nThe willingness to use by the respondents, and to recommend to others by registration type\n†Adjusted for demographic and socio-economic characteristics of the respondents.\n\nDiscussion:\n Major findings The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\nThe findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\n Strengths and limitations A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\nA particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\n Comparisons with existing literature Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\nUnlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\n Implications for research and practice Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\nLocal residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\n\nMajor findings:\nThe findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\n\nStrengths and limitations:\nA particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\n\nComparisons with existing literature:\nUnlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\n\nImplications for research and practice:\nLocal residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\n\nConclusions:\nIn summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contribution:\nXLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nProviding good quality primary health care to all inhabitants is one of the Chinese Government's health care objectives. However, information is scarce regarding the difference in quality of primary health care delivered to migrants and local residents respectively. This study aimed to compare patients' perceptions of quality of primary health care between migrants and local patients, and their willingness to use and recommend primary health care to others.\n\nMethods:\nA cross-sectional survey was conducted. 787 patients in total were chosen from four randomly drawn Community Health Centers (CHCs) for interviews.\n\nResults:\nLocal residents scored higher than migrants in terms of their satisfaction with types of drugs available (3.62 vs. 3.45, p=0.035), attitude of health workers (4.41 vs. 4.14, p=0.042) and waiting time (4.30 vs. 3.86, p<0.001). Even though there was no significant difference in overall satisfaction between local residents and migrants (4.16 vs. 3.91, p=0.159), migrants were more likely to utilize primary health care as the first choice for their usual health problems (94.1% vs. 87.1%, p=0.032), while local residents were more inclined to recommend Traditional Chinese Medicine to others (65.6% vs. 56.6%, p=0.026).\n\nConclusions:\nQuality of primary health care given to migrants is less satisfactory than to local residents in terms of attitude of health workers and waiting time. Our study suggests quality of care could be improved through extending opening hours of CHCs and strengthening professional ethics education. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals' recommendations for traditional Chinese medicine were possibly because of cultural differences."},"background_conclusion_text":{"kind":"string","value":"Background:\nChina’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.\n\nConclusions:\nIn summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nProviding good quality primary health care to all inhabitants is one of the Chinese Government's health care objectives. However, information is scarce regarding the difference in quality of primary health care delivered to migrants and local residents respectively. This study aimed to compare patients' perceptions of quality of primary health care between migrants and local patients, and their willingness to use and recommend primary health care to others.\n\nMethods:\nA cross-sectional survey was conducted. 787 patients in total were chosen from four randomly drawn Community Health Centers (CHCs) for interviews.\n\nResults:\nLocal residents scored higher than migrants in terms of their satisfaction with types of drugs available (3.62 vs. 3.45, p=0.035), attitude of health workers (4.41 vs. 4.14, p=0.042) and waiting time (4.30 vs. 3.86, p<0.001). Even though there was no significant difference in overall satisfaction between local residents and migrants (4.16 vs. 3.91, p=0.159), migrants were more likely to utilize primary health care as the first choice for their usual health problems (94.1% vs. 87.1%, p=0.032), while local residents were more inclined to recommend Traditional Chinese Medicine to others (65.6% vs. 56.6%, p=0.026).\n\nConclusions:\nQuality of primary health care given to migrants is less satisfactory than to local residents in terms of attitude of health workers and waiting time. Our study suggests quality of care could be improved through extending opening hours of CHCs and strengthening professional ethics education. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals' recommendations for traditional Chinese medicine were possibly because of cultural differences."},"whole_article_text_length":{"kind":"number","value":9543,"string":"9,543"},"whole_article_abstract_length":{"kind":"number","value":316,"string":"316"},"other_sections_lengths":{"kind":"list like","value":[880,279,519,202,33,89,255,903,290,10,81,16],"string":"[\n 880,\n 279,\n 519,\n 202,\n 33,\n 89,\n 255,\n 903,\n 290,\n 10,\n 81,\n 16\n]"},"num_sections":{"kind":"number","value":16,"string":"16"},"most_frequent_words":{"kind":"list like","value":["health","care","migrants","health care","chcs","satisfaction","study","primary","primary health care","primary health"],"string":"[\n \"health\",\n \"care\",\n \"migrants\",\n \"health care\",\n \"chcs\",\n \"satisfaction\",\n \"study\",\n \"primary\",\n \"primary health care\",\n \"primary health\"\n]"},"keybert_topics":{"kind":"list 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[SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] the Chinese Government's ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 787 | four | Community Health Centers [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 3.62 | 3.45 | 4.41 | 4.14 | 4.30 | 3.86 ||| 4.16 | 3.91 | first | 94.1% | 87.1% | Traditional Chinese Medicine | 65.6% | 56.6% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| opening hours ||| first | Chinese [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] the Chinese Government's ||| ||| ||| ||| 787 | four | Community Health Centers ||| 3.62 | 3.45 | 4.41 | 4.14 | 4.30 | 3.86 ||| 4.16 | 3.91 | first | 94.1% | 87.1% | Traditional Chinese Medicine | 65.6% | 56.6% ||| ||| opening hours ||| first | Chinese [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] the Chinese Government's ||| ||| ||| ||| 787 | four | Community Health Centers ||| 3.62 | 3.45 | 4.41 | 4.14 | 4.30 | 3.86 ||| 4.16 | 3.91 | first | 94.1% | 87.1% | Traditional Chinese Medicine | 65.6% | 56.6% ||| ||| opening hours ||| first | Chinese [SUMMARY]"}}},{"rowIdx":3390,"cells":{"title":{"kind":"string","value":"Simultaneous resection of coexisting pulmonary and mediastinal lesions by video-assisted thoracic surgery: a case-series study."},"pmid":{"kind":"string","value":"35725438"},"background_abstract":{"kind":"string","value":"With the growing number of patients with coexisting pulmonary and mediastinal lesions detected, reports about simultaneous video-assisted thoracic surgery (VATS) for these concurrent diseases are still rare. To further explore the safety and effectiveness of simultaneous resection of pulmonary and mediastinal lesions by uniportal or biportal VATS, we retrospectively analyzed the clinical data of the largest series of cases to date."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"From July 2018 to July 2021, all patients whose pulmonary lesions and mediastinal tumors were resected simultaneously in our institution were retrospectively reviewed. Their demographic and clinical data were collected and analyzed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 54 patients were enrolled, of whom 44 underwent unilateral uniportal VATS, 3 underwent bilateral uniportal VATS and 7 underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. For the remaining 47 patients with various demographic and clinical characteristics, most of the operations were completed within 3 h (n = 33, 70.2%) with blood loss of no more than 100 mL (n = 43, 91.5%). The duration of chest tube drainage was 5.66 ± 3.34 days, and the average daily volume was 196.90 ± 122.31 mL. Four cases of postoperative complications occurred during hospitalization. The length of postoperative hospital stay was 8.60 ± 3.63 days. No severe complications or deaths were observed during follow-up."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Uniportal and biportal VATS are safe and effective for simultaneous resection of selected coexisting pulmonary and mediastinal lesions, but the indications and operational details need more evaluation."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Lung Neoplasms","Pneumonectomy","Retrospective Studies","Thoracic Surgery, Video-Assisted","Thoracotomy"],"string":"[\n \"Humans\",\n \"Lung Neoplasms\",\n \"Pneumonectomy\",\n \"Retrospective Studies\",\n \"Thoracic Surgery, Video-Assisted\",\n \"Thoracotomy\"\n]"},"pmcid":{"kind":"string","value":"9208703"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.\n\nTable 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nGeneral information of the patients\nASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nFor the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.\n\nTable 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.\nLung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1\nPulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nLocations and pathological diagnosis of isolated pulmonary lesions\nAIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\n\nTable 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone\nLocations and pathological diagnosis of isolated mediastinal lesions\nACTH adrenocorticotropic hormone\nNineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.\n\nTable 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nPathological diagnosis of lesions suspected to invade surrounding tissues\nLUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nSurgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.\n\nTable 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3\nFor the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18\nOperative details and characteristics\nAfter thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.\n\nTable 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1\nGeneral postoperative conditions of the 47 cases of non-converted VATS"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy."},"other_sections_titles":{"kind":"list like","value":["Background","Methods","Study design and patient collection","Preoperative assessment","Surgical technique","Data collection and analysis"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design and patient collection\",\n \"Preoperative assessment\",\n \"Surgical technique\",\n \"Data collection and analysis\"\n]"},"other_sections_texts":{"kind":"list like","value":["Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.","Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.","Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.","All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy","General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.","Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out."],"string":"[\n \"Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.\",\n \"Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\\n\\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\\n\\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\\n\\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\\n\\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\",\n \"Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\",\n \"All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\\n\\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\",\n \"General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\\n\\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\",\n \"Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study design and patient collection","Preoperative assessment","Surgical technique","Data collection and analysis","Results","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study design and patient collection\",\n \"Preoperative assessment\",\n \"Surgical technique\",\n \"Data collection and analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.","Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.","Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.","All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy","General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.","Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.","The general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.\n\nTable 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nGeneral information of the patients\nASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nFor the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.\n\nTable 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.\nLung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1\nPulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nLocations and pathological diagnosis of isolated pulmonary lesions\nAIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\n\nTable 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone\nLocations and pathological diagnosis of isolated mediastinal lesions\nACTH adrenocorticotropic hormone\nNineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.\n\nTable 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nPathological diagnosis of lesions suspected to invade surrounding tissues\nLUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nSurgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.\n\nTable 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3\nFor the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18\nOperative details and characteristics\nAfter thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.\n\nTable 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1\nGeneral postoperative conditions of the 47 cases of non-converted VATS","Yoon et al. indicated that the expected prevalence of anterior mediastinal nodular lesions is approximately 1% in populations aged 55–74 at high risk for lung cancer during low-dose chest CT screening [11]. Similarly, combined surgery for coexisting pulmonary and mediastinal lesions accounted for approximately 0.93% of the pulmonary operations between July 2018 and July 2021 in our hospital and 6.64% of the mediastinal operations. Although the mechanism has not been clarified, thymoma is associated with an increased risk of a secondary neoplasm, such as primary lung cancer [12, 13]. With the help of rapidly developed medical imaging technology and regular examinations before thoracic surgery, coexisting pulmonary and mediastinal lesions are being increasingly detected clinically, but their management remains a challenge due to the lack of surgical guidelines or reports of mass cases.\nThe adjacent anatomical locations of the lung and mediastinum allow the possibility of combined resection, and some surgeons have already tried this approach and shared their valuable experience [1–7]. As a minimally invasive surgery, VATS has been applied widely in the diagnosis and treatment of thoracic diseases. Since first introduced by Migliore in 2001, uniportal VATS has been increasingly adopted by virtue of its advantages, such as a smaller incision, less intraoperative bleeding, rapider recovery, and a shorter duration of surgery, drainage, and hospitalization than multiportal VATS [14–17]. Taken together, VATS, especially uniportal VATS, seems to be an optimal choice for coexisting pulmonary and mediastinal lesions, but the potential risks from complex techniques and prolonged surgery make it necessary for more assessment of its feasibility and safety.\nFor the 54 cases enrolled in this study, simultaneous VATS was successfully performed in 47. Most of the non-converted VATS were completed within 3 h (n = 33, 70.2%) and with bleeding no more than 100 mL (n = 43, 91.5%), which is comparable to the data shown in other studies [4–6]. Specifically, 12 cases (25.5%) of combined surgery were completed within 2 h, 29 cases (61.7%) were between 2 and 4 h, and 6 cases (12.8%) took more than 4 h. The pleural adhesions, large sizes of lesions, and dissection of lymph nodes and invaded tissues were the main causes of prolonged surgical duration. Besides severe pleural adhesions, another factor that converted VATS into thoracotomy was the large size (54.67 ± 14.99 mm vs. 32.87 ± 17.95 mm) or extensive invasion of the mediastinal lesions, especially when the number of local metastases was more than two. In addition to the surgeons’ habits, biportal VATS was usually chosen instead of uniportal VATS when both coexisting lesions were relatively large but had no severe local invasion.\nTo ensure that all lesions are resected, median sternotomy is generally chosen for invasive thymoma [18]. We gathered the data of combined thoracotomy as well, from which it could be found that the average size of the mediastinal lesions was 79.91 ± 26.99 mm, and almost all of them (84.8%) had invaded the surrounding lung tissues (Fig. 1G, H). As shown in Fig. 3, a diameter of 50 mm for mediastinal lesions can be chosen as a turning point by comparing the data between successful VATS and open surgery (including the operations converted to thoracotomy). Therefore, for mediastinal lesions no larger than 50 mm coexisting with pulmonary lesions, if there is no severe local invasion on imaging, it will be feasible to perform uniportal VATS exploration first and decide whether to continue thoracoscopic resection or convert to thoracotomy.\n\nFig. 3Sizes of mediastinal lesions between VATS and thoracotomy\nSizes of mediastinal lesions between VATS and thoracotomy\nFor the resection of pulmonary lesions, the incision should be located in the midaxillary line, slightly anterior for segmental resection near the anterior hilum and slightly posterior for segmental resection near the posterior hilum. For combined VATS, because the location of the single port needed to take both the pulmonary and mediastinal lesions into account, the operation was more difficult. It is necessary to choose the location of the surgical incision with flexibly, and typically a single incision is made in the midaxillary line of the 5th ICS near the posterior axillary line. For the right thoracic incision, if the incision is slightly anterior, the internal thoracic vein will block the visual field and affect the exposure of the left innominate vein. In this case, the internal thoracic vein can be cut (Fig. 2C). For left thoracic incision, surgical instruments are susceptible to interference from the heart if the incision is too far posterior, and resection of segments or lobes at certain sites may not be easy. None of our patients who underwent uniportal VATS had a thoracotomy or auxiliary port added because of poor placement of the incision.\nIt seems that our drainage duration (5.66 ± 3.34 days) was longer than that in other studies, but the difference in standards for the removal of chest tubes should not be ignored. In our department, the last 24-h drainage volume should be less than 200 mL. Most cases of massive drainage occurred after systematic lymph node dissection, and the other case of pleural effusion was mostly due to the large inner wound surface left by a 78 mm mediastinal lesion. All of them were resolved by conservative treatment. Besides, the standards for discharge also delayed some patients’ leaving. The chest imaging (X-ray photograph or CT) and routine blood test (especially the WBC count and neutrophil proportion) were required to become normal before discharge, otherwise the patients should stay to receive further observation and treatment.\nTwo cases of severe postoperative complications occurred during hospitalization. One case of ectopic ACTH syndrome broke out acutely 3 days after uniportal VATS. Fortunately, the condition gradually became controlled, and the patient left the hospital 15 days after surgery. The other case showed difficultly weaning from mechanical ventilation. Although MG had not been diagnosed before surgery, the cause was discovered to be postoperative myasthenic crisis (PMC). The patient recovered and left the hospital 23 days after surgery. In sum, these severe complications were mainly caused by the primary diseases of the patients rather than the operations.\nTelephone follow-up was performed for all 47 non-converted VATS cases, and 2 were lost to follow-up. One case of new-onset bronchospasm and associated paroxysmal hypoxemia was reported. According to the Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score, this case of long-duration (290 min) intrathoracic surgery can be leveled as high risk for PPCs [8, 9]. For combined surgery, relative extension of operation time and trauma is inevitable, but the risk remains controllable. Other complaints reported during follow-up included pain, cough, and small amounts of pleural effusion, which were gradually improved over time.\nConsistent with normal thoracoscopic operations, combined uniportal or biportal VATS does not have many absolute contraindications. Besides enough physical toleration and relatively early clinical stages required by most thoracic tumor operations, coexisting lesions that are difficult to be resected through the same surgical incision might be a relative contraindication, although the clinical judgment lacks quantitative criteria and needs abundant operational experience. The history of thoracic surgery should also be taken into account in view of the fact that severe pleural adhesion is one of the main factors that turned VATS into thoracotomy.\nIt is worth discussing whether pulmonary lesions or mediastinal tumors should be removed first. Normally, surgeons tend to first resect the pulmonary lesions [4–6], which is the same in our study (38:9). Technically, the resection of pulmonary lesions can provide more space for the exposure of mediastinal lesions. When the mediastinal lesions are large but have not invaded surrounding tissues, mediastinum-first resection is preferred. However, if mediastinal tumor invades or adheres closely to the surrounding tissues, the invaded tissues should be removed first. In general, lung-first VATS helps to maintain a clear field of vision for both pulmonary and mediastinal surgery. If mediastinal lesions are treated first before pulmonary lesions, the exudation of mediastinal surgical wounds will flow to the lung, thus affecting the clear field of pulmonary surgery.\nAlthough the patients enrolled in this study were various, its reliability is limited by the retrospective design and relatively small sample size. Items such as the incision length were not analyzed due to the missing of records, and it failed to establish a case-control study since there lacked enough comparable cases of multiportal VATS or staged operations for coexisting pulmonary and mediastinal lesions in our hospital. We tried to assess the indications by comparing the characteristics of cases between VATS and thoracotomy, but this was inevitably influenced by the subjective bias and habits of surgeons.\nIn brief, this is thus far the largest case series analysis about combined resection of pulmonary and mediastinal lesions through uniportal VATS. We analyzed the clinical pathological characteristics of these patients and the efficacy and safety of the operations. The surgical indications and selection of the appropriate incision site were also explored. The reliability of this study is limited due to the small number of cases and non-comparative design. Further researches with larger sample sizes are needed.","We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy."],"string":"[\n \"Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.\",\n \"Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\\n\\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\\n\\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\\n\\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\\n\\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\",\n \"Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\",\n \"All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\\n\\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\",\n \"General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\\n\\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\",\n \"Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\",\n \"The general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.\\n\\nTable 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\\nGeneral information of the patients\\nASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\\nFor the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.\\n\\nTable 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.\\nLung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1\\nPulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\\nLocations and pathological diagnosis of isolated pulmonary lesions\\nAIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\\n\\nTable 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone\\nLocations and pathological diagnosis of isolated mediastinal lesions\\nACTH adrenocorticotropic hormone\\nNineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.\\n\\nTable 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\\nPathological diagnosis of lesions suspected to invade surrounding tissues\\nLUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\\nSurgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.\\n\\nTable 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3\\nFor the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18\\nOperative details and characteristics\\nAfter thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.\\n\\nTable 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1\\nGeneral postoperative conditions of the 47 cases of non-converted VATS\",\n \"Yoon et al. indicated that the expected prevalence of anterior mediastinal nodular lesions is approximately 1% in populations aged 55–74 at high risk for lung cancer during low-dose chest CT screening [11]. Similarly, combined surgery for coexisting pulmonary and mediastinal lesions accounted for approximately 0.93% of the pulmonary operations between July 2018 and July 2021 in our hospital and 6.64% of the mediastinal operations. Although the mechanism has not been clarified, thymoma is associated with an increased risk of a secondary neoplasm, such as primary lung cancer [12, 13]. With the help of rapidly developed medical imaging technology and regular examinations before thoracic surgery, coexisting pulmonary and mediastinal lesions are being increasingly detected clinically, but their management remains a challenge due to the lack of surgical guidelines or reports of mass cases.\\nThe adjacent anatomical locations of the lung and mediastinum allow the possibility of combined resection, and some surgeons have already tried this approach and shared their valuable experience [1–7]. As a minimally invasive surgery, VATS has been applied widely in the diagnosis and treatment of thoracic diseases. Since first introduced by Migliore in 2001, uniportal VATS has been increasingly adopted by virtue of its advantages, such as a smaller incision, less intraoperative bleeding, rapider recovery, and a shorter duration of surgery, drainage, and hospitalization than multiportal VATS [14–17]. Taken together, VATS, especially uniportal VATS, seems to be an optimal choice for coexisting pulmonary and mediastinal lesions, but the potential risks from complex techniques and prolonged surgery make it necessary for more assessment of its feasibility and safety.\\nFor the 54 cases enrolled in this study, simultaneous VATS was successfully performed in 47. Most of the non-converted VATS were completed within 3 h (n = 33, 70.2%) and with bleeding no more than 100 mL (n = 43, 91.5%), which is comparable to the data shown in other studies [4–6]. Specifically, 12 cases (25.5%) of combined surgery were completed within 2 h, 29 cases (61.7%) were between 2 and 4 h, and 6 cases (12.8%) took more than 4 h. The pleural adhesions, large sizes of lesions, and dissection of lymph nodes and invaded tissues were the main causes of prolonged surgical duration. Besides severe pleural adhesions, another factor that converted VATS into thoracotomy was the large size (54.67 ± 14.99 mm vs. 32.87 ± 17.95 mm) or extensive invasion of the mediastinal lesions, especially when the number of local metastases was more than two. In addition to the surgeons’ habits, biportal VATS was usually chosen instead of uniportal VATS when both coexisting lesions were relatively large but had no severe local invasion.\\nTo ensure that all lesions are resected, median sternotomy is generally chosen for invasive thymoma [18]. We gathered the data of combined thoracotomy as well, from which it could be found that the average size of the mediastinal lesions was 79.91 ± 26.99 mm, and almost all of them (84.8%) had invaded the surrounding lung tissues (Fig. 1G, H). As shown in Fig. 3, a diameter of 50 mm for mediastinal lesions can be chosen as a turning point by comparing the data between successful VATS and open surgery (including the operations converted to thoracotomy). Therefore, for mediastinal lesions no larger than 50 mm coexisting with pulmonary lesions, if there is no severe local invasion on imaging, it will be feasible to perform uniportal VATS exploration first and decide whether to continue thoracoscopic resection or convert to thoracotomy.\\n\\nFig. 3Sizes of mediastinal lesions between VATS and thoracotomy\\nSizes of mediastinal lesions between VATS and thoracotomy\\nFor the resection of pulmonary lesions, the incision should be located in the midaxillary line, slightly anterior for segmental resection near the anterior hilum and slightly posterior for segmental resection near the posterior hilum. For combined VATS, because the location of the single port needed to take both the pulmonary and mediastinal lesions into account, the operation was more difficult. It is necessary to choose the location of the surgical incision with flexibly, and typically a single incision is made in the midaxillary line of the 5th ICS near the posterior axillary line. For the right thoracic incision, if the incision is slightly anterior, the internal thoracic vein will block the visual field and affect the exposure of the left innominate vein. In this case, the internal thoracic vein can be cut (Fig. 2C). For left thoracic incision, surgical instruments are susceptible to interference from the heart if the incision is too far posterior, and resection of segments or lobes at certain sites may not be easy. None of our patients who underwent uniportal VATS had a thoracotomy or auxiliary port added because of poor placement of the incision.\\nIt seems that our drainage duration (5.66 ± 3.34 days) was longer than that in other studies, but the difference in standards for the removal of chest tubes should not be ignored. In our department, the last 24-h drainage volume should be less than 200 mL. Most cases of massive drainage occurred after systematic lymph node dissection, and the other case of pleural effusion was mostly due to the large inner wound surface left by a 78 mm mediastinal lesion. All of them were resolved by conservative treatment. Besides, the standards for discharge also delayed some patients’ leaving. The chest imaging (X-ray photograph or CT) and routine blood test (especially the WBC count and neutrophil proportion) were required to become normal before discharge, otherwise the patients should stay to receive further observation and treatment.\\nTwo cases of severe postoperative complications occurred during hospitalization. One case of ectopic ACTH syndrome broke out acutely 3 days after uniportal VATS. Fortunately, the condition gradually became controlled, and the patient left the hospital 15 days after surgery. The other case showed difficultly weaning from mechanical ventilation. Although MG had not been diagnosed before surgery, the cause was discovered to be postoperative myasthenic crisis (PMC). The patient recovered and left the hospital 23 days after surgery. In sum, these severe complications were mainly caused by the primary diseases of the patients rather than the operations.\\nTelephone follow-up was performed for all 47 non-converted VATS cases, and 2 were lost to follow-up. One case of new-onset bronchospasm and associated paroxysmal hypoxemia was reported. According to the Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score, this case of long-duration (290 min) intrathoracic surgery can be leveled as high risk for PPCs [8, 9]. For combined surgery, relative extension of operation time and trauma is inevitable, but the risk remains controllable. Other complaints reported during follow-up included pain, cough, and small amounts of pleural effusion, which were gradually improved over time.\\nConsistent with normal thoracoscopic operations, combined uniportal or biportal VATS does not have many absolute contraindications. Besides enough physical toleration and relatively early clinical stages required by most thoracic tumor operations, coexisting lesions that are difficult to be resected through the same surgical incision might be a relative contraindication, although the clinical judgment lacks quantitative criteria and needs abundant operational experience. The history of thoracic surgery should also be taken into account in view of the fact that severe pleural adhesion is one of the main factors that turned VATS into thoracotomy.\\nIt is worth discussing whether pulmonary lesions or mediastinal tumors should be removed first. Normally, surgeons tend to first resect the pulmonary lesions [4–6], which is the same in our study (38:9). Technically, the resection of pulmonary lesions can provide more space for the exposure of mediastinal lesions. When the mediastinal lesions are large but have not invaded surrounding tissues, mediastinum-first resection is preferred. However, if mediastinal tumor invades or adheres closely to the surrounding tissues, the invaded tissues should be removed first. In general, lung-first VATS helps to maintain a clear field of vision for both pulmonary and mediastinal surgery. If mediastinal lesions are treated first before pulmonary lesions, the exudation of mediastinal surgical wounds will flow to the lung, thus affecting the clear field of pulmonary surgery.\\nAlthough the patients enrolled in this study were various, its reliability is limited by the retrospective design and relatively small sample size. Items such as the incision length were not analyzed due to the missing of records, and it failed to establish a case-control study since there lacked enough comparable cases of multiportal VATS or staged operations for coexisting pulmonary and mediastinal lesions in our hospital. We tried to assess the indications by comparing the characteristics of cases between VATS and thoracotomy, but this was inevitably influenced by the subjective bias and habits of surgeons.\\nIn brief, this is thus far the largest case series analysis about combined resection of pulmonary and mediastinal lesions through uniportal VATS. We analyzed the clinical pathological characteristics of these patients and the efficacy and safety of the operations. The surgical indications and selection of the appropriate incision site were also explored. The reliability of this study is limited due to the small number of cases and non-comparative design. Further researches with larger sample sizes are needed.\",\n \"We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,"results","discussion","conclusion"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusion\"\n]"},"keywords":{"kind":"list like","value":["Lung neoplasms","Mediastinal neoplasms","Simultaneous operation","Thoracic surgery","Video-assisted thoracoscopic surgery"],"string":"[\n \"Lung neoplasms\",\n \"Mediastinal neoplasms\",\n \"Simultaneous operation\",\n \"Thoracic surgery\",\n \"Video-assisted thoracoscopic surgery\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nLung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.\n\nMethods:\nStudy design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\n\nStudy design and patient collection:\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\n\nPreoperative assessment:\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\n\nSurgical technique:\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\n\nData collection and analysis:\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\n\nResults:\nThe general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.\n\nTable 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nGeneral information of the patients\nASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nFor the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.\n\nTable 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.\nLung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1\nPulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nLocations and pathological diagnosis of isolated pulmonary lesions\nAIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\n\nTable 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone\nLocations and pathological diagnosis of isolated mediastinal lesions\nACTH adrenocorticotropic hormone\nNineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.\n\nTable 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nPathological diagnosis of lesions suspected to invade surrounding tissues\nLUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nSurgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.\n\nTable 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3\nFor the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18\nOperative details and characteristics\nAfter thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.\n\nTable 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1\nGeneral postoperative conditions of the 47 cases of non-converted VATS\n\nDiscussion:\nYoon et al. indicated that the expected prevalence of anterior mediastinal nodular lesions is approximately 1% in populations aged 55–74 at high risk for lung cancer during low-dose chest CT screening [11]. Similarly, combined surgery for coexisting pulmonary and mediastinal lesions accounted for approximately 0.93% of the pulmonary operations between July 2018 and July 2021 in our hospital and 6.64% of the mediastinal operations. Although the mechanism has not been clarified, thymoma is associated with an increased risk of a secondary neoplasm, such as primary lung cancer [12, 13]. With the help of rapidly developed medical imaging technology and regular examinations before thoracic surgery, coexisting pulmonary and mediastinal lesions are being increasingly detected clinically, but their management remains a challenge due to the lack of surgical guidelines or reports of mass cases.\nThe adjacent anatomical locations of the lung and mediastinum allow the possibility of combined resection, and some surgeons have already tried this approach and shared their valuable experience [1–7]. As a minimally invasive surgery, VATS has been applied widely in the diagnosis and treatment of thoracic diseases. Since first introduced by Migliore in 2001, uniportal VATS has been increasingly adopted by virtue of its advantages, such as a smaller incision, less intraoperative bleeding, rapider recovery, and a shorter duration of surgery, drainage, and hospitalization than multiportal VATS [14–17]. Taken together, VATS, especially uniportal VATS, seems to be an optimal choice for coexisting pulmonary and mediastinal lesions, but the potential risks from complex techniques and prolonged surgery make it necessary for more assessment of its feasibility and safety.\nFor the 54 cases enrolled in this study, simultaneous VATS was successfully performed in 47. Most of the non-converted VATS were completed within 3 h (n = 33, 70.2%) and with bleeding no more than 100 mL (n = 43, 91.5%), which is comparable to the data shown in other studies [4–6]. Specifically, 12 cases (25.5%) of combined surgery were completed within 2 h, 29 cases (61.7%) were between 2 and 4 h, and 6 cases (12.8%) took more than 4 h. The pleural adhesions, large sizes of lesions, and dissection of lymph nodes and invaded tissues were the main causes of prolonged surgical duration. Besides severe pleural adhesions, another factor that converted VATS into thoracotomy was the large size (54.67 ± 14.99 mm vs. 32.87 ± 17.95 mm) or extensive invasion of the mediastinal lesions, especially when the number of local metastases was more than two. In addition to the surgeons’ habits, biportal VATS was usually chosen instead of uniportal VATS when both coexisting lesions were relatively large but had no severe local invasion.\nTo ensure that all lesions are resected, median sternotomy is generally chosen for invasive thymoma [18]. We gathered the data of combined thoracotomy as well, from which it could be found that the average size of the mediastinal lesions was 79.91 ± 26.99 mm, and almost all of them (84.8%) had invaded the surrounding lung tissues (Fig. 1G, H). As shown in Fig. 3, a diameter of 50 mm for mediastinal lesions can be chosen as a turning point by comparing the data between successful VATS and open surgery (including the operations converted to thoracotomy). Therefore, for mediastinal lesions no larger than 50 mm coexisting with pulmonary lesions, if there is no severe local invasion on imaging, it will be feasible to perform uniportal VATS exploration first and decide whether to continue thoracoscopic resection or convert to thoracotomy.\n\nFig. 3Sizes of mediastinal lesions between VATS and thoracotomy\nSizes of mediastinal lesions between VATS and thoracotomy\nFor the resection of pulmonary lesions, the incision should be located in the midaxillary line, slightly anterior for segmental resection near the anterior hilum and slightly posterior for segmental resection near the posterior hilum. For combined VATS, because the location of the single port needed to take both the pulmonary and mediastinal lesions into account, the operation was more difficult. It is necessary to choose the location of the surgical incision with flexibly, and typically a single incision is made in the midaxillary line of the 5th ICS near the posterior axillary line. For the right thoracic incision, if the incision is slightly anterior, the internal thoracic vein will block the visual field and affect the exposure of the left innominate vein. In this case, the internal thoracic vein can be cut (Fig. 2C). For left thoracic incision, surgical instruments are susceptible to interference from the heart if the incision is too far posterior, and resection of segments or lobes at certain sites may not be easy. None of our patients who underwent uniportal VATS had a thoracotomy or auxiliary port added because of poor placement of the incision.\nIt seems that our drainage duration (5.66 ± 3.34 days) was longer than that in other studies, but the difference in standards for the removal of chest tubes should not be ignored. In our department, the last 24-h drainage volume should be less than 200 mL. Most cases of massive drainage occurred after systematic lymph node dissection, and the other case of pleural effusion was mostly due to the large inner wound surface left by a 78 mm mediastinal lesion. All of them were resolved by conservative treatment. Besides, the standards for discharge also delayed some patients’ leaving. The chest imaging (X-ray photograph or CT) and routine blood test (especially the WBC count and neutrophil proportion) were required to become normal before discharge, otherwise the patients should stay to receive further observation and treatment.\nTwo cases of severe postoperative complications occurred during hospitalization. One case of ectopic ACTH syndrome broke out acutely 3 days after uniportal VATS. Fortunately, the condition gradually became controlled, and the patient left the hospital 15 days after surgery. The other case showed difficultly weaning from mechanical ventilation. Although MG had not been diagnosed before surgery, the cause was discovered to be postoperative myasthenic crisis (PMC). The patient recovered and left the hospital 23 days after surgery. In sum, these severe complications were mainly caused by the primary diseases of the patients rather than the operations.\nTelephone follow-up was performed for all 47 non-converted VATS cases, and 2 were lost to follow-up. One case of new-onset bronchospasm and associated paroxysmal hypoxemia was reported. According to the Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score, this case of long-duration (290 min) intrathoracic surgery can be leveled as high risk for PPCs [8, 9]. For combined surgery, relative extension of operation time and trauma is inevitable, but the risk remains controllable. Other complaints reported during follow-up included pain, cough, and small amounts of pleural effusion, which were gradually improved over time.\nConsistent with normal thoracoscopic operations, combined uniportal or biportal VATS does not have many absolute contraindications. Besides enough physical toleration and relatively early clinical stages required by most thoracic tumor operations, coexisting lesions that are difficult to be resected through the same surgical incision might be a relative contraindication, although the clinical judgment lacks quantitative criteria and needs abundant operational experience. The history of thoracic surgery should also be taken into account in view of the fact that severe pleural adhesion is one of the main factors that turned VATS into thoracotomy.\nIt is worth discussing whether pulmonary lesions or mediastinal tumors should be removed first. Normally, surgeons tend to first resect the pulmonary lesions [4–6], which is the same in our study (38:9). Technically, the resection of pulmonary lesions can provide more space for the exposure of mediastinal lesions. When the mediastinal lesions are large but have not invaded surrounding tissues, mediastinum-first resection is preferred. However, if mediastinal tumor invades or adheres closely to the surrounding tissues, the invaded tissues should be removed first. In general, lung-first VATS helps to maintain a clear field of vision for both pulmonary and mediastinal surgery. If mediastinal lesions are treated first before pulmonary lesions, the exudation of mediastinal surgical wounds will flow to the lung, thus affecting the clear field of pulmonary surgery.\nAlthough the patients enrolled in this study were various, its reliability is limited by the retrospective design and relatively small sample size. Items such as the incision length were not analyzed due to the missing of records, and it failed to establish a case-control study since there lacked enough comparable cases of multiportal VATS or staged operations for coexisting pulmonary and mediastinal lesions in our hospital. We tried to assess the indications by comparing the characteristics of cases between VATS and thoracotomy, but this was inevitably influenced by the subjective bias and habits of surgeons.\nIn brief, this is thus far the largest case series analysis about combined resection of pulmonary and mediastinal lesions through uniportal VATS. We analyzed the clinical pathological characteristics of these patients and the efficacy and safety of the operations. The surgical indications and selection of the appropriate incision site were also explored. The reliability of this study is limited due to the small number of cases and non-comparative design. Further researches with larger sample sizes are needed.\n\nConclusions:\nWe found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWith the growing number of patients with coexisting pulmonary and mediastinal lesions detected, reports about simultaneous video-assisted thoracic surgery (VATS) for these concurrent diseases are still rare. To further explore the safety and effectiveness of simultaneous resection of pulmonary and mediastinal lesions by uniportal or biportal VATS, we retrospectively analyzed the clinical data of the largest series of cases to date.\n\nMethods:\nFrom July 2018 to July 2021, all patients whose pulmonary lesions and mediastinal tumors were resected simultaneously in our institution were retrospectively reviewed. Their demographic and clinical data were collected and analyzed.\n\nResults:\nA total of 54 patients were enrolled, of whom 44 underwent unilateral uniportal VATS, 3 underwent bilateral uniportal VATS and 7 underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. For the remaining 47 patients with various demographic and clinical characteristics, most of the operations were completed within 3 h (n = 33, 70.2%) with blood loss of no more than 100 mL (n = 43, 91.5%). The duration of chest tube drainage was 5.66 ± 3.34 days, and the average daily volume was 196.90 ± 122.31 mL. Four cases of postoperative complications occurred during hospitalization. The length of postoperative hospital stay was 8.60 ± 3.63 days. No severe complications or deaths were observed during follow-up.\n\nConclusions:\nUniportal and biportal VATS are safe and effective for simultaneous resection of selected coexisting pulmonary and mediastinal lesions, but the indications and operational details need more evaluation."},"background_conclusion_text":{"kind":"string","value":"Background:\nLung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.\n\nConclusions:\nWe found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWith the growing number of patients with coexisting pulmonary and mediastinal lesions detected, reports about simultaneous video-assisted thoracic surgery (VATS) for these concurrent diseases are still rare. To further explore the safety and effectiveness of simultaneous resection of pulmonary and mediastinal lesions by uniportal or biportal VATS, we retrospectively analyzed the clinical data of the largest series of cases to date.\n\nMethods:\nFrom July 2018 to July 2021, all patients whose pulmonary lesions and mediastinal tumors were resected simultaneously in our institution were retrospectively reviewed. Their demographic and clinical data were collected and analyzed.\n\nResults:\nA total of 54 patients were enrolled, of whom 44 underwent unilateral uniportal VATS, 3 underwent bilateral uniportal VATS and 7 underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. For the remaining 47 patients with various demographic and clinical characteristics, most of the operations were completed within 3 h (n = 33, 70.2%) with blood loss of no more than 100 mL (n = 43, 91.5%). The duration of chest tube drainage was 5.66 ± 3.34 days, and the average daily volume was 196.90 ± 122.31 mL. Four cases of postoperative complications occurred during hospitalization. The length of postoperative hospital stay was 8.60 ± 3.63 days. No severe complications or deaths were observed during follow-up.\n\nConclusions:\nUniportal and biportal VATS are safe and effective for simultaneous resection of selected coexisting pulmonary and mediastinal lesions, but the indications and operational details need more evaluation."},"whole_article_text_length":{"kind":"number","value":8547,"string":"8,547"},"whole_article_abstract_length":{"kind":"number","value":298,"string":"298"},"other_sections_lengths":{"kind":"list like","value":[243,2930,162,607,586,100],"string":"[\n 243,\n 2930,\n 162,\n 607,\n 586,\n 100\n]"},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["mediastinal","lesions","vats","patients","mm","pulmonary","lobe","uniportal","left","uniportal vats"],"string":"[\n \"mediastinal\",\n \"lesions\",\n \"vats\",\n \"patients\",\n \"mm\",\n \"pulmonary\",\n \"lobe\",\n \"uniportal\",\n \"left\",\n \"uniportal vats\"\n]"},"keybert_topics":{"kind":"list like","value":["resection pulmonary mediastinal","pulmonary mediastinal surgery","video assisted thoracic","simultaneous thoracoscopic resection","thoracic surgery vats"],"string":"[\n \"resection pulmonary mediastinal\",\n \"pulmonary mediastinal surgery\",\n \"video assisted thoracic\",\n \"simultaneous thoracoscopic resection\",\n \"thoracic surgery vats\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | lesions | invasive | invasive lesions | largest | sample | resection | operations | diseases | pulmonary mediastinal lesions [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lobe | table | right | pathological | lower | lesions | range | lower lobe | thymic | cases [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] lesions | mm local invasion pulmonary | vats safe | local invasion pulmonary lesions | method certain | surgical procedures noteworthy | method certain indications | method certain indications general | vats safe feasible surgical | vats safe feasible [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | uniportal | lobe | uniportal vats | resection [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | uniportal | lobe | uniportal vats | resection [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] VATS ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 54 | 44 | VATS | 3 | VATS | 7 | VATS ||| Seven ||| 47 | 3 | 33 | 70.2% | no more than 100 mL (n = | 43 | 91.5% ||| 5.66 | 3.34 days | daily | 196.90 | 122.31 mL. Four ||| 8.60 | 3.63 days ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] VATS ||| ||| July 2018 to July 2021 ||| ||| 54 | 44 | VATS | 3 | VATS | 7 | VATS ||| Seven ||| 47 | 3 | 33 | 70.2% | no more than 100 mL (n = | 43 | 91.5% ||| 5.66 | 3.34 days | daily | 196.90 | 122.31 mL. Four ||| 8.60 | 3.63 days ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] VATS ||| ||| July 2018 to July 2021 ||| ||| 54 | 44 | VATS | 3 | VATS | 7 | VATS ||| Seven ||| 47 | 3 | 33 | 70.2% | no more than 100 mL (n = | 43 | 91.5% ||| 5.66 | 3.34 days | daily | 196.90 | 122.31 mL. Four ||| 8.60 | 3.63 days ||| ||| [SUMMARY]"}}},{"rowIdx":3391,"cells":{"title":{"kind":"string","value":"Histologic features and genomic alterations of primary colorectal adenocarcinoma predict growth patterns of liver metastasis."},"pmid":{"kind":"string","value":"31341365"},"background_abstract":{"kind":"string","value":"Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adenocarcinoma","Adult","Aged","Aged, 80 and over","B7-H1 Antigen","Class I Phosphatidylinositol 3-Kinases","Colon","Colorectal Neoplasms","DNA Mismatch Repair","Female","Humans","Kaplan-Meier Estimate","Liver","Liver Neoplasms","Male","Middle Aged","Mutation","Predictive Value of Tests","Prognosis","Proto-Oncogene Proteins B-raf","Rectum","Retrospective Studies","Exome Sequencing"],"string":"[\n \"Adenocarcinoma\",\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"B7-H1 Antigen\",\n \"Class I Phosphatidylinositol 3-Kinases\",\n \"Colon\",\n \"Colorectal Neoplasms\",\n \"DNA Mismatch Repair\",\n \"Female\",\n \"Humans\",\n \"Kaplan-Meier Estimate\",\n \"Liver\",\n \"Liver Neoplasms\",\n \"Male\",\n \"Middle Aged\",\n \"Mutation\",\n \"Predictive Value of Tests\",\n \"Prognosis\",\n \"Proto-Oncogene Proteins B-raf\",\n \"Rectum\",\n \"Retrospective Studies\",\n \"Exome Sequencing\"\n]"},"pmcid":{"kind":"string","value":"6639555"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\nWith the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\n Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\nFor each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\n Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\nThe tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\n Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\nAll 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\n Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\nFormalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\n Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\nStatistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Research conclusion"},"conclusions_text":{"kind":"string","value":"We thank Dr. Logan Walsh at Department of Human Genetics, Rosalind and Morris Goodman Cancer Research Center, McGill University for his assistance in analyzing the whole exome sequencing data, and associate professor Jianfeng Luo at Department of Biostatistics, School of Public Health, Fudan University for the help of biostatistical analysis."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Patients","Evaluation of HGPs of primary and metastatic CRC","Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC","Immunohistochemistry","Whole exome sequencing","Statistical analysis","RESULTS","Clinical parameters","Pathological features","Value of selected pathological features in predicting the HGPs of CRCLMs","Whole-exome sequencing","DISCUSSION","ARTICLE HIGHLIGHTS","Research background","Research motivation","Research objective","Research methods","Research results","Research conclusion"],"string":"[\n \"INTRODUCTION\",\n \"Patients\",\n \"Evaluation of HGPs of primary and metastatic CRC\",\n \"Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC\",\n \"Immunohistochemistry\",\n \"Whole exome sequencing\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Clinical parameters\",\n \"Pathological features\",\n \"Value of selected pathological features in predicting the HGPs of CRCLMs\",\n \"Whole-exome sequencing\",\n \"DISCUSSION\",\n \"ARTICLE HIGHLIGHTS\",\n \"Research background\",\n \"Research motivation\",\n \"Research objective\",\n \"Research methods\",\n \"Research results\",\n \"Research conclusion\"\n]"},"other_sections_texts":{"kind":"list like","value":["Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.","With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.","For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).","The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].","All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.","Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).","Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis."," Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.","The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).","Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.","EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.","Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.","Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering."," Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.","Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.","Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis","To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.","A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.","In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).","The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern."],"string":"[\n \"Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.\",\n \"With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\",\n \"For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\",\n \"The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\",\n \"All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\",\n \"Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\",\n \"Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\",\n \" Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\\nAssociation between clinicopathological characteristics and growth patterns\\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\\nAssociation between clinicopathological characteristics and growth patterns\\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\",\n \"The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\\nAssociation between clinicopathological characteristics and growth patterns\\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\",\n \"Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\",\n \"EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\",\n \"Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\",\n \"Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\",\n \" Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\",\n \"Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\",\n \"Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\",\n \"To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\",\n \"A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\",\n \"In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\",\n \"The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Patients","Evaluation of HGPs of primary and metastatic CRC","Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC","Immunohistochemistry","Whole exome sequencing","Statistical analysis","RESULTS","Clinical parameters","Pathological features","Value of selected pathological features in predicting the HGPs of CRCLMs","Whole-exome sequencing","DISCUSSION","ARTICLE HIGHLIGHTS","Research background","Research motivation","Research objective","Research methods","Research results","Research conclusion"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Patients\",\n \"Evaluation of HGPs of primary and metastatic CRC\",\n \"Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC\",\n \"Immunohistochemistry\",\n \"Whole exome sequencing\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Clinical parameters\",\n \"Pathological features\",\n \"Value of selected pathological features in predicting the HGPs of CRCLMs\",\n \"Whole-exome sequencing\",\n \"DISCUSSION\",\n \"ARTICLE HIGHLIGHTS\",\n \"Research background\",\n \"Research motivation\",\n \"Research objective\",\n \"Research methods\",\n \"Research results\",\n \"Research conclusion\"\n]"},"all_sections_texts":{"kind":"list like","value":["Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis."," Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\nWith the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\n Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\nFor each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\n Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\nThe tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\n Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\nAll 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\n Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\nFormalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\n Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\nStatistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.","With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.","For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).","The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].","All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.","Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).","Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis."," Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.","The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).","Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.","EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.","Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.","Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering."," Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.","Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.","Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis","To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.","A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.","In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).","The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern."],"string":"[\n \"Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.\",\n \" Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\\nWith the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\\n Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\\nFor each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\\n Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\\nThe tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\\n Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\\nAll 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\\n Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\\nFormalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\\n Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\\nStatistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\",\n \"With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\",\n \"For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\",\n \"The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\",\n \"All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\",\n \"Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\",\n \"Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\",\n \" Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\\nAssociation between clinicopathological characteristics and growth patterns\\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\\nAssociation between clinicopathological characteristics and growth patterns\\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\",\n \"The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\\nAssociation between clinicopathological characteristics and growth patterns\\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\",\n \"Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\",\n \"EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\",\n \"Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\",\n \"Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\",\n \" Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\",\n \"Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\",\n \"Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\",\n \"To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\",\n \"A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\",\n \"In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\",\n \"The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Colorectal carcinoma","Liver metastasis","Histologic growth pattern","Clinicopathological characteristics","Whole exome sequencing"],"string":"[\n \"Colorectal carcinoma\",\n \"Liver metastasis\",\n \"Histologic growth pattern\",\n \"Clinicopathological characteristics\",\n \"Whole exome sequencing\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nColorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.\n\nMATERIALS AND METHODS:\n Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\nWith the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\n Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\nFor each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\n Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\nThe tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\n Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\nAll 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\n Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\nFormalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\n Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\nStatistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\n\nPatients:\nWith the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\n\nEvaluation of HGPs of primary and metastatic CRC:\nFor each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\n\nEvaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC:\nThe tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\n\nImmunohistochemistry:\nAll 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\n\nWhole exome sequencing:\nFormalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\n\nStatistical analysis:\nStatistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\n\nRESULTS:\n Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\n\nClinical parameters:\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\n\nPathological features:\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\n\nValue of selected pathological features in predicting the HGPs of CRCLMs:\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\n\nWhole-exome sequencing:\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\n\nDISCUSSION:\nColorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\n\nARTICLE HIGHLIGHTS:\n Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\n\nResearch background:\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\n\nResearch motivation:\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\n\nResearch objective:\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\n\nResearch methods:\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\n\nResearch results:\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n\nResearch conclusion:\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established.\n\nMethods:\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis.\n\nResults:\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n\nConclusions:\nThe HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nColorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.\n\nResearch conclusion:\nWe thank Dr. Logan Walsh at Department of Human Genetics, Rosalind and Morris Goodman Cancer Research Center, McGill University for his assistance in analyzing the whole exome sequencing data, and associate professor Jianfeng Luo at Department of Biostatistics, School of Public Health, Fudan University for the help of biostatistical analysis."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established.\n\nMethods:\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis.\n\nResults:\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n\nConclusions:\nThe HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis."},"whole_article_text_length":{"kind":"number","value":14357,"string":"14,357"},"whole_article_abstract_length":{"kind":"number","value":335,"string":"335"},"other_sections_lengths":{"kind":"list like","value":[520,210,474,144,366,419,132,3721,526,789,217,313,1398,1027,27,40,37,77,144,91],"string":"[\n 520,\n 210,\n 474,\n 144,\n 366,\n 419,\n 132,\n 3721,\n 526,\n 789,\n 217,\n 313,\n 1398,\n 1027,\n 27,\n 40,\n 37,\n 77,\n 144,\n 91\n]"},"num_sections":{"kind":"number","value":21,"string":"21"},"most_frequent_words":{"kind":"list like","value":["tumor","liver","metastasis","group","primary","growth","liver metastasis","pattern","growth pattern","cases"],"string":"[\n \"tumor\",\n \"liver\",\n \"metastasis\",\n \"group\",\n \"primary\",\n \"growth\",\n \"liver metastasis\",\n \"pattern\",\n \"growth pattern\",\n \"cases\"\n]"},"keybert_topics":{"kind":"list like","value":["cancer liver metastasis","liver metastasis statistical","liver metastases crclms","colorectal adenocarcinoma liver","colorectal carcinoma crc"],"string":"[\n \"cancer liver metastasis\",\n \"liver metastasis statistical\",\n \"liver metastases crclms\",\n \"colorectal adenocarcinoma liver\",\n \"colorectal carcinoma crc\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] liver | liver metastases | metastases | hgps | distinct | patients | metastasis | cancer | liver metastasis | crclms [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] version | tissue | tumor | arrows | normal | icb | dna | growth | pattern | tumor normal [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] crcs | growth pattern | primary crcs | crcs expanding growth pattern | primary crcs expanding growth | crcs expanding growth | crcs expanding | primary crcs expanding | pattern | growth [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] liver | metastasis | liver metastasis | primary | tumor | group | growth | pattern | growth pattern | hgps [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] liver | metastasis | liver metastasis | primary | tumor | group | growth | pattern | growth pattern | hgps [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 29 | CRC | two | 15 | 14 ||| ||| ||| Five | WES [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] CDR | CRC [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| ||| 29 | CRC | two | 15 | 14 ||| ||| ||| Five | WES ||| ||| Crohn | CDR ||| WES | APC | 4/5 | 3/5 | KRAS | PIK3CA | 2/5 | BRCA2 | BRAF | DNAH5 (1/5 | APC | 3/5 | KRAS | DNH5 | SMAD | ERBB2 | ERBB3 | 1/5 ||| CDR | CRC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] CRC ||| ||| 29 | CRC | two | 15 | 14 ||| ||| ||| Five | WES ||| ||| Crohn | CDR ||| WES | APC | 4/5 | 3/5 | KRAS | PIK3CA | 2/5 | BRCA2 | BRAF | DNAH5 (1/5 | APC | 3/5 | KRAS | DNH5 | SMAD | ERBB2 | ERBB3 | 1/5 ||| CDR | CRC [SUMMARY]"}}},{"rowIdx":3392,"cells":{"title":{"kind":"string","value":"Eradication rate and safety of a \"simplified rescue therapy\": 14-day vonoprazan and amoxicillin dual regimen as rescue therapy on treatment of Helicobacter pylori infection previously failed in eradication: A real-world, retrospective clinical study in China."},"pmid":{"kind":"string","value":"35877765"},"background_abstract":{"kind":"string","value":"The currently recommended quadruple regimens as rescue therapy on Helicobacter pylori infection were not as effective as being supposed, especially in those who had failed two or more times. Dual regimen composed of vonoprazan (a potassium-competitive acid blocker) and amoxicillin might be an option since it's effective in eradication therapy as first-line treatment."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"From May 2020 to June 2021, the clinical data of patients who had failed in Helicobacter pylori infection treatment were collected in GI department of Peking University First Hospital, Beijing, China. Patients were given vonoprazan 20 mg or 40 mg per day and amoxicillin 3000 mg per day (VA dual therapy) for 14 days as rescue treatment. Helicobacter pylori status was evaluated by 13 C-urease breath test 6 weeks after treatment. All adverse effects during treatment were recorded."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 186 patients were enrolled, including 67 males and 119 females. All of them had failed for 1 ~ 7 times in their previous treatment. Successful eradication was achieved in 172 patients (92.5%, 172/186). The adverse effects (referring to skin rash, abdominal pain, diarrhea, and headache), mainly mild and did not cause quit of treatment, occurred in 14 patients (7.5%, 14/186) and all symptoms relieved spontaneously."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Dual regimen composed of vonoprazan and amoxicillin for 14 days was effective and safe as rescue therapy in Helicobacter pylori infection treatment. It could be chosen as a \"simplified rescue therapy\" with relatively high eradication rate no matter how many times the patients had failed and what regimens they had used previously."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Amoxicillin","Anti-Bacterial Agents","Clarithromycin","Drug Therapy, Combination","Female","Helicobacter Infections","Helicobacter pylori","Humans","Male","Potassium","Proton Pump Inhibitors","Pyrroles","Retrospective Studies","Sulfonamides","Treatment Outcome","Urease"],"string":"[\n \"Amoxicillin\",\n \"Anti-Bacterial Agents\",\n \"Clarithromycin\",\n \"Drug Therapy, Combination\",\n \"Female\",\n \"Helicobacter Infections\",\n \"Helicobacter pylori\",\n \"Humans\",\n \"Male\",\n \"Potassium\",\n \"Proton Pump Inhibitors\",\n \"Pyrroles\",\n \"Retrospective Studies\",\n \"Sulfonamides\",\n \"Treatment Outcome\",\n \"Urease\"\n]"},"pmcid":{"kind":"string","value":"9542484"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Patients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nA total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nEradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\nAll the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\n\nMIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nTwenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nCompliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\nOf all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg)."},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Study design and participants","Diagnosis of H. pylori infection and treatment regimen","Antibiotic susceptibility test","Statistical analysis","Patients enrolled and baseline characteristics","Eradication of H. pylori infection","\nMIC to antibiotics of isolated H. pylori strains","Compliance and adverse events"],"string":"[\n \"INTRODUCTION\",\n \"Study design and participants\",\n \"Diagnosis of H. pylori infection and treatment regimen\",\n \"Antibiotic susceptibility test\",\n \"Statistical analysis\",\n \"Patients enrolled and baseline characteristics\",\n \"Eradication of H. pylori infection\",\n \"\\nMIC to antibiotics of isolated H. pylori strains\",\n \"Compliance and adverse events\"\n]"},"other_sections_texts":{"kind":"list like","value":["\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.","A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.","\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.","Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n","Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.","A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.","All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).","Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.","Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg)."],"string":"[\n \"\\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\\n1\\n, \\n2\\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\\n3\\n, \\n4\\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\\n5\\n, \\n6\\n, \\n7\\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\\n8\\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.\",\n \"A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\\n\\nNote: Data are n (%), or mean (SD, standard deviation).\\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\\n*Compliance, taken >80% of tablets.\",\n \"\\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\",\n \"Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\\n9\\n, \\n10\\n\\n\",\n \"Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\",\n \"A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\",\n \"All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\\n).\",\n \"Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\",\n \"Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\\nAdverse events happened\\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Study design and participants","Diagnosis of H. pylori infection and treatment regimen","Antibiotic susceptibility test","Statistical analysis","RESULTS","Patients enrolled and baseline characteristics","Eradication of H. pylori infection","\nMIC to antibiotics of isolated H. pylori strains","Compliance and adverse events","DISCUSSION","CONCLUSION","CONFLICT OF INTEREST"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Study design and participants\",\n \"Diagnosis of H. pylori infection and treatment regimen\",\n \"Antibiotic susceptibility test\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Patients enrolled and baseline characteristics\",\n \"Eradication of H. pylori infection\",\n \"\\nMIC to antibiotics of isolated H. pylori strains\",\n \"Compliance and adverse events\",\n \"DISCUSSION\",\n \"CONCLUSION\",\n \"CONFLICT OF INTEREST\"\n]"},"all_sections_texts":{"kind":"list like","value":["\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.","Study design and participants A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\nA real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\nDiagnosis of H. pylori infection and treatment regimen \nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\n\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\nAntibiotic susceptibility test Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\nSome of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\nStatistical analysis Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\nData collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.","A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.","\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.","Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n","Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.","Patients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nA total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nEradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\nAll the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\n\nMIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nTwenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nCompliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\nOf all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).","A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.","All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).","Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.","Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).","In our study, VA dual regimen was designed as rescue treatment used in patients who failed one or more times before, no matter what regimen they had used, including those who had used PPI + amoxicillin dual therapy. The overall eradication rate was 92.5% (95% CI 87.4%–95.7%) with minimal side effects (7.5%, 95% CI 4.3%–12.6%).\nCauses of treatment failure of anti‐H. pylori therapy include antibiotics resistance, poor compliance of patients, low gastric pH, and high bacterial load.\n11\n, \n12\n The prevalence of multidrug‐resistant H. pylori strains is increasing, especially in cases with multiple eradication failure, which makes rescue treatment difficult.\n13\n, \n14\n However, since the resistance rate to amoxicillin is low even after the failure of eradication, amoxicillin can be a candidate of antimicrobial agent for the rescue therapy.\n8\n, \n15\n, \n16\n, \n17\n\n\nBeyond the traditional quadruple therapy, dual therapy, which was composed of PPI + amoxicillin, was testified to be an effective regimen used in treatment of H. pylori infection in recent years.\n5\n, \n16\n, \n18\nAfter the first report in 1989, the efficacy of AMX‐contained dual therapy was unstable and being abandoned for many years.\n19\n, \n20\nWhile in recent 10 years, there were more and more studies showing that it could be pretty effective.\n21\n It was believed that there were two critical variables/factors that affected the efficacy of the treatment.\n21\n One was to achieve and maintain a relatively high intragastric pH value, in which the antibactericidal effect of amoxicillin would be stable to get a better bioavailability in gastric cavity. The second was the concentration of amoxicillin in stomach.\n21\n Amoxicillin is a time‐dependent antibiotic, which is rapidly absorbed into plasma and then to be excreted in 6 ~ 8 h after administration. Comparing with 1000 mg twice daily, a dosage of 500 ~ 750 mg per 6 h might be more likely to maintain a higher plasma concentration.\n6\n, \n22\n\n\nAccording to the choice of acid inhibitors, since the intragastric pH value might vary according to the potency of different PPIs and ethnic difference in PPI metabolism (cytochrome P450 [CYP2C19] pharmacogenetic polymorphism)\n8\n, vonoprazan was chosen as part of the combination. Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker, which could provide fast and powerful acid inhibition, suggesting it might be possible to sustain a higher intragastric pH value. It was observed that a pH >4.0 status could be obtained at 4 h and to sustain for 24 h after the first administration of VPZ.\n13\n, \n23\n The effectiveness of VPZ and amoxicillin dual therapy used as first‐line treatment was pretty good with a eradication rates varied as 85%–90% in Japan, while there was little data on its effect on rescue treatment.\n3\n, \n13\n In our study, the dual therapy was composed of vonoprazan 20/40 mg per day (10/20 mg b.i.d) and amoxicillin 3000 mg per day (1000 mg t.i.d or 750 mg q.i.d). It was used as rescue therapy for patients who failed in their previous treatment, no matter how many times they had failed and what eradication regimens they had used before.\nIn this study, all patients had not used VA dual therapy, while some of them had used PPI + AMX dual therapy before (3/186). Altogether 186 subjects were enrolled, the eradication rate was 92.5% (172/186). Fourteen patients failed in VA dual therapy. We tried to identify factors that might be potential risk factors of eradication failure, such as gender, BMI, smoking, alcohol drinking, previous use of amoxicillin, or previous treatment failure times. It seemed that the previous use of amoxicillin and smoking might influence the eradication efficacy, but not statistically (Table 1). A recent meta‐analysis showed that although smoking increases the failure rate of H. pylori eradication treatment, however, when vonoprazan is used to treat the H. pylori infection, smoking has no effect on the eradication rate.\n24\n Although it inclined the more times the patients had failed, the less possibility they succeeded in VA dual therapy, previous failure times did not influence the efficacy of rescue treatment statistically (Figure 1).\nThe eradication rate of VA dual treatment according to different failure times previously. All patients had endured previous treatment failure. Most of them had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). Forty‐eight patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). The overall eradication rate of VA dual rescue therapy was 92.5% (172/186). The eradication rate was 92.9% (78 of 84) in patients who had failed one time, 96.3% (52 of 54) in patients who had failed two times, and 87.5% (42 of 48) in patients who had failed three or more times previously\nPrevalence of antimicrobial resistance of 25 cases who had been performed MIC tests. 5 cases had accepted the MIC test, most of H. pylori strains isolated from them were resistant to CLA (92%, 23 of 25), MTZ (96%, 24 of 25), LEV (88%, 22 of 25), and MOX (88%, 22 of 25). Three of 25 cases were resistant to AMX (12%) and none of them resistant to TET. Resistance to antibiotics was defined as: AMX resistant at MIC >0.125 mg/L; TET resistant at MIC >1 mg/L; CLA resistant at MIC >0.5 mg/L; MTZ resistant at MIC >8 mg/L; LEV resistant at MIC>1 mg/L; MOX resistant at MIC >1 mg/L. CLA: clarithromycin; MTZ: metronidazole; LEV: levofloxacin AMX: amoxicillin; TET: tetracycline; MOX: moxifloxacinMIC: Minimum Inhibitory Concentration\nEradication rates of patients who had or had not used amoxicillin in their previous treatment. In 186 patients, 83.3% of them (n = 155) had accepted previously treatments in which amoxicillin was included, while 16.7% of them (n = 31) did not get a regimen contained amoxicillin before. In all 186 patients, the overall eradication rate was 92.5%(172 of 186). In patients who had not used amoxicillin in their previous treatment, all of them got a successful eradication (100%, 31 of 31). While 91.0% (141 of 155) of patients who had used amoxicillin before eliminated the bacteria\nEradication rates of total 186 patients and patients who had accepted regimens with 10 mg bid or 20 mg b.i.d vonoprazan. In 186 patients, 23.1% of them (n = 43) had accepted 20 mg/d vonoprazan while 76.9% (n = 143) with 40 mg/d vonoprazan in their rescue treatment. In all 186 patients, the overall eradication rate was 92.5% (172 of 186). The eradication rates of different dose of vonoprazan were 95.3% (41 of 43) in 10 mg b.i.d and 91.6% (131 of 143) in 20 mg b.i.d, respectively\nIn 25 cases who got an MIC test during treatment, three cases were resistant to AMX with an MIC of .25 mg/L (resistance defined as a MIC >0.125 mg/L). Among them, one case failed in the VA dual treatment while two of them succeeded in eradication. All 14 who failed had used amoxicillin in their previous treatment, three of them got MIC test with results of .125 mg/L, .125 mg/L and .25 mg/L to AMX, with one of three was defined as resistant to AMX theoretically. Although we did not know the detail about it, it seemed that resistance to AMX was not absolute contraindication in VA dual therapy. There was an inclination that VA dual therapy might be more effective in patients who had not used amoxicillin before.\nTo our knowledge, this is the first real‐world study to reveal the efficacy of vonoprazan and amoxicillin dual regimen (VA dual regimen) as rescue treatment in China. In previous studies, dual therapy with VPZ was mostly used as first‐line treatment, and the eradication rates varied as 85% ~ 90% in Japan.\n13\n, \n25\n In clinical practices from Japan, VPZ was mostly used as component of triple therapy in first‐line (VPZ + amoxicillin + clarithromycin), second‐line (VPZ + amoxicillin + metronidazole), and third‐line (VPZ + amoxicillin + sitafloxacin) treatment of H. pylori infection. VPZ‐contained triple regimens had a relatively high eradication rate as 88.1%, 80.1%, and 75.8%, respectively.\n26\n Although vonoprazan appeared to restore the effectiveness of triple therapy, the improvement was almost entirely to improved effectiveness of amoxicillin dual therapy componentand resulted in the majority (>85% currently in Japan) of those receiving vonoprazan + amoxicillin plus a second antibiotic (e.g., clarithromycin, metronidazole, fluoroquinolone, or rifabutin) receiving no benefit from the second antibiotic.\n27\n It seemed that the only contribution of the second antibiotic is to increase global antimicrobial resistance.\n27\n, \n28\n, \n29\n\n\nFrom the data of our study, it was supposed that the VA dual therapy was effective as rescue treatment no matter what regimen had been given and which antibiotics had been used before. The VA dual therapy could be defined as a “simplified rescue therapy.”\nDuring the treatment course of the VA dual therapy, the adverse events happened were mild and did not cause quitting or failure of treatment. The administration mode was suitable since the compliance of patients was pretty good.\nThere were many limitations in our study. As a real‐world retrospective study, it's not a randomized controlled trial. The regimens were not consistent, whereas components of dual therapy were the same as vonoprazan + amoxicillin, the administration frequency or doses in VA dual therapy varied. The frequency of amoxicillin was given either t.i.d or q.i.d casually more than well‐designed, although the total dose was the same as 3000 mg per day. The total doses of vonoprazan also varied (20mg or 40 mg per day) with the body weight of patient. As to the MIC analysis, there was only 25 cases who got a successful H. pylori culture and antibacterial susceptibility test, depending on the patients' willingness. The MIC data were limited and not very representative. Based on the results of this retrospective study, a well‐designed random controlled prospective clinical trial could be anticipated being performed in the future.","The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before.","The authors have no competing interests."],"string":"[\n \"\\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\\n1\\n, \\n2\\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\\n3\\n, \\n4\\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\\n5\\n, \\n6\\n, \\n7\\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\\n8\\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.\",\n \"Study design and participants A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\\n\\nNote: Data are n (%), or mean (SD, standard deviation).\\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\\n*Compliance, taken >80% of tablets.\\nA real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\\n\\nNote: Data are n (%), or mean (SD, standard deviation).\\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\\n*Compliance, taken >80% of tablets.\\nDiagnosis of H. pylori infection and treatment regimen \\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\\n\\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\\nAntibiotic susceptibility test Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\\n9\\n, \\n10\\n\\n\\nSome of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\\n9\\n, \\n10\\n\\n\\nStatistical analysis Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\\nData collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\",\n \"A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\\n\\nNote: Data are n (%), or mean (SD, standard deviation).\\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\\n*Compliance, taken >80% of tablets.\",\n \"\\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\",\n \"Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\\n9\\n, \\n10\\n\\n\",\n \"Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\",\n \"Patients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\\nA total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\\nEradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\\n).\\nAll the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\\n).\\n\\nMIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\\nTwenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\\nCompliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\\nAdverse events happened\\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\\nOf all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\\nAdverse events happened\\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\",\n \"A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\",\n \"All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\\n).\",\n \"Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\",\n \"Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\\nAdverse events happened\\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\",\n \"In our study, VA dual regimen was designed as rescue treatment used in patients who failed one or more times before, no matter what regimen they had used, including those who had used PPI + amoxicillin dual therapy. The overall eradication rate was 92.5% (95% CI 87.4%–95.7%) with minimal side effects (7.5%, 95% CI 4.3%–12.6%).\\nCauses of treatment failure of anti‐H. pylori therapy include antibiotics resistance, poor compliance of patients, low gastric pH, and high bacterial load.\\n11\\n, \\n12\\n The prevalence of multidrug‐resistant H. pylori strains is increasing, especially in cases with multiple eradication failure, which makes rescue treatment difficult.\\n13\\n, \\n14\\n However, since the resistance rate to amoxicillin is low even after the failure of eradication, amoxicillin can be a candidate of antimicrobial agent for the rescue therapy.\\n8\\n, \\n15\\n, \\n16\\n, \\n17\\n\\n\\nBeyond the traditional quadruple therapy, dual therapy, which was composed of PPI + amoxicillin, was testified to be an effective regimen used in treatment of H. pylori infection in recent years.\\n5\\n, \\n16\\n, \\n18\\nAfter the first report in 1989, the efficacy of AMX‐contained dual therapy was unstable and being abandoned for many years.\\n19\\n, \\n20\\nWhile in recent 10 years, there were more and more studies showing that it could be pretty effective.\\n21\\n It was believed that there were two critical variables/factors that affected the efficacy of the treatment.\\n21\\n One was to achieve and maintain a relatively high intragastric pH value, in which the antibactericidal effect of amoxicillin would be stable to get a better bioavailability in gastric cavity. The second was the concentration of amoxicillin in stomach.\\n21\\n Amoxicillin is a time‐dependent antibiotic, which is rapidly absorbed into plasma and then to be excreted in 6 ~ 8 h after administration. Comparing with 1000 mg twice daily, a dosage of 500 ~ 750 mg per 6 h might be more likely to maintain a higher plasma concentration.\\n6\\n, \\n22\\n\\n\\nAccording to the choice of acid inhibitors, since the intragastric pH value might vary according to the potency of different PPIs and ethnic difference in PPI metabolism (cytochrome P450 [CYP2C19] pharmacogenetic polymorphism)\\n8\\n, vonoprazan was chosen as part of the combination. Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker, which could provide fast and powerful acid inhibition, suggesting it might be possible to sustain a higher intragastric pH value. It was observed that a pH >4.0 status could be obtained at 4 h and to sustain for 24 h after the first administration of VPZ.\\n13\\n, \\n23\\n The effectiveness of VPZ and amoxicillin dual therapy used as first‐line treatment was pretty good with a eradication rates varied as 85%–90% in Japan, while there was little data on its effect on rescue treatment.\\n3\\n, \\n13\\n In our study, the dual therapy was composed of vonoprazan 20/40 mg per day (10/20 mg b.i.d) and amoxicillin 3000 mg per day (1000 mg t.i.d or 750 mg q.i.d). It was used as rescue therapy for patients who failed in their previous treatment, no matter how many times they had failed and what eradication regimens they had used before.\\nIn this study, all patients had not used VA dual therapy, while some of them had used PPI + AMX dual therapy before (3/186). Altogether 186 subjects were enrolled, the eradication rate was 92.5% (172/186). Fourteen patients failed in VA dual therapy. We tried to identify factors that might be potential risk factors of eradication failure, such as gender, BMI, smoking, alcohol drinking, previous use of amoxicillin, or previous treatment failure times. It seemed that the previous use of amoxicillin and smoking might influence the eradication efficacy, but not statistically (Table 1). A recent meta‐analysis showed that although smoking increases the failure rate of H. pylori eradication treatment, however, when vonoprazan is used to treat the H. pylori infection, smoking has no effect on the eradication rate.\\n24\\n Although it inclined the more times the patients had failed, the less possibility they succeeded in VA dual therapy, previous failure times did not influence the efficacy of rescue treatment statistically (Figure 1).\\nThe eradication rate of VA dual treatment according to different failure times previously. All patients had endured previous treatment failure. Most of them had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). Forty‐eight patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). The overall eradication rate of VA dual rescue therapy was 92.5% (172/186). The eradication rate was 92.9% (78 of 84) in patients who had failed one time, 96.3% (52 of 54) in patients who had failed two times, and 87.5% (42 of 48) in patients who had failed three or more times previously\\nPrevalence of antimicrobial resistance of 25 cases who had been performed MIC tests. 5 cases had accepted the MIC test, most of H. pylori strains isolated from them were resistant to CLA (92%, 23 of 25), MTZ (96%, 24 of 25), LEV (88%, 22 of 25), and MOX (88%, 22 of 25). Three of 25 cases were resistant to AMX (12%) and none of them resistant to TET. Resistance to antibiotics was defined as: AMX resistant at MIC >0.125 mg/L; TET resistant at MIC >1 mg/L; CLA resistant at MIC >0.5 mg/L; MTZ resistant at MIC >8 mg/L; LEV resistant at MIC>1 mg/L; MOX resistant at MIC >1 mg/L. CLA: clarithromycin; MTZ: metronidazole; LEV: levofloxacin AMX: amoxicillin; TET: tetracycline; MOX: moxifloxacinMIC: Minimum Inhibitory Concentration\\nEradication rates of patients who had or had not used amoxicillin in their previous treatment. In 186 patients, 83.3% of them (n = 155) had accepted previously treatments in which amoxicillin was included, while 16.7% of them (n = 31) did not get a regimen contained amoxicillin before. In all 186 patients, the overall eradication rate was 92.5%(172 of 186). In patients who had not used amoxicillin in their previous treatment, all of them got a successful eradication (100%, 31 of 31). While 91.0% (141 of 155) of patients who had used amoxicillin before eliminated the bacteria\\nEradication rates of total 186 patients and patients who had accepted regimens with 10 mg bid or 20 mg b.i.d vonoprazan. In 186 patients, 23.1% of them (n = 43) had accepted 20 mg/d vonoprazan while 76.9% (n = 143) with 40 mg/d vonoprazan in their rescue treatment. In all 186 patients, the overall eradication rate was 92.5% (172 of 186). The eradication rates of different dose of vonoprazan were 95.3% (41 of 43) in 10 mg b.i.d and 91.6% (131 of 143) in 20 mg b.i.d, respectively\\nIn 25 cases who got an MIC test during treatment, three cases were resistant to AMX with an MIC of .25 mg/L (resistance defined as a MIC >0.125 mg/L). Among them, one case failed in the VA dual treatment while two of them succeeded in eradication. All 14 who failed had used amoxicillin in their previous treatment, three of them got MIC test with results of .125 mg/L, .125 mg/L and .25 mg/L to AMX, with one of three was defined as resistant to AMX theoretically. Although we did not know the detail about it, it seemed that resistance to AMX was not absolute contraindication in VA dual therapy. There was an inclination that VA dual therapy might be more effective in patients who had not used amoxicillin before.\\nTo our knowledge, this is the first real‐world study to reveal the efficacy of vonoprazan and amoxicillin dual regimen (VA dual regimen) as rescue treatment in China. In previous studies, dual therapy with VPZ was mostly used as first‐line treatment, and the eradication rates varied as 85% ~ 90% in Japan.\\n13\\n, \\n25\\n In clinical practices from Japan, VPZ was mostly used as component of triple therapy in first‐line (VPZ + amoxicillin + clarithromycin), second‐line (VPZ + amoxicillin + metronidazole), and third‐line (VPZ + amoxicillin + sitafloxacin) treatment of H. pylori infection. VPZ‐contained triple regimens had a relatively high eradication rate as 88.1%, 80.1%, and 75.8%, respectively.\\n26\\n Although vonoprazan appeared to restore the effectiveness of triple therapy, the improvement was almost entirely to improved effectiveness of amoxicillin dual therapy componentand resulted in the majority (>85% currently in Japan) of those receiving vonoprazan + amoxicillin plus a second antibiotic (e.g., clarithromycin, metronidazole, fluoroquinolone, or rifabutin) receiving no benefit from the second antibiotic.\\n27\\n It seemed that the only contribution of the second antibiotic is to increase global antimicrobial resistance.\\n27\\n, \\n28\\n, \\n29\\n\\n\\nFrom the data of our study, it was supposed that the VA dual therapy was effective as rescue treatment no matter what regimen had been given and which antibiotics had been used before. The VA dual therapy could be defined as a “simplified rescue therapy.”\\nDuring the treatment course of the VA dual therapy, the adverse events happened were mild and did not cause quitting or failure of treatment. The administration mode was suitable since the compliance of patients was pretty good.\\nThere were many limitations in our study. As a real‐world retrospective study, it's not a randomized controlled trial. The regimens were not consistent, whereas components of dual therapy were the same as vonoprazan + amoxicillin, the administration frequency or doses in VA dual therapy varied. The frequency of amoxicillin was given either t.i.d or q.i.d casually more than well‐designed, although the total dose was the same as 3000 mg per day. The total doses of vonoprazan also varied (20mg or 40 mg per day) with the body weight of patient. As to the MIC analysis, there was only 25 cases who got a successful H. pylori culture and antibacterial susceptibility test, depending on the patients' willingness. The MIC data were limited and not very representative. Based on the results of this retrospective study, a well‐designed random controlled prospective clinical trial could be anticipated being performed in the future.\",\n \"The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before.\",\n \"The authors have no competing interests.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials-and-methods",null,null,null,null,"results",null,null,null,null,"discussion","conclusions","COI-statement"],"string":"[\n null,\n \"materials-and-methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n \"COI-statement\"\n]"},"keywords":{"kind":"list like","value":["amoxicillin","dual therapy","\nHelicobacter pylori infection","simplified rescue therapy","vonoprazan"],"string":"[\n \"amoxicillin\",\n \"dual therapy\",\n \"\\nHelicobacter pylori infection\",\n \"simplified rescue therapy\",\n \"vonoprazan\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\n\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.\n\nMATERIALS AND METHODS:\nStudy design and participants A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\nA real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\nDiagnosis of H. pylori infection and treatment regimen \nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\n\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\nAntibiotic susceptibility test Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\nSome of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\nStatistical analysis Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\nData collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\n\nStudy design and participants:\nA real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\n\nDiagnosis of H. pylori infection and treatment regimen:\n\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\n\nAntibiotic susceptibility test:\nSome of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\n\nStatistical analysis:\nData collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\n\nRESULTS:\nPatients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nA total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nEradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\nAll the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\n\nMIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nTwenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nCompliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\nOf all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\n\nPatients enrolled and baseline characteristics:\nA total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\n\nEradication of H. pylori infection:\nAll the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\n\n\nMIC to antibiotics of isolated H. pylori strains:\nTwenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\n\nCompliance and adverse events:\nOf all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\n\nDISCUSSION:\nIn our study, VA dual regimen was designed as rescue treatment used in patients who failed one or more times before, no matter what regimen they had used, including those who had used PPI + amoxicillin dual therapy. The overall eradication rate was 92.5% (95% CI 87.4%–95.7%) with minimal side effects (7.5%, 95% CI 4.3%–12.6%).\nCauses of treatment failure of anti‐H. pylori therapy include antibiotics resistance, poor compliance of patients, low gastric pH, and high bacterial load.\n11\n, \n12\n The prevalence of multidrug‐resistant H. pylori strains is increasing, especially in cases with multiple eradication failure, which makes rescue treatment difficult.\n13\n, \n14\n However, since the resistance rate to amoxicillin is low even after the failure of eradication, amoxicillin can be a candidate of antimicrobial agent for the rescue therapy.\n8\n, \n15\n, \n16\n, \n17\n\n\nBeyond the traditional quadruple therapy, dual therapy, which was composed of PPI + amoxicillin, was testified to be an effective regimen used in treatment of H. pylori infection in recent years.\n5\n, \n16\n, \n18\nAfter the first report in 1989, the efficacy of AMX‐contained dual therapy was unstable and being abandoned for many years.\n19\n, \n20\nWhile in recent 10 years, there were more and more studies showing that it could be pretty effective.\n21\n It was believed that there were two critical variables/factors that affected the efficacy of the treatment.\n21\n One was to achieve and maintain a relatively high intragastric pH value, in which the antibactericidal effect of amoxicillin would be stable to get a better bioavailability in gastric cavity. The second was the concentration of amoxicillin in stomach.\n21\n Amoxicillin is a time‐dependent antibiotic, which is rapidly absorbed into plasma and then to be excreted in 6 ~ 8 h after administration. Comparing with 1000 mg twice daily, a dosage of 500 ~ 750 mg per 6 h might be more likely to maintain a higher plasma concentration.\n6\n, \n22\n\n\nAccording to the choice of acid inhibitors, since the intragastric pH value might vary according to the potency of different PPIs and ethnic difference in PPI metabolism (cytochrome P450 [CYP2C19] pharmacogenetic polymorphism)\n8\n, vonoprazan was chosen as part of the combination. Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker, which could provide fast and powerful acid inhibition, suggesting it might be possible to sustain a higher intragastric pH value. It was observed that a pH >4.0 status could be obtained at 4 h and to sustain for 24 h after the first administration of VPZ.\n13\n, \n23\n The effectiveness of VPZ and amoxicillin dual therapy used as first‐line treatment was pretty good with a eradication rates varied as 85%–90% in Japan, while there was little data on its effect on rescue treatment.\n3\n, \n13\n In our study, the dual therapy was composed of vonoprazan 20/40 mg per day (10/20 mg b.i.d) and amoxicillin 3000 mg per day (1000 mg t.i.d or 750 mg q.i.d). It was used as rescue therapy for patients who failed in their previous treatment, no matter how many times they had failed and what eradication regimens they had used before.\nIn this study, all patients had not used VA dual therapy, while some of them had used PPI + AMX dual therapy before (3/186). Altogether 186 subjects were enrolled, the eradication rate was 92.5% (172/186). Fourteen patients failed in VA dual therapy. We tried to identify factors that might be potential risk factors of eradication failure, such as gender, BMI, smoking, alcohol drinking, previous use of amoxicillin, or previous treatment failure times. It seemed that the previous use of amoxicillin and smoking might influence the eradication efficacy, but not statistically (Table 1). A recent meta‐analysis showed that although smoking increases the failure rate of H. pylori eradication treatment, however, when vonoprazan is used to treat the H. pylori infection, smoking has no effect on the eradication rate.\n24\n Although it inclined the more times the patients had failed, the less possibility they succeeded in VA dual therapy, previous failure times did not influence the efficacy of rescue treatment statistically (Figure 1).\nThe eradication rate of VA dual treatment according to different failure times previously. All patients had endured previous treatment failure. Most of them had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). Forty‐eight patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). The overall eradication rate of VA dual rescue therapy was 92.5% (172/186). The eradication rate was 92.9% (78 of 84) in patients who had failed one time, 96.3% (52 of 54) in patients who had failed two times, and 87.5% (42 of 48) in patients who had failed three or more times previously\nPrevalence of antimicrobial resistance of 25 cases who had been performed MIC tests. 5 cases had accepted the MIC test, most of H. pylori strains isolated from them were resistant to CLA (92%, 23 of 25), MTZ (96%, 24 of 25), LEV (88%, 22 of 25), and MOX (88%, 22 of 25). Three of 25 cases were resistant to AMX (12%) and none of them resistant to TET. Resistance to antibiotics was defined as: AMX resistant at MIC >0.125 mg/L; TET resistant at MIC >1 mg/L; CLA resistant at MIC >0.5 mg/L; MTZ resistant at MIC >8 mg/L; LEV resistant at MIC>1 mg/L; MOX resistant at MIC >1 mg/L. CLA: clarithromycin; MTZ: metronidazole; LEV: levofloxacin AMX: amoxicillin; TET: tetracycline; MOX: moxifloxacinMIC: Minimum Inhibitory Concentration\nEradication rates of patients who had or had not used amoxicillin in their previous treatment. In 186 patients, 83.3% of them (n = 155) had accepted previously treatments in which amoxicillin was included, while 16.7% of them (n = 31) did not get a regimen contained amoxicillin before. In all 186 patients, the overall eradication rate was 92.5%(172 of 186). In patients who had not used amoxicillin in their previous treatment, all of them got a successful eradication (100%, 31 of 31). While 91.0% (141 of 155) of patients who had used amoxicillin before eliminated the bacteria\nEradication rates of total 186 patients and patients who had accepted regimens with 10 mg bid or 20 mg b.i.d vonoprazan. In 186 patients, 23.1% of them (n = 43) had accepted 20 mg/d vonoprazan while 76.9% (n = 143) with 40 mg/d vonoprazan in their rescue treatment. In all 186 patients, the overall eradication rate was 92.5% (172 of 186). The eradication rates of different dose of vonoprazan were 95.3% (41 of 43) in 10 mg b.i.d and 91.6% (131 of 143) in 20 mg b.i.d, respectively\nIn 25 cases who got an MIC test during treatment, three cases were resistant to AMX with an MIC of .25 mg/L (resistance defined as a MIC >0.125 mg/L). Among them, one case failed in the VA dual treatment while two of them succeeded in eradication. All 14 who failed had used amoxicillin in their previous treatment, three of them got MIC test with results of .125 mg/L, .125 mg/L and .25 mg/L to AMX, with one of three was defined as resistant to AMX theoretically. Although we did not know the detail about it, it seemed that resistance to AMX was not absolute contraindication in VA dual therapy. There was an inclination that VA dual therapy might be more effective in patients who had not used amoxicillin before.\nTo our knowledge, this is the first real‐world study to reveal the efficacy of vonoprazan and amoxicillin dual regimen (VA dual regimen) as rescue treatment in China. In previous studies, dual therapy with VPZ was mostly used as first‐line treatment, and the eradication rates varied as 85% ~ 90% in Japan.\n13\n, \n25\n In clinical practices from Japan, VPZ was mostly used as component of triple therapy in first‐line (VPZ + amoxicillin + clarithromycin), second‐line (VPZ + amoxicillin + metronidazole), and third‐line (VPZ + amoxicillin + sitafloxacin) treatment of H. pylori infection. VPZ‐contained triple regimens had a relatively high eradication rate as 88.1%, 80.1%, and 75.8%, respectively.\n26\n Although vonoprazan appeared to restore the effectiveness of triple therapy, the improvement was almost entirely to improved effectiveness of amoxicillin dual therapy componentand resulted in the majority (>85% currently in Japan) of those receiving vonoprazan + amoxicillin plus a second antibiotic (e.g., clarithromycin, metronidazole, fluoroquinolone, or rifabutin) receiving no benefit from the second antibiotic.\n27\n It seemed that the only contribution of the second antibiotic is to increase global antimicrobial resistance.\n27\n, \n28\n, \n29\n\n\nFrom the data of our study, it was supposed that the VA dual therapy was effective as rescue treatment no matter what regimen had been given and which antibiotics had been used before. The VA dual therapy could be defined as a “simplified rescue therapy.”\nDuring the treatment course of the VA dual therapy, the adverse events happened were mild and did not cause quitting or failure of treatment. The administration mode was suitable since the compliance of patients was pretty good.\nThere were many limitations in our study. As a real‐world retrospective study, it's not a randomized controlled trial. The regimens were not consistent, whereas components of dual therapy were the same as vonoprazan + amoxicillin, the administration frequency or doses in VA dual therapy varied. The frequency of amoxicillin was given either t.i.d or q.i.d casually more than well‐designed, although the total dose was the same as 3000 mg per day. The total doses of vonoprazan also varied (20mg or 40 mg per day) with the body weight of patient. As to the MIC analysis, there was only 25 cases who got a successful H. pylori culture and antibacterial susceptibility test, depending on the patients' willingness. The MIC data were limited and not very representative. Based on the results of this retrospective study, a well‐designed random controlled prospective clinical trial could be anticipated being performed in the future.\n\nCONCLUSION:\nThe utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before.\n\nCONFLICT OF INTEREST:\nThe authors have no competing interests.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe currently recommended quadruple regimens as rescue therapy on Helicobacter pylori infection were not as effective as being supposed, especially in those who had failed two or more times. Dual regimen composed of vonoprazan (a potassium-competitive acid blocker) and amoxicillin might be an option since it's effective in eradication therapy as first-line treatment.\n\nMethods:\nFrom May 2020 to June 2021, the clinical data of patients who had failed in Helicobacter pylori infection treatment were collected in GI department of Peking University First Hospital, Beijing, China. Patients were given vonoprazan 20 mg or 40 mg per day and amoxicillin 3000 mg per day (VA dual therapy) for 14 days as rescue treatment. Helicobacter pylori status was evaluated by 13 C-urease breath test 6 weeks after treatment. All adverse effects during treatment were recorded.\n\nResults:\nA total of 186 patients were enrolled, including 67 males and 119 females. All of them had failed for 1 ~ 7 times in their previous treatment. Successful eradication was achieved in 172 patients (92.5%, 172/186). The adverse effects (referring to skin rash, abdominal pain, diarrhea, and headache), mainly mild and did not cause quit of treatment, occurred in 14 patients (7.5%, 14/186) and all symptoms relieved spontaneously.\n\nConclusions:\nDual regimen composed of vonoprazan and amoxicillin for 14 days was effective and safe as rescue therapy in Helicobacter pylori infection treatment. It could be chosen as a \"simplified rescue therapy\" with relatively high eradication rate no matter how many times the patients had failed and what regimens they had used previously."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\n\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.\n\nCONCLUSION:\nThe utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe currently recommended quadruple regimens as rescue therapy on Helicobacter pylori infection were not as effective as being supposed, especially in those who had failed two or more times. Dual regimen composed of vonoprazan (a potassium-competitive acid blocker) and amoxicillin might be an option since it's effective in eradication therapy as first-line treatment.\n\nMethods:\nFrom May 2020 to June 2021, the clinical data of patients who had failed in Helicobacter pylori infection treatment were collected in GI department of Peking University First Hospital, Beijing, China. Patients were given vonoprazan 20 mg or 40 mg per day and amoxicillin 3000 mg per day (VA dual therapy) for 14 days as rescue treatment. Helicobacter pylori status was evaluated by 13 C-urease breath test 6 weeks after treatment. All adverse effects during treatment were recorded.\n\nResults:\nA total of 186 patients were enrolled, including 67 males and 119 females. All of them had failed for 1 ~ 7 times in their previous treatment. Successful eradication was achieved in 172 patients (92.5%, 172/186). The adverse effects (referring to skin rash, abdominal pain, diarrhea, and headache), mainly mild and did not cause quit of treatment, occurred in 14 patients (7.5%, 14/186) and all symptoms relieved spontaneously.\n\nConclusions:\nDual regimen composed of vonoprazan and amoxicillin for 14 days was effective and safe as rescue therapy in Helicobacter pylori infection treatment. It could be chosen as a \"simplified rescue therapy\" with relatively high eradication rate no matter how many times the patients had failed and what regimens they had used previously."},"whole_article_text_length":{"kind":"number","value":8712,"string":"8,712"},"whole_article_abstract_length":{"kind":"number","value":319,"string":"319"},"other_sections_lengths":{"kind":"list like","value":[392,161,225,246,56,285,531,159,294],"string":"[\n 392,\n 161,\n 225,\n 246,\n 56,\n 285,\n 531,\n 159,\n 294\n]"},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["treatment","mg","patients","amoxicillin","therapy","failed","eradication","times","mic","dual"],"string":"[\n \"treatment\",\n \"mg\",\n \"patients\",\n \"amoxicillin\",\n \"therapy\",\n \"failed\",\n \"eradication\",\n \"times\",\n \"mic\",\n \"dual\"\n]"},"keybert_topics":{"kind":"list like","value":["antibiotic resistance pylori","pylori therapy include","regimen treatment pylori","outcome treatment pylori","previous pylori treatment"],"string":"[\n \"antibiotic resistance pylori\",\n \"pylori therapy include\",\n \"regimen treatment pylori\",\n \"outcome treatment pylori\",\n \"previous pylori treatment\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] amoxicillin | dual therapy | \nHelicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] amoxicillin | dual therapy | \nHelicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] amoxicillin | dual therapy | \nHelicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] amoxicillin | dual therapy | \nHelicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] amoxicillin | dual therapy | \nHelicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] different | ph | line | treatment | therapy | pylori | regimen | widely | plus amoxicillin dual | level [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] times | treatment | patients | 95 | events | adverse | adverse events | failed | 186 | eradication [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] effective safe | treatment pylori infection | safe | especially | effective | pylori | regimen | treatment pylori | treatment | day [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mg | treatment | patients | times | mic | amoxicillin | therapy | failed | eradication | dual [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mg | treatment | patients | times | mic | amoxicillin | therapy | failed | eradication | dual [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] two ||| first [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 186 | 67 | 119 ||| 1 | 7 ||| 172 | 92.5% | 172/186 ||| 14 | 7.5% | 14/186 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 14 days ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] two ||| first ||| May 2020 to June 2021 | GI | Peking University First Hospital | Beijing | China ||| 20 mg | 40 mg | 14 days ||| 13 | 6 weeks ||| ||| 186 | 67 | 119 ||| 1 | 7 ||| 172 | 92.5% | 172/186 ||| 14 | 7.5% | 14/186 ||| 14 days ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] two ||| first ||| May 2020 to June 2021 | GI | Peking University First Hospital | Beijing | China ||| 20 mg | 40 mg | 14 days ||| 13 | 6 weeks ||| ||| 186 | 67 | 119 ||| 1 | 7 ||| 172 | 92.5% | 172/186 ||| 14 | 7.5% | 14/186 ||| 14 days ||| [SUMMARY]"}}},{"rowIdx":3393,"cells":{"title":{"kind":"string","value":"Nationwide survey to evaluate the decision-making process in euthanasia requests in Belgium: do specifically trained 2nd physicians improve quality of consultation?"},"pmid":{"kind":"string","value":"25030375"},"background_abstract":{"kind":"string","value":"Following the 2002 enactment of the Belgian law on euthanasia, which requires the consultation of an independent second physician before proceeding with euthanasia, the Life End Information Forum (LEIF) was founded which provides specifically trained physicians who can act as mandatory consultants in euthanasia requests. This study assesses quality of consultations in Flanders and Brussels and compares these between LEIF and non-LEIF consultants."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A questionnaire was sent in 2009 to a random sample of 3,006 physicians in Belgium from specialties likely involved in the care of dying patients. Several questions about the last euthanasia request of one of their patients were asked. As LEIF serves the Flemish speaking community (i.e. region of Flanders and the bilingual Brussels Capital Region) and no similar counterpart is present in Wallonia, analyses were limited to Flemish speaking physicians in Flanders and Brussels."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Response was 34%. Of the 244 physicians who indicated having received a euthanasia request seventy percent consulted a second physician in their last request; in 30% this was with a LEIF physician. Compared to non-LEIF physicians, LEIF physicians were more often not a colleague (69% vs 42%) and not a co-attending physician (89% vs 66%). They tended to more often discuss the request with the attending physician (100% vs 95%) and with the family (76% vs 69%), and also more frequently helped the attending physician with performing euthanasia (44% vs 24%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%) and examined the patient file (94% vs 97%)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In cases of explicit euthanasia requests in Belgium, the consultation procedure of another physician by the attending physician is not optimal and can be improved. Training and putting at disposal consultants through forums such as LEIF seems able to improve this situation. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Irrespective of whether euthanasia is a legal practice within a country, similar services may prove useful to also improve quality of consultations in various other difficult end-of-life decision-making situations."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Belgium","Decision Making","Euthanasia","Humans","Practice Patterns, Physicians'","Referral and Consultation","Surveys and Questionnaires"],"string":"[\n \"Belgium\",\n \"Decision Making\",\n \"Euthanasia\",\n \"Humans\",\n \"Practice Patterns, Physicians'\",\n \"Referral and Consultation\",\n \"Surveys and Questionnaires\"\n]"},"pmcid":{"kind":"string","value":"4114442"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?"},"methods_title":{"kind":"string","value":"Method"},"methods_text":{"kind":"string","value":" Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\nIn March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\n Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\n Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\nFor all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\nOf the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\n Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\nTwo hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\n Quality of consultation Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\nTable \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\n Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\nThe consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table)."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.\nAs we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making."},"other_sections_titles":{"kind":"list like","value":["Background","Study design","Questionnaire","\nList of quality criteria as outlined by the Dutch consultation protocol\n","Statistical analysis","Response rate and response bias","Physicians receiving euthanasia requests and consulting a second physician","Quality of consultation","Advice of the consultant and outcome of the request","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Study design\",\n \"Questionnaire\",\n \"\\nList of quality criteria as outlined by the Dutch consultation protocol\\n\",\n \"Statistical analysis\",\n \"Response rate and response bias\",\n \"Physicians receiving euthanasia requests and consulting a second physician\",\n \"Quality of consultation\",\n \"Advice of the consultant and outcome of the request\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?","In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].","The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.","The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.","For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.","Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.","Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.","Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.","The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).","Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.","JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\n"],"string":"[\n \"Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\\n[5].\\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?\",\n \"In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\\n[17].\",\n \"The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\\n \\nList of quality criteria as outlined by the Dutch consultation protocol\\n The consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\\nThe consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\",\n \"The consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\",\n \"For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\",\n \"Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\",\n \"Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \\n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\",\n \"Table \\n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\\n*1 to 16 missing cases.\\n†1 to 3 missing cases.\\n‡1 to 13 missing cases.\\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \\n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\\n*2 to 22 missing cases.\\n†1 to 4 missing cases.\\n‡1 to 16 missing cases.\",\n \"The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \\n4).\\nAdvice of consultant according to whether the second physician is a LEIF physician or not\\n*3 missing cases.\\n†1 to 3 missing cases.\\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\",\n \"Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.\",\n \"JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Method","Study design","Questionnaire","\nList of quality criteria as outlined by the Dutch consultation protocol\n","Statistical analysis","Results","Response rate and response bias","Physicians receiving euthanasia requests and consulting a second physician","Quality of consultation","Advice of the consultant and outcome of the request","Discussion","Conclusion","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Method\",\n \"Study design\",\n \"Questionnaire\",\n \"\\nList of quality criteria as outlined by the Dutch consultation protocol\\n\",\n \"Statistical analysis\",\n \"Results\",\n \"Response rate and response bias\",\n \"Physicians receiving euthanasia requests and consulting a second physician\",\n \"Quality of consultation\",\n \"Advice of the consultant and outcome of the request\",\n \"Discussion\",\n \"Conclusion\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?"," Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\nIn March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\n Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\n Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\nFor all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.","In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].","The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.","The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.","For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0."," Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\nOf the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\n Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\nTwo hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\n Quality of consultation Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\nTable \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\n Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\nThe consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).","Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.","Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.","Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.","The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).","This study is the first to examine the quality of consultation between attending physicians and consultants in euthanasia requests in Belgium. We found that in 70% of the euthanasia requests described the attending physician had consulted with a second physician. Of the (Dutch) criteria for good practice, independence of the consultant, either from the physician or the patients, is the one most often unmet. Life End Information Forum (LEIF) physicians, who had undergone training as consultants in euthanasia requests, were significantly more often independent from the attending physician and from the patient compared with those who had not received the LEIF training.\nAn important strength of this study is that we used a large sample of physicians from diverse specialties to ask questions about a very sensitive subject. To this end, we used a rigorous sampling and mailing procedure. The questionnaire was comprehensively tested. There are however also some limitations. First, the low response percentage makes it difficult to generalize the results to all physicians in Flanders and Brussels, although analyses of the non-response survey indicate that the sample of responders was comparable to the sample of non-responders regarding having received a euthanasia request. As the survey is retrospective, there may be recall bias, especially for requests from more than a year earlier. Furthermore, the information provided in this survey on the activities of the consultants stems only from the attending physician. An additional limitation is that, with the absence of an initiative of similar magnitude and organisation present in Wallonia or for French speaking Belgium, the analyses had to be limited to Flemish speaking physicians in Flanders and Brussels. The presence of an initiative for Flemish speaking physicians but absence of one for French speaking physicians may reflect the fact that Flanders shares the language of and is culturally closer to the Netherlands. In addition, a previous study also indicated more positive attitudes among Flemish physicians towards the due care criteria of the euthanasia law and a larger willingness to comply with these criteria\n[20]. As regional differences in the euthanasia practice likely also reflect cultural differences\n[20] a comparison of LEIF physicians versus non-LEIF physicians for the whole of Belgium (with all French speaking physicians thus being in the non-LEIF group) would have been contaminated by these differences. Hence, although it is likely that a service like LEIF would have similar positive effects in French speaking Belgium, our results cannot just be generalized to French speaking Belgium.\nWe found that 70% of the physicians who had received a euthanasia request had consulted with a mandatory second physician and for the cases where euthanasia was eventually performed this was 92%. A previous study in the Netherlands found consultation to take place in 87% of requests reported by GPs in the period 2000–2002 and in 97% of cases where euthanasia was performed\n[21]. Compared with the Netherlands, consultation in Flanders is thus rather low, especially when taking into account that the percentages in the Netherlands date from a period before 2002, when euthanasia was tolerated but not yet legalized in this country. That attending physicians do not consult a second physician in 30% of euthanasia requests can partly be attributed to the fact that in some cases they have already decided not to grant the request and consider they do not need a colleague to confirm their decision. It is open to discussion whether physicians need to consult with colleague physicians about every euthanasia request even if they feel it is not serious enough or feel reluctant to comply with it. The 8% of performed cases of euthanasia where no consultation took place are, however, problematic and do not comply with the legal due care criteria. Not consulting in these cases can possibly be attributed to a lack of knowledge of the procedure or to the reluctance of attending physicians to be scrutinized by a colleague\n[22].\nConsultants in Flanders and Brussels were found not always to have examined or talked to the patient and a written report was not made in over one third of consultations. As these are prescribed in the euthanasia law as tasks of the consultant, it is surprising that these are not always met. It may not be possible to talk to all patients, especially if they have lost competency in decision-making (a situation that can also qualify for euthanasia if there is a previous written request or euthanasia declaration); however, our question also asked about an examination of the patient and it could be expected that at least this would have been done in patients who lost competency. The number of cases where neither were done is, however, very low. The cases without a legally required written report is a more frequent problem and may be due to the lack of knowledge about this legal criterion and a lack of legal control mechanisms to ascertain that a written report is made. An additional potential problem is that independence in relation to the physician and patient seemed often not guaranteed. In one third of cases the consultant was a co-attending physician of the patient. While the law does not describe precisely what is meant by independence\n[1], being a co-attending physician seems incongruent with the intention of the law that the consultant is able to give independent advice without influence from the attending physician or the patient. The fact that attending physicians do sometimes not seek an independent consultant could indicate that they consider the consultation merely a formality.\nOur study indicated that involving consultants who followed a special training instead of those who did not could contribute to various aspects of the quality of consultation. The most important contribution seems to be in terms of the legally required independence: the LEIF physicians in our study were more often independent than non-LEIF physicians from the attending physician and the patient (i.e. they were more often unacquainted with them). The contact procedure through the central telephone number of LEIF, which is intended to assign a random LEIF physician (preferably from the region) to the attending physician, seems to create a better guarantee of independence as opposed to simply consulting a colleague from the same hospital or practice. However, this service appears to be called upon in particular by general practitioners, and much less often by specialists in hospitals who often call on their direct colleagues, which may be more practical and private but jeopardizes independence. Specialists therefore might especially benefit from a service such as LEIF in order to comply fully with this aspect of the law.\nAlthough differences were not statistically significant, LEIF physicians also tended to more often discuss the request with the attending physician and the family than non-LEIF physicians. While these are not strict due care requirements stated in the law or the Dutch consultation protocol\n[19] they can also be seen as aspects determining the quality of a consultation, contributing to a more careful and qualitative consultation practice.\nInvolving specifically trained consultants can thus benefit euthanasia consultations in a number of aspects. A number of found differences between LEIF and non-LEIF consultants are perhaps not necessarily indicative of differences in the quality of consultation but nevertheless suggest relevant differences in consultation practice that merit further discussion. While the fact that LEIF-physicians less frequently discussed euthanasia requests with other attending physicians (i.e. colleague-attending physicians) is a result of the fact that LEIF physicians are more often consulted by GPs rather than by hospital specialists, the most striking difference with non-LEIF consultations is that LEIF consultations significantly more often led to a positive advice on the euthanasia request (i.e. a judgement that the conditions for euthanasia have been met) and significantly more often involved help with performing euthanasia. It may be that these differences reflect a more positive attitude towards euthanasia in general in LEIF physicians. Not being fundamentally against euthanasia is a prerequisite to being admitted to the LEIF training programme\n[6]. It has to be acknowledged that underlying attitudinal aspects may result in different outcomes of a consultation, which is inherent to the fact that assessing the nature and validity of a euthanasia request does not merely involve checking off objective criteria but also involves a great deal of subjective and compassionate judgement. While some may find in this finding a proof for too high a compliance with euthanasia requests in LEIF physicians, others will argue that the higher reluctance of non-LEIF consultants to give a positive advice about a euthanasia request is due to their larger uncertainty about the due care criteria and the procedure since they lack the specific training and similar experience.\nSimilarly some will argue that helping with the performance of euthanasia is not and cannot be the responsibility of a consultant whereas others will see it as a pedagogic assistance for attending physicians. The function of role models in medical education, particularly in end-of-life care, and their influence on ethical decision-making has been described in other studies\n[23-27] and previous research indicated that LEIF consultants help with euthanasia for medico-technical reasons, because they consider it important that a more experienced colleague can show the attending physician how to perform a delicate act they are not prepared for through the medical curriculum\n[7].\nThe model of LEIF, as a training and consultation service, is strongly based and inspired on the model of SCEN (Support and Consultation for Euthanasia in the Netherlands) in the Netherlands\n[6], which was already developed prior to the euthanasia legalisation in 2002 in the Netherlands\n[28]. The fact that SCEN is more regulated, organised on a larger scale, more endorsed by government, and puts more focus on formal aspects of the consultation (e.g. the written consultation report)\n[6], but also the more specific stipulations in the Dutch euthanasia law about the activities that are required by a consultant in a euthanasia request may be explanations for the larger compliance with legal requirements in SCEN physicians and in the Netherlands in general\n[29].\nThe euthanasia law in Belgium has recently (and only after our study was conducted) been adapted to include competent minor patients\n[30]. Although the practice of euthanasia in minor patients will likely continue to be a very rare and marginal practice (a previous study including all deaths of minor persons in Flanders during one and a half year identified no case of euthanasia\n[31]), the specific difficulties and challenges a careful euthanasia practice in this population poses will require LEIF to pay specific attention to euthanasia requests in minor patients in its trainings.","In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.\nAs we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making.","Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.","JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\n"],"string":"[\n \"Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\\n[5].\\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?\",\n \" Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\\n[17].\\nIn March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\\n[17].\\n Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\\n \\nList of quality criteria as outlined by the Dutch consultation protocol\\n The consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\\nThe consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\\nThe pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\\n \\nList of quality criteria as outlined by the Dutch consultation protocol\\n The consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\\nThe consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\\n Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\\nFor all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\",\n \"In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\\n[17].\",\n \"The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\\n \\nList of quality criteria as outlined by the Dutch consultation protocol\\n The consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\\nThe consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\",\n \"The consultant\\n– was not a colleague of the attending physician\\n– was not a co-attending physician\\n– did not know the patient\\n– talked to/examined the patient\\n– considered the request*\\n– talked about possible alternatives†\\n– made a written report\\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\",\n \"For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\",\n \" Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\\nOf the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\\n Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \\n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\\nTwo hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \\n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\\n Quality of consultation Table \\n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\\n*1 to 16 missing cases.\\n†1 to 3 missing cases.\\n‡1 to 13 missing cases.\\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \\n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\\n*2 to 22 missing cases.\\n†1 to 4 missing cases.\\n‡1 to 16 missing cases.\\nTable \\n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\\n*1 to 16 missing cases.\\n†1 to 3 missing cases.\\n‡1 to 13 missing cases.\\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \\n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\\n*2 to 22 missing cases.\\n†1 to 4 missing cases.\\n‡1 to 16 missing cases.\\n Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \\n4).\\nAdvice of consultant according to whether the second physician is a LEIF physician or not\\n*3 missing cases.\\n†1 to 3 missing cases.\\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\\nThe consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \\n4).\\nAdvice of consultant according to whether the second physician is a LEIF physician or not\\n*3 missing cases.\\n†1 to 3 missing cases.\\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\",\n \"Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\",\n \"Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \\n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\",\n \"Table \\n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\\n*1 to 16 missing cases.\\n†1 to 3 missing cases.\\n‡1 to 13 missing cases.\\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \\n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\\n*2 to 22 missing cases.\\n†1 to 4 missing cases.\\n‡1 to 16 missing cases.\",\n \"The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \\n4).\\nAdvice of consultant according to whether the second physician is a LEIF physician or not\\n*3 missing cases.\\n†1 to 3 missing cases.\\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\",\n \"This study is the first to examine the quality of consultation between attending physicians and consultants in euthanasia requests in Belgium. We found that in 70% of the euthanasia requests described the attending physician had consulted with a second physician. Of the (Dutch) criteria for good practice, independence of the consultant, either from the physician or the patients, is the one most often unmet. Life End Information Forum (LEIF) physicians, who had undergone training as consultants in euthanasia requests, were significantly more often independent from the attending physician and from the patient compared with those who had not received the LEIF training.\\nAn important strength of this study is that we used a large sample of physicians from diverse specialties to ask questions about a very sensitive subject. To this end, we used a rigorous sampling and mailing procedure. The questionnaire was comprehensively tested. There are however also some limitations. First, the low response percentage makes it difficult to generalize the results to all physicians in Flanders and Brussels, although analyses of the non-response survey indicate that the sample of responders was comparable to the sample of non-responders regarding having received a euthanasia request. As the survey is retrospective, there may be recall bias, especially for requests from more than a year earlier. Furthermore, the information provided in this survey on the activities of the consultants stems only from the attending physician. An additional limitation is that, with the absence of an initiative of similar magnitude and organisation present in Wallonia or for French speaking Belgium, the analyses had to be limited to Flemish speaking physicians in Flanders and Brussels. The presence of an initiative for Flemish speaking physicians but absence of one for French speaking physicians may reflect the fact that Flanders shares the language of and is culturally closer to the Netherlands. In addition, a previous study also indicated more positive attitudes among Flemish physicians towards the due care criteria of the euthanasia law and a larger willingness to comply with these criteria\\n[20]. As regional differences in the euthanasia practice likely also reflect cultural differences\\n[20] a comparison of LEIF physicians versus non-LEIF physicians for the whole of Belgium (with all French speaking physicians thus being in the non-LEIF group) would have been contaminated by these differences. Hence, although it is likely that a service like LEIF would have similar positive effects in French speaking Belgium, our results cannot just be generalized to French speaking Belgium.\\nWe found that 70% of the physicians who had received a euthanasia request had consulted with a mandatory second physician and for the cases where euthanasia was eventually performed this was 92%. A previous study in the Netherlands found consultation to take place in 87% of requests reported by GPs in the period 2000–2002 and in 97% of cases where euthanasia was performed\\n[21]. Compared with the Netherlands, consultation in Flanders is thus rather low, especially when taking into account that the percentages in the Netherlands date from a period before 2002, when euthanasia was tolerated but not yet legalized in this country. That attending physicians do not consult a second physician in 30% of euthanasia requests can partly be attributed to the fact that in some cases they have already decided not to grant the request and consider they do not need a colleague to confirm their decision. It is open to discussion whether physicians need to consult with colleague physicians about every euthanasia request even if they feel it is not serious enough or feel reluctant to comply with it. The 8% of performed cases of euthanasia where no consultation took place are, however, problematic and do not comply with the legal due care criteria. Not consulting in these cases can possibly be attributed to a lack of knowledge of the procedure or to the reluctance of attending physicians to be scrutinized by a colleague\\n[22].\\nConsultants in Flanders and Brussels were found not always to have examined or talked to the patient and a written report was not made in over one third of consultations. As these are prescribed in the euthanasia law as tasks of the consultant, it is surprising that these are not always met. It may not be possible to talk to all patients, especially if they have lost competency in decision-making (a situation that can also qualify for euthanasia if there is a previous written request or euthanasia declaration); however, our question also asked about an examination of the patient and it could be expected that at least this would have been done in patients who lost competency. The number of cases where neither were done is, however, very low. The cases without a legally required written report is a more frequent problem and may be due to the lack of knowledge about this legal criterion and a lack of legal control mechanisms to ascertain that a written report is made. An additional potential problem is that independence in relation to the physician and patient seemed often not guaranteed. In one third of cases the consultant was a co-attending physician of the patient. While the law does not describe precisely what is meant by independence\\n[1], being a co-attending physician seems incongruent with the intention of the law that the consultant is able to give independent advice without influence from the attending physician or the patient. The fact that attending physicians do sometimes not seek an independent consultant could indicate that they consider the consultation merely a formality.\\nOur study indicated that involving consultants who followed a special training instead of those who did not could contribute to various aspects of the quality of consultation. The most important contribution seems to be in terms of the legally required independence: the LEIF physicians in our study were more often independent than non-LEIF physicians from the attending physician and the patient (i.e. they were more often unacquainted with them). The contact procedure through the central telephone number of LEIF, which is intended to assign a random LEIF physician (preferably from the region) to the attending physician, seems to create a better guarantee of independence as opposed to simply consulting a colleague from the same hospital or practice. However, this service appears to be called upon in particular by general practitioners, and much less often by specialists in hospitals who often call on their direct colleagues, which may be more practical and private but jeopardizes independence. Specialists therefore might especially benefit from a service such as LEIF in order to comply fully with this aspect of the law.\\nAlthough differences were not statistically significant, LEIF physicians also tended to more often discuss the request with the attending physician and the family than non-LEIF physicians. While these are not strict due care requirements stated in the law or the Dutch consultation protocol\\n[19] they can also be seen as aspects determining the quality of a consultation, contributing to a more careful and qualitative consultation practice.\\nInvolving specifically trained consultants can thus benefit euthanasia consultations in a number of aspects. A number of found differences between LEIF and non-LEIF consultants are perhaps not necessarily indicative of differences in the quality of consultation but nevertheless suggest relevant differences in consultation practice that merit further discussion. While the fact that LEIF-physicians less frequently discussed euthanasia requests with other attending physicians (i.e. colleague-attending physicians) is a result of the fact that LEIF physicians are more often consulted by GPs rather than by hospital specialists, the most striking difference with non-LEIF consultations is that LEIF consultations significantly more often led to a positive advice on the euthanasia request (i.e. a judgement that the conditions for euthanasia have been met) and significantly more often involved help with performing euthanasia. It may be that these differences reflect a more positive attitude towards euthanasia in general in LEIF physicians. Not being fundamentally against euthanasia is a prerequisite to being admitted to the LEIF training programme\\n[6]. It has to be acknowledged that underlying attitudinal aspects may result in different outcomes of a consultation, which is inherent to the fact that assessing the nature and validity of a euthanasia request does not merely involve checking off objective criteria but also involves a great deal of subjective and compassionate judgement. While some may find in this finding a proof for too high a compliance with euthanasia requests in LEIF physicians, others will argue that the higher reluctance of non-LEIF consultants to give a positive advice about a euthanasia request is due to their larger uncertainty about the due care criteria and the procedure since they lack the specific training and similar experience.\\nSimilarly some will argue that helping with the performance of euthanasia is not and cannot be the responsibility of a consultant whereas others will see it as a pedagogic assistance for attending physicians. The function of role models in medical education, particularly in end-of-life care, and their influence on ethical decision-making has been described in other studies\\n[23-27] and previous research indicated that LEIF consultants help with euthanasia for medico-technical reasons, because they consider it important that a more experienced colleague can show the attending physician how to perform a delicate act they are not prepared for through the medical curriculum\\n[7].\\nThe model of LEIF, as a training and consultation service, is strongly based and inspired on the model of SCEN (Support and Consultation for Euthanasia in the Netherlands) in the Netherlands\\n[6], which was already developed prior to the euthanasia legalisation in 2002 in the Netherlands\\n[28]. The fact that SCEN is more regulated, organised on a larger scale, more endorsed by government, and puts more focus on formal aspects of the consultation (e.g. the written consultation report)\\n[6], but also the more specific stipulations in the Dutch euthanasia law about the activities that are required by a consultant in a euthanasia request may be explanations for the larger compliance with legal requirements in SCEN physicians and in the Netherlands in general\\n[29].\\nThe euthanasia law in Belgium has recently (and only after our study was conducted) been adapted to include competent minor patients\\n[30]. Although the practice of euthanasia in minor patients will likely continue to be a very rare and marginal practice (a previous study including all deaths of minor persons in Flanders during one and a half year identified no case of euthanasia\\n[31]), the specific difficulties and challenges a careful euthanasia practice in this population poses will require LEIF to pay specific attention to euthanasia requests in minor patients in its trainings.\",\n \"In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.\\nAs we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making.\",\n \"Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.\",\n \"JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results",null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Euthanasia","Consultation","Referral practice","Terminal care"],"string":"[\n \"Euthanasia\",\n \"Consultation\",\n \"Referral practice\",\n \"Terminal care\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nEuthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?\n\nMethod:\n Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\nIn March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\n Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\n Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\nFor all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\n\nStudy design:\nIn March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\n\nQuestionnaire:\nThe pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\n\n\nList of quality criteria as outlined by the Dutch consultation protocol\n:\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\n\nStatistical analysis:\nFor all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\n\nResults:\n Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\nOf the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\n Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\nTwo hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\n Quality of consultation Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\nTable \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\n Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\nThe consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\n\nResponse rate and response bias:\nOf the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\n\nPhysicians receiving euthanasia requests and consulting a second physician:\nTwo hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\n\nQuality of consultation:\nTable \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\n\nAdvice of the consultant and outcome of the request:\nThe consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\n\nDiscussion:\nThis study is the first to examine the quality of consultation between attending physicians and consultants in euthanasia requests in Belgium. We found that in 70% of the euthanasia requests described the attending physician had consulted with a second physician. Of the (Dutch) criteria for good practice, independence of the consultant, either from the physician or the patients, is the one most often unmet. Life End Information Forum (LEIF) physicians, who had undergone training as consultants in euthanasia requests, were significantly more often independent from the attending physician and from the patient compared with those who had not received the LEIF training.\nAn important strength of this study is that we used a large sample of physicians from diverse specialties to ask questions about a very sensitive subject. To this end, we used a rigorous sampling and mailing procedure. The questionnaire was comprehensively tested. There are however also some limitations. First, the low response percentage makes it difficult to generalize the results to all physicians in Flanders and Brussels, although analyses of the non-response survey indicate that the sample of responders was comparable to the sample of non-responders regarding having received a euthanasia request. As the survey is retrospective, there may be recall bias, especially for requests from more than a year earlier. Furthermore, the information provided in this survey on the activities of the consultants stems only from the attending physician. An additional limitation is that, with the absence of an initiative of similar magnitude and organisation present in Wallonia or for French speaking Belgium, the analyses had to be limited to Flemish speaking physicians in Flanders and Brussels. The presence of an initiative for Flemish speaking physicians but absence of one for French speaking physicians may reflect the fact that Flanders shares the language of and is culturally closer to the Netherlands. In addition, a previous study also indicated more positive attitudes among Flemish physicians towards the due care criteria of the euthanasia law and a larger willingness to comply with these criteria\n[20]. As regional differences in the euthanasia practice likely also reflect cultural differences\n[20] a comparison of LEIF physicians versus non-LEIF physicians for the whole of Belgium (with all French speaking physicians thus being in the non-LEIF group) would have been contaminated by these differences. Hence, although it is likely that a service like LEIF would have similar positive effects in French speaking Belgium, our results cannot just be generalized to French speaking Belgium.\nWe found that 70% of the physicians who had received a euthanasia request had consulted with a mandatory second physician and for the cases where euthanasia was eventually performed this was 92%. A previous study in the Netherlands found consultation to take place in 87% of requests reported by GPs in the period 2000–2002 and in 97% of cases where euthanasia was performed\n[21]. Compared with the Netherlands, consultation in Flanders is thus rather low, especially when taking into account that the percentages in the Netherlands date from a period before 2002, when euthanasia was tolerated but not yet legalized in this country. That attending physicians do not consult a second physician in 30% of euthanasia requests can partly be attributed to the fact that in some cases they have already decided not to grant the request and consider they do not need a colleague to confirm their decision. It is open to discussion whether physicians need to consult with colleague physicians about every euthanasia request even if they feel it is not serious enough or feel reluctant to comply with it. The 8% of performed cases of euthanasia where no consultation took place are, however, problematic and do not comply with the legal due care criteria. Not consulting in these cases can possibly be attributed to a lack of knowledge of the procedure or to the reluctance of attending physicians to be scrutinized by a colleague\n[22].\nConsultants in Flanders and Brussels were found not always to have examined or talked to the patient and a written report was not made in over one third of consultations. As these are prescribed in the euthanasia law as tasks of the consultant, it is surprising that these are not always met. It may not be possible to talk to all patients, especially if they have lost competency in decision-making (a situation that can also qualify for euthanasia if there is a previous written request or euthanasia declaration); however, our question also asked about an examination of the patient and it could be expected that at least this would have been done in patients who lost competency. The number of cases where neither were done is, however, very low. The cases without a legally required written report is a more frequent problem and may be due to the lack of knowledge about this legal criterion and a lack of legal control mechanisms to ascertain that a written report is made. An additional potential problem is that independence in relation to the physician and patient seemed often not guaranteed. In one third of cases the consultant was a co-attending physician of the patient. While the law does not describe precisely what is meant by independence\n[1], being a co-attending physician seems incongruent with the intention of the law that the consultant is able to give independent advice without influence from the attending physician or the patient. The fact that attending physicians do sometimes not seek an independent consultant could indicate that they consider the consultation merely a formality.\nOur study indicated that involving consultants who followed a special training instead of those who did not could contribute to various aspects of the quality of consultation. The most important contribution seems to be in terms of the legally required independence: the LEIF physicians in our study were more often independent than non-LEIF physicians from the attending physician and the patient (i.e. they were more often unacquainted with them). The contact procedure through the central telephone number of LEIF, which is intended to assign a random LEIF physician (preferably from the region) to the attending physician, seems to create a better guarantee of independence as opposed to simply consulting a colleague from the same hospital or practice. However, this service appears to be called upon in particular by general practitioners, and much less often by specialists in hospitals who often call on their direct colleagues, which may be more practical and private but jeopardizes independence. Specialists therefore might especially benefit from a service such as LEIF in order to comply fully with this aspect of the law.\nAlthough differences were not statistically significant, LEIF physicians also tended to more often discuss the request with the attending physician and the family than non-LEIF physicians. While these are not strict due care requirements stated in the law or the Dutch consultation protocol\n[19] they can also be seen as aspects determining the quality of a consultation, contributing to a more careful and qualitative consultation practice.\nInvolving specifically trained consultants can thus benefit euthanasia consultations in a number of aspects. A number of found differences between LEIF and non-LEIF consultants are perhaps not necessarily indicative of differences in the quality of consultation but nevertheless suggest relevant differences in consultation practice that merit further discussion. While the fact that LEIF-physicians less frequently discussed euthanasia requests with other attending physicians (i.e. colleague-attending physicians) is a result of the fact that LEIF physicians are more often consulted by GPs rather than by hospital specialists, the most striking difference with non-LEIF consultations is that LEIF consultations significantly more often led to a positive advice on the euthanasia request (i.e. a judgement that the conditions for euthanasia have been met) and significantly more often involved help with performing euthanasia. It may be that these differences reflect a more positive attitude towards euthanasia in general in LEIF physicians. Not being fundamentally against euthanasia is a prerequisite to being admitted to the LEIF training programme\n[6]. It has to be acknowledged that underlying attitudinal aspects may result in different outcomes of a consultation, which is inherent to the fact that assessing the nature and validity of a euthanasia request does not merely involve checking off objective criteria but also involves a great deal of subjective and compassionate judgement. While some may find in this finding a proof for too high a compliance with euthanasia requests in LEIF physicians, others will argue that the higher reluctance of non-LEIF consultants to give a positive advice about a euthanasia request is due to their larger uncertainty about the due care criteria and the procedure since they lack the specific training and similar experience.\nSimilarly some will argue that helping with the performance of euthanasia is not and cannot be the responsibility of a consultant whereas others will see it as a pedagogic assistance for attending physicians. The function of role models in medical education, particularly in end-of-life care, and their influence on ethical decision-making has been described in other studies\n[23-27] and previous research indicated that LEIF consultants help with euthanasia for medico-technical reasons, because they consider it important that a more experienced colleague can show the attending physician how to perform a delicate act they are not prepared for through the medical curriculum\n[7].\nThe model of LEIF, as a training and consultation service, is strongly based and inspired on the model of SCEN (Support and Consultation for Euthanasia in the Netherlands) in the Netherlands\n[6], which was already developed prior to the euthanasia legalisation in 2002 in the Netherlands\n[28]. The fact that SCEN is more regulated, organised on a larger scale, more endorsed by government, and puts more focus on formal aspects of the consultation (e.g. the written consultation report)\n[6], but also the more specific stipulations in the Dutch euthanasia law about the activities that are required by a consultant in a euthanasia request may be explanations for the larger compliance with legal requirements in SCEN physicians and in the Netherlands in general\n[29].\nThe euthanasia law in Belgium has recently (and only after our study was conducted) been adapted to include competent minor patients\n[30]. Although the practice of euthanasia in minor patients will likely continue to be a very rare and marginal practice (a previous study including all deaths of minor persons in Flanders during one and a half year identified no case of euthanasia\n[31]), the specific difficulties and challenges a careful euthanasia practice in this population poses will require LEIF to pay specific attention to euthanasia requests in minor patients in its trainings.\n\nConclusion:\nIn cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.\nAs we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making.\n\nCompeting interests:\nNon-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.\n\nAuthors’ contributions:\nJC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nFollowing the 2002 enactment of the Belgian law on euthanasia, which requires the consultation of an independent second physician before proceeding with euthanasia, the Life End Information Forum (LEIF) was founded which provides specifically trained physicians who can act as mandatory consultants in euthanasia requests. This study assesses quality of consultations in Flanders and Brussels and compares these between LEIF and non-LEIF consultants.\n\nMethods:\nA questionnaire was sent in 2009 to a random sample of 3,006 physicians in Belgium from specialties likely involved in the care of dying patients. Several questions about the last euthanasia request of one of their patients were asked. As LEIF serves the Flemish speaking community (i.e. region of Flanders and the bilingual Brussels Capital Region) and no similar counterpart is present in Wallonia, analyses were limited to Flemish speaking physicians in Flanders and Brussels.\n\nResults:\nResponse was 34%. Of the 244 physicians who indicated having received a euthanasia request seventy percent consulted a second physician in their last request; in 30% this was with a LEIF physician. Compared to non-LEIF physicians, LEIF physicians were more often not a colleague (69% vs 42%) and not a co-attending physician (89% vs 66%). They tended to more often discuss the request with the attending physician (100% vs 95%) and with the family (76% vs 69%), and also more frequently helped the attending physician with performing euthanasia (44% vs 24%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%) and examined the patient file (94% vs 97%).\n\nConclusions:\nIn cases of explicit euthanasia requests in Belgium, the consultation procedure of another physician by the attending physician is not optimal and can be improved. Training and putting at disposal consultants through forums such as LEIF seems able to improve this situation. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Irrespective of whether euthanasia is a legal practice within a country, similar services may prove useful to also improve quality of consultations in various other difficult end-of-life decision-making situations."},"background_conclusion_text":{"kind":"string","value":"Background:\nEuthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?\n\nConclusion:\nIn cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.\nAs we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nFollowing the 2002 enactment of the Belgian law on euthanasia, which requires the consultation of an independent second physician before proceeding with euthanasia, the Life End Information Forum (LEIF) was founded which provides specifically trained physicians who can act as mandatory consultants in euthanasia requests. This study assesses quality of consultations in Flanders and Brussels and compares these between LEIF and non-LEIF consultants.\n\nMethods:\nA questionnaire was sent in 2009 to a random sample of 3,006 physicians in Belgium from specialties likely involved in the care of dying patients. Several questions about the last euthanasia request of one of their patients were asked. As LEIF serves the Flemish speaking community (i.e. region of Flanders and the bilingual Brussels Capital Region) and no similar counterpart is present in Wallonia, analyses were limited to Flemish speaking physicians in Flanders and Brussels.\n\nResults:\nResponse was 34%. Of the 244 physicians who indicated having received a euthanasia request seventy percent consulted a second physician in their last request; in 30% this was with a LEIF physician. Compared to non-LEIF physicians, LEIF physicians were more often not a colleague (69% vs 42%) and not a co-attending physician (89% vs 66%). They tended to more often discuss the request with the attending physician (100% vs 95%) and with the family (76% vs 69%), and also more frequently helped the attending physician with performing euthanasia (44% vs 24%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%) and examined the patient file (94% vs 97%).\n\nConclusions:\nIn cases of explicit euthanasia requests in Belgium, the consultation procedure of another physician by the attending physician is not optimal and can be improved. Training and putting at disposal consultants through forums such as LEIF seems able to improve this situation. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Irrespective of whether euthanasia is a legal practice within a country, similar services may prove useful to also improve quality of consultations in various other difficult end-of-life decision-making situations."},"whole_article_text_length":{"kind":"number","value":8968,"string":"8,968"},"whole_article_abstract_length":{"kind":"number","value":427,"string":"427"},"other_sections_lengths":{"kind":"list like","value":[936,349,381,108,76,111,241,373,261,73,71,16],"string":"[\n 936,\n 349,\n 381,\n 108,\n 76,\n 111,\n 241,\n 373,\n 261,\n 73,\n 71,\n 16\n]"},"num_sections":{"kind":"number","value":16,"string":"16"},"most_frequent_words":{"kind":"list like","value":["physician","leif","euthanasia","physicians","request","attending","non","consultant","attending physician","cases"],"string":"[\n \"physician\",\n \"leif\",\n \"euthanasia\",\n \"physicians\",\n \"request\",\n \"attending\",\n \"non\",\n \"consultant\",\n \"attending physician\",\n \"cases\"\n]"},"keybert_topics":{"kind":"list like","value":["physician consulted euthanasia","cases euthanasia consultation","person requesting euthanasia","euthanasia request physicians","consultant euthanasia request"],"string":"[\n \"physician consulted euthanasia\",\n \"cases euthanasia consultation\",\n \"person requesting euthanasia\",\n \"euthanasia request physicians\",\n \"consultant euthanasia request\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Euthanasia | Consultation | Referral practice | Terminal care [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Euthanasia | Consultation | Referral practice | Terminal care [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Euthanasia | Consultation | Referral practice | Terminal care [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Euthanasia | Consultation | Referral practice | Terminal care [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Euthanasia | Consultation | Referral practice | Terminal care [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Euthanasia | Consultation | Referral practice | Terminal care [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Belgium | Decision Making | Euthanasia | Humans | Practice Patterns, Physicians' | Referral 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[SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Belgium | Decision Making | Euthanasia | Humans | Practice Patterns, Physicians' | Referral and Consultation | Surveys and Questionnaires [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] physician consulted euthanasia | cases euthanasia consultation | person requesting euthanasia | euthanasia request physicians | consultant euthanasia request [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] physician consulted euthanasia | cases euthanasia consultation | person requesting euthanasia | euthanasia request physicians | consultant euthanasia request [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] physician consulted euthanasia | cases euthanasia consultation | person requesting euthanasia | euthanasia request physicians | consultant euthanasia request 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| physicians | request | attending | non | consultant | attending physician | cases [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] physician | euthanasia | leif | care | person | leif physician | palliative | palliative care | training | request [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] belgian | physician | questionnaire | euthanasia | register | questions | patient | criterion questioned belgian | criterion questioned | questioned belgian [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] leif | physician | cases | leif physician | missing cases | missing | physicians | vs | euthanasia | table [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] findings | consultation | relevance | end | life | belgium | contribute | euthanasia | service | end life [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] physician | leif | euthanasia | physicians | belgian | attending | request | non | attending physician | cases [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] physician | leif | euthanasia | physicians | belgian | attending | request | non | attending physician | cases [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2002 | Belgian | second | the Life End Information Forum | euthanasia ||| Flanders | Brussels [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 2009 | 3,006 | Belgium ||| one ||| Flemish | Flanders | Brussels Capital Region | Wallonia | Flemish | Flanders | Brussels [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Response | 34% ||| 244 | seventy percent | second | 30% ||| 69% | 42% | 89% | 66% ||| 100% | 95% | 76% | 69% | 44% | 24% ||| 96% | 93% | 94% | 97% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Belgium ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2002 | Belgian | second | the Life End Information Forum | euthanasia ||| Flanders | Brussels ||| 2009 | 3,006 | Belgium ||| one ||| Flemish | Flanders | Brussels Capital Region | Wallonia | Flemish | Flanders | Brussels ||| Response | 34% ||| 244 | seventy percent | second | 30% ||| 69% | 42% | 89% | 66% ||| 100% | 95% | 76% | 69% | 44% | 24% ||| 96% | 93% | 94% | 97% ||| Belgium ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] 2002 | Belgian | second | the Life End Information Forum | euthanasia ||| Flanders | Brussels ||| 2009 | 3,006 | Belgium ||| one ||| Flemish | Flanders | Brussels Capital Region | Wallonia | Flemish | Flanders | Brussels ||| Response | 34% ||| 244 | seventy percent | second | 30% ||| 69% | 42% | 89% | 66% ||| 100% | 95% | 76% | 69% | 44% | 24% ||| 96% | 93% | 94% | 97% ||| Belgium ||| ||| ||| [SUMMARY]"}}},{"rowIdx":3394,"cells":{"title":{"kind":"string","value":"Knowledge, preventive behavior and risk perception regarding COVID-19: a self-reported study on college students."},"pmid":{"kind":"string","value":"33623599"},"background_abstract":{"kind":"string","value":"There are a limited number of studies on the issues associated with the knowledge and self-practice preventive measures for COVID-19 among medical students. We aimed to determine the extent of knowledge, self-reported preventive behavior, and risk perception of the COVID-19 outbreak among college students in Libya."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"A cross-sectional study was conducted from April 20 to April 30, 2020. The participants were students of medical and non-medical subjects from Libyan educational institutes. Data on participants' characteristics, knowledge, preventive behavior, and risk perception were collected."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Approximately 3669 participants completed the questionnaire, of which 2547 (69.4) were medical students and 1122 (30.6%) were non-medical students. The mean knowledge score on COVID-19 was 8.62 (SD: 1.26, range: 0-12), corresponding to 71.8% correct answers. A significant difference was observed between medical and non-medical students in terms of knowledge (p < 0.001). Overall, the knowledge score of the students differed significantly with respect to age, current year of study, and financial source (p < 0.05). The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Notably, college students were observed to have substantial knowledge, preventive behavior, and a positive attitude toward COVID-19. Government programs should aim to educate individuals from other sectors of the society to ensure the proper dissemination of knowledge on preventive safety measures, as this will help restrict and control the pandemic."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","COVID-19","Cross-Sectional Studies","Disease Outbreaks","Female","Health Knowledge, Attitudes, Practice","Humans","Libya","Male","Perception","Self Report","Students","Students, Medical","Surveys and Questionnaires","Universities","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"COVID-19\",\n \"Cross-Sectional Studies\",\n \"Disease Outbreaks\",\n \"Female\",\n \"Health Knowledge, Attitudes, Practice\",\n \"Humans\",\n \"Libya\",\n \"Male\",\n \"Perception\",\n \"Self Report\",\n \"Students\",\n \"Students, Medical\",\n \"Surveys and Questionnaires\",\n \"Universities\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"7875787"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].\nThe development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.\nMedical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Study setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.\nCOVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.\nPreventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.\nStatistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.\nEthical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Out of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.\nCharacteristics of individuals in the study\nQuestionnaire on knowledge toward COVID-19\nDifferences in knowledge score according to study characteristics\nThe mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.\nQuestionnaire on preventive behavior and attitude toward COVID-19\nTable 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).\nDifferences in preventive score according to study characteristics"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.\nWhat is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nWhat this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups."},"other_sections_titles":{"kind":"list like","value":["What is known about this topic","What this study adds"],"string":"[\n \"What is known about this topic\",\n \"What this study adds\"\n]"},"other_sections_texts":{"kind":"list like","value":["The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.","Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups."],"string":"[\n \"The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\",\n \"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null],"string":"[\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Results","Discussion","Conclusion","What is known about this topic","What this study adds","Competing interests"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusion\",\n \"What is known about this topic\",\n \"What this study adds\",\n \"Competing interests\"\n]"},"all_sections_texts":{"kind":"list like","value":["The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].\nThe development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.\nMedical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.","Study setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.\nCOVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.\nPreventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.\nStatistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.\nEthical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study.","Out of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.\nCharacteristics of individuals in the study\nQuestionnaire on knowledge toward COVID-19\nDifferences in knowledge score according to study characteristics\nThe mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.\nQuestionnaire on preventive behavior and attitude toward COVID-19\nTable 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).\nDifferences in preventive score according to study characteristics","To the best of our knowledge, this is the first study conducted in Africa that discusses knowledge, self-preventive measures, and attitudes toward COVID-19 among college students, who constitute an integral part of an educated society. Overall, 71.8% of the study participants were aware of COVID-19, while more than 92.7% of both medical and non-medical students were taking preventive measures against COVID-19 to reduce the risk of infection transmission. Our study provides an overview of the knowledge of medical students, who will work as physicians in future, and are currently engaged in hospitals and at a high risk of infection transmission; the study compared their knowledge, preventive measures, and attitude toward COVID-19 to that of non-medical students to determine the difference. A majority of the participants (87.7% of non-medical students and 84.8% of medical students) opined that the COVID-19 pandemic could be controlled successfully, while a lower percentage of participants (74.6% non-medical students and 73.5% medical students) believed that the disease could be controlled in Libya. Contrastingly, the preventive behavior score among students was encouraging, as indicated by a high mean score of 7.4 ± 0.93 in non-medical students and 7.43 ± 0.96 of medical student out of 8, which corresponds to approximately 7.4/8 *100 = 92.5% of non-medical student and 7.43/8 *100 = 92.87% of medical students; this suggests that the students have a positive preventive behavior and attitude toward COVID-19. Students in their internship year had better knowledge and greater preventive behavior owing to the extent of time they spend at the medical department, where they work at a higher risk of exposure and encounter more cases. Therefore, their knowledge and attitude are attributed to their experience.\nApproximately 97% participants from both groups reported avoidance of crowds and shopping, which displays the high levels of caution among the individuals. In addition, we identified several factors that can be associated with the preventive behavior and knowledge of the participants toward COVID-19. Our findings are necessary to understand the current state of public health awareness and to determine the need for proper dissemination of knowledge and awareness among students who are medical practitioners of the future and may possibly come in contact with infected individuals during their rotation in the hospitals. As medical students are at a higher risk of contracting infection and disease transmission owing to the course of study in hospitals, their knowledge and awareness of preventive measures are crucial for reducing the risk of community spread of SARS-CoV-2. Despite the healthcare situation and civil war in Libya, college students achieved high scores in knowledge, preventive behavior, and attitude toward COVID-19 infection. There was no significant difference between the rates of correct answers to most questions asked to either medical or non-medical students, which displays the substantial overall knowledge among college students. Their level of knowledge, preventive behavior, and attitude owing to their active learning and sources of information on COVID-19 yielded positive results. A similar study conducted on a Chinese population reported an overall knowledge of 90% [18]. Another unpublished study conducted by Haque et al. on a Bangladeshi population reported an overall knowledge score of 54.87% toward COVID-19 [19]. In addition, another study conducted on the Nepalese population reported an overall knowledge score ranging from 60.0-98.7% and attitude scores ranging from 77.9-96.4%. The study also confirmed that participants with a medical degree had greater knowledge than non-medical participants [20]. Another study conducted on an Egyptian population comprising 559 participants reported the mean and standard deviation of knowledge score as 16.39±2.63, ranging from 7 to 22, which corresponds to approximately 74.5% overall knowledge among participants regarding COVID-19 [17]. Another study in Thailand reported 67.9% as the overall level of knowledge toward COVID-19 prevention and only 29.5% were reported to have substantial knowledge about the preventive measures for COVID-19, which is lower than that in our study findings [21]. Another study conducted among Iranian medical students showed that 79.6% of medical students had adequate knowledge about COVID-19 [14].\nOur study reported high scores of attitude and preventive behavior toward COVID-19, which corresponds to a lower potential risk rate for COVID-19. In addition, more than 88% of the study participants reported discussing the preventive measures with their families and friends, which can effectively increase awareness about the current situation. This indicates the importance of health education that could improve the prevention behavior toward COVID-19 in society. The health education approach is a common method adopted for reducing the risk of transmission of infectious diseases. The study had a large sample size and involved surveying students from approximately 15 colleges and universities during the COVID-19 outbreak in Libya. In addition, our sample was representative of those from major cities, and the study yielded a generalized result. However, our sample predominantly comprised female students, 2923 (79.7%) overall, between the two groups. In addition, we included only college students who are likely to have better knowledge and information about COVID-19. Therefore, this may have limited our estimation of the knowledge of people who do not have access to a college education or the general population. Additionally, the comparison between medical and non-medical students did not display significant differences for most questions despite the fact that medical students were more likely to have greater knowledge owing to the nature of their profession and academic pursuit; this could be attributed to the larger sample size of medical students, where 69.4% of the sample size comprised medical students and 30.6% were non-medical students. However, our study covered populations from major cities and universities and can be considered as a generalized representation of the Libyan population irrespective of residence state.","Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.\nWhat is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nWhat this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.","The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.","Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.","The authors declare no competing interests."],"string":"[\n \"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].\\nThe development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.\\nMedical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.\",\n \"Study setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.\\nCOVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.\\nPreventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.\\nStatistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.\\nEthical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study.\",\n \"Out of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.\\nCharacteristics of individuals in the study\\nQuestionnaire on knowledge toward COVID-19\\nDifferences in knowledge score according to study characteristics\\nThe mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.\\nQuestionnaire on preventive behavior and attitude toward COVID-19\\nTable 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).\\nDifferences in preventive score according to study characteristics\",\n \"To the best of our knowledge, this is the first study conducted in Africa that discusses knowledge, self-preventive measures, and attitudes toward COVID-19 among college students, who constitute an integral part of an educated society. Overall, 71.8% of the study participants were aware of COVID-19, while more than 92.7% of both medical and non-medical students were taking preventive measures against COVID-19 to reduce the risk of infection transmission. Our study provides an overview of the knowledge of medical students, who will work as physicians in future, and are currently engaged in hospitals and at a high risk of infection transmission; the study compared their knowledge, preventive measures, and attitude toward COVID-19 to that of non-medical students to determine the difference. A majority of the participants (87.7% of non-medical students and 84.8% of medical students) opined that the COVID-19 pandemic could be controlled successfully, while a lower percentage of participants (74.6% non-medical students and 73.5% medical students) believed that the disease could be controlled in Libya. Contrastingly, the preventive behavior score among students was encouraging, as indicated by a high mean score of 7.4 ± 0.93 in non-medical students and 7.43 ± 0.96 of medical student out of 8, which corresponds to approximately 7.4/8 *100 = 92.5% of non-medical student and 7.43/8 *100 = 92.87% of medical students; this suggests that the students have a positive preventive behavior and attitude toward COVID-19. Students in their internship year had better knowledge and greater preventive behavior owing to the extent of time they spend at the medical department, where they work at a higher risk of exposure and encounter more cases. Therefore, their knowledge and attitude are attributed to their experience.\\nApproximately 97% participants from both groups reported avoidance of crowds and shopping, which displays the high levels of caution among the individuals. In addition, we identified several factors that can be associated with the preventive behavior and knowledge of the participants toward COVID-19. Our findings are necessary to understand the current state of public health awareness and to determine the need for proper dissemination of knowledge and awareness among students who are medical practitioners of the future and may possibly come in contact with infected individuals during their rotation in the hospitals. As medical students are at a higher risk of contracting infection and disease transmission owing to the course of study in hospitals, their knowledge and awareness of preventive measures are crucial for reducing the risk of community spread of SARS-CoV-2. Despite the healthcare situation and civil war in Libya, college students achieved high scores in knowledge, preventive behavior, and attitude toward COVID-19 infection. There was no significant difference between the rates of correct answers to most questions asked to either medical or non-medical students, which displays the substantial overall knowledge among college students. Their level of knowledge, preventive behavior, and attitude owing to their active learning and sources of information on COVID-19 yielded positive results. A similar study conducted on a Chinese population reported an overall knowledge of 90% [18]. Another unpublished study conducted by Haque et al. on a Bangladeshi population reported an overall knowledge score of 54.87% toward COVID-19 [19]. In addition, another study conducted on the Nepalese population reported an overall knowledge score ranging from 60.0-98.7% and attitude scores ranging from 77.9-96.4%. The study also confirmed that participants with a medical degree had greater knowledge than non-medical participants [20]. Another study conducted on an Egyptian population comprising 559 participants reported the mean and standard deviation of knowledge score as 16.39±2.63, ranging from 7 to 22, which corresponds to approximately 74.5% overall knowledge among participants regarding COVID-19 [17]. Another study in Thailand reported 67.9% as the overall level of knowledge toward COVID-19 prevention and only 29.5% were reported to have substantial knowledge about the preventive measures for COVID-19, which is lower than that in our study findings [21]. Another study conducted among Iranian medical students showed that 79.6% of medical students had adequate knowledge about COVID-19 [14].\\nOur study reported high scores of attitude and preventive behavior toward COVID-19, which corresponds to a lower potential risk rate for COVID-19. In addition, more than 88% of the study participants reported discussing the preventive measures with their families and friends, which can effectively increase awareness about the current situation. This indicates the importance of health education that could improve the prevention behavior toward COVID-19 in society. The health education approach is a common method adopted for reducing the risk of transmission of infectious diseases. The study had a large sample size and involved surveying students from approximately 15 colleges and universities during the COVID-19 outbreak in Libya. In addition, our sample was representative of those from major cities, and the study yielded a generalized result. However, our sample predominantly comprised female students, 2923 (79.7%) overall, between the two groups. In addition, we included only college students who are likely to have better knowledge and information about COVID-19. Therefore, this may have limited our estimation of the knowledge of people who do not have access to a college education or the general population. Additionally, the comparison between medical and non-medical students did not display significant differences for most questions despite the fact that medical students were more likely to have greater knowledge owing to the nature of their profession and academic pursuit; this could be attributed to the larger sample size of medical students, where 69.4% of the sample size comprised medical students and 30.6% were non-medical students. However, our study covered populations from major cities and universities and can be considered as a generalized representation of the Libyan population irrespective of residence state.\",\n \"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.\\nWhat is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\\nWhat this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\",\n \"The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\",\n \"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\",\n \"The authors declare no competing interests.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results","discussion","conclusion",null,null,"COI-statement"],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n \"discussion\",\n \"conclusion\",\n null,\n null,\n \"COI-statement\"\n]"},"keywords":{"kind":"list like","value":["SARS-CoV-2","knowledge","public health","COVID-19","behavior","pandemic","awareness"],"string":"[\n \"SARS-CoV-2\",\n \"knowledge\",\n \"public health\",\n \"COVID-19\",\n \"behavior\",\n \"pandemic\",\n \"awareness\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].\nThe development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.\nMedical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.\n\nMethods:\nStudy setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.\nCOVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.\nPreventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.\nStatistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.\nEthical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study.\n\nResults:\nOut of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.\nCharacteristics of individuals in the study\nQuestionnaire on knowledge toward COVID-19\nDifferences in knowledge score according to study characteristics\nThe mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.\nQuestionnaire on preventive behavior and attitude toward COVID-19\nTable 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).\nDifferences in preventive score according to study characteristics\n\nDiscussion:\nTo the best of our knowledge, this is the first study conducted in Africa that discusses knowledge, self-preventive measures, and attitudes toward COVID-19 among college students, who constitute an integral part of an educated society. Overall, 71.8% of the study participants were aware of COVID-19, while more than 92.7% of both medical and non-medical students were taking preventive measures against COVID-19 to reduce the risk of infection transmission. Our study provides an overview of the knowledge of medical students, who will work as physicians in future, and are currently engaged in hospitals and at a high risk of infection transmission; the study compared their knowledge, preventive measures, and attitude toward COVID-19 to that of non-medical students to determine the difference. A majority of the participants (87.7% of non-medical students and 84.8% of medical students) opined that the COVID-19 pandemic could be controlled successfully, while a lower percentage of participants (74.6% non-medical students and 73.5% medical students) believed that the disease could be controlled in Libya. Contrastingly, the preventive behavior score among students was encouraging, as indicated by a high mean score of 7.4 ± 0.93 in non-medical students and 7.43 ± 0.96 of medical student out of 8, which corresponds to approximately 7.4/8 *100 = 92.5% of non-medical student and 7.43/8 *100 = 92.87% of medical students; this suggests that the students have a positive preventive behavior and attitude toward COVID-19. Students in their internship year had better knowledge and greater preventive behavior owing to the extent of time they spend at the medical department, where they work at a higher risk of exposure and encounter more cases. Therefore, their knowledge and attitude are attributed to their experience.\nApproximately 97% participants from both groups reported avoidance of crowds and shopping, which displays the high levels of caution among the individuals. In addition, we identified several factors that can be associated with the preventive behavior and knowledge of the participants toward COVID-19. Our findings are necessary to understand the current state of public health awareness and to determine the need for proper dissemination of knowledge and awareness among students who are medical practitioners of the future and may possibly come in contact with infected individuals during their rotation in the hospitals. As medical students are at a higher risk of contracting infection and disease transmission owing to the course of study in hospitals, their knowledge and awareness of preventive measures are crucial for reducing the risk of community spread of SARS-CoV-2. Despite the healthcare situation and civil war in Libya, college students achieved high scores in knowledge, preventive behavior, and attitude toward COVID-19 infection. There was no significant difference between the rates of correct answers to most questions asked to either medical or non-medical students, which displays the substantial overall knowledge among college students. Their level of knowledge, preventive behavior, and attitude owing to their active learning and sources of information on COVID-19 yielded positive results. A similar study conducted on a Chinese population reported an overall knowledge of 90% [18]. Another unpublished study conducted by Haque et al. on a Bangladeshi population reported an overall knowledge score of 54.87% toward COVID-19 [19]. In addition, another study conducted on the Nepalese population reported an overall knowledge score ranging from 60.0-98.7% and attitude scores ranging from 77.9-96.4%. The study also confirmed that participants with a medical degree had greater knowledge than non-medical participants [20]. Another study conducted on an Egyptian population comprising 559 participants reported the mean and standard deviation of knowledge score as 16.39±2.63, ranging from 7 to 22, which corresponds to approximately 74.5% overall knowledge among participants regarding COVID-19 [17]. Another study in Thailand reported 67.9% as the overall level of knowledge toward COVID-19 prevention and only 29.5% were reported to have substantial knowledge about the preventive measures for COVID-19, which is lower than that in our study findings [21]. Another study conducted among Iranian medical students showed that 79.6% of medical students had adequate knowledge about COVID-19 [14].\nOur study reported high scores of attitude and preventive behavior toward COVID-19, which corresponds to a lower potential risk rate for COVID-19. In addition, more than 88% of the study participants reported discussing the preventive measures with their families and friends, which can effectively increase awareness about the current situation. This indicates the importance of health education that could improve the prevention behavior toward COVID-19 in society. The health education approach is a common method adopted for reducing the risk of transmission of infectious diseases. The study had a large sample size and involved surveying students from approximately 15 colleges and universities during the COVID-19 outbreak in Libya. In addition, our sample was representative of those from major cities, and the study yielded a generalized result. However, our sample predominantly comprised female students, 2923 (79.7%) overall, between the two groups. In addition, we included only college students who are likely to have better knowledge and information about COVID-19. Therefore, this may have limited our estimation of the knowledge of people who do not have access to a college education or the general population. Additionally, the comparison between medical and non-medical students did not display significant differences for most questions despite the fact that medical students were more likely to have greater knowledge owing to the nature of their profession and academic pursuit; this could be attributed to the larger sample size of medical students, where 69.4% of the sample size comprised medical students and 30.6% were non-medical students. However, our study covered populations from major cities and universities and can be considered as a generalized representation of the Libyan population irrespective of residence state.\n\nConclusion:\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.\nWhat is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nWhat this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\n\nWhat is known about this topic:\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\n\nWhat this study adds:\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\n\nCompeting interests:\nThe authors declare no competing interests.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThere are a limited number of studies on the issues associated with the knowledge and self-practice preventive measures for COVID-19 among medical students. We aimed to determine the extent of knowledge, self-reported preventive behavior, and risk perception of the COVID-19 outbreak among college students in Libya.\n\nMethods:\nA cross-sectional study was conducted from April 20 to April 30, 2020. The participants were students of medical and non-medical subjects from Libyan educational institutes. Data on participants' characteristics, knowledge, preventive behavior, and risk perception were collected.\n\nResults:\nApproximately 3669 participants completed the questionnaire, of which 2547 (69.4) were medical students and 1122 (30.6%) were non-medical students. The mean knowledge score on COVID-19 was 8.62 (SD: 1.26, range: 0-12), corresponding to 71.8% correct answers. A significant difference was observed between medical and non-medical students in terms of knowledge (p < 0.001). Overall, the knowledge score of the students differed significantly with respect to age, current year of study, and financial source (p < 0.05). The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students.\n\nConclusions:\nNotably, college students were observed to have substantial knowledge, preventive behavior, and a positive attitude toward COVID-19. Government programs should aim to educate individuals from other sectors of the society to ensure the proper dissemination of knowledge on preventive safety measures, as this will help restrict and control the pandemic."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].\nThe development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.\nMedical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.\n\nConclusion:\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.\nWhat is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nWhat this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThere are a limited number of studies on the issues associated with the knowledge and self-practice preventive measures for COVID-19 among medical students. We aimed to determine the extent of knowledge, self-reported preventive behavior, and risk perception of the COVID-19 outbreak among college students in Libya.\n\nMethods:\nA cross-sectional study was conducted from April 20 to April 30, 2020. The participants were students of medical and non-medical subjects from Libyan educational institutes. Data on participants' characteristics, knowledge, preventive behavior, and risk perception were collected.\n\nResults:\nApproximately 3669 participants completed the questionnaire, of which 2547 (69.4) were medical students and 1122 (30.6%) were non-medical students. The mean knowledge score on COVID-19 was 8.62 (SD: 1.26, range: 0-12), corresponding to 71.8% correct answers. A significant difference was observed between medical and non-medical students in terms of knowledge (p < 0.001). Overall, the knowledge score of the students differed significantly with respect to age, current year of study, and financial source (p < 0.05). The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students.\n\nConclusions:\nNotably, college students were observed to have substantial knowledge, preventive behavior, and a positive attitude toward COVID-19. Government programs should aim to educate individuals from other sectors of the society to ensure the proper dissemination of knowledge on preventive safety measures, as this will help restrict and control the pandemic."},"whole_article_text_length":{"kind":"number","value":3748,"string":"3,748"},"whole_article_abstract_length":{"kind":"number","value":335,"string":"335"},"other_sections_lengths":{"kind":"list like","value":[38,47],"string":"[\n 38,\n 47\n]"},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["medical","students","19","covid","covid 19","knowledge","preventive","medical students","study","non"],"string":"[\n \"medical\",\n \"students\",\n \"19\",\n \"covid\",\n \"covid 19\",\n \"knowledge\",\n \"preventive\",\n \"medical students\",\n \"study\",\n \"non\"\n]"},"keybert_topics":{"kind":"list like","value":["coronavirus disease covid","awareness covid 19","exposure covid 19","prevention covid","covid 19 outbreaks"],"string":"[\n \"coronavirus disease covid\",\n \"awareness covid 19\",\n \"exposure covid 19\",\n \"prevention covid\",\n \"covid 19 outbreaks\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 | covid | 19 | risk | transmission | measures | preventive measures | preventive | infection | self [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] study | related | questions | score | covid 19 | 19 | covid | calculated | added | survey [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] medical students | medical | students | non medical students | non medical | non | characteristics | score | approximately | table [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] preventive | positive attitude | knowledge | attitude | preventive behavior | behavior | knowledge attitude preventive | knowledge attitude preventive behavior | college | college students [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | knowledge | preventive | students | 19 | covid 19 | covid | study | medical students | behavior [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] medical | knowledge | preventive | students | 19 | covid 19 | covid | study | medical students | behavior [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| COVID-19 | Libya [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] April 20 to April 30, 2020 ||| Libyan ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Approximately 3669 | 2547 | 69.4 | 1122 | 30.6% ||| COVID-19 | 8.62 | 1.26 | 0-12 | 71.8% ||| ||| ||| COVID-19 | 8) | 7.42 | 0.95 | 0-8) | approximately 7.42/8*100 | 92.7% [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| COVID-19 | Libya | April 20 to April 30, 2020 ||| Libyan ||| ||| Approximately 3669 | 2547 | 69.4 | 1122 | 30.6% ||| COVID-19 | 8.62 | 1.26 | 0-12 | 71.8% ||| ||| ||| COVID-19 | 8) | 7.42 | 0.95 | 0-8) | approximately 7.42/8*100 | 92.7% ||| COVID-19 ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 ||| COVID-19 | Libya | April 20 to April 30, 2020 ||| Libyan ||| ||| Approximately 3669 | 2547 | 69.4 | 1122 | 30.6% ||| COVID-19 | 8.62 | 1.26 | 0-12 | 71.8% ||| ||| ||| COVID-19 | 8) | 7.42 | 0.95 | 0-8) | approximately 7.42/8*100 | 92.7% ||| COVID-19 ||| [SUMMARY]"}}},{"rowIdx":3395,"cells":{"title":{"kind":"string","value":"Excessive proinflammatory cytokine and chemokine responses of human monocyte-derived macrophages to enterovirus 71 infection."},"pmid":{"kind":"string","value":"22994237"},"background_abstract":{"kind":"string","value":"The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Cells, Cultured","Cytokines","DEAD Box Protein 58","DEAD-box RNA Helicases","Enterovirus A, Human","Gene Expression Profiling","Humans","Interferon-Induced Helicase, IFIH1","Macrophages","Real-Time Polymerase Chain Reaction","Receptors, Immunologic","Toll-Like Receptors","Young Adult"],"string":"[\n \"Adult\",\n \"Cells, Cultured\",\n \"Cytokines\",\n \"DEAD Box Protein 58\",\n \"DEAD-box RNA Helicases\",\n \"Enterovirus A, Human\",\n \"Gene Expression Profiling\",\n \"Humans\",\n \"Interferon-Induced Helicase, IFIH1\",\n \"Macrophages\",\n \"Real-Time Polymerase Chain Reaction\",\n \"Receptors, Immunologic\",\n \"Toll-Like Receptors\",\n \"Young Adult\"\n]"},"pmcid":{"kind":"string","value":"3519709"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\nThe study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\n Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\nEnterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\n Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\nMDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\n Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\nInfected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\n Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\nConcentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\n Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\nStatistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\nWe first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\n Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nWe subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nChemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\nIn order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed."},"other_sections_titles":{"kind":"list like","value":["Background","Effective infection and viral replication of EV71 in human MDMs","Enhanced proinflammatory cytokine responses of human MDMs to EV71","Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71","Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs","Isolation and culture of monocyte-derived macrophages","Infection of MDMs with EV71","Immunofluorescence assay of viral VP1 protein in infected cells","Quantification of mRNA by real-time RT-PCR","Measurement of cytokines","Statistical analysis","Abbreviations","Competing interests","Authors' contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Effective infection and viral replication of EV71 in human MDMs\",\n \"Enhanced proinflammatory cytokine responses of human MDMs to EV71\",\n \"Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71\",\n \"Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs\",\n \"Isolation and culture of monocyte-derived macrophages\",\n \"Infection of MDMs with EV71\",\n \"Immunofluorescence assay of viral VP1 protein in infected cells\",\n \"Quantification of mRNA by real-time RT-PCR\",\n \"Measurement of cytokines\",\n \"Statistical analysis\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors' contributions\",\n \"Pre-publication history\"\n]"},"other_sections_texts":{"kind":"list like","value":["Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.","We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.","We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).","Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).","In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).","The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.","Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.","MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.","Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay","Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).","Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.","(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.","The authors declare that they have no competing interests.","Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\n"],"string":"[\n \"Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.\",\n \"We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\",\n \"We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\",\n \"Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\",\n \"In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\",\n \"The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\",\n \"Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\",\n \"MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\",\n \"Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\\nPrimer sequences and probes used in real-time PCR assay\",\n \"Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\",\n \"Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\",\n \"(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.\",\n \"The authors declare that they have no competing interests.\",\n \"Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\\n\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Results","Effective infection and viral replication of EV71 in human MDMs","Enhanced proinflammatory cytokine responses of human MDMs to EV71","Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71","Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs","Discussion","Conclusions","Methods","Isolation and culture of monocyte-derived macrophages","Infection of MDMs with EV71","Immunofluorescence assay of viral VP1 protein in infected cells","Quantification of mRNA by real-time RT-PCR","Measurement of cytokines","Statistical analysis","Abbreviations","Competing interests","Authors' contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Results\",\n \"Effective infection and viral replication of EV71 in human MDMs\",\n \"Enhanced proinflammatory cytokine responses of human MDMs to EV71\",\n \"Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71\",\n \"Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs\",\n \"Discussion\",\n \"Conclusions\",\n \"Methods\",\n \"Isolation and culture of monocyte-derived macrophages\",\n \"Infection of MDMs with EV71\",\n \"Immunofluorescence assay of viral VP1 protein in infected cells\",\n \"Quantification of mRNA by real-time RT-PCR\",\n \"Measurement of cytokines\",\n \"Statistical analysis\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors' contributions\",\n \"Pre-publication history\"\n]"},"all_sections_texts":{"kind":"list like","value":["Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection."," Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\nWe first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\n Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nWe subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nChemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\nIn order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).","We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.","We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).","Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).","In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).","Macrophages, which are shown to support the infection of various viruses including HIV-1, influenza viruses, and poliovirus and so on, play a critical role in presentation of antigens, pathogen clearance, and induction of inflammation during the early phase of viral infection [21-23]. In this study, both viral gene and antigen of EV71 were detected. The increases of virus yields and the number of viral gene copies were observed in EV71-infected MDMs between the 6-h and 48-h POI. Excess cytokine and chemokine responses of MDMs were triggered by EV71. These findings suggested that macrophages may be not only the target cells but also the effectors during EV71 infection.\nIt is controversial whether TNF-α was involved in fatal EV71 infection. Significant or minor change of blood TNF-α in EV71 patient with both encephalitis and PE was reported by different clinical studies [24,25]. Significant stronger release of TNF-α from EV71-infected MDMs at 12-h POI than 24-h POI in the study, implying that TNF-α was induced by EV71 infection at early stage and maybe involved in its pathology. Consistent with clinical features of EV71 patients with encephalitis and PE, who are presented with higher levels of the proinflammatory cytokines in blood [9,24], IL-6, IL-1β and TNF-α were induced in EV71-infected MDMs. These proinflammatory cytokines are thought to be the potent pyrogens inducing fever, and the magnitude of febrile response correlates with the level of virus shedding in human and animals [26]. Notably, a transient increase of blood brain barrier (BBB) permeability and its injury were found at early stage of EV71 infection [27]. The pathology may be due to an augmentation of systemic and local TNF-α production, which exhibits detrimental effects by enhancing cell infiltration, cytopathic damage, or functioning as a paracellular pathway for the virus across the BBB [28]. Furthermore, the subsequent responses of acute phase proteins and chemokine activations mediated by IL-6, IL-1β and TNF-α could exacerbate virus induced inflammation and pathology [29]. Therefore, the rapid and strong proinflammatory response of MDMs to EV71 may partially explain the clinical severity.\nMacrophages or plasmacytoid DCs, specialized in secreting large amounts of type I IFN after virus infection, play an important role in viral infection. However, minor change of IFN-α in EV71-infected MDMs was detected in our study. It is likely that the 3C protein of EV71 virus inhibits type I interferon activation by viral nuclear acid or RIG-I signalling [30]. Although elevated level of IFN-γ in both plasma and cerebrospinal fluid was found in patients with PE [10], the IFN-γ from EV71-infected MDMs was undetectable here. The discrepancy may be a result of the main cellular source of IFN-γ in vivo from activated T and NK cells other than macrophages. A series of IFN-γ-responsive and inflammatory chemokines such as RANTES, IP-10 and IL-8 in MDMs were triggered by EV71 virus. Not only live- but also UV-inactivated EV71 can induce IL-8 releasing from MDMs, which suggested that viral proteins may also be involved in inducing of IL-8. IL-8 is a potent chemoattractant and activator of neutrophils, one of the major immune cells responsible for inflammation of CNS during meningitis or encephalitis [31]. Our in vitro findings support the clinical findings that a higher total WBC count, absolute neutrophil count and elevated IL-8 and IP-10 level in patients with BE or PE [11,25].\nTLRs and RLHs recognize distinct ligands and trigger host immune response in different virus infection [32]. The recognition of human rhinovirus, human parechovirus 1, rotavirus or coxsackie B virus by different host cells are mediated through elevated TLR2, 7, 8 and (or) Mda5 expression, which induce secretion of proinflammatory cytokines, chemokines, and interferons [16-19]. When TLR2, TLR7 and TLR8 were silenced, there was a considerable decrease in cytokine secretion in human airway epithelial cells with HRV-6 infection [18]. In our study, elevated TLR2, TLR7 and TLR8 expressions as well as increased proinflammatory cytokines and chemokines were observed in EV71-infected MDMs. Furthermore, enhanced IL-8 and TLR2 mRNA expression were also found in UV-inactivated virus treated-MDMs. It is likely that the interaction between TLR2 on cell surface and viral proteins rather than viral RNAs is necessary for the activation of MDMs. Significant up-regulations of mRNA for TLR7 and TLR8 were observed at different time points, and it suggested that there were differential kinetics between TLR7 and TLR8 involvements in EV71-infected MDMs. These results indicated the cytokine productions in EV71-infected MDMs may be partly through the activation of TLR2, TLR7 and (or) TLR8. Further investigations such as gene knockout experiments are needed to determine the exact roles of them.\nAs documented previously, younger children less than 5-year-old were the most susceptible groups to EV71 and usually presented with severe infection [3]. The underlying mechanisms for the severity remain unknown. And enhanced proinflammatory cytokines and chemokines were indeed found in children patients with encephalitis or PE [9,11,24]. Although there is differential expression level of chemokine receptors on adult and neonatal MDMs [33], one of possible factors for the age-related severity in avian influenza virus infection, a similar cytokines/chemokines profile was found in influenza virus-infected adult and neonatal MDMs and the levels of most of the cytokines/chemokines were comparable [23]. Moreover, the adults can also be infected with EV71 and the clinical severity in adult patients with acute encephalitis was similar to those of EV71 infection in children [34]. Therefore, the findings on adult MDMs infection model here may partially reflect natural in vivo infection.","In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed."," Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\nThe study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\n Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\nEnterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\n Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\nMDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\n Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\nInfected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\n Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\nConcentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\n Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\nStatistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.","The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.","Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.","MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.","Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay","Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).","Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.","(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.","The authors declare that they have no competing interests.","Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\n"],"string":"[\n \"Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.\",\n \" Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\\nWe first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\\n Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\\nWe subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\\n Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\\nChemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\\n Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\\nIn order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\",\n \"We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\",\n \"We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\",\n \"Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\",\n \"In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\",\n \"Macrophages, which are shown to support the infection of various viruses including HIV-1, influenza viruses, and poliovirus and so on, play a critical role in presentation of antigens, pathogen clearance, and induction of inflammation during the early phase of viral infection [21-23]. In this study, both viral gene and antigen of EV71 were detected. The increases of virus yields and the number of viral gene copies were observed in EV71-infected MDMs between the 6-h and 48-h POI. Excess cytokine and chemokine responses of MDMs were triggered by EV71. These findings suggested that macrophages may be not only the target cells but also the effectors during EV71 infection.\\nIt is controversial whether TNF-α was involved in fatal EV71 infection. Significant or minor change of blood TNF-α in EV71 patient with both encephalitis and PE was reported by different clinical studies [24,25]. Significant stronger release of TNF-α from EV71-infected MDMs at 12-h POI than 24-h POI in the study, implying that TNF-α was induced by EV71 infection at early stage and maybe involved in its pathology. Consistent with clinical features of EV71 patients with encephalitis and PE, who are presented with higher levels of the proinflammatory cytokines in blood [9,24], IL-6, IL-1β and TNF-α were induced in EV71-infected MDMs. These proinflammatory cytokines are thought to be the potent pyrogens inducing fever, and the magnitude of febrile response correlates with the level of virus shedding in human and animals [26]. Notably, a transient increase of blood brain barrier (BBB) permeability and its injury were found at early stage of EV71 infection [27]. The pathology may be due to an augmentation of systemic and local TNF-α production, which exhibits detrimental effects by enhancing cell infiltration, cytopathic damage, or functioning as a paracellular pathway for the virus across the BBB [28]. Furthermore, the subsequent responses of acute phase proteins and chemokine activations mediated by IL-6, IL-1β and TNF-α could exacerbate virus induced inflammation and pathology [29]. Therefore, the rapid and strong proinflammatory response of MDMs to EV71 may partially explain the clinical severity.\\nMacrophages or plasmacytoid DCs, specialized in secreting large amounts of type I IFN after virus infection, play an important role in viral infection. However, minor change of IFN-α in EV71-infected MDMs was detected in our study. It is likely that the 3C protein of EV71 virus inhibits type I interferon activation by viral nuclear acid or RIG-I signalling [30]. Although elevated level of IFN-γ in both plasma and cerebrospinal fluid was found in patients with PE [10], the IFN-γ from EV71-infected MDMs was undetectable here. The discrepancy may be a result of the main cellular source of IFN-γ in vivo from activated T and NK cells other than macrophages. A series of IFN-γ-responsive and inflammatory chemokines such as RANTES, IP-10 and IL-8 in MDMs were triggered by EV71 virus. Not only live- but also UV-inactivated EV71 can induce IL-8 releasing from MDMs, which suggested that viral proteins may also be involved in inducing of IL-8. IL-8 is a potent chemoattractant and activator of neutrophils, one of the major immune cells responsible for inflammation of CNS during meningitis or encephalitis [31]. Our in vitro findings support the clinical findings that a higher total WBC count, absolute neutrophil count and elevated IL-8 and IP-10 level in patients with BE or PE [11,25].\\nTLRs and RLHs recognize distinct ligands and trigger host immune response in different virus infection [32]. The recognition of human rhinovirus, human parechovirus 1, rotavirus or coxsackie B virus by different host cells are mediated through elevated TLR2, 7, 8 and (or) Mda5 expression, which induce secretion of proinflammatory cytokines, chemokines, and interferons [16-19]. When TLR2, TLR7 and TLR8 were silenced, there was a considerable decrease in cytokine secretion in human airway epithelial cells with HRV-6 infection [18]. In our study, elevated TLR2, TLR7 and TLR8 expressions as well as increased proinflammatory cytokines and chemokines were observed in EV71-infected MDMs. Furthermore, enhanced IL-8 and TLR2 mRNA expression were also found in UV-inactivated virus treated-MDMs. It is likely that the interaction between TLR2 on cell surface and viral proteins rather than viral RNAs is necessary for the activation of MDMs. Significant up-regulations of mRNA for TLR7 and TLR8 were observed at different time points, and it suggested that there were differential kinetics between TLR7 and TLR8 involvements in EV71-infected MDMs. These results indicated the cytokine productions in EV71-infected MDMs may be partly through the activation of TLR2, TLR7 and (or) TLR8. Further investigations such as gene knockout experiments are needed to determine the exact roles of them.\\nAs documented previously, younger children less than 5-year-old were the most susceptible groups to EV71 and usually presented with severe infection [3]. The underlying mechanisms for the severity remain unknown. And enhanced proinflammatory cytokines and chemokines were indeed found in children patients with encephalitis or PE [9,11,24]. Although there is differential expression level of chemokine receptors on adult and neonatal MDMs [33], one of possible factors for the age-related severity in avian influenza virus infection, a similar cytokines/chemokines profile was found in influenza virus-infected adult and neonatal MDMs and the levels of most of the cytokines/chemokines were comparable [23]. Moreover, the adults can also be infected with EV71 and the clinical severity in adult patients with acute encephalitis was similar to those of EV71 infection in children [34]. Therefore, the findings on adult MDMs infection model here may partially reflect natural in vivo infection.\",\n \"In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed.\",\n \" Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\\nThe study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\\n Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\\nEnterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\\n Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\\nMDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\\n Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\\nPrimer sequences and probes used in real-time PCR assay\\nInfected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\\nPrimer sequences and probes used in real-time PCR assay\\n Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\\nConcentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\\n Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\\nStatistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\",\n \"The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\",\n \"Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\",\n \"MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\",\n \"Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\\nPrimer sequences and probes used in real-time PCR assay\",\n \"Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\",\n \"Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\",\n \"(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.\",\n \"The authors declare that they have no competing interests.\",\n \"Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"results",null,null,null,null,"discussion","conclusions","methods",null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["Enterovirus 71","Macrophages","Proinflammatory cytokines","Chemokines","Toll-like receptors"],"string":"[\n \"Enterovirus 71\",\n \"Macrophages\",\n \"Proinflammatory cytokines\",\n \"Chemokines\",\n \"Toll-like receptors\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nEnterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.\n\nResults:\n Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\nWe first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\n Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nWe subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nChemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\nIn order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\n\nEffective infection and viral replication of EV71 in human MDMs:\nWe first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\n\nEnhanced proinflammatory cytokine responses of human MDMs to EV71:\nWe subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n\nEnhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71:\nChemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n\nUp-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs:\nIn order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\n\nDiscussion:\nMacrophages, which are shown to support the infection of various viruses including HIV-1, influenza viruses, and poliovirus and so on, play a critical role in presentation of antigens, pathogen clearance, and induction of inflammation during the early phase of viral infection [21-23]. In this study, both viral gene and antigen of EV71 were detected. The increases of virus yields and the number of viral gene copies were observed in EV71-infected MDMs between the 6-h and 48-h POI. Excess cytokine and chemokine responses of MDMs were triggered by EV71. These findings suggested that macrophages may be not only the target cells but also the effectors during EV71 infection.\nIt is controversial whether TNF-α was involved in fatal EV71 infection. Significant or minor change of blood TNF-α in EV71 patient with both encephalitis and PE was reported by different clinical studies [24,25]. Significant stronger release of TNF-α from EV71-infected MDMs at 12-h POI than 24-h POI in the study, implying that TNF-α was induced by EV71 infection at early stage and maybe involved in its pathology. Consistent with clinical features of EV71 patients with encephalitis and PE, who are presented with higher levels of the proinflammatory cytokines in blood [9,24], IL-6, IL-1β and TNF-α were induced in EV71-infected MDMs. These proinflammatory cytokines are thought to be the potent pyrogens inducing fever, and the magnitude of febrile response correlates with the level of virus shedding in human and animals [26]. Notably, a transient increase of blood brain barrier (BBB) permeability and its injury were found at early stage of EV71 infection [27]. The pathology may be due to an augmentation of systemic and local TNF-α production, which exhibits detrimental effects by enhancing cell infiltration, cytopathic damage, or functioning as a paracellular pathway for the virus across the BBB [28]. Furthermore, the subsequent responses of acute phase proteins and chemokine activations mediated by IL-6, IL-1β and TNF-α could exacerbate virus induced inflammation and pathology [29]. Therefore, the rapid and strong proinflammatory response of MDMs to EV71 may partially explain the clinical severity.\nMacrophages or plasmacytoid DCs, specialized in secreting large amounts of type I IFN after virus infection, play an important role in viral infection. However, minor change of IFN-α in EV71-infected MDMs was detected in our study. It is likely that the 3C protein of EV71 virus inhibits type I interferon activation by viral nuclear acid or RIG-I signalling [30]. Although elevated level of IFN-γ in both plasma and cerebrospinal fluid was found in patients with PE [10], the IFN-γ from EV71-infected MDMs was undetectable here. The discrepancy may be a result of the main cellular source of IFN-γ in vivo from activated T and NK cells other than macrophages. A series of IFN-γ-responsive and inflammatory chemokines such as RANTES, IP-10 and IL-8 in MDMs were triggered by EV71 virus. Not only live- but also UV-inactivated EV71 can induce IL-8 releasing from MDMs, which suggested that viral proteins may also be involved in inducing of IL-8. IL-8 is a potent chemoattractant and activator of neutrophils, one of the major immune cells responsible for inflammation of CNS during meningitis or encephalitis [31]. Our in vitro findings support the clinical findings that a higher total WBC count, absolute neutrophil count and elevated IL-8 and IP-10 level in patients with BE or PE [11,25].\nTLRs and RLHs recognize distinct ligands and trigger host immune response in different virus infection [32]. The recognition of human rhinovirus, human parechovirus 1, rotavirus or coxsackie B virus by different host cells are mediated through elevated TLR2, 7, 8 and (or) Mda5 expression, which induce secretion of proinflammatory cytokines, chemokines, and interferons [16-19]. When TLR2, TLR7 and TLR8 were silenced, there was a considerable decrease in cytokine secretion in human airway epithelial cells with HRV-6 infection [18]. In our study, elevated TLR2, TLR7 and TLR8 expressions as well as increased proinflammatory cytokines and chemokines were observed in EV71-infected MDMs. Furthermore, enhanced IL-8 and TLR2 mRNA expression were also found in UV-inactivated virus treated-MDMs. It is likely that the interaction between TLR2 on cell surface and viral proteins rather than viral RNAs is necessary for the activation of MDMs. Significant up-regulations of mRNA for TLR7 and TLR8 were observed at different time points, and it suggested that there were differential kinetics between TLR7 and TLR8 involvements in EV71-infected MDMs. These results indicated the cytokine productions in EV71-infected MDMs may be partly through the activation of TLR2, TLR7 and (or) TLR8. Further investigations such as gene knockout experiments are needed to determine the exact roles of them.\nAs documented previously, younger children less than 5-year-old were the most susceptible groups to EV71 and usually presented with severe infection [3]. The underlying mechanisms for the severity remain unknown. And enhanced proinflammatory cytokines and chemokines were indeed found in children patients with encephalitis or PE [9,11,24]. Although there is differential expression level of chemokine receptors on adult and neonatal MDMs [33], one of possible factors for the age-related severity in avian influenza virus infection, a similar cytokines/chemokines profile was found in influenza virus-infected adult and neonatal MDMs and the levels of most of the cytokines/chemokines were comparable [23]. Moreover, the adults can also be infected with EV71 and the clinical severity in adult patients with acute encephalitis was similar to those of EV71 infection in children [34]. Therefore, the findings on adult MDMs infection model here may partially reflect natural in vivo infection.\n\nConclusions:\nIn summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed.\n\nMethods:\n Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\nThe study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\n Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\nEnterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\n Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\nMDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\n Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\nInfected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\n Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\nConcentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\n Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\nStatistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\n\nIsolation and culture of monocyte-derived macrophages:\nThe study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\n\nInfection of MDMs with EV71:\nEnterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\n\nImmunofluorescence assay of viral VP1 protein in infected cells:\nMDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\n\nQuantification of mRNA by real-time RT-PCR:\nInfected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\n\nMeasurement of cytokines:\nConcentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\n\nStatistical analysis:\nStatistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\n\nAbbreviations:\n(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors' contributions:\nConceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms.\n\nMethods:\nHuman primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR.\n\nResults:\nEffective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs.\n\nConclusions:\nOur findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs."},"background_conclusion_text":{"kind":"string","value":"Background:\nEnterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.\n\nConclusions:\nIn summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms.\n\nMethods:\nHuman primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR.\n\nResults:\nEffective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs.\n\nConclusions:\nOur findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs."},"whole_article_text_length":{"kind":"number","value":9660,"string":"9,660"},"whole_article_abstract_length":{"kind":"number","value":335,"string":"335"},"other_sections_lengths":{"kind":"list like","value":[434,569,318,339,360,199,194,72,213,189,54,113,10,67,16],"string":"[\n 434,\n 569,\n 318,\n 339,\n 360,\n 199,\n 194,\n 72,\n 213,\n 189,\n 54,\n 113,\n 10,\n 67,\n 16\n]"},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["ev71","mdms","infected","infection","poi","il","24","viral","infected mdms","12"],"string":"[\n \"ev71\",\n \"mdms\",\n \"infected\",\n \"infection\",\n \"poi\",\n \"il\",\n \"24\",\n \"viral\",\n \"infected mdms\",\n \"12\"\n]"},"keybert_topics":{"kind":"list like","value":["virus inflammation pathology","cytokines chemokines ev71","macrophages ev71","enterovirus 71 positive","exacerbate virus inflammation"],"string":"[\n \"virus inflammation pathology\",\n \"cytokines chemokines ev71\",\n \"macrophages ev71\",\n \"enterovirus 71 positive\",\n \"exacerbate virus inflammation\"\n]"},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | cells | ev71 infection | cells human | macrophages | infection | immune | proinflammatory | system | responses [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ml | pg ml | pg | il | usa | cells | pcr | assay | island ny usa | island ny [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | poi | infected | 24 | mock | mrna | expression | 24 poi | infection [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] response | ev71 | inflammatory | chemokine | important | mdms | infected | macrophages | ev71 infected mdms | viral [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | il | infection | pg ml | pg | ml | poi | cells [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ev71 | mdms | infected | il | infection | pg ml | pg | ml | poi | cells [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 71 ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Cytometric Bead Array(CBA ||| 5 | RT-PCR [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 6 | 48 | POI ||| IL-1 | IL-6 | TNF | IFN | EV71 ||| IP-10 | 12 | 24-h | POI ||| TLR8 | TLR3 | TLR4 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 71 ||| ||| ||| Cytometric Bead Array(CBA ||| 5 | RT-PCR ||| ||| 6 | 48 | POI ||| IL-1 | IL-6 | TNF | IFN | EV71 ||| IP-10 | 12 | 24-h | POI ||| TLR8 | TLR3 | TLR4 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] 71 ||| ||| ||| Cytometric Bead Array(CBA ||| 5 | RT-PCR ||| ||| 6 | 48 | POI ||| IL-1 | IL-6 | TNF | IFN | EV71 ||| IP-10 | 12 | 24-h | POI ||| TLR8 | TLR3 | TLR4 ||| ||| [SUMMARY]"}}},{"rowIdx":3396,"cells":{"title":{"kind":"string","value":"Association of vancomycin trough concentration on the treatment outcome of patients with bacteremia caused by Enterococcus species."},"pmid":{"kind":"string","value":"34702193"},"background_abstract":{"kind":"string","value":"Pharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"In this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Anti-Bacterial Agents","Bacteremia","Enterococcus","Humans","Methicillin-Resistant Staphylococcus aureus","Microbial Sensitivity Tests","Retrospective Studies","Staphylococcal Infections","Treatment Outcome","Vancomycin"],"string":"[\n \"Anti-Bacterial Agents\",\n \"Bacteremia\",\n \"Enterococcus\",\n \"Humans\",\n \"Methicillin-Resistant Staphylococcus aureus\",\n \"Microbial Sensitivity Tests\",\n \"Retrospective Studies\",\n \"Staphylococcal Infections\",\n \"Treatment Outcome\",\n \"Vancomycin\"\n]"},"pmcid":{"kind":"string","value":"8547083"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"A total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).\n\nTable 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality\nCharacteristics\nTotal (n = 37)Survivors (n = 29)Non-survivors (n = 8)\nP value\n\nDemographics\nAge (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nBaseline demographic and clinical features of patients with enterococcal bacteremia\nBMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis\nContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nThe 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.\nIn univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).\n\nTable 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval\nMultivariate logistic regression analysis for factors associated with 28-day mortality\nOR, odds ratio; CI, confidence interval\n\nFig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level\nKaplan-Meier survival analysis of patients according to vancomycin trough level"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species."},"other_sections_titles":{"kind":"list like","value":["Background","Methods","Study population and design","Definitions","Microbiological data","Vancomycin dosing and pharmacodynamics data","Statistical analysis",""],"string":"[\n \"Background\",\n \"Methods\",\n \"Study population and design\",\n \"Definitions\",\n \"Microbiological data\",\n \"Vancomycin dosing and pharmacodynamics data\",\n \"Statistical analysis\",\n \"\"\n]"},"other_sections_texts":{"kind":"list like","value":["Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.","Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).","A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.","Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.","The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].","To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.","The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).","\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia."],"string":"[\n \"Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.\",\n \"Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\",\n \"A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\",\n \"Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\",\n \"The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\",\n \"To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\",\n \"The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\",\n \"\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study population and design","Definitions","Microbiological data","Vancomycin dosing and pharmacodynamics data","Statistical analysis","Results","Discussion","Conclusions","Supplementary Information",""],"string":"[\n \"Background\",\n \"Methods\",\n \"Study population and design\",\n \"Definitions\",\n \"Microbiological data\",\n \"Vancomycin dosing and pharmacodynamics data\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Supplementary Information\",\n \"\"\n]"},"all_sections_texts":{"kind":"list like","value":["Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.","Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).","A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.","Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.","The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].","To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.","The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).","A total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).\n\nTable 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality\nCharacteristics\nTotal (n = 37)Survivors (n = 29)Non-survivors (n = 8)\nP value\n\nDemographics\nAge (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nBaseline demographic and clinical features of patients with enterococcal bacteremia\nBMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis\nContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nThe 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.\nIn univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).\n\nTable 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval\nMultivariate logistic regression analysis for factors associated with 28-day mortality\nOR, odds ratio; CI, confidence interval\n\nFig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level\nKaplan-Meier survival analysis of patients according to vancomycin trough level","In this study, we found that vancomycin trough concentration (≤ 15 µg/mL) was significantly associated with increased mortality in patients with Enterococcus bacteremia.\nMany studies have examined pharmacokinetic/pharmacodynamic (PK/PD) parameters to maintain the efficiency of vancomycin and to minimize its adverse effects, but most of these studies have examined MRSA infection. Of the PK/PD parameters, the trough level and AUC24/MIC have been reported to describe the effectiveness of vancomycin [15, 16]. The clinical success and renal toxicity of vancomycin treatment are exposure-dependent and characterized by the AUC24. The exact range targeted by clinicians is influenced by the AUC24 estimation and bacterial MIC [15, 17]. Using AUC-based vancomycin therapeutic drug monitoring (TDM) helps to individualize the estimation of the AUC24/MIC and minimize the risk of toxicity due to unnecessary overexposure.\nAlthough trough concentration is a known surrogate marker for AUC24/MIC, the method using only trough-based vancomycin TDM is controversial for several reasons. First, the vancomycin trough concentration poorly characterizes the AUC24, and adequate vancomycin AUC24 levels can be obtained at trough concentrations < 15 mg/L. Vancomycin-associated nephrotoxicity also increases when the vancomycin trough concentration is >15 mg/L. These factors provide evidence for an AUC-based approach to vancomycin TDM [18]. Thus, according to the Infectious Diseases Society of America guidelines, the trough level does not correlate well with the AUC, and trough-only monitoring with a target of 15–20 µg/mL is no longer recommended based on its efficacy in patients with MRSA infections [10]. However, because it may be difficult to determine the AUC24/MIC in a clinical setting, trough serum concentrations are still monitored clinically. Moreover, there is insufficient evidence to recommend whether trough level-only or AUC/MIC-guided vancomycin monitoring should be used for patients with non-MRSA infections.\nIn one study of PK/PD determinants of vancomycin efficacy in enterococcal bacteremia, a vancomycin AUC/MIC ≥ 389 achieved within 72 h was associated with reduced mortality. However, the study found that vancomycin trough concentrations differed significantly between survivors and non-survivors [19]. According to Nakamura et al. [20] neither the AUC24/MIC nor the trough concentration of vancomycin is significantly associated with mortality in patients with E. faecium bacteremia. The trough concentration was higher in non-survivors than in survivors, and AUC24/MIC did not differ significantly between non-survivors and survivors.\nIn our study, vancomycin trough levels of 15 µg/mL or lower were associated with mortality in patients with enterococcal bacteremia, but the AUC24/MIC was not. Zelenitsky et al. reported that the survival rate was higher when the vancomycin trough concentration was maintained above 15 mg/L than when low trough concentrations were maintained [21]. In addition, Kullar et al. reported that the vancomycin treatment period was significantly shorter in the group in which the vancomycin trough concentration was higher than the group to which the vancomycin trough concentration was applied at 5 to 20 mg/L, and the rate at which the bacteria were negatively converted was also found to be significantly higher in the group that maintained the higher trough concentration [22]. According to a meta-analysis comparing high and low vancomycin serum trough regimen groups with gram-positive bacterial infections, there was no significant difference between the two in all-cause mortality or risk of clinical failure; however, a subgroup analysis confirmed that treatment failure was reduced in high vancomycin trough regimen groups [23]. Therefore, in enterococcal bacteremia, the trough level may be a good parameter for monitoring the therapeutic effects of vancomycin.\nAll enterococcal BSIs treated with vancomycin were included in this study to examine the therapeutic effect of vancomycin on Enterococcus species. Therefore, strains susceptible to ampicillin were added in this study. In most cases, vancomycin was used for ampicillin-susceptible Enterococcus species when beta-lactam antibiotics could not be used due to its side effects, or was used without checking the results of antimicrobial susceptibility tests. Ampicillin is the preferred antibiotic used to treat ampicillin-sensitive enterococcal infections. However, according to several studies comparing the therapeutic effects of beta-lactam antibiotics and vancomycin on ampicillin-susceptible enterococcal BSI, there was no significant difference in mortality [24]. Therefore, when needed, or when beta-lactam antibiotics are difficult to use, vancomycin can be considered as a treatment option for ampicillin-susceptible strains.\nAn important concern when using vancomycin is the occurrence of acute kidney injury. A higher vancomycin trough concentration increases the risk of VIN [20, 25]. Although most vancomycin-induced nephrotoxic events are reversible, many studies support there is increased mortality from AKI of any cause, including vancomycin-induced nephropathy [26]. While 6 of our 37 patients developed VIN, it was not statistically related to trough concentration (p = 0.09) or 28-day mortality. (p= 0.446).\nIn this study, mortality was higher at younger ages. Aging process causes structural and functional changes in multiple organ systems, especially the kidneys, and is known to affect PK/PD of drugs by causing changes in body composition, drug absorption, distribution, and clearance [27]. These changes may have affected the patient’s prognosis or treatment outcome.\nThis study has several limitations that are inherent to its retrospective design. As with any observational study, there remains a possibility that unmeasured confounders influenced our findings. According to several studies, it is well known that sepsis or septic shock affects drug absorption, distribution and other pharmacological processes due to pathophysiological changes such as decreased perfusion to body organs, interstitial edema or increased capillary permeability [28]. Therefore, the PK/PD alterations may not have been reflected depending on the patients’ disease severity. In addition, in the early stage of sepsis, extravascular volume expansion with fluid loading and capillary leak causes a change in volume distribution, which can cause hyperfiltration in the kidney and lead to an alteration of drug concentration. Thereby, it may have affected the prognosis of non-survivors with low trough levels. For these reasons, we cannot exclude the possibility that residual bias affected our findings. Another limitation of this study was that only patients with bacteremia were enrolled, and patients with non-bacteremic sepsis were not analyzed. In addition, the single-center nature of a referral tertiary hospital leads to selection bias in severe cases admitted to our hospital. A multicenter study with a larger sample size is needed.","In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species."," \nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\n\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.","\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia."],"string":"[\n \"Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.\",\n \"Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\",\n \"A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\",\n \"Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\",\n \"The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\",\n \"To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\",\n \"The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\",\n \"A total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).\\n\\nTable 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality\\nCharacteristics\\nTotal (n = 37)Survivors (n = 29)Non-survivors (n = 8)\\nP value\\n\\nDemographics\\nAge (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\\nBaseline demographic and clinical features of patients with enterococcal bacteremia\\nBMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis\\nContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\\nThe 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.\\nIn univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).\\n\\nTable 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval\\nMultivariate logistic regression analysis for factors associated with 28-day mortality\\nOR, odds ratio; CI, confidence interval\\n\\nFig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level\\nKaplan-Meier survival analysis of patients according to vancomycin trough level\",\n \"In this study, we found that vancomycin trough concentration (≤ 15 µg/mL) was significantly associated with increased mortality in patients with Enterococcus bacteremia.\\nMany studies have examined pharmacokinetic/pharmacodynamic (PK/PD) parameters to maintain the efficiency of vancomycin and to minimize its adverse effects, but most of these studies have examined MRSA infection. Of the PK/PD parameters, the trough level and AUC24/MIC have been reported to describe the effectiveness of vancomycin [15, 16]. The clinical success and renal toxicity of vancomycin treatment are exposure-dependent and characterized by the AUC24. The exact range targeted by clinicians is influenced by the AUC24 estimation and bacterial MIC [15, 17]. Using AUC-based vancomycin therapeutic drug monitoring (TDM) helps to individualize the estimation of the AUC24/MIC and minimize the risk of toxicity due to unnecessary overexposure.\\nAlthough trough concentration is a known surrogate marker for AUC24/MIC, the method using only trough-based vancomycin TDM is controversial for several reasons. First, the vancomycin trough concentration poorly characterizes the AUC24, and adequate vancomycin AUC24 levels can be obtained at trough concentrations < 15 mg/L. Vancomycin-associated nephrotoxicity also increases when the vancomycin trough concentration is >15 mg/L. These factors provide evidence for an AUC-based approach to vancomycin TDM [18]. Thus, according to the Infectious Diseases Society of America guidelines, the trough level does not correlate well with the AUC, and trough-only monitoring with a target of 15–20 µg/mL is no longer recommended based on its efficacy in patients with MRSA infections [10]. However, because it may be difficult to determine the AUC24/MIC in a clinical setting, trough serum concentrations are still monitored clinically. Moreover, there is insufficient evidence to recommend whether trough level-only or AUC/MIC-guided vancomycin monitoring should be used for patients with non-MRSA infections.\\nIn one study of PK/PD determinants of vancomycin efficacy in enterococcal bacteremia, a vancomycin AUC/MIC ≥ 389 achieved within 72 h was associated with reduced mortality. However, the study found that vancomycin trough concentrations differed significantly between survivors and non-survivors [19]. According to Nakamura et al. [20] neither the AUC24/MIC nor the trough concentration of vancomycin is significantly associated with mortality in patients with E. faecium bacteremia. The trough concentration was higher in non-survivors than in survivors, and AUC24/MIC did not differ significantly between non-survivors and survivors.\\nIn our study, vancomycin trough levels of 15 µg/mL or lower were associated with mortality in patients with enterococcal bacteremia, but the AUC24/MIC was not. Zelenitsky et al. reported that the survival rate was higher when the vancomycin trough concentration was maintained above 15 mg/L than when low trough concentrations were maintained [21]. In addition, Kullar et al. reported that the vancomycin treatment period was significantly shorter in the group in which the vancomycin trough concentration was higher than the group to which the vancomycin trough concentration was applied at 5 to 20 mg/L, and the rate at which the bacteria were negatively converted was also found to be significantly higher in the group that maintained the higher trough concentration [22]. According to a meta-analysis comparing high and low vancomycin serum trough regimen groups with gram-positive bacterial infections, there was no significant difference between the two in all-cause mortality or risk of clinical failure; however, a subgroup analysis confirmed that treatment failure was reduced in high vancomycin trough regimen groups [23]. Therefore, in enterococcal bacteremia, the trough level may be a good parameter for monitoring the therapeutic effects of vancomycin.\\nAll enterococcal BSIs treated with vancomycin were included in this study to examine the therapeutic effect of vancomycin on Enterococcus species. Therefore, strains susceptible to ampicillin were added in this study. In most cases, vancomycin was used for ampicillin-susceptible Enterococcus species when beta-lactam antibiotics could not be used due to its side effects, or was used without checking the results of antimicrobial susceptibility tests. Ampicillin is the preferred antibiotic used to treat ampicillin-sensitive enterococcal infections. However, according to several studies comparing the therapeutic effects of beta-lactam antibiotics and vancomycin on ampicillin-susceptible enterococcal BSI, there was no significant difference in mortality [24]. Therefore, when needed, or when beta-lactam antibiotics are difficult to use, vancomycin can be considered as a treatment option for ampicillin-susceptible strains.\\nAn important concern when using vancomycin is the occurrence of acute kidney injury. A higher vancomycin trough concentration increases the risk of VIN [20, 25]. Although most vancomycin-induced nephrotoxic events are reversible, many studies support there is increased mortality from AKI of any cause, including vancomycin-induced nephropathy [26]. While 6 of our 37 patients developed VIN, it was not statistically related to trough concentration (p = 0.09) or 28-day mortality. (p= 0.446).\\nIn this study, mortality was higher at younger ages. Aging process causes structural and functional changes in multiple organ systems, especially the kidneys, and is known to affect PK/PD of drugs by causing changes in body composition, drug absorption, distribution, and clearance [27]. These changes may have affected the patient’s prognosis or treatment outcome.\\nThis study has several limitations that are inherent to its retrospective design. As with any observational study, there remains a possibility that unmeasured confounders influenced our findings. According to several studies, it is well known that sepsis or septic shock affects drug absorption, distribution and other pharmacological processes due to pathophysiological changes such as decreased perfusion to body organs, interstitial edema or increased capillary permeability [28]. Therefore, the PK/PD alterations may not have been reflected depending on the patients’ disease severity. In addition, in the early stage of sepsis, extravascular volume expansion with fluid loading and capillary leak causes a change in volume distribution, which can cause hyperfiltration in the kidney and lead to an alteration of drug concentration. Thereby, it may have affected the prognosis of non-survivors with low trough levels. For these reasons, we cannot exclude the possibility that residual bias affected our findings. Another limitation of this study was that only patients with bacteremia were enrolled, and patients with non-bacteremic sepsis were not analyzed. In addition, the single-center nature of a referral tertiary hospital leads to selection bias in severe cases admitted to our hospital. A multicenter study with a larger sample size is needed.\",\n \"In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species.\",\n \" \\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\\n\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\",\n \"\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,"results","discussion","conclusion","supplementary-material",null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusion\",\n \"supplementary-material\",\n null\n]"},"keywords":{"kind":"list like","value":["Vancomycin","\nEnterococcus\n","Trough level","AUC/MIC"],"string":"[\n \"Vancomycin\",\n \"\\nEnterococcus\\n\",\n \"Trough level\",\n \"AUC/MIC\"\n]"},"whole_article_text":{"kind":"string","value":"Background:\nEnterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.\n\nMethods:\nStudy population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\n\nStudy population and design:\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\n\nDefinitions:\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\n\nMicrobiological data:\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\n\nVancomycin dosing and pharmacodynamics data:\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\n\nStatistical analysis:\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\n\nResults:\nA total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).\n\nTable 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality\nCharacteristics\nTotal (n = 37)Survivors (n = 29)Non-survivors (n = 8)\nP value\n\nDemographics\nAge (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nBaseline demographic and clinical features of patients with enterococcal bacteremia\nBMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis\nContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nThe 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.\nIn univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).\n\nTable 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval\nMultivariate logistic regression analysis for factors associated with 28-day mortality\nOR, odds ratio; CI, confidence interval\n\nFig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level\nKaplan-Meier survival analysis of patients according to vancomycin trough level\n\nDiscussion:\nIn this study, we found that vancomycin trough concentration (≤ 15 µg/mL) was significantly associated with increased mortality in patients with Enterococcus bacteremia.\nMany studies have examined pharmacokinetic/pharmacodynamic (PK/PD) parameters to maintain the efficiency of vancomycin and to minimize its adverse effects, but most of these studies have examined MRSA infection. Of the PK/PD parameters, the trough level and AUC24/MIC have been reported to describe the effectiveness of vancomycin [15, 16]. The clinical success and renal toxicity of vancomycin treatment are exposure-dependent and characterized by the AUC24. The exact range targeted by clinicians is influenced by the AUC24 estimation and bacterial MIC [15, 17]. Using AUC-based vancomycin therapeutic drug monitoring (TDM) helps to individualize the estimation of the AUC24/MIC and minimize the risk of toxicity due to unnecessary overexposure.\nAlthough trough concentration is a known surrogate marker for AUC24/MIC, the method using only trough-based vancomycin TDM is controversial for several reasons. First, the vancomycin trough concentration poorly characterizes the AUC24, and adequate vancomycin AUC24 levels can be obtained at trough concentrations < 15 mg/L. Vancomycin-associated nephrotoxicity also increases when the vancomycin trough concentration is >15 mg/L. These factors provide evidence for an AUC-based approach to vancomycin TDM [18]. Thus, according to the Infectious Diseases Society of America guidelines, the trough level does not correlate well with the AUC, and trough-only monitoring with a target of 15–20 µg/mL is no longer recommended based on its efficacy in patients with MRSA infections [10]. However, because it may be difficult to determine the AUC24/MIC in a clinical setting, trough serum concentrations are still monitored clinically. Moreover, there is insufficient evidence to recommend whether trough level-only or AUC/MIC-guided vancomycin monitoring should be used for patients with non-MRSA infections.\nIn one study of PK/PD determinants of vancomycin efficacy in enterococcal bacteremia, a vancomycin AUC/MIC ≥ 389 achieved within 72 h was associated with reduced mortality. However, the study found that vancomycin trough concentrations differed significantly between survivors and non-survivors [19]. According to Nakamura et al. [20] neither the AUC24/MIC nor the trough concentration of vancomycin is significantly associated with mortality in patients with E. faecium bacteremia. The trough concentration was higher in non-survivors than in survivors, and AUC24/MIC did not differ significantly between non-survivors and survivors.\nIn our study, vancomycin trough levels of 15 µg/mL or lower were associated with mortality in patients with enterococcal bacteremia, but the AUC24/MIC was not. Zelenitsky et al. reported that the survival rate was higher when the vancomycin trough concentration was maintained above 15 mg/L than when low trough concentrations were maintained [21]. In addition, Kullar et al. reported that the vancomycin treatment period was significantly shorter in the group in which the vancomycin trough concentration was higher than the group to which the vancomycin trough concentration was applied at 5 to 20 mg/L, and the rate at which the bacteria were negatively converted was also found to be significantly higher in the group that maintained the higher trough concentration [22]. According to a meta-analysis comparing high and low vancomycin serum trough regimen groups with gram-positive bacterial infections, there was no significant difference between the two in all-cause mortality or risk of clinical failure; however, a subgroup analysis confirmed that treatment failure was reduced in high vancomycin trough regimen groups [23]. Therefore, in enterococcal bacteremia, the trough level may be a good parameter for monitoring the therapeutic effects of vancomycin.\nAll enterococcal BSIs treated with vancomycin were included in this study to examine the therapeutic effect of vancomycin on Enterococcus species. Therefore, strains susceptible to ampicillin were added in this study. In most cases, vancomycin was used for ampicillin-susceptible Enterococcus species when beta-lactam antibiotics could not be used due to its side effects, or was used without checking the results of antimicrobial susceptibility tests. Ampicillin is the preferred antibiotic used to treat ampicillin-sensitive enterococcal infections. However, according to several studies comparing the therapeutic effects of beta-lactam antibiotics and vancomycin on ampicillin-susceptible enterococcal BSI, there was no significant difference in mortality [24]. Therefore, when needed, or when beta-lactam antibiotics are difficult to use, vancomycin can be considered as a treatment option for ampicillin-susceptible strains.\nAn important concern when using vancomycin is the occurrence of acute kidney injury. A higher vancomycin trough concentration increases the risk of VIN [20, 25]. Although most vancomycin-induced nephrotoxic events are reversible, many studies support there is increased mortality from AKI of any cause, including vancomycin-induced nephropathy [26]. While 6 of our 37 patients developed VIN, it was not statistically related to trough concentration (p = 0.09) or 28-day mortality. (p= 0.446).\nIn this study, mortality was higher at younger ages. Aging process causes structural and functional changes in multiple organ systems, especially the kidneys, and is known to affect PK/PD of drugs by causing changes in body composition, drug absorption, distribution, and clearance [27]. These changes may have affected the patient’s prognosis or treatment outcome.\nThis study has several limitations that are inherent to its retrospective design. As with any observational study, there remains a possibility that unmeasured confounders influenced our findings. According to several studies, it is well known that sepsis or septic shock affects drug absorption, distribution and other pharmacological processes due to pathophysiological changes such as decreased perfusion to body organs, interstitial edema or increased capillary permeability [28]. Therefore, the PK/PD alterations may not have been reflected depending on the patients’ disease severity. In addition, in the early stage of sepsis, extravascular volume expansion with fluid loading and capillary leak causes a change in volume distribution, which can cause hyperfiltration in the kidney and lead to an alteration of drug concentration. Thereby, it may have affected the prognosis of non-survivors with low trough levels. For these reasons, we cannot exclude the possibility that residual bias affected our findings. Another limitation of this study was that only patients with bacteremia were enrolled, and patients with non-bacteremic sepsis were not analyzed. In addition, the single-center nature of a referral tertiary hospital leads to selection bias in severe cases admitted to our hospital. A multicenter study with a larger sample size is needed.\n\nConclusions:\nIn conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species.\n\nSupplementary Information:\n \nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\n\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\n\n:\n\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia.\n\nMethods:\nA retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality.\n\nResults:\nA total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia.\n\nConclusions:\nIn this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia."},"background_conclusion_text":{"kind":"string","value":"Background:\nEnterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.\n\nConclusions:\nIn conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nPharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia.\n\nMethods:\nA retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality.\n\nResults:\nA total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia.\n\nConclusions:\nIn this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia."},"whole_article_text_length":{"kind":"number","value":6069,"string":"6,069"},"whole_article_abstract_length":{"kind":"number","value":328,"string":"328"},"other_sections_lengths":{"kind":"list like","value":[495,1925,218,259,89,225,160,46],"string":"[\n 495,\n 1925,\n 218,\n 259,\n 89,\n 225,\n 160,\n 46\n]"},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["vancomycin","bacteremia","trough","patients","study","concentration","mortality","blood","enterococcus","mic"],"string":"[\n \"vancomycin\",\n \"bacteremia\",\n \"trough\",\n \"patients\",\n \"study\",\n \"concentration\",\n \"mortality\",\n \"blood\",\n \"enterococcus\",\n \"mic\"\n]"},"keybert_topics":{"kind":"list like","value":["pathogens especially enterococcus","nosocomial environments enterococcus","ampicillin 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[SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Vancomycin | \nEnterococcus\n | Trough level | AUC/MIC [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Vancomycin | \nEnterococcus\n | Trough level | AUC/MIC [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Vancomycin | \nEnterococcus\n | Trough level | AUC/MIC [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Vancomycin | \nEnterococcus\n | Trough level | AUC/MIC [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Anti-Bacterial Agents | Bacteremia | Enterococcus | Humans | Methicillin-Resistant Staphylococcus aureus | Microbial Sensitivity Tests | Retrospective Studies | Staphylococcal Infections | Treatment Outcome | Vancomycin 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concentration | mortality | blood | enterococcus | mic [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] vancomycin | bacteremia | trough | patients | study | concentration | mortality | blood | enterococcus | mic [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] vancomycin | bacteremia | trough | patients | study | concentration | mortality | blood | enterococcus | mic [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vancomycin | bacteremia | trough | patients | study | concentration | mortality | blood | enterococcus | mic [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vancomycin | bacteremia | trough | patients | study | concentration | mortality | blood | enterococcus | mic [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | vancomycin | enterococcus | resistant | concentration | environments | nosocomial | enterococcus species | mrsa | ampicillin [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 37 | 12 | 24 | 21 | 25 | 15 | mean | ab | day | infection [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] especially enterococcus species | vancomycin pk | prospective studies | prospective studies needed | prospective studies needed examine | day mortality patients enterococcal | vancomycin pk pd parameters | vancomycin pk pd | pathogens especially enterococcus species | conclusion vancomycin trough [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] vancomycin | bacteremia | trough | enterococcus | patients | blood | test | clinical | mortality | study [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] vancomycin | bacteremia | trough | enterococcus | patients | blood | test | clinical | mortality | study [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] methicillin | Staphylococcus ||| AUC/MIC [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 37 | enrolled-26 | 11 ||| 28-day | 21.6 % ||| 15 | 0.042 | 0.044 | 0.049 | 28-day | 389 | 0.479 ||| 15 | 0.041 | 0.031 | 28-day [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 15 | 28-day ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] methicillin | Staphylococcus ||| AUC/MIC ||| Enterococcus | January 2007 to December 2017 | tertiary | Seoul | South Korea ||| ||| 28-day ||| 37 | enrolled-26 | 11 ||| 28-day | 21.6 % ||| 15 | 0.042 | 0.044 | 0.049 | 28-day | 389 | 0.479 ||| 15 | 0.041 | 0.031 | 28-day ||| 15 | 28-day ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] methicillin | Staphylococcus ||| AUC/MIC ||| Enterococcus | January 2007 to December 2017 | tertiary | Seoul | South Korea ||| ||| 28-day ||| 37 | enrolled-26 | 11 ||| 28-day | 21.6 % ||| 15 | 0.042 | 0.044 | 0.049 | 28-day | 389 | 0.479 ||| 15 | 0.041 | 0.031 | 28-day ||| 15 | 28-day ||| [SUMMARY]"}}},{"rowIdx":3397,"cells":{"title":{"kind":"string","value":"Risk factors for prolonged virus shedding of respiratory tract and fecal in adults with severe acute respiratory syndrome coronavirus-2 infection."},"pmid":{"kind":"string","value":"34390043"},"background_abstract":{"kind":"string","value":"The dynamic alteration and comparative study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding pattern during treatment are limited. This study explores the potential risk factors influencing prolonged viral shedding in COVID-19."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A total of 126 COVID-19 patients were enrolled in this retrospective longitudinal study. A multivariate logistic regression analysis was carried out to estimate the potential risk factors."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"38.1% (48/126) cases presented prolonged respiratory tract viral shedding, and 30 (23.8%) cases presented prolonged rectal swab viral shedding. Obesity (OR, 3.31; 95% CI, 1.08-10.09), positive rectal swab (OR, 3.43; 95% CI, 1.53-7.7), treatment by lopinavir/ritonavir with chloroquine phosphate (OR, 2.5; 95% CI, 1.04-6.03), the interval from onset to antiviral treatment more than 7 days (OR, 2.26; 95% CI, 1.04-4.93), lower CD4+ T cell (OR, 0.92; 95% CI, 0.86-0.99) and higher NK cells (OR, 1.11; 95% CI, 1.02-1.20) were significantly associated with prolonged respiratory tract viral shedding. CD3-CD56+ NK cells (OR, 0.87; 95% CI, 0.76-0.99) were related with prolonged fecal shedding."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Obesity, delayed antiviral treatment, and positive SARS-CoV-2 for stool were independent risk factors for prolonged SARS-CoV-2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with chloroquine phosphate. CD4+ T cell and NK cells were significantly associated with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Animals","Antiviral Agents","CD4 Lymphocyte Count","COVID-19","Chloroquine","Feces","Female","Humans","Killer Cells, Natural","Longitudinal Studies","Lopinavir","Lynx","Male","Obesity","Respiratory System","Retrospective Studies","Risk Factors","Ritonavir","SARS-CoV-2","Time Factors","Virus Shedding"],"string":"[\n \"Adult\",\n \"Animals\",\n \"Antiviral Agents\",\n \"CD4 Lymphocyte Count\",\n \"COVID-19\",\n \"Chloroquine\",\n \"Feces\",\n \"Female\",\n \"Humans\",\n \"Killer Cells, Natural\",\n \"Longitudinal Studies\",\n \"Lopinavir\",\n \"Lynx\",\n \"Male\",\n \"Obesity\",\n \"Respiratory System\",\n \"Retrospective Studies\",\n \"Risk Factors\",\n \"Ritonavir\",\n \"SARS-CoV-2\",\n \"Time Factors\",\n \"Virus Shedding\"\n]"},"pmcid":{"kind":"string","value":"8418473"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Demographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nA total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nLaboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nBaseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nTreatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nAll patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nRisk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nThe univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nRisk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\nPatients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Study design and participants","Data collection","Cutoff of respiratory and fecal viral shedding","Statistical analysis","Demographic and clinical manifestations","Laboratory findings and immunological indicators","Treatment and outcome","Risk factors with prolonged respiratory tract viral shedding","Risk factors with prolonged fecal viral shedding","AUTHOR CONTRIBUTIONS"],"string":"[\n \"INTRODUCTION\",\n \"Study design and participants\",\n \"Data collection\",\n \"Cutoff of respiratory and fecal viral shedding\",\n \"Statistical analysis\",\n \"Demographic and clinical manifestations\",\n \"Laboratory findings and immunological indicators\",\n \"Treatment and outcome\",\n \"Risk factors with prolonged respiratory tract viral shedding\",\n \"Risk factors with prolonged fecal viral shedding\",\n \"AUTHOR CONTRIBUTIONS\"\n]"},"other_sections_texts":{"kind":"list like","value":["Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.","This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.","Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.","The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.","Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.","A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.","Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).","All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset","The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.","Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).","Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content."],"string":"[\n \"Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.\",\n \"This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\\nThe privacy rights of human subjects are always observed.\",\n \"Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\\n\\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\",\n \"The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\",\n \"Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\",\n \"A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\\nAbbreviations: BMI, body mass index; CT, computed tomography.\\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\",\n \"Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\",\n \"All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\\nTreatment and outcomes of COVID‐19 patients\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\",\n \"The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\",\n \"Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\",\n \"Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Study design and participants","Data collection","Cutoff of respiratory and fecal viral shedding","Statistical analysis","RESULTS","Demographic and clinical manifestations","Laboratory findings and immunological indicators","Treatment and outcome","Risk factors with prolonged respiratory tract viral shedding","Risk factors with prolonged fecal viral shedding","DISCUSSION","CONFLICT OF INTEREST","AUTHOR CONTRIBUTIONS","Supporting information"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Study design and participants\",\n \"Data collection\",\n \"Cutoff of respiratory and fecal viral shedding\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Demographic and clinical manifestations\",\n \"Laboratory findings and immunological indicators\",\n \"Treatment and outcome\",\n \"Risk factors with prolonged respiratory tract viral shedding\",\n \"Risk factors with prolonged fecal viral shedding\",\n \"DISCUSSION\",\n \"CONFLICT OF INTEREST\",\n \"AUTHOR CONTRIBUTIONS\",\n \"Supporting information\"\n]"},"all_sections_texts":{"kind":"list like","value":["Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.","Study design and participants This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\nThis longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\nData collection Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\nDemographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\nCutoff of respiratory and fecal viral shedding The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\nThe duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\nStatistical analysis Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\nContinuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.","This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.","Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.","The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.","Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.","Demographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nA total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nLaboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nBaseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nTreatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nAll patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nRisk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nThe univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nRisk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\nPatients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).","A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.","Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).","All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset","The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.","Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).","The viral shedding window of COVID‐19 remains largely uncharacterized, which poses challenges to reappraising discontinuation of quarantine. In our study, the dynamic detection of SARS‐CoV‐2 RNA on the respiratory tract and rectal swabs among 126 infected cases was conducted. We found that 38.1% (48/126) of confirmed cases presented prolonged respiratory tract viral shedding, and 23.8% (30/126) of cases presented prolonged fecal viral shedding. Obesity, positive detection of rectal swab species for viral RNA, delayed antiviral treatment, elevated NK, and decreased CD4+ T cell cells were significantly related to prolonged respiratory viral shedding, and patients treated by LPV/r combined with arbidol had remarkably shortened duration of respiratory viral shedding compared with those treated by LPV/r combined with CQ. We also identified that decreased CD3−CD56+ NK cells on admission were related to shorten fecal shedding.\nIn this study, the median duration from onset of symptoms to antiviral therapy was 3 days (IQR, 1–7.3 days), and the interval from onset to antiviral treatment more than 7 days was an independent risk factor for prolonged viral shedding. Our results were in line with the current report that timely detection and supportive care for symptomatic patients with COVID‐19 contributed to the clearance of viral RNA.15 Previous data indicated the complex interaction between obesity and increased pneumonia risk, and the underlying mechanism might involve immune system dysregulation.16, 17 A retrospective cohort study in French reported that the proportion of COVID‐19 patients who required invasive mechanical ventilation increased with BMI.18 Additionally, a previous study on influenza A H1N1 found obesity was associated with prolonged hospitalization and the need for IMV.19 In our cohort, the obesity rate was 13.5%, and obesity was an independent predisposition factor for prolonged respiratory tract viral RNA shedding after adjustment for age and gender. Obesity‐related low‐grade inflammation appeared to be associated with the prognosis of virus infection, and further study is to be warranted to elucidate etiological mechanisms.\nNo specific antiviral drugs have been approved to be safe and effective for the treatment of COVID‐19 so far. Some drugs with antiviral properties and immunomodulatory effects were officially recommended for COVID‐19, including LPV/r, arbidol, CQ, etc.12, 20 LPV/r and arbidol were previously recommended for patients with SARS and MERS‐CoV.21, 22 Some studies implied that the use of LPV/r and arbidol was associated with an apparent favorable clinical response to COVID‐19,23, 24 but controversy existed in different studies. For example, Cheng and his/her colleagues reported LPV/r had no advantage in shortening the duration of SARS‐CoV‐2 shedding in five mild patients in Taiwan.25 The US Center for Disease Control clarified that CQ inhibited viral replication by interfering with SARS‐CoV‐2 binding to the ACE2 receptor.26 CQ has been used to treat COVID‐19 with no benefits having been observed in a multinational registry analysis.27 Our findings demonstrated that the use of LPV/r in combination with arbidol was linked with a shorter hospital stay and duration of viral shedding compared with the use of LPV/r in combination with CQ.\nT lymphocyte plays a vital role in SARS‐CoV‐2 elimination by stimulating antigen‐(Ag) specific T lymphocytes.28 Emerging data indicated that lymphocytopenia was one of the important predictor factors influencing exacerbation and mortality of patients with COVID‐19.29, 30, 31 Likewise, our data identified that decreased CD4+ T cell was a significant risk factor for prolonged viral shedding of the respiratory tract. NK cells were one of the predominant lymphocyte subsets and accounted for the third of intrahepatic lymphocytes.32 Viral infection‐induced NK cells present activated and mature phenotypes with stronger cytotoxic capabilities in a murine model.33 However, our study showed the increased NK cell was a risk factor for prolonged viral shedding of the respiratory tract but a protective factor for fecal viral shedding. Our study also showed that 60 (47.6%) of 126 COVID‐19 patients detected positive fecal viral results, and 30 (23.8%) presented prolonged fecal viral shedding, and the positive SARS‐CoV‐2 for stool was related to prolonged duration of respiratory viral shedding. However, GI symptoms just occurred in 11(8.7%) of patients in our study. Likewise, a meta‐analysis reported that approximately 7.4% of patients with COVID‐19 experienced diarrhea, and about 30% to 50% of patients were detected for positive SARS‐CoV‐2 RNA.34 Chen et al.15 found diarrhea was independently associated with prolonged SARS‐CoV‐2 RNA shedding in the respiratory tract among 267 confirmed cases. It has been proven that ACE2 is abundantly expressed in the GI tract.35, 36 Positive detection of SARS‐CoV‐2 in digestive tract specimens suggested that pathogenicity of SARS‐CoV‐2 might be transmitted by the fecal‐oral route, not limited by droplet transmission.37 In our study, the viral shedding patterns of the respiratory and digestive tract were not consistent. Similarly, Wang et al.38 reported that the median duration of positive SARS‐CoV‐2 in fecal samples was 25 days, significantly longer than that in respiratory tract samples by approximately 9 days and that patients with SARS‐CoV‐2 RNA in fecal presented higher proportions of complete absorption and no lesion progression of chest CT results.\nThere were some limitations in this study. First, most patients enrolled in this study had mild clinical symptoms, so our findings may not apply to severe and critical patients. Second, immune indicators including T lymphocyte subsets and cytokines were not conducted in all patients, so the role of immunomodulatory effects in eliminating SARS‐CoV‐2 might be underestimated. Third, our study was a single‐center study and limited by a relatively small sample size. Fourth, the underlying reason for the unparallel detection results between the respiratory tract and fecal samples needs to be clarified in the future.\nIn conclusion, obesity, delayed antiviral treatment, and positive SARS‐CoV‐2 for stool were independent risk factors for prolonged SARS‐CoV‐2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with CQ. Decreased CD4+ T cell and elevated CD3−CD56+ NK cells were associated with prolonged respiratory tract RNA shedding, while decreased CD3−CD56+ NK cells might be linked with shortened fecal RNA shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.","None.","Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.","Fig S1\nClick here for additional data file.\nTab S1\nClick here for additional data file.\nTab S2\nClick here for additional data file.\nTab S3\nClick here for additional data file.\nTab S4\nClick here for additional data file."],"string":"[\n \"Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.\",\n \"Study design and participants This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\\nThe privacy rights of human subjects are always observed.\\nThis longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\\nThe privacy rights of human subjects are always observed.\\nData collection Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\\n\\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\\nDemographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\\n\\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\\nCutoff of respiratory and fecal viral shedding The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\\nThe duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\\nStatistical analysis Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\\nContinuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\",\n \"This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\\nThe privacy rights of human subjects are always observed.\",\n \"Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\\n\\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\",\n \"The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\",\n \"Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\",\n \"Demographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\\nAbbreviations: BMI, body mass index; CT, computed tomography.\\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\\nA total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\\nAbbreviations: BMI, body mass index; CT, computed tomography.\\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\\nLaboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\\nBaseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\\nTreatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\\nTreatment and outcomes of COVID‐19 patients\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\\nAll patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\\nTreatment and outcomes of COVID‐19 patients\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\\nRisk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\\nThe univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\\nRisk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\\nPatients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\",\n \"A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\\nAbbreviations: BMI, body mass index; CT, computed tomography.\\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\",\n \"Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\",\n \"All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\\nTreatment and outcomes of COVID‐19 patients\\nData are presented as the median and inter‐quartile range (IQR) and n (%).\\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\",\n \"The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\",\n \"Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\",\n \"The viral shedding window of COVID‐19 remains largely uncharacterized, which poses challenges to reappraising discontinuation of quarantine. In our study, the dynamic detection of SARS‐CoV‐2 RNA on the respiratory tract and rectal swabs among 126 infected cases was conducted. We found that 38.1% (48/126) of confirmed cases presented prolonged respiratory tract viral shedding, and 23.8% (30/126) of cases presented prolonged fecal viral shedding. Obesity, positive detection of rectal swab species for viral RNA, delayed antiviral treatment, elevated NK, and decreased CD4+ T cell cells were significantly related to prolonged respiratory viral shedding, and patients treated by LPV/r combined with arbidol had remarkably shortened duration of respiratory viral shedding compared with those treated by LPV/r combined with CQ. We also identified that decreased CD3−CD56+ NK cells on admission were related to shorten fecal shedding.\\nIn this study, the median duration from onset of symptoms to antiviral therapy was 3 days (IQR, 1–7.3 days), and the interval from onset to antiviral treatment more than 7 days was an independent risk factor for prolonged viral shedding. Our results were in line with the current report that timely detection and supportive care for symptomatic patients with COVID‐19 contributed to the clearance of viral RNA.15 Previous data indicated the complex interaction between obesity and increased pneumonia risk, and the underlying mechanism might involve immune system dysregulation.16, 17 A retrospective cohort study in French reported that the proportion of COVID‐19 patients who required invasive mechanical ventilation increased with BMI.18 Additionally, a previous study on influenza A H1N1 found obesity was associated with prolonged hospitalization and the need for IMV.19 In our cohort, the obesity rate was 13.5%, and obesity was an independent predisposition factor for prolonged respiratory tract viral RNA shedding after adjustment for age and gender. Obesity‐related low‐grade inflammation appeared to be associated with the prognosis of virus infection, and further study is to be warranted to elucidate etiological mechanisms.\\nNo specific antiviral drugs have been approved to be safe and effective for the treatment of COVID‐19 so far. Some drugs with antiviral properties and immunomodulatory effects were officially recommended for COVID‐19, including LPV/r, arbidol, CQ, etc.12, 20 LPV/r and arbidol were previously recommended for patients with SARS and MERS‐CoV.21, 22 Some studies implied that the use of LPV/r and arbidol was associated with an apparent favorable clinical response to COVID‐19,23, 24 but controversy existed in different studies. For example, Cheng and his/her colleagues reported LPV/r had no advantage in shortening the duration of SARS‐CoV‐2 shedding in five mild patients in Taiwan.25 The US Center for Disease Control clarified that CQ inhibited viral replication by interfering with SARS‐CoV‐2 binding to the ACE2 receptor.26 CQ has been used to treat COVID‐19 with no benefits having been observed in a multinational registry analysis.27 Our findings demonstrated that the use of LPV/r in combination with arbidol was linked with a shorter hospital stay and duration of viral shedding compared with the use of LPV/r in combination with CQ.\\nT lymphocyte plays a vital role in SARS‐CoV‐2 elimination by stimulating antigen‐(Ag) specific T lymphocytes.28 Emerging data indicated that lymphocytopenia was one of the important predictor factors influencing exacerbation and mortality of patients with COVID‐19.29, 30, 31 Likewise, our data identified that decreased CD4+ T cell was a significant risk factor for prolonged viral shedding of the respiratory tract. NK cells were one of the predominant lymphocyte subsets and accounted for the third of intrahepatic lymphocytes.32 Viral infection‐induced NK cells present activated and mature phenotypes with stronger cytotoxic capabilities in a murine model.33 However, our study showed the increased NK cell was a risk factor for prolonged viral shedding of the respiratory tract but a protective factor for fecal viral shedding. Our study also showed that 60 (47.6%) of 126 COVID‐19 patients detected positive fecal viral results, and 30 (23.8%) presented prolonged fecal viral shedding, and the positive SARS‐CoV‐2 for stool was related to prolonged duration of respiratory viral shedding. However, GI symptoms just occurred in 11(8.7%) of patients in our study. Likewise, a meta‐analysis reported that approximately 7.4% of patients with COVID‐19 experienced diarrhea, and about 30% to 50% of patients were detected for positive SARS‐CoV‐2 RNA.34 Chen et al.15 found diarrhea was independently associated with prolonged SARS‐CoV‐2 RNA shedding in the respiratory tract among 267 confirmed cases. It has been proven that ACE2 is abundantly expressed in the GI tract.35, 36 Positive detection of SARS‐CoV‐2 in digestive tract specimens suggested that pathogenicity of SARS‐CoV‐2 might be transmitted by the fecal‐oral route, not limited by droplet transmission.37 In our study, the viral shedding patterns of the respiratory and digestive tract were not consistent. Similarly, Wang et al.38 reported that the median duration of positive SARS‐CoV‐2 in fecal samples was 25 days, significantly longer than that in respiratory tract samples by approximately 9 days and that patients with SARS‐CoV‐2 RNA in fecal presented higher proportions of complete absorption and no lesion progression of chest CT results.\\nThere were some limitations in this study. First, most patients enrolled in this study had mild clinical symptoms, so our findings may not apply to severe and critical patients. Second, immune indicators including T lymphocyte subsets and cytokines were not conducted in all patients, so the role of immunomodulatory effects in eliminating SARS‐CoV‐2 might be underestimated. Third, our study was a single‐center study and limited by a relatively small sample size. Fourth, the underlying reason for the unparallel detection results between the respiratory tract and fecal samples needs to be clarified in the future.\\nIn conclusion, obesity, delayed antiviral treatment, and positive SARS‐CoV‐2 for stool were independent risk factors for prolonged SARS‐CoV‐2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with CQ. Decreased CD4+ T cell and elevated CD3−CD56+ NK cells were associated with prolonged respiratory tract RNA shedding, while decreased CD3−CD56+ NK cells might be linked with shortened fecal RNA shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.\",\n \"None.\",\n \"Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.\",\n \"Fig S1\\nClick here for additional data file.\\nTab S1\\nClick here for additional data file.\\nTab S2\\nClick here for additional data file.\\nTab S3\\nClick here for additional data file.\\nTab S4\\nClick here for additional data file.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":[null,"materials-and-methods",null,null,null,null,"results",null,null,null,null,null,"discussion","COI-statement",null,"supplementary-material"],"string":"[\n null,\n \"materials-and-methods\",\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"COI-statement\",\n null,\n \"supplementary-material\"\n]"},"keywords":{"kind":"list like","value":["COVID‐19","longitudinal study","prolonged viral shedding","risk factor","SARS‐CoV‐2"],"string":"[\n \"COVID‐19\",\n \"longitudinal study\",\n \"prolonged viral shedding\",\n \"risk factor\",\n \"SARS‐CoV‐2\"\n]"},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.\n\nMATERIALS AND METHODS:\nStudy design and participants This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\nThis longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\nData collection Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\nDemographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\nCutoff of respiratory and fecal viral shedding The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\nThe duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\nStatistical analysis Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\nContinuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\n\nStudy design and participants:\nThis longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\n\nData collection:\nDemographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\n\nCutoff of respiratory and fecal viral shedding:\nThe duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\n\nStatistical analysis:\nContinuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\n\nRESULTS:\nDemographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nA total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nLaboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nBaseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nTreatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nAll patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nRisk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nThe univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nRisk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\nPatients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\n\nDemographic and clinical manifestations:\nA total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\n\nLaboratory findings and immunological indicators:\nBaseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\n\nTreatment and outcome:\nAll patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\n\nRisk factors with prolonged respiratory tract viral shedding:\nThe univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\n\nRisk factors with prolonged fecal viral shedding:\nPatients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\n\nDISCUSSION:\nThe viral shedding window of COVID‐19 remains largely uncharacterized, which poses challenges to reappraising discontinuation of quarantine. In our study, the dynamic detection of SARS‐CoV‐2 RNA on the respiratory tract and rectal swabs among 126 infected cases was conducted. We found that 38.1% (48/126) of confirmed cases presented prolonged respiratory tract viral shedding, and 23.8% (30/126) of cases presented prolonged fecal viral shedding. Obesity, positive detection of rectal swab species for viral RNA, delayed antiviral treatment, elevated NK, and decreased CD4+ T cell cells were significantly related to prolonged respiratory viral shedding, and patients treated by LPV/r combined with arbidol had remarkably shortened duration of respiratory viral shedding compared with those treated by LPV/r combined with CQ. We also identified that decreased CD3−CD56+ NK cells on admission were related to shorten fecal shedding.\nIn this study, the median duration from onset of symptoms to antiviral therapy was 3 days (IQR, 1–7.3 days), and the interval from onset to antiviral treatment more than 7 days was an independent risk factor for prolonged viral shedding. Our results were in line with the current report that timely detection and supportive care for symptomatic patients with COVID‐19 contributed to the clearance of viral RNA.15 Previous data indicated the complex interaction between obesity and increased pneumonia risk, and the underlying mechanism might involve immune system dysregulation.16, 17 A retrospective cohort study in French reported that the proportion of COVID‐19 patients who required invasive mechanical ventilation increased with BMI.18 Additionally, a previous study on influenza A H1N1 found obesity was associated with prolonged hospitalization and the need for IMV.19 In our cohort, the obesity rate was 13.5%, and obesity was an independent predisposition factor for prolonged respiratory tract viral RNA shedding after adjustment for age and gender. Obesity‐related low‐grade inflammation appeared to be associated with the prognosis of virus infection, and further study is to be warranted to elucidate etiological mechanisms.\nNo specific antiviral drugs have been approved to be safe and effective for the treatment of COVID‐19 so far. Some drugs with antiviral properties and immunomodulatory effects were officially recommended for COVID‐19, including LPV/r, arbidol, CQ, etc.12, 20 LPV/r and arbidol were previously recommended for patients with SARS and MERS‐CoV.21, 22 Some studies implied that the use of LPV/r and arbidol was associated with an apparent favorable clinical response to COVID‐19,23, 24 but controversy existed in different studies. For example, Cheng and his/her colleagues reported LPV/r had no advantage in shortening the duration of SARS‐CoV‐2 shedding in five mild patients in Taiwan.25 The US Center for Disease Control clarified that CQ inhibited viral replication by interfering with SARS‐CoV‐2 binding to the ACE2 receptor.26 CQ has been used to treat COVID‐19 with no benefits having been observed in a multinational registry analysis.27 Our findings demonstrated that the use of LPV/r in combination with arbidol was linked with a shorter hospital stay and duration of viral shedding compared with the use of LPV/r in combination with CQ.\nT lymphocyte plays a vital role in SARS‐CoV‐2 elimination by stimulating antigen‐(Ag) specific T lymphocytes.28 Emerging data indicated that lymphocytopenia was one of the important predictor factors influencing exacerbation and mortality of patients with COVID‐19.29, 30, 31 Likewise, our data identified that decreased CD4+ T cell was a significant risk factor for prolonged viral shedding of the respiratory tract. NK cells were one of the predominant lymphocyte subsets and accounted for the third of intrahepatic lymphocytes.32 Viral infection‐induced NK cells present activated and mature phenotypes with stronger cytotoxic capabilities in a murine model.33 However, our study showed the increased NK cell was a risk factor for prolonged viral shedding of the respiratory tract but a protective factor for fecal viral shedding. Our study also showed that 60 (47.6%) of 126 COVID‐19 patients detected positive fecal viral results, and 30 (23.8%) presented prolonged fecal viral shedding, and the positive SARS‐CoV‐2 for stool was related to prolonged duration of respiratory viral shedding. However, GI symptoms just occurred in 11(8.7%) of patients in our study. Likewise, a meta‐analysis reported that approximately 7.4% of patients with COVID‐19 experienced diarrhea, and about 30% to 50% of patients were detected for positive SARS‐CoV‐2 RNA.34 Chen et al.15 found diarrhea was independently associated with prolonged SARS‐CoV‐2 RNA shedding in the respiratory tract among 267 confirmed cases. It has been proven that ACE2 is abundantly expressed in the GI tract.35, 36 Positive detection of SARS‐CoV‐2 in digestive tract specimens suggested that pathogenicity of SARS‐CoV‐2 might be transmitted by the fecal‐oral route, not limited by droplet transmission.37 In our study, the viral shedding patterns of the respiratory and digestive tract were not consistent. Similarly, Wang et al.38 reported that the median duration of positive SARS‐CoV‐2 in fecal samples was 25 days, significantly longer than that in respiratory tract samples by approximately 9 days and that patients with SARS‐CoV‐2 RNA in fecal presented higher proportions of complete absorption and no lesion progression of chest CT results.\nThere were some limitations in this study. First, most patients enrolled in this study had mild clinical symptoms, so our findings may not apply to severe and critical patients. Second, immune indicators including T lymphocyte subsets and cytokines were not conducted in all patients, so the role of immunomodulatory effects in eliminating SARS‐CoV‐2 might be underestimated. Third, our study was a single‐center study and limited by a relatively small sample size. Fourth, the underlying reason for the unparallel detection results between the respiratory tract and fecal samples needs to be clarified in the future.\nIn conclusion, obesity, delayed antiviral treatment, and positive SARS‐CoV‐2 for stool were independent risk factors for prolonged SARS‐CoV‐2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with CQ. Decreased CD4+ T cell and elevated CD3−CD56+ NK cells were associated with prolonged respiratory tract RNA shedding, while decreased CD3−CD56+ NK cells might be linked with shortened fecal RNA shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.\n\nCONFLICT OF INTEREST:\nNone.\n\nAUTHOR CONTRIBUTIONS:\nTing Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.\n\nSupporting information:\nFig S1\nClick here for additional data file.\nTab S1\nClick here for additional data file.\nTab S2\nClick here for additional data file.\nTab S3\nClick here for additional data file.\nTab S4\nClick here for additional data file.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe dynamic alteration and comparative study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding pattern during treatment are limited. This study explores the potential risk factors influencing prolonged viral shedding in COVID-19.\n\nMethods:\nA total of 126 COVID-19 patients were enrolled in this retrospective longitudinal study. A multivariate logistic regression analysis was carried out to estimate the potential risk factors.\n\nResults:\n38.1% (48/126) cases presented prolonged respiratory tract viral shedding, and 30 (23.8%) cases presented prolonged rectal swab viral shedding. Obesity (OR, 3.31; 95% CI, 1.08-10.09), positive rectal swab (OR, 3.43; 95% CI, 1.53-7.7), treatment by lopinavir/ritonavir with chloroquine phosphate (OR, 2.5; 95% CI, 1.04-6.03), the interval from onset to antiviral treatment more than 7 days (OR, 2.26; 95% CI, 1.04-4.93), lower CD4+ T cell (OR, 0.92; 95% CI, 0.86-0.99) and higher NK cells (OR, 1.11; 95% CI, 1.02-1.20) were significantly associated with prolonged respiratory tract viral shedding. CD3-CD56+ NK cells (OR, 0.87; 95% CI, 0.76-0.99) were related with prolonged fecal shedding.\n\nConclusions:\nObesity, delayed antiviral treatment, and positive SARS-CoV-2 for stool were independent risk factors for prolonged SARS-CoV-2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with chloroquine phosphate. CD4+ T cell and NK cells were significantly associated with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":7980,"string":"7,980"},"whole_article_abstract_length":{"kind":"number","value":356,"string":"356"},"other_sections_lengths":{"kind":"list like","value":[239,137,190,144,146,384,126,547,234,196,70],"string":"[\n 239,\n 137,\n 190,\n 144,\n 146,\n 384,\n 126,\n 547,\n 234,\n 196,\n 70\n]"},"num_sections":{"kind":"number","value":16,"string":"16"},"most_frequent_words":{"kind":"list like","value":["shedding","days","viral","patients","respiratory","positive","viral shedding","cov","sars","sars cov"],"string":"[\n \"shedding\",\n \"days\",\n \"viral\",\n \"patients\",\n \"respiratory\",\n \"positive\",\n \"viral shedding\",\n \"cov\",\n 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19 | covid | gi | respiratory | cov | sars | sars cov | viral | manifestation [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] days | 95 ci | 95 | ci | 28 | patients | positive | 28 days | day | shedding [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] days | viral | patients | shedding | respiratory | positive | 28 | viral shedding | sars | cov [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2 | RNA ||| COVID-19 [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 38.1% | 48/126 | 30 | 23.8% ||| 3.31 | 95% | CI | 1.08 | 3.43 | 95% | CI | 1.53 | 2.5 | 95% | CI | 1.04 | more than 7 days | 2.26 | 95% | CI | 1.04-4.93 | 0.92 | 95% | CI | 0.86 | 1.11 | 95% | CI | 1.02 ||| 0.87 | 95% | CI | 0.76-0.99 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2 | RNA ||| COVID-19 ||| 126 ||| ||| 38.1% | 48/126 | 30 | 23.8% ||| 3.31 | 95% | CI | 1.08 | 3.43 | 95% | CI | 1.53 | 2.5 | 95% | CI | 1.04 | more than 7 days | 2.26 | 95% | CI | 1.04-4.93 | 0.92 | 95% | CI | 0.86 | 1.11 | 95% | CI | 1.02 ||| 0.87 | 95% | CI | 0.76-0.99 ||| ||| LPV | LPV ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":3398,"cells":{"title":{"kind":"string","value":"Characterizing Help-Seeking Searches for Substance Use Treatment From Google Trends and Assessing Their Use for Infoveillance: Longitudinal Descriptive and Validation Statistical Analysis."},"pmid":{"kind":"string","value":"36454620"},"background_abstract":{"kind":"string","value":"There is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUDs) seeking help within a given geographical area. This presents a challenge to policy makers in the effective deployment of resources for the treatment of SUDs. Internet search queries related to help seeking for SUDs using Google Trends may represent a low-cost, real-time, and data-driven infoveillance tool to address this shortfall in information."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We used negative binomial regression models to examine temporal trends in the annual SUD help-seeking internet search queries from Google Trends by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol from 2010 to 2020. To validate the value of these data for surveillance purposes, we then used negative binomial regression models to investigate the relationship between SUD help-seeking searches and state-level outcomes across the continuum of care (including lack of care). We started by looking at associations with self-reported treatment need using data from the National Survey on Drug Use and Health, a national survey of the US general population. Next, we explored associations with treatment admission rates from the Treatment Episode Data Set, a national data system on SUD treatment facilities. Finally, we studied associations with state-level rates of people experiencing and dying from an opioid overdose, using data from the Agency for Healthcare Research and Quality and the CDC WONDER database."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Statistically significant differences in help-seeking searches were observed over time between 2010 and 2020 (based on P<.05 for the corresponding Wald tests). We were able to identify outlier states for each drug over time (eg, West Virginia for both opioids and methamphetamine), indicating significantly higher help-seeking behaviors compared to national trends. Results from our validation analyses across different outcomes showed positive, statistically significant associations for the models relating to treatment need for alcohol use, treatment admissions for opioid and methamphetamine use, emergency department visits related to opioid use, and opioid overdose mortality data (based on regression coefficients having P≤.05)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study demonstrates the clear potential for using internet search queries from Google Trends as an infoveillance tool to predict the demand for substance use treatment spatially and temporally, especially for opioid use disorders."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["United States","Humans","Analgesics, Opioid","Opiate Overdose","Infodemiology","Search Engine","Opioid-Related Disorders","Methamphetamine"],"string":"[\n \"United States\",\n \"Humans\",\n \"Analgesics, Opioid\",\n \"Opiate Overdose\",\n \"Infodemiology\",\n \"Search Engine\",\n \"Opioid-Related Disorders\",\n \"Methamphetamine\"\n]"},"pmcid":{"kind":"string","value":"9756118"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].\nIndirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.\nA promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].\nWhile the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.\nWe sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.\nWe tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Extraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nWe obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nStatistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nNegative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nValidation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nFirst, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nValidation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nWe investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nValidation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nWe investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nEthical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\nEthical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Descriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nFigure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nStatistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nThe negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nThe analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nThe analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nValidation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs."},"other_sections_titles":{"kind":"list like","value":["Extraction of Google Search Query Data","Statistical Analysis of Google Search Query Data","Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1)","Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2)","Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)","Ethical Considerations","Descriptive Analysis of Google Search Query Data for SUD Help Seeking","Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking","Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1)","Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2)","Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)","Principal Findings","Limitations","Conclusions"],"string":"[\n \"Extraction of Google Search Query Data\",\n \"Statistical Analysis of Google Search Query Data\",\n \"Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1)\",\n \"Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2)\",\n \"Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)\",\n \"Ethical Considerations\",\n \"Descriptive Analysis of Google Search Query Data for SUD Help Seeking\",\n \"Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking\",\n \"Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1)\",\n \"Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2)\",\n \"Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)\",\n \"Principal Findings\",\n \"Limitations\",\n \"Conclusions\"\n]"},"other_sections_texts":{"kind":"list like","value":["We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.","Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.","First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.","We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.","We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].","Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).","Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.","The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.","The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.","The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.","The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.","To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].","Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].","This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs."],"string":"[\n \"We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\",\n \"Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\\nbMeth: methamphetamine.\\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\",\n \"First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\",\n \"We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\",\n \"We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\",\n \"Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\",\n \"Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\",\n \"The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\\nGini coefficient estimates from query fractions (QF) variables across substances and years.\\nBox and whisker plot of help-seeking searches for opioid use.\",\n \"The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\",\n \"The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\\naQF: query fraction.\\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\",\n \"The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\\naQF: query fraction.\",\n \"To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\",\n \"Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\",\n \"This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\"\n]"},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]"},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Extraction of Google Search Query Data","Statistical Analysis of Google Search Query Data","Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1)","Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2)","Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)","Ethical Considerations","Results","Descriptive Analysis of Google Search Query Data for SUD Help Seeking","Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking","Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1)","Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2)","Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)","Discussion","Principal Findings","Limitations","Conclusions"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Extraction of Google Search Query Data\",\n \"Statistical Analysis of Google Search Query Data\",\n \"Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1)\",\n \"Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2)\",\n \"Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)\",\n \"Ethical Considerations\",\n \"Results\",\n \"Descriptive Analysis of Google Search Query Data for SUD Help Seeking\",\n \"Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking\",\n \"Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1)\",\n \"Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2)\",\n \"Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)\",\n \"Discussion\",\n \"Principal Findings\",\n \"Limitations\",\n \"Conclusions\"\n]"},"all_sections_texts":{"kind":"list like","value":["Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].\nIndirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.\nA promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].\nWhile the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.\nWe sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.\nWe tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.","Extraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nWe obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nStatistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nNegative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nValidation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nFirst, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nValidation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nWe investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nValidation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nWe investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nEthical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\nEthical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).","We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.","Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.","First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.","We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.","We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].","Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).","Descriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nFigure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nStatistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nThe negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nThe analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nThe analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nValidation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.","Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.","The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.","The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.","The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.","The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.","Principal Findings To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\nTo our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\nLimitations Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\nOur study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\nConclusions This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\nThis study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.","To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].","Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].","This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs."],"string":"[\n \"Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].\\nIndirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.\\nA promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].\\nWhile the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.\\nWe sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.\\nWe tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.\",\n \"Extraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\\nWe obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\\nStatistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\\nbMeth: methamphetamine.\\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\\nNegative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\\nbMeth: methamphetamine.\\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\\nValidation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\\nFirst, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\\nValidation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\\nWe investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\\nValidation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\\nWe investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\\nEthical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\\nEthical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\",\n \"We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\",\n \"Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\\nbMeth: methamphetamine.\\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\",\n \"First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\",\n \"We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\",\n \"We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\",\n \"Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\",\n \"Descriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\\nFigure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\\nStatistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\\nGini coefficient estimates from query fractions (QF) variables across substances and years.\\nBox and whisker plot of help-seeking searches for opioid use.\\nThe negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\\nGini coefficient estimates from query fractions (QF) variables across substances and years.\\nBox and whisker plot of help-seeking searches for opioid use.\\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\\nThe analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\\naQF: query fraction.\\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\\nThe analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\\naQF: query fraction.\\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\\nValidation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\\naQF: query fraction.\\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\\naQF: query fraction.\",\n \"Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\",\n \"The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\\nGini coefficient estimates from query fractions (QF) variables across substances and years.\\nBox and whisker plot of help-seeking searches for opioid use.\",\n \"The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\",\n \"The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\\naQF: query fraction.\\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\",\n \"The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\\naQF: query fraction.\",\n \"Principal Findings To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\\nTo our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\\nLimitations Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\\nOur study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\\nConclusions This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\\nThis study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\",\n \"To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\",\n \"Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\",\n \"This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["introduction","methods",null,null,null,null,null,null,"results",null,null,null,null,null,"discussion",null,null,null],"string":"[\n \"introduction\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null\n]"},"keywords":{"kind":"list like","value":["internet","search","help-seeking","substance use treatment","surveillance","infoveillance","google trends"],"string":"[\n \"internet\",\n \"search\",\n \"help-seeking\",\n \"substance use treatment\",\n \"surveillance\",\n \"infoveillance\",\n \"google trends\"\n]"},"whole_article_text":{"kind":"string","value":"Introduction:\nUnderstanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].\nIndirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.\nA promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].\nWhile the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.\nWe sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.\nWe tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.\n\nMethods:\nExtraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nWe obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nStatistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nNegative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nValidation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nFirst, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nValidation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nWe investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nValidation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nWe investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nEthical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\nEthical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\n\nExtraction of Google Search Query Data:\nWe obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\n\nStatistical Analysis of Google Search Query Data:\nNegative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\n\nValidation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1):\nFirst, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\n\nValidation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2):\nWe investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\n\nValidation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3):\nWe investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\n\nEthical Considerations:\nEthical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\n\nResults:\nDescriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nFigure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nStatistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nThe negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nThe analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nThe analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nValidation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.\n\nDescriptive Analysis of Google Search Query Data for SUD Help Seeking:\nFigure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\n\nStatistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking:\nThe negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\n\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1):\nThe analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\n\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2):\nThe analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\n\nValidation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3):\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.\n\nDiscussion:\nPrincipal Findings To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\nTo our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\nLimitations Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\nOur study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\nConclusions This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\nThis study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\n\nPrincipal Findings:\nTo our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\n\nLimitations:\nOur study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\n\nConclusions:\nThis study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThere is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUDs) seeking help within a given geographical area. This presents a challenge to policy makers in the effective deployment of resources for the treatment of SUDs. Internet search queries related to help seeking for SUDs using Google Trends may represent a low-cost, real-time, and data-driven infoveillance tool to address this shortfall in information.\n\nMethods:\nWe used negative binomial regression models to examine temporal trends in the annual SUD help-seeking internet search queries from Google Trends by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol from 2010 to 2020. To validate the value of these data for surveillance purposes, we then used negative binomial regression models to investigate the relationship between SUD help-seeking searches and state-level outcomes across the continuum of care (including lack of care). We started by looking at associations with self-reported treatment need using data from the National Survey on Drug Use and Health, a national survey of the US general population. Next, we explored associations with treatment admission rates from the Treatment Episode Data Set, a national data system on SUD treatment facilities. Finally, we studied associations with state-level rates of people experiencing and dying from an opioid overdose, using data from the Agency for Healthcare Research and Quality and the CDC WONDER database.\n\nResults:\nStatistically significant differences in help-seeking searches were observed over time between 2010 and 2020 (based on P<.05 for the corresponding Wald tests). We were able to identify outlier states for each drug over time (eg, West Virginia for both opioids and methamphetamine), indicating significantly higher help-seeking behaviors compared to national trends. Results from our validation analyses across different outcomes showed positive, statistically significant associations for the models relating to treatment need for alcohol use, treatment admissions for opioid and methamphetamine use, emergency department visits related to opioid use, and opioid overdose mortality data (based on regression coefficients having P≤.05).\n\nConclusions:\nThis study demonstrates the clear potential for using internet search queries from Google Trends as an infoveillance tool to predict the demand for substance use treatment spatially and temporally, especially for opioid use disorders."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nUnderstanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].\nIndirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.\nA promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].\nWhile the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.\nWe sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.\nWe tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.\n\nConclusions:\nThis study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThere is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUDs) seeking help within a given geographical area. This presents a challenge to policy makers in the effective deployment of resources for the treatment of SUDs. Internet search queries related to help seeking for SUDs using Google Trends may represent a low-cost, real-time, and data-driven infoveillance tool to address this shortfall in information.\n\nMethods:\nWe used negative binomial regression models to examine temporal trends in the annual SUD help-seeking internet search queries from Google Trends by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol from 2010 to 2020. To validate the value of these data for surveillance purposes, we then used negative binomial regression models to investigate the relationship between SUD help-seeking searches and state-level outcomes across the continuum of care (including lack of care). We started by looking at associations with self-reported treatment need using data from the National Survey on Drug Use and Health, a national survey of the US general population. Next, we explored associations with treatment admission rates from the Treatment Episode Data Set, a national data system on SUD treatment facilities. Finally, we studied associations with state-level rates of people experiencing and dying from an opioid overdose, using data from the Agency for Healthcare Research and Quality and the CDC WONDER database.\n\nResults:\nStatistically significant differences in help-seeking searches were observed over time between 2010 and 2020 (based on P<.05 for the corresponding Wald tests). We were able to identify outlier states for each drug over time (eg, West Virginia for both opioids and methamphetamine), indicating significantly higher help-seeking behaviors compared to national trends. Results from our validation analyses across different outcomes showed positive, statistically significant associations for the models relating to treatment need for alcohol use, treatment admissions for opioid and methamphetamine use, emergency department visits related to opioid use, and opioid overdose mortality data (based on regression coefficients having P≤.05).\n\nConclusions:\nThis study demonstrates the clear potential for using internet search queries from Google Trends as an infoveillance tool to predict the demand for substance use treatment spatially and temporally, especially for opioid use disorders."},"whole_article_text_length":{"kind":"number","value":18201,"string":"18,201"},"whole_article_abstract_length":{"kind":"number","value":433,"string":"433"},"other_sections_lengths":{"kind":"list like","value":[566,265,336,223,242,65,192,363,475,587,460,629,1032,126],"string":"[\n 566,\n 265,\n 336,\n 223,\n 242,\n 65,\n 192,\n 363,\n 475,\n 587,\n 460,\n 629,\n 1032,\n 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surveillance | infoveillance | google trends [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] internet | search | help-seeking | substance use treatment | surveillance | infoveillance | google trends [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] internet | search | help-seeking | substance use treatment | surveillance | infoveillance | google trends [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] internet | search | help-seeking | substance use treatment | surveillance | infoveillance | google trends [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] United States | Humans | Analgesics, Opioid | Opiate Overdose | Infodemiology | Search Engine | Opioid-Related Disorders | Methamphetamine [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] United States | Humans | Analgesics, Opioid | Opiate Overdose | Infodemiology | Search Engine | Opioid-Related Disorders | Methamphetamine [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] United States | Humans | Analgesics, Opioid | Opiate Overdose | Infodemiology | Search Engine | Opioid-Related Disorders | Methamphetamine [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] United States | Humans | Analgesics, Opioid | Opiate Overdose | Infodemiology | Search Engine | Opioid-Related Disorders | Methamphetamine [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] United States | Humans | Analgesics, Opioid | Opiate Overdose | Infodemiology | Search Engine | Opioid-Related Disorders | Methamphetamine [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] United States | Humans | Analgesics, Opioid | Opiate Overdose | Infodemiology | Search Engine | Opioid-Related Disorders | Methamphetamine [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] individuals substance use | seeking substance use | harms overdose statistics | opioid related data | survey drug use [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] individuals substance use | seeking substance use | harms overdose statistics | opioid related data | survey drug use [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] individuals substance use | seeking substance use | harms overdose statistics | opioid related data | survey drug use [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] individuals substance use | seeking substance use | harms overdose statistics | opioid related data | survey drug use 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[SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] multimedia appendix | multimedia | appendix | qf | increase | variable | use | performance | predictive performance | opioid [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] public health | use | public | treatment | health | trends | substance use | internet search queries state | approach understand evolving | approach understand evolving public [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | data | use | seeking | qf | states | help | opioid | search | alcohol [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] treatment | data | use | seeking | qf | states | help | opioid | search | alcohol [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Google Trends [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] annual | Google Trends | US | 2010 | 2020 ||| ||| the National Survey on Drug Use and Health | US ||| the Treatment Episode Data Set ||| Agency for Healthcare Research and Quality | CDC [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] between 2010 and 2020 | P<.05 | Wald ||| West Virginia ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Google Trends [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Google Trends ||| annual | Google Trends | US | 2010 | 2020 ||| ||| the National Survey on Drug Use and Health | US ||| the Treatment Episode Data Set ||| Agency for Healthcare Research and Quality | CDC ||| between 2010 and 2020 | P<.05 | Wald ||| West Virginia ||| ||| Google Trends [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Google Trends ||| annual | Google Trends | US | 2010 | 2020 ||| ||| the National Survey on Drug Use and Health | US ||| the Treatment Episode Data Set ||| Agency for Healthcare Research and Quality | CDC ||| between 2010 and 2020 | P<.05 | Wald ||| West Virginia ||| ||| Google Trends [SUMMARY]"}}},{"rowIdx":3399,"cells":{"title":{"kind":"string","value":"Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action."},"pmid":{"kind":"string","value":"35508030"},"background_abstract":{"kind":"string","value":"Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain)."},"results_abstract_label":{"kind":"string","value":"FINDINGS"},"conclusions_abstract":{"kind":"string","value":"Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models."},"conclusions_abstract_label":{"kind":"string","value":"MAIN CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Antiprotozoal Agents","Flow Cytometry","Humans","Ketoconazole","Leishmania","Leishmaniasis","Mice","Mice, Inbred BALB C","Mitochondria"],"string":"[\n \"Animals\",\n \"Antiprotozoal Agents\",\n \"Flow Cytometry\",\n \"Humans\",\n \"Ketoconazole\",\n \"Leishmania\",\n \"Leishmaniasis\",\n \"Mice\",\n \"Mice, Inbred BALB C\",\n \"Mitochondria\"\n]"},"pmcid":{"kind":"string","value":"9060495"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"\nKetoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.\n\nFig. 1:\nLeishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).\n\n\nKetoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.\n\nFig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).\n\n\nKetoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.\nAutophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.\n\nFig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).\n\n\nFig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).\n\n\nKetoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).\n\nFig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).\n\n\nKetoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.\n\nFig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.\n\n\nKetoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.\n\nFig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.\n"},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":[],"string":"[]"},"other_sections_texts":{"kind":"list like","value":[],"string":"[]"},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]"},"all_sections_titles":{"kind":"list like","value":["MATERIALS AND METHODS","RESULTS","DISCUSSION"],"string":"[\n \"MATERIALS AND METHODS\",\n \"RESULTS\",\n \"DISCUSSION\"\n]"},"all_sections_texts":{"kind":"list like","value":["\nChemicals - Ketoconazole, dimethylsulfoxide (DMSO), penicillin, streptomycin, triton x-100, rhodamine 123, monodansylcadaverine (MDC), RPMI 1640 medium from Sigma Chemical Co. (USA), annexin V FITC apoptosis detection kit from BD Pharmingen, MitoTrackerR RedCMXRos from Invitrogen (USA), foetal bovine serum (FBS) from Cultilab (Brazil), and Schneider’s insect medium from LGC Biotecnologia (Brazil) were used in this study. Ketoconazole was dissolved in a 1 M stock of DMSO and stored at -20ºC. During assays new dilutions were carried out to ensure that the DMSO concentration in culture medium did not exceed 0.1%.\n\nAnimals - Male BALB/c mice (six-eight weeks old) had access to water and standard chow ad libitum. They were kept in a room with controlled temperature (25ºC) and luminosity (12 h light/dark cycles). The experimental procedures were analysed and approved by the Animal Ethics Committee of the Federal University of Uberlândia (CEUA/UFU) under the number 36/2013.\n\nCells culture - Promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8 strain) were cultured in Schneider’s insect medium pH 7.4 containing 10% heat-inactivated FBS, 1% penicillin (100 UI x mL−1), and streptomycin (100 µg x mL−1). Parasites were kept in a BOD chamber at 23 ± 0.5ºC.\nMurine macrophage cell line RAW264.7 (Rio de Janeiro Bank Cell) was cultured in RPMI 1640 medium pH 7.4 supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) in 75-cm2 flasks. All cell cultures were done at 37ºC in humidified air with 5% CO2.\nFree amastigotes were obtained from footpad of BALB/c mice previously infected with promastigote forms (1 × 107 cells/footpad) for five to six weeks.\n29\n These parasites were cultured in complete Schneider’s insect medium, at 32 ± 0.5ºC pH 5, ensuring that these parasites remained as axenic amastigote forms.\n30\n\n\n\nAntiproliferative and viability assays - The effect of ketoconazole on cellular proliferation was evaluated. Promastigotes (5 × 106 cells x mL−1) were cultured in 25 cm2 cell culture flasks containing medium with different drug concentrations (300 to 0.001 μM). After 24, 48, 72, 96 and 120 h of incubation with drug, the parasite concentrations were determined by blind counting using a Neubauer chamber. Three independent experiments were performed, each one in triplicate. The EC50 values with 95% confidence limits were calculated by GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, USA).\nViability assay was carried out on promastigote forms using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent. Parasites (5 × 106 cells x mL−1) were incubated in the absence (viability control) or presence of ketoconazole (200 to 0.097 µM) for 24, 48, and 72 h into 96 wells plate. The internal controls of the experiment were: culture medium only, DMSO only, parasites cultured in the presence of Triton X-100 (death control) and parasites cultured in the presence of culture medium (viability control) (data not shown to make the figure clean). Three independent experiments were performed, all in triplicate. The effective concentration of drug able to cause 50% of citotoxicity (EC50) was graphically determined by non-linear regression log in each independent experiment using the Graphpad Prism 5 software (United States); for this, all treatments and the viability control had their absorbances deducted from the absorbance value of the culture medium. Finally, the absorbance of the viability control deducted from the absorbance of the culture medium was used as reference parameter (100% of viability) and the percentages of treatments were calculated from then on.\nMoreover, another viability assay was performed: Trypan Blue exclusion test. Parasites (5 x 106 cells x mL−1) were cultured in medium for 48 h. Subsequently, the cells were treated with 10 µM ketoconazole (2xEC50 of 72 h growth curve) and the number of promastigotes was determined by blind counting using a Neubauer chamber for three consecutive days (72 h) and Trypan blue stain. Three independent experiments were performed, all in triplicate.\n\nMorphological and ultrastructural analysis - For evaluating ultrastructural changes, transmission electron (TEM) and scanning electron (SEM) microscopies were performed. For TEM, promastigotes (5 x 106 cells x mL−1, log phase) were incubated with or without (control) ketoconazole 10 μM for 72 h. After being treated, the parasites were fixed at 4ºC with 2.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS) pH 7.2 for 24 h, washed in PBS, post-fixed in PBS containing 0.8% potassium ferrocyanide and 1% osmium tetroxide (OsO4) for 1 h and washed once again. Subsequently, the parasites were dehydrated in a gradual series of acetone and set in resin. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate and analysed using a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany). For SEM, promastigotes treated or not with ketoconazole 10 μM for 72 h were dehydrated in ethanol, critical point-dried in CO2, mounted in stubs, sputtered with a thin gold layer, and analysed using a scanning electron microscope (Zeiss EVO MA10).\n\nEvaluation of mitochondrial damage - The fluorescent stain Rhodamine 123 was employed to evaluate the mitochondrial transmembrane potential (∆Ψ\nm\n ) of promastigotes. Initially, parasites (5 x 106 cells x mL−1) were treated with 10 µM ketoconazole or not (control) for 24, 48, and 72 h. Aliquots were incubated with Rhodamine 123 (15 µg x mL−1) for 15 min at room temperature and protected from light (dark). Subsequently, the parasites were washed twice with PBS. The cell population analysis was carried out in a flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. Three independent experiments were performed.\nFurthermore, promastigotes (5 x 106 cells x mL−1) treated or not (control) with ketoconazole (10 µM, 72 h) were incubated with MitoTrackerR RedCMXRos (300 nM) for 30 min in the dark. Subsequently, parasites were washed with PBS, fixed in paraformaldehyde 1% in cacodylate buffer, and washed twice with PBS. The cell population analysis was performed with a confocal fluorescence microscopy (Zeiss LSM510 Meta).\n\nDetection of autophagic vacuoles - The monodansylcadaverine (MDC) labeling was carried out to evaluate the autophagic vacuoles formation. Promastigotes (5 x 106 cells x mL−1, log phase) were cultured in absence (control) or presence of ketoconazole 10 μM for 24, 48, and 72 h. Subsequently, 100 µM MDC was added to parasites and incubated for 2 h in the dark. Afterwards, the parasites were washed with PBS, fixed in 1% paraformaldehyde in cacodylate buffer, washed twice with PBS, mounted on microscope slides, and analysed by fluorescence microscopy (excitation wavelength 358 nm and emission wavelength 463 nm). Two independent experiments were performed and the fluorescence intensity was quantified by the software Image J version 1.48.\n\nCell cycle analysis - Initially, the interference of ketoconazole on the cell cycle was evaluated by counting the number of nucleous, kinetoplast and flagella by parasite. Promastigotes (5 x 106 cells x mL-1) treated or not (control) with ketoconazole (10 uM, 72 h) labelled with DAPI (1:500, for 1 h) were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta). For a more detailed study, parasites (5 x 106 cells x mL−1, log phase) were incubated for 72 h with or without (control) ketoconazole (10 μM, 72 h) in medium containing 10% FBS. After incubation, parasites were washed and resuspended in ice-cold 70% ethanol in PBS and fixed for 18 h at 4ºC. Subsequently, the parasites were incubated with PBS containing 10 µg x mL−1 propidium iodide and 100 µg x mL−1 RNAse A for 45 min at 37ºC, protected from light. The cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. For each phase of the cell cycle, a respective percentage was obtained using the FlowJov10.0.7 software.\n\nCell death analysis - Promastigotes (5 x 106 cells x mL−1) were incubated for 72 h with or without (negative control) ketoconazole (10 μM, 72 h); while parasites incubated with formaldehyde 4% for 15 min were considered positive control. Subsequently, a binding buffer containing annexin V-FITC and 2 μg x mL−1 propidium iodide (BD Bioscience, Brazil) was added to the parasites and incubated for 15 min at 25ºC in the dark, according to the manufacturer’s instructions. Then, the parasites were washed and the cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA). Thirty thousand events were obtained from the region corresponding to the parasites. The percentages of apoptotic cells were determined using the FlowJo v10.0.7 software.\nFor Propidium Iodide (PI) and Annexin V (AV) labeling, promastigotes (5 x 106 cells x mL−1) treated with ketoconazole (10 μM, 72 h), medium (negative control) or formaldehyde 4% (positive control) were incubated in presence of PI (2 µg x mL-1), AV (1 µg x mL-1) or both for 15 min at 25ºC, protected from the light. Subsequently, parasites were fixed with 1% paraformaldehyde in a cacodylate buffer, washed and placed on glass coverslips. Finally, the parasites were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta).\n\nIntracellular amastigote assay - Murine macrophage cell line RAW264.7 (4 × 105/well) cultured in RPMI 1640 medium, supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) was placed in 24-well plates containing 13-mm diameter glass coverslips and infected with L. (L.) amazonensis free amastigotes isolated just before their use (two free amastigotes: 1 macrophage). After 1 h 30, plates were washed with PBS to remove non-internalised amastigotes and RPMI medium alone (control) or containing drugs (200 to 12,5 µM for ketoconazole, double serial dilutions) was added. Experiments were conducted at 37ºC in a 5% CO2 humidified incubator for 48 h. Cells on coverslips were fixed and stained with Giemsa stain modified solution for evaluation of the infectivity index; 100 cells per coverslip were blind counted and the infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage. This assay was carried out in quadruplicate and two independent experiments were performed.\nIt is worth highlighting the cytotoxic effect of ketoconazole on murine macrophages cell line RAW264.7 was, previously, performed by MTT test at the same drug concentrations used to assess the viability of promastigotes (section 2.4), but within 48 h. Since the macrophages were infected with amastigotes isolated from animal paw just before use and it was not necessary time to promastigote-amastigote differentiation, the infectivity assay was conducted at 48 h, time that allowed better visualisation and counting of the intramacrophage amastigotes.\n\nStatistics - Statistical analysis was conducted using GraphPad Prism 5. Each set of results was firstly checked for normal distribution using KolmogorovSmirnov, D’Agostinho and Pearson, and ShapiroWalk tests. Normally distributed data were analysed through one-way-ANOVA followed by Tukey’s post-test or Student test T. Statistically significant differences were assumed when at least p < 0.05.","\nKetoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.\n\nFig. 1:\nLeishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).\n\n\nKetoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.\n\nFig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).\n\n\nKetoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.\nAutophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.\n\nFig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).\n\n\nFig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).\n\n\nKetoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).\n\nFig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).\n\n\nKetoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.\n\nFig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.\n\n\nKetoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.\n\nFig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.\n","Considering the importance of ergosterol for the biology and pathogenesis of the Leishmania parasite, herein, we certified the antiproliferative/viability effects of ketoconazole against L. (L.) amazonensis both promastigotes\n31\n and intracellular amastigotes\n32\n in another parasite strain: IFLA/BR/67/PH8. Furthermore, we show in more detail the biological effects of the inhibitor, aiming at understanding the drug mechanism of action.\nRegarding ultrastructure, our results revealed rounded up shape, mitochondrial swelling, augmented accumulation of lipid droplets and acidocalcisomes, presence of multivesicular bodies and frequent truncated and/or double flagella. These data are similar to those previously obtained for L. amazonensis promastigotes treated with ketoconazole\n31\n\n,\n\n33\n\n,\n\n34\n and suggest mitochondrial damage.\nWhen parasites (Trypanosoma cruzi, Leishmania spp, Toxoplasma gondii) are treated with inhibitors of important enzymes of the ergosterol biosynthesis pathway, the mitochondrion is the organelle primarily affected.\n35\n In our study, the ketoconazole-treated parasites presented hyperpolarisation of the mitochondrial membrane potential, time-dependent up to 72 h. Mitochondrial membrane potential variations could be the result of diverse events: inhibition of electron transport chain (decrease); blockage of ATP synthase (increase); stimulation of uncoupling proteins (decrease); or permeabilisation of the inner membrane (decrease).\n36\n Macedo et al.\n37\n demonstrated that L. (L.) amazonensis promastigotes treated with itraconazole and posaconazole for 48 h presented collapse of the mitochondrial membrane potential associated with intense mitochondrial swelling, disorganisation and rupture of mitochondrial membranes. Another study showed that T. cruzi treated with ketoconazole (EC50 = 32 µM for 72 h) presented a gradual increase in Rho 123 time-dependent fluorescence and a confocal microscopy of parasites confirmed an intense proliferation of the inner mitochondrial membrane. This organelle became highly branched and compact and elevated levels of Rho 123 were accumulated within the cells.\n38\n In many cases, a transient hyperpolarisation occurs before mitochondrial depolarisation, as if it were an attempt of the cells to avoid death. This effect can be seen in great part of the heat-shocked Leishmania promastigotes.\n39\n\n\nThe mitochondrial membrane is hyperpolarised does not mean that the organelle is functioning normally. Our results showed that MitoTrackerR labeling, a probe which passively diffuses across the plasma membrane and accumulates in active mitochondria, revealed an outstanding intensity decrease in ketoconazole-treated promastigotes, indicating loss of mitochondrial activity in these promastigotes. This result was also confirmed by the MTT viability assay, which measures the activity of mitochondrial enzymes, and by the proliferation curve using Trypan blue, which takes into account only viable cells. Mitochondrion is essential for generating the necessary energy for the survival and proliferation of eukaryotic cells.\n40\n\n) In this way, malfunctioning mitochondria could impair the ATP synthesis and inorganic phosphate would accumulate in acidocalcisomes, which explains the increase in the amount of this organelle 72 h after ketoconazole treatment. Acidocalcisomes are organelles of acidic nature and high electron density important due to their storage of polyphosphates, calcium, magnesium, and other elements.\n41\n The augmented number of this organelle was already reported for ketoconazole and L. amazonensis (MHOM/Josefa/75/Br strain).\n42\n\n\nThe treatment of L. amazonensis with ketoconazole stressed the cells, especially early after the treatment (24 and 48 h), leading to the appearance of autophagic vacuoles. Itraconazole and posaconazole induced appearance of autophagosome-like structures in L. (L.) amazonensis\n\n37\n and Rodrigues et al.\n43\n described the effect of autophagy on L. amazonensis (MHOM/BR/75/Josefa strain) treated with 22,26-azasterol. Thus, the confirmed presence of autophagic vacuoles might represent an adaptive response of the parasite to treatment (stress condition). Initially, parasites would resort to autophagic vacuoles in an attempt to remodel/remove abnormal cellular constituents, degrading damaged structures.\n44\n Over time, directing energy to the parasite single mitochondrion may be more important than repairing cell damage. This would explain the fact that at 72 h there was less labeling for autophagic vacuoles and higher mitochondrial hyperpolarisation occurred. Autophagy is involved in turnover and recycling by removal of damaged cellular components, regulating homeostasis during crucial processes such as cell growth and differentiation and plays fundamental role in mitochondrial functionality.\n44\n\n,\n\n45\n\n\nResults of the growth curve and frequent ultrastructural alterations in flagellum (truncated and/or double flagella), despite the intact kinetoplast, suggest that ketoconazole interferes with the L. amazonensis cell cycle. Therefore, the cell cycle of the parasites was evaluated both by nucleus, kinetoplast, and flagellum (n-k-f) counts and by flow cytometry. Different from some data in the literature involving ketoconazole acting on other cell types\n46\n\n,\n\n47\n and azole compounds on L. amazonensis,\n37\n our results of the n-k-f score did not reveal differences between the control and ketoconazole-treated parasites. In addition, the cell cycle assay showed that the treatment was not able to alter the cycle phases (Sub G1, G1, S, and G2/M) in relation to control parasites suggesting that ketoconazole does not interfere with parasite replication under the tested conditions. When L. (L.) amazonensis was treated with itraconazole and posaconazole, some cells presented more than two flagella and significant alterations in kinetoplast were observed by ultrastructural analysis; however, the possible alteration in the cell cycle was not evaluated.\n37\n\n\nKetoconazole has been related to apoptosis in several normal and tumoral cells.\n48\n\n,\n\n49\n\n,\n\n50\n\n,\n\n51\n Haegler et al.\n52\n showed that ketoconazole and posaconazole presented hepatocellular toxicity, impairing the ∆Ψm, the function of enzyme complexes of electron transport chain, accumulating mitochondrial superoxide and inducing apoptosis. Possibly, mitochondrial dysfunction could be associated with hepatotoxicity caused by those azole compounds. Our data revealed that treatment of the parasites with ketoconazole, under the conditions previously described, showed a trend towards apoptosis, although there was a slight positive result for PI. Furthermore, few autophagic vacuoles were observed after treatment. Other researchers reported that treatment of T. cruzi with ketoconazole (EC50/72 h) resulted in neither necrosis nor apoptosis. However, high concentrations of the drug (EC100/24h) resulted in fast cell death due to necrosis.\n38\n\n\nSBIs effects on extracellular L. amazonensis parasites (promastigotes) appear to be more gradual than that observed with the intracellular form (amastigote);\n31\n thus, SBIs effects profile allows a more detailed study of the events in the promastigote form. In view of the effects provoked by ketoconazole on the L. (L.) amazonensis promastigote, the interference of drug on infectivity capacity of amastigotes, the clinically relevant form of the parasite, was evaluated and evidenced a susceptibility to treatment. Taken together, the infectivity results added to the data of mitochondrial damage, increase in the acidocalcisome and autophagic vacuoles amounts may be evidence that the susceptibility observed in vitro is related to mitochondrial dysfunction provoked by ketoconazole. However, future studies should be carried out on amastigotes to confirm this hypothesis.\nTo our knowledge, this study demonstrates the effects of ketoconazole on mitochondrion functioning, autophagic compartments, cell cycle and death of L. (L.) amazonensis promastigotes for the first time. We suggest that some damages are occurring in the parasite, mainly in the mitochondrion. In an attempt to remodel and/or destroy eventual damaged structures, parasites hyperpolarise mitochondrion and resort to autophagic vacuoles. However, this survival strategy adopted by the cell to direct efforts to maintain the cell energy is not sufficient because of the decrease in cell and mitochondria viabilities and increase in acidocalcisomes. Although these damages do not reflect directly in the parasite cell cycle, they lead the parasites to death, making them susceptible to in vitro treatment (Fig. 8).\n\nFig. 8:proposed mode action of ketoconazole on Leishmania (Leishmania) amazonensis. Parasites resort to autophagic vacuoles and mitochondrion hyperpolarisation in attempt to survive to damages mainly in the mitochondrion. The efforts to maintain the cell energy are not sufficient since the decrease of cell and mitochondria viability and increase of acidocalcisomes. These damages do not interfere with the parasite cell cycle, but they lead the parasites to death, making them susceptible to in vitro treatment conditions.\n"],"string":"[\n \"\\nChemicals - Ketoconazole, dimethylsulfoxide (DMSO), penicillin, streptomycin, triton x-100, rhodamine 123, monodansylcadaverine (MDC), RPMI 1640 medium from Sigma Chemical Co. (USA), annexin V FITC apoptosis detection kit from BD Pharmingen, MitoTrackerR RedCMXRos from Invitrogen (USA), foetal bovine serum (FBS) from Cultilab (Brazil), and Schneider’s insect medium from LGC Biotecnologia (Brazil) were used in this study. Ketoconazole was dissolved in a 1 M stock of DMSO and stored at -20ºC. During assays new dilutions were carried out to ensure that the DMSO concentration in culture medium did not exceed 0.1%.\\n\\nAnimals - Male BALB/c mice (six-eight weeks old) had access to water and standard chow ad libitum. They were kept in a room with controlled temperature (25ºC) and luminosity (12 h light/dark cycles). The experimental procedures were analysed and approved by the Animal Ethics Committee of the Federal University of Uberlândia (CEUA/UFU) under the number 36/2013.\\n\\nCells culture - Promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8 strain) were cultured in Schneider’s insect medium pH 7.4 containing 10% heat-inactivated FBS, 1% penicillin (100 UI x mL−1), and streptomycin (100 µg x mL−1). Parasites were kept in a BOD chamber at 23 ± 0.5ºC.\\nMurine macrophage cell line RAW264.7 (Rio de Janeiro Bank Cell) was cultured in RPMI 1640 medium pH 7.4 supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) in 75-cm2 flasks. All cell cultures were done at 37ºC in humidified air with 5% CO2.\\nFree amastigotes were obtained from footpad of BALB/c mice previously infected with promastigote forms (1 × 107 cells/footpad) for five to six weeks.\\n29\\n These parasites were cultured in complete Schneider’s insect medium, at 32 ± 0.5ºC pH 5, ensuring that these parasites remained as axenic amastigote forms.\\n30\\n\\n\\n\\nAntiproliferative and viability assays - The effect of ketoconazole on cellular proliferation was evaluated. Promastigotes (5 × 106 cells x mL−1) were cultured in 25 cm2 cell culture flasks containing medium with different drug concentrations (300 to 0.001 μM). After 24, 48, 72, 96 and 120 h of incubation with drug, the parasite concentrations were determined by blind counting using a Neubauer chamber. Three independent experiments were performed, each one in triplicate. The EC50 values with 95% confidence limits were calculated by GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, USA).\\nViability assay was carried out on promastigote forms using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent. Parasites (5 × 106 cells x mL−1) were incubated in the absence (viability control) or presence of ketoconazole (200 to 0.097 µM) for 24, 48, and 72 h into 96 wells plate. The internal controls of the experiment were: culture medium only, DMSO only, parasites cultured in the presence of Triton X-100 (death control) and parasites cultured in the presence of culture medium (viability control) (data not shown to make the figure clean). Three independent experiments were performed, all in triplicate. The effective concentration of drug able to cause 50% of citotoxicity (EC50) was graphically determined by non-linear regression log in each independent experiment using the Graphpad Prism 5 software (United States); for this, all treatments and the viability control had their absorbances deducted from the absorbance value of the culture medium. Finally, the absorbance of the viability control deducted from the absorbance of the culture medium was used as reference parameter (100% of viability) and the percentages of treatments were calculated from then on.\\nMoreover, another viability assay was performed: Trypan Blue exclusion test. Parasites (5 x 106 cells x mL−1) were cultured in medium for 48 h. Subsequently, the cells were treated with 10 µM ketoconazole (2xEC50 of 72 h growth curve) and the number of promastigotes was determined by blind counting using a Neubauer chamber for three consecutive days (72 h) and Trypan blue stain. Three independent experiments were performed, all in triplicate.\\n\\nMorphological and ultrastructural analysis - For evaluating ultrastructural changes, transmission electron (TEM) and scanning electron (SEM) microscopies were performed. For TEM, promastigotes (5 x 106 cells x mL−1, log phase) were incubated with or without (control) ketoconazole 10 μM for 72 h. After being treated, the parasites were fixed at 4ºC with 2.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS) pH 7.2 for 24 h, washed in PBS, post-fixed in PBS containing 0.8% potassium ferrocyanide and 1% osmium tetroxide (OsO4) for 1 h and washed once again. Subsequently, the parasites were dehydrated in a gradual series of acetone and set in resin. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate and analysed using a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany). For SEM, promastigotes treated or not with ketoconazole 10 μM for 72 h were dehydrated in ethanol, critical point-dried in CO2, mounted in stubs, sputtered with a thin gold layer, and analysed using a scanning electron microscope (Zeiss EVO MA10).\\n\\nEvaluation of mitochondrial damage - The fluorescent stain Rhodamine 123 was employed to evaluate the mitochondrial transmembrane potential (∆Ψ\\nm\\n ) of promastigotes. Initially, parasites (5 x 106 cells x mL−1) were treated with 10 µM ketoconazole or not (control) for 24, 48, and 72 h. Aliquots were incubated with Rhodamine 123 (15 µg x mL−1) for 15 min at room temperature and protected from light (dark). Subsequently, the parasites were washed twice with PBS. The cell population analysis was carried out in a flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. Three independent experiments were performed.\\nFurthermore, promastigotes (5 x 106 cells x mL−1) treated or not (control) with ketoconazole (10 µM, 72 h) were incubated with MitoTrackerR RedCMXRos (300 nM) for 30 min in the dark. Subsequently, parasites were washed with PBS, fixed in paraformaldehyde 1% in cacodylate buffer, and washed twice with PBS. The cell population analysis was performed with a confocal fluorescence microscopy (Zeiss LSM510 Meta).\\n\\nDetection of autophagic vacuoles - The monodansylcadaverine (MDC) labeling was carried out to evaluate the autophagic vacuoles formation. Promastigotes (5 x 106 cells x mL−1, log phase) were cultured in absence (control) or presence of ketoconazole 10 μM for 24, 48, and 72 h. Subsequently, 100 µM MDC was added to parasites and incubated for 2 h in the dark. Afterwards, the parasites were washed with PBS, fixed in 1% paraformaldehyde in cacodylate buffer, washed twice with PBS, mounted on microscope slides, and analysed by fluorescence microscopy (excitation wavelength 358 nm and emission wavelength 463 nm). Two independent experiments were performed and the fluorescence intensity was quantified by the software Image J version 1.48.\\n\\nCell cycle analysis - Initially, the interference of ketoconazole on the cell cycle was evaluated by counting the number of nucleous, kinetoplast and flagella by parasite. Promastigotes (5 x 106 cells x mL-1) treated or not (control) with ketoconazole (10 uM, 72 h) labelled with DAPI (1:500, for 1 h) were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta). For a more detailed study, parasites (5 x 106 cells x mL−1, log phase) were incubated for 72 h with or without (control) ketoconazole (10 μM, 72 h) in medium containing 10% FBS. After incubation, parasites were washed and resuspended in ice-cold 70% ethanol in PBS and fixed for 18 h at 4ºC. Subsequently, the parasites were incubated with PBS containing 10 µg x mL−1 propidium iodide and 100 µg x mL−1 RNAse A for 45 min at 37ºC, protected from light. The cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. For each phase of the cell cycle, a respective percentage was obtained using the FlowJov10.0.7 software.\\n\\nCell death analysis - Promastigotes (5 x 106 cells x mL−1) were incubated for 72 h with or without (negative control) ketoconazole (10 μM, 72 h); while parasites incubated with formaldehyde 4% for 15 min were considered positive control. Subsequently, a binding buffer containing annexin V-FITC and 2 μg x mL−1 propidium iodide (BD Bioscience, Brazil) was added to the parasites and incubated for 15 min at 25ºC in the dark, according to the manufacturer’s instructions. Then, the parasites were washed and the cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA). Thirty thousand events were obtained from the region corresponding to the parasites. The percentages of apoptotic cells were determined using the FlowJo v10.0.7 software.\\nFor Propidium Iodide (PI) and Annexin V (AV) labeling, promastigotes (5 x 106 cells x mL−1) treated with ketoconazole (10 μM, 72 h), medium (negative control) or formaldehyde 4% (positive control) were incubated in presence of PI (2 µg x mL-1), AV (1 µg x mL-1) or both for 15 min at 25ºC, protected from the light. Subsequently, parasites were fixed with 1% paraformaldehyde in a cacodylate buffer, washed and placed on glass coverslips. Finally, the parasites were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta).\\n\\nIntracellular amastigote assay - Murine macrophage cell line RAW264.7 (4 × 105/well) cultured in RPMI 1640 medium, supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) was placed in 24-well plates containing 13-mm diameter glass coverslips and infected with L. (L.) amazonensis free amastigotes isolated just before their use (two free amastigotes: 1 macrophage). After 1 h 30, plates were washed with PBS to remove non-internalised amastigotes and RPMI medium alone (control) or containing drugs (200 to 12,5 µM for ketoconazole, double serial dilutions) was added. Experiments were conducted at 37ºC in a 5% CO2 humidified incubator for 48 h. Cells on coverslips were fixed and stained with Giemsa stain modified solution for evaluation of the infectivity index; 100 cells per coverslip were blind counted and the infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage. This assay was carried out in quadruplicate and two independent experiments were performed.\\nIt is worth highlighting the cytotoxic effect of ketoconazole on murine macrophages cell line RAW264.7 was, previously, performed by MTT test at the same drug concentrations used to assess the viability of promastigotes (section 2.4), but within 48 h. Since the macrophages were infected with amastigotes isolated from animal paw just before use and it was not necessary time to promastigote-amastigote differentiation, the infectivity assay was conducted at 48 h, time that allowed better visualisation and counting of the intramacrophage amastigotes.\\n\\nStatistics - Statistical analysis was conducted using GraphPad Prism 5. Each set of results was firstly checked for normal distribution using KolmogorovSmirnov, D’Agostinho and Pearson, and ShapiroWalk tests. Normally distributed data were analysed through one-way-ANOVA followed by Tukey’s post-test or Student test T. Statistically significant differences were assumed when at least p < 0.05.\",\n \"\\nKetoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.\\n\\nFig. 1:\\nLeishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).\\n\\n\\nKetoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.\\n\\nFig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).\\n\\n\\nKetoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.\\nAutophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.\\n\\nFig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).\\n\\n\\nFig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).\\n\\n\\nKetoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).\\n\\nFig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).\\n\\n\\nKetoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.\\n\\nFig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.\\n\\n\\nKetoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.\\n\\nFig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.\\n\",\n \"Considering the importance of ergosterol for the biology and pathogenesis of the Leishmania parasite, herein, we certified the antiproliferative/viability effects of ketoconazole against L. (L.) amazonensis both promastigotes\\n31\\n and intracellular amastigotes\\n32\\n in another parasite strain: IFLA/BR/67/PH8. Furthermore, we show in more detail the biological effects of the inhibitor, aiming at understanding the drug mechanism of action.\\nRegarding ultrastructure, our results revealed rounded up shape, mitochondrial swelling, augmented accumulation of lipid droplets and acidocalcisomes, presence of multivesicular bodies and frequent truncated and/or double flagella. These data are similar to those previously obtained for L. amazonensis promastigotes treated with ketoconazole\\n31\\n\\n,\\n\\n33\\n\\n,\\n\\n34\\n and suggest mitochondrial damage.\\nWhen parasites (Trypanosoma cruzi, Leishmania spp, Toxoplasma gondii) are treated with inhibitors of important enzymes of the ergosterol biosynthesis pathway, the mitochondrion is the organelle primarily affected.\\n35\\n In our study, the ketoconazole-treated parasites presented hyperpolarisation of the mitochondrial membrane potential, time-dependent up to 72 h. Mitochondrial membrane potential variations could be the result of diverse events: inhibition of electron transport chain (decrease); blockage of ATP synthase (increase); stimulation of uncoupling proteins (decrease); or permeabilisation of the inner membrane (decrease).\\n36\\n Macedo et al.\\n37\\n demonstrated that L. (L.) amazonensis promastigotes treated with itraconazole and posaconazole for 48 h presented collapse of the mitochondrial membrane potential associated with intense mitochondrial swelling, disorganisation and rupture of mitochondrial membranes. Another study showed that T. cruzi treated with ketoconazole (EC50 = 32 µM for 72 h) presented a gradual increase in Rho 123 time-dependent fluorescence and a confocal microscopy of parasites confirmed an intense proliferation of the inner mitochondrial membrane. This organelle became highly branched and compact and elevated levels of Rho 123 were accumulated within the cells.\\n38\\n In many cases, a transient hyperpolarisation occurs before mitochondrial depolarisation, as if it were an attempt of the cells to avoid death. This effect can be seen in great part of the heat-shocked Leishmania promastigotes.\\n39\\n\\n\\nThe mitochondrial membrane is hyperpolarised does not mean that the organelle is functioning normally. Our results showed that MitoTrackerR labeling, a probe which passively diffuses across the plasma membrane and accumulates in active mitochondria, revealed an outstanding intensity decrease in ketoconazole-treated promastigotes, indicating loss of mitochondrial activity in these promastigotes. This result was also confirmed by the MTT viability assay, which measures the activity of mitochondrial enzymes, and by the proliferation curve using Trypan blue, which takes into account only viable cells. Mitochondrion is essential for generating the necessary energy for the survival and proliferation of eukaryotic cells.\\n40\\n\\n) In this way, malfunctioning mitochondria could impair the ATP synthesis and inorganic phosphate would accumulate in acidocalcisomes, which explains the increase in the amount of this organelle 72 h after ketoconazole treatment. Acidocalcisomes are organelles of acidic nature and high electron density important due to their storage of polyphosphates, calcium, magnesium, and other elements.\\n41\\n The augmented number of this organelle was already reported for ketoconazole and L. amazonensis (MHOM/Josefa/75/Br strain).\\n42\\n\\n\\nThe treatment of L. amazonensis with ketoconazole stressed the cells, especially early after the treatment (24 and 48 h), leading to the appearance of autophagic vacuoles. Itraconazole and posaconazole induced appearance of autophagosome-like structures in L. (L.) amazonensis\\n\\n37\\n and Rodrigues et al.\\n43\\n described the effect of autophagy on L. amazonensis (MHOM/BR/75/Josefa strain) treated with 22,26-azasterol. Thus, the confirmed presence of autophagic vacuoles might represent an adaptive response of the parasite to treatment (stress condition). Initially, parasites would resort to autophagic vacuoles in an attempt to remodel/remove abnormal cellular constituents, degrading damaged structures.\\n44\\n Over time, directing energy to the parasite single mitochondrion may be more important than repairing cell damage. This would explain the fact that at 72 h there was less labeling for autophagic vacuoles and higher mitochondrial hyperpolarisation occurred. Autophagy is involved in turnover and recycling by removal of damaged cellular components, regulating homeostasis during crucial processes such as cell growth and differentiation and plays fundamental role in mitochondrial functionality.\\n44\\n\\n,\\n\\n45\\n\\n\\nResults of the growth curve and frequent ultrastructural alterations in flagellum (truncated and/or double flagella), despite the intact kinetoplast, suggest that ketoconazole interferes with the L. amazonensis cell cycle. Therefore, the cell cycle of the parasites was evaluated both by nucleus, kinetoplast, and flagellum (n-k-f) counts and by flow cytometry. Different from some data in the literature involving ketoconazole acting on other cell types\\n46\\n\\n,\\n\\n47\\n and azole compounds on L. amazonensis,\\n37\\n our results of the n-k-f score did not reveal differences between the control and ketoconazole-treated parasites. In addition, the cell cycle assay showed that the treatment was not able to alter the cycle phases (Sub G1, G1, S, and G2/M) in relation to control parasites suggesting that ketoconazole does not interfere with parasite replication under the tested conditions. When L. (L.) amazonensis was treated with itraconazole and posaconazole, some cells presented more than two flagella and significant alterations in kinetoplast were observed by ultrastructural analysis; however, the possible alteration in the cell cycle was not evaluated.\\n37\\n\\n\\nKetoconazole has been related to apoptosis in several normal and tumoral cells.\\n48\\n\\n,\\n\\n49\\n\\n,\\n\\n50\\n\\n,\\n\\n51\\n Haegler et al.\\n52\\n showed that ketoconazole and posaconazole presented hepatocellular toxicity, impairing the ∆Ψm, the function of enzyme complexes of electron transport chain, accumulating mitochondrial superoxide and inducing apoptosis. Possibly, mitochondrial dysfunction could be associated with hepatotoxicity caused by those azole compounds. Our data revealed that treatment of the parasites with ketoconazole, under the conditions previously described, showed a trend towards apoptosis, although there was a slight positive result for PI. Furthermore, few autophagic vacuoles were observed after treatment. Other researchers reported that treatment of T. cruzi with ketoconazole (EC50/72 h) resulted in neither necrosis nor apoptosis. However, high concentrations of the drug (EC100/24h) resulted in fast cell death due to necrosis.\\n38\\n\\n\\nSBIs effects on extracellular L. amazonensis parasites (promastigotes) appear to be more gradual than that observed with the intracellular form (amastigote);\\n31\\n thus, SBIs effects profile allows a more detailed study of the events in the promastigote form. In view of the effects provoked by ketoconazole on the L. (L.) amazonensis promastigote, the interference of drug on infectivity capacity of amastigotes, the clinically relevant form of the parasite, was evaluated and evidenced a susceptibility to treatment. Taken together, the infectivity results added to the data of mitochondrial damage, increase in the acidocalcisome and autophagic vacuoles amounts may be evidence that the susceptibility observed in vitro is related to mitochondrial dysfunction provoked by ketoconazole. However, future studies should be carried out on amastigotes to confirm this hypothesis.\\nTo our knowledge, this study demonstrates the effects of ketoconazole on mitochondrion functioning, autophagic compartments, cell cycle and death of L. (L.) amazonensis promastigotes for the first time. We suggest that some damages are occurring in the parasite, mainly in the mitochondrion. In an attempt to remodel and/or destroy eventual damaged structures, parasites hyperpolarise mitochondrion and resort to autophagic vacuoles. However, this survival strategy adopted by the cell to direct efforts to maintain the cell energy is not sufficient because of the decrease in cell and mitochondria viabilities and increase in acidocalcisomes. Although these damages do not reflect directly in the parasite cell cycle, they lead the parasites to death, making them susceptible to in vitro treatment (Fig. 8).\\n\\nFig. 8:proposed mode action of ketoconazole on Leishmania (Leishmania) amazonensis. Parasites resort to autophagic vacuoles and mitochondrion hyperpolarisation in attempt to survive to damages mainly in the mitochondrion. The efforts to maintain the cell energy are not sufficient since the decrease of cell and mitochondria viability and increase of acidocalcisomes. These damages do not interfere with the parasite cell cycle, but they lead the parasites to death, making them susceptible to in vitro treatment conditions.\\n\"\n]"},"all_sections_sec_types":{"kind":"list like","value":["materials|methods","results","discussion"],"string":"[\n \"materials|methods\",\n \"results\",\n \"discussion\"\n]"},"keywords":{"kind":"list like","value":["cutaneous leishmaniasis","ergosterol","mitochondrial damage","sterol biosynthesis inhibitor"],"string":"[\n \"cutaneous leishmaniasis\",\n \"ergosterol\",\n \"mitochondrial damage\",\n \"sterol biosynthesis inhibitor\"\n]"},"whole_article_text":{"kind":"string","value":"MATERIALS AND METHODS:\n\nChemicals - Ketoconazole, dimethylsulfoxide (DMSO), penicillin, streptomycin, triton x-100, rhodamine 123, monodansylcadaverine (MDC), RPMI 1640 medium from Sigma Chemical Co. (USA), annexin V FITC apoptosis detection kit from BD Pharmingen, MitoTrackerR RedCMXRos from Invitrogen (USA), foetal bovine serum (FBS) from Cultilab (Brazil), and Schneider’s insect medium from LGC Biotecnologia (Brazil) were used in this study. Ketoconazole was dissolved in a 1 M stock of DMSO and stored at -20ºC. During assays new dilutions were carried out to ensure that the DMSO concentration in culture medium did not exceed 0.1%.\n\nAnimals - Male BALB/c mice (six-eight weeks old) had access to water and standard chow ad libitum. They were kept in a room with controlled temperature (25ºC) and luminosity (12 h light/dark cycles). The experimental procedures were analysed and approved by the Animal Ethics Committee of the Federal University of Uberlândia (CEUA/UFU) under the number 36/2013.\n\nCells culture - Promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8 strain) were cultured in Schneider’s insect medium pH 7.4 containing 10% heat-inactivated FBS, 1% penicillin (100 UI x mL−1), and streptomycin (100 µg x mL−1). Parasites were kept in a BOD chamber at 23 ± 0.5ºC.\nMurine macrophage cell line RAW264.7 (Rio de Janeiro Bank Cell) was cultured in RPMI 1640 medium pH 7.4 supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) in 75-cm2 flasks. All cell cultures were done at 37ºC in humidified air with 5% CO2.\nFree amastigotes were obtained from footpad of BALB/c mice previously infected with promastigote forms (1 × 107 cells/footpad) for five to six weeks.\n29\n These parasites were cultured in complete Schneider’s insect medium, at 32 ± 0.5ºC pH 5, ensuring that these parasites remained as axenic amastigote forms.\n30\n\n\n\nAntiproliferative and viability assays - The effect of ketoconazole on cellular proliferation was evaluated. Promastigotes (5 × 106 cells x mL−1) were cultured in 25 cm2 cell culture flasks containing medium with different drug concentrations (300 to 0.001 μM). After 24, 48, 72, 96 and 120 h of incubation with drug, the parasite concentrations were determined by blind counting using a Neubauer chamber. Three independent experiments were performed, each one in triplicate. The EC50 values with 95% confidence limits were calculated by GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, USA).\nViability assay was carried out on promastigote forms using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent. Parasites (5 × 106 cells x mL−1) were incubated in the absence (viability control) or presence of ketoconazole (200 to 0.097 µM) for 24, 48, and 72 h into 96 wells plate. The internal controls of the experiment were: culture medium only, DMSO only, parasites cultured in the presence of Triton X-100 (death control) and parasites cultured in the presence of culture medium (viability control) (data not shown to make the figure clean). Three independent experiments were performed, all in triplicate. The effective concentration of drug able to cause 50% of citotoxicity (EC50) was graphically determined by non-linear regression log in each independent experiment using the Graphpad Prism 5 software (United States); for this, all treatments and the viability control had their absorbances deducted from the absorbance value of the culture medium. Finally, the absorbance of the viability control deducted from the absorbance of the culture medium was used as reference parameter (100% of viability) and the percentages of treatments were calculated from then on.\nMoreover, another viability assay was performed: Trypan Blue exclusion test. Parasites (5 x 106 cells x mL−1) were cultured in medium for 48 h. Subsequently, the cells were treated with 10 µM ketoconazole (2xEC50 of 72 h growth curve) and the number of promastigotes was determined by blind counting using a Neubauer chamber for three consecutive days (72 h) and Trypan blue stain. Three independent experiments were performed, all in triplicate.\n\nMorphological and ultrastructural analysis - For evaluating ultrastructural changes, transmission electron (TEM) and scanning electron (SEM) microscopies were performed. For TEM, promastigotes (5 x 106 cells x mL−1, log phase) were incubated with or without (control) ketoconazole 10 μM for 72 h. After being treated, the parasites were fixed at 4ºC with 2.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS) pH 7.2 for 24 h, washed in PBS, post-fixed in PBS containing 0.8% potassium ferrocyanide and 1% osmium tetroxide (OsO4) for 1 h and washed once again. Subsequently, the parasites were dehydrated in a gradual series of acetone and set in resin. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate and analysed using a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany). For SEM, promastigotes treated or not with ketoconazole 10 μM for 72 h were dehydrated in ethanol, critical point-dried in CO2, mounted in stubs, sputtered with a thin gold layer, and analysed using a scanning electron microscope (Zeiss EVO MA10).\n\nEvaluation of mitochondrial damage - The fluorescent stain Rhodamine 123 was employed to evaluate the mitochondrial transmembrane potential (∆Ψ\nm\n ) of promastigotes. Initially, parasites (5 x 106 cells x mL−1) were treated with 10 µM ketoconazole or not (control) for 24, 48, and 72 h. Aliquots were incubated with Rhodamine 123 (15 µg x mL−1) for 15 min at room temperature and protected from light (dark). Subsequently, the parasites were washed twice with PBS. The cell population analysis was carried out in a flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. Three independent experiments were performed.\nFurthermore, promastigotes (5 x 106 cells x mL−1) treated or not (control) with ketoconazole (10 µM, 72 h) were incubated with MitoTrackerR RedCMXRos (300 nM) for 30 min in the dark. Subsequently, parasites were washed with PBS, fixed in paraformaldehyde 1% in cacodylate buffer, and washed twice with PBS. The cell population analysis was performed with a confocal fluorescence microscopy (Zeiss LSM510 Meta).\n\nDetection of autophagic vacuoles - The monodansylcadaverine (MDC) labeling was carried out to evaluate the autophagic vacuoles formation. Promastigotes (5 x 106 cells x mL−1, log phase) were cultured in absence (control) or presence of ketoconazole 10 μM for 24, 48, and 72 h. Subsequently, 100 µM MDC was added to parasites and incubated for 2 h in the dark. Afterwards, the parasites were washed with PBS, fixed in 1% paraformaldehyde in cacodylate buffer, washed twice with PBS, mounted on microscope slides, and analysed by fluorescence microscopy (excitation wavelength 358 nm and emission wavelength 463 nm). Two independent experiments were performed and the fluorescence intensity was quantified by the software Image J version 1.48.\n\nCell cycle analysis - Initially, the interference of ketoconazole on the cell cycle was evaluated by counting the number of nucleous, kinetoplast and flagella by parasite. Promastigotes (5 x 106 cells x mL-1) treated or not (control) with ketoconazole (10 uM, 72 h) labelled with DAPI (1:500, for 1 h) were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta). For a more detailed study, parasites (5 x 106 cells x mL−1, log phase) were incubated for 72 h with or without (control) ketoconazole (10 μM, 72 h) in medium containing 10% FBS. After incubation, parasites were washed and resuspended in ice-cold 70% ethanol in PBS and fixed for 18 h at 4ºC. Subsequently, the parasites were incubated with PBS containing 10 µg x mL−1 propidium iodide and 100 µg x mL−1 RNAse A for 45 min at 37ºC, protected from light. The cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. For each phase of the cell cycle, a respective percentage was obtained using the FlowJov10.0.7 software.\n\nCell death analysis - Promastigotes (5 x 106 cells x mL−1) were incubated for 72 h with or without (negative control) ketoconazole (10 μM, 72 h); while parasites incubated with formaldehyde 4% for 15 min were considered positive control. Subsequently, a binding buffer containing annexin V-FITC and 2 μg x mL−1 propidium iodide (BD Bioscience, Brazil) was added to the parasites and incubated for 15 min at 25ºC in the dark, according to the manufacturer’s instructions. Then, the parasites were washed and the cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA). Thirty thousand events were obtained from the region corresponding to the parasites. The percentages of apoptotic cells were determined using the FlowJo v10.0.7 software.\nFor Propidium Iodide (PI) and Annexin V (AV) labeling, promastigotes (5 x 106 cells x mL−1) treated with ketoconazole (10 μM, 72 h), medium (negative control) or formaldehyde 4% (positive control) were incubated in presence of PI (2 µg x mL-1), AV (1 µg x mL-1) or both for 15 min at 25ºC, protected from the light. Subsequently, parasites were fixed with 1% paraformaldehyde in a cacodylate buffer, washed and placed on glass coverslips. Finally, the parasites were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta).\n\nIntracellular amastigote assay - Murine macrophage cell line RAW264.7 (4 × 105/well) cultured in RPMI 1640 medium, supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) was placed in 24-well plates containing 13-mm diameter glass coverslips and infected with L. (L.) amazonensis free amastigotes isolated just before their use (two free amastigotes: 1 macrophage). After 1 h 30, plates were washed with PBS to remove non-internalised amastigotes and RPMI medium alone (control) or containing drugs (200 to 12,5 µM for ketoconazole, double serial dilutions) was added. Experiments were conducted at 37ºC in a 5% CO2 humidified incubator for 48 h. Cells on coverslips were fixed and stained with Giemsa stain modified solution for evaluation of the infectivity index; 100 cells per coverslip were blind counted and the infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage. This assay was carried out in quadruplicate and two independent experiments were performed.\nIt is worth highlighting the cytotoxic effect of ketoconazole on murine macrophages cell line RAW264.7 was, previously, performed by MTT test at the same drug concentrations used to assess the viability of promastigotes (section 2.4), but within 48 h. Since the macrophages were infected with amastigotes isolated from animal paw just before use and it was not necessary time to promastigote-amastigote differentiation, the infectivity assay was conducted at 48 h, time that allowed better visualisation and counting of the intramacrophage amastigotes.\n\nStatistics - Statistical analysis was conducted using GraphPad Prism 5. Each set of results was firstly checked for normal distribution using KolmogorovSmirnov, D’Agostinho and Pearson, and ShapiroWalk tests. Normally distributed data were analysed through one-way-ANOVA followed by Tukey’s post-test or Student test T. Statistically significant differences were assumed when at least p < 0.05.\n\nRESULTS:\n\nKetoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.\n\nFig. 1:\nLeishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).\n\n\nKetoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.\n\nFig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).\n\n\nKetoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.\nAutophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.\n\nFig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).\n\n\nFig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).\n\n\nKetoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).\n\nFig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).\n\n\nKetoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.\n\nFig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.\n\n\nKetoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.\n\nFig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.\n\n\nDISCUSSION:\nConsidering the importance of ergosterol for the biology and pathogenesis of the Leishmania parasite, herein, we certified the antiproliferative/viability effects of ketoconazole against L. (L.) amazonensis both promastigotes\n31\n and intracellular amastigotes\n32\n in another parasite strain: IFLA/BR/67/PH8. Furthermore, we show in more detail the biological effects of the inhibitor, aiming at understanding the drug mechanism of action.\nRegarding ultrastructure, our results revealed rounded up shape, mitochondrial swelling, augmented accumulation of lipid droplets and acidocalcisomes, presence of multivesicular bodies and frequent truncated and/or double flagella. These data are similar to those previously obtained for L. amazonensis promastigotes treated with ketoconazole\n31\n\n,\n\n33\n\n,\n\n34\n and suggest mitochondrial damage.\nWhen parasites (Trypanosoma cruzi, Leishmania spp, Toxoplasma gondii) are treated with inhibitors of important enzymes of the ergosterol biosynthesis pathway, the mitochondrion is the organelle primarily affected.\n35\n In our study, the ketoconazole-treated parasites presented hyperpolarisation of the mitochondrial membrane potential, time-dependent up to 72 h. Mitochondrial membrane potential variations could be the result of diverse events: inhibition of electron transport chain (decrease); blockage of ATP synthase (increase); stimulation of uncoupling proteins (decrease); or permeabilisation of the inner membrane (decrease).\n36\n Macedo et al.\n37\n demonstrated that L. (L.) amazonensis promastigotes treated with itraconazole and posaconazole for 48 h presented collapse of the mitochondrial membrane potential associated with intense mitochondrial swelling, disorganisation and rupture of mitochondrial membranes. Another study showed that T. cruzi treated with ketoconazole (EC50 = 32 µM for 72 h) presented a gradual increase in Rho 123 time-dependent fluorescence and a confocal microscopy of parasites confirmed an intense proliferation of the inner mitochondrial membrane. This organelle became highly branched and compact and elevated levels of Rho 123 were accumulated within the cells.\n38\n In many cases, a transient hyperpolarisation occurs before mitochondrial depolarisation, as if it were an attempt of the cells to avoid death. This effect can be seen in great part of the heat-shocked Leishmania promastigotes.\n39\n\n\nThe mitochondrial membrane is hyperpolarised does not mean that the organelle is functioning normally. Our results showed that MitoTrackerR labeling, a probe which passively diffuses across the plasma membrane and accumulates in active mitochondria, revealed an outstanding intensity decrease in ketoconazole-treated promastigotes, indicating loss of mitochondrial activity in these promastigotes. This result was also confirmed by the MTT viability assay, which measures the activity of mitochondrial enzymes, and by the proliferation curve using Trypan blue, which takes into account only viable cells. Mitochondrion is essential for generating the necessary energy for the survival and proliferation of eukaryotic cells.\n40\n\n) In this way, malfunctioning mitochondria could impair the ATP synthesis and inorganic phosphate would accumulate in acidocalcisomes, which explains the increase in the amount of this organelle 72 h after ketoconazole treatment. Acidocalcisomes are organelles of acidic nature and high electron density important due to their storage of polyphosphates, calcium, magnesium, and other elements.\n41\n The augmented number of this organelle was already reported for ketoconazole and L. amazonensis (MHOM/Josefa/75/Br strain).\n42\n\n\nThe treatment of L. amazonensis with ketoconazole stressed the cells, especially early after the treatment (24 and 48 h), leading to the appearance of autophagic vacuoles. Itraconazole and posaconazole induced appearance of autophagosome-like structures in L. (L.) amazonensis\n\n37\n and Rodrigues et al.\n43\n described the effect of autophagy on L. amazonensis (MHOM/BR/75/Josefa strain) treated with 22,26-azasterol. Thus, the confirmed presence of autophagic vacuoles might represent an adaptive response of the parasite to treatment (stress condition). Initially, parasites would resort to autophagic vacuoles in an attempt to remodel/remove abnormal cellular constituents, degrading damaged structures.\n44\n Over time, directing energy to the parasite single mitochondrion may be more important than repairing cell damage. This would explain the fact that at 72 h there was less labeling for autophagic vacuoles and higher mitochondrial hyperpolarisation occurred. Autophagy is involved in turnover and recycling by removal of damaged cellular components, regulating homeostasis during crucial processes such as cell growth and differentiation and plays fundamental role in mitochondrial functionality.\n44\n\n,\n\n45\n\n\nResults of the growth curve and frequent ultrastructural alterations in flagellum (truncated and/or double flagella), despite the intact kinetoplast, suggest that ketoconazole interferes with the L. amazonensis cell cycle. Therefore, the cell cycle of the parasites was evaluated both by nucleus, kinetoplast, and flagellum (n-k-f) counts and by flow cytometry. Different from some data in the literature involving ketoconazole acting on other cell types\n46\n\n,\n\n47\n and azole compounds on L. amazonensis,\n37\n our results of the n-k-f score did not reveal differences between the control and ketoconazole-treated parasites. In addition, the cell cycle assay showed that the treatment was not able to alter the cycle phases (Sub G1, G1, S, and G2/M) in relation to control parasites suggesting that ketoconazole does not interfere with parasite replication under the tested conditions. When L. (L.) amazonensis was treated with itraconazole and posaconazole, some cells presented more than two flagella and significant alterations in kinetoplast were observed by ultrastructural analysis; however, the possible alteration in the cell cycle was not evaluated.\n37\n\n\nKetoconazole has been related to apoptosis in several normal and tumoral cells.\n48\n\n,\n\n49\n\n,\n\n50\n\n,\n\n51\n Haegler et al.\n52\n showed that ketoconazole and posaconazole presented hepatocellular toxicity, impairing the ∆Ψm, the function of enzyme complexes of electron transport chain, accumulating mitochondrial superoxide and inducing apoptosis. Possibly, mitochondrial dysfunction could be associated with hepatotoxicity caused by those azole compounds. Our data revealed that treatment of the parasites with ketoconazole, under the conditions previously described, showed a trend towards apoptosis, although there was a slight positive result for PI. Furthermore, few autophagic vacuoles were observed after treatment. Other researchers reported that treatment of T. cruzi with ketoconazole (EC50/72 h) resulted in neither necrosis nor apoptosis. However, high concentrations of the drug (EC100/24h) resulted in fast cell death due to necrosis.\n38\n\n\nSBIs effects on extracellular L. amazonensis parasites (promastigotes) appear to be more gradual than that observed with the intracellular form (amastigote);\n31\n thus, SBIs effects profile allows a more detailed study of the events in the promastigote form. In view of the effects provoked by ketoconazole on the L. (L.) amazonensis promastigote, the interference of drug on infectivity capacity of amastigotes, the clinically relevant form of the parasite, was evaluated and evidenced a susceptibility to treatment. Taken together, the infectivity results added to the data of mitochondrial damage, increase in the acidocalcisome and autophagic vacuoles amounts may be evidence that the susceptibility observed in vitro is related to mitochondrial dysfunction provoked by ketoconazole. However, future studies should be carried out on amastigotes to confirm this hypothesis.\nTo our knowledge, this study demonstrates the effects of ketoconazole on mitochondrion functioning, autophagic compartments, cell cycle and death of L. (L.) amazonensis promastigotes for the first time. We suggest that some damages are occurring in the parasite, mainly in the mitochondrion. In an attempt to remodel and/or destroy eventual damaged structures, parasites hyperpolarise mitochondrion and resort to autophagic vacuoles. However, this survival strategy adopted by the cell to direct efforts to maintain the cell energy is not sufficient because of the decrease in cell and mitochondria viabilities and increase in acidocalcisomes. Although these damages do not reflect directly in the parasite cell cycle, they lead the parasites to death, making them susceptible to in vitro treatment (Fig. 8).\n\nFig. 8:proposed mode action of ketoconazole on Leishmania (Leishmania) amazonensis. Parasites resort to autophagic vacuoles and mitochondrion hyperpolarisation in attempt to survive to damages mainly in the mitochondrion. The efforts to maintain the cell energy are not sufficient since the decrease of cell and mitochondria viability and increase of acidocalcisomes. These damages do not interfere with the parasite cell cycle, but they lead the parasites to death, making them susceptible to in vitro treatment conditions.\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nLeishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase.\n\nMethods:\nHerein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment.\n\nResults:\nThe study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain).\n\nConclusions:\nKetoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. 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CCl4-induced hepatotoxicity: protective effect of rutin on p53, CYP2E1 and the antioxidative status in rat. | 23043521 | Rutin is a polyphenolic natural flavonoid which possesses antioxidant and anticancer activity. In the present study the hepatoprotective effect of rutin was evaluated against carbon tetrachloride (CCl₄)-induced liver injuries in rats. | BACKGROUND | 24 Sprague-Dawley male rats were equally divided into 4 groups for the assessment of hepatoprotective potential of rutin. Rats of group I (control) received only vehicles; 1 ml/kg bw of saline (0.85%) and olive oil (3 ml/kg) and had free access to food and water. Rats of group II, III and IV were treated with CCl₄ (30% in olive oil, 3 ml/kg bw) via the intraperitoneal route twice a week for four weeks. The rutin at the doses of 50 and 70 mg/kg were administered intragastrically after 48 h of CCl₄ treatment to group III and IV, respectively. Protective effect of rutin on serum enzyme level, lipid profile, activities of antioxidant enzymes and molecular markers were calculated in CCl₄-induced hepatotoxicity in rat. | METHODS AND MATERIALS | Rutin showed significant protection with the depletion of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT) in serum as was raised by the induction of CCl₄. Concentration of serum triglycerides, total cholesterol and low density lipoproteins was increased while high-density lipoprotein was decreased with rutin in a dose dependent manner. Activity level of endogenous liver antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHpx), glutathione-S-transferase (GST) and glutathione reductase (GSR) and glutathione (GSH) contents were increased while lipid peroxidation (TBARS) was decreased dose dependently with rutin. Moreover, increase in DNA fragmentation and oxo8dG damages while decrease in p53 and CYP 2E1 expression induced with CCl₄ was restored with the treatment of rutin. | RESULTS | From these results, it is suggested that rutin possesses hepatoprotective properties. | CONCLUSION | [
"Animals",
"Antioxidants",
"Carbon Tetrachloride",
"Chemical and Drug Induced Liver Injury",
"Cytochrome P-450 CYP2E1",
"DNA Fragmentation",
"Dose-Response Relationship, Drug",
"Enzymes",
"Lipid Peroxidation",
"Lipids",
"Liver",
"Male",
"Oxidative Stress",
"Phytotherapy",
"Plant Extracts",
"Rats",
"Rats, Sprague-Dawley",
"Rutin",
"Tumor Suppressor Protein p53"
] | 3519517 | Background | Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl4) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2-4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl4 metabolites and cytokines, which propagate inflammatory response [6]. CCl4 is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7,8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (·CCl3) and peroxy trichloromethyl radicals (·OOCCl3). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl4 exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9,10].
An increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11,12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].
Flavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18,19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20,21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl4-induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity. | Methods | Drugs and chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.
Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.
Animals and treatment Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.
After 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.
Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.
After 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.
Assessment of hepatotoxicity Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.
Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.
Assessment of oxidative stress For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].
For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].
DNA damages Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.
Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.
Statistical analysis To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.
To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability. | Results | Body weight, liver weight Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).
Effect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).
Effect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Lipids profile Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.
Effect of Rutin on Lipids profile
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.
Effect of Rutin on Lipids profile
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Genotoxicity studies Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).
Effect of Rutin on Genotoxicity studies
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Protective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl
4
treated rats), 9,10 (CCl
4
+50 mg/kg b. w. rutin), 11,12 (CCl
4
+70 mg/kg b.w. rutin).
Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).
Effect of Rutin on Genotoxicity studies
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Protective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl
4
treated rats), 9,10 (CCl
4
+50 mg/kg b. w. rutin), 11,12 (CCl
4
+70 mg/kg b.w. rutin).
Indices of hepatotoxicity Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.
Effect of Rutin on Indices of hepatotoxicity
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.
Effect of Rutin on Indices of hepatotoxicity
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Assessment of oxidative stress CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).
Effect of Rutin on assessment of oxidative stress
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).
Effect of Rutin on assessment of oxidative stress
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level. | Conclusion | These results demonstrate that administration of rutin may be useful in the treatment and prevention of hepatic stress. | [
"Background",
"Drugs and chemicals",
"Animals and treatment",
"Assessment of hepatotoxicity",
"Assessment of oxidative stress",
"DNA damages",
"Statistical analysis",
"Body weight, liver weight",
"Lipids profile",
"Genotoxicity studies",
"Indices of hepatotoxicity",
"Assessment of oxidative stress",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl4) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2-4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl4 metabolites and cytokines, which propagate inflammatory response [6]. CCl4 is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7,8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (·CCl3) and peroxy trichloromethyl radicals (·OOCCl3). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl4 exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9,10].\nAn increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11,12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].\nFlavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18,19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20,21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl4-induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity.",
"Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.",
"Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.\nAfter 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.",
"Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.",
"For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].",
"Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.",
"To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.",
"Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).\nEffect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.\nEffect of Rutin on Lipids profile\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).\nEffect of Rutin on Genotoxicity studies\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n\nProtective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl\n\n4\n\ntreated rats), 9,10 (CCl\n\n4\n\n+50 mg/kg b. w. rutin), 11,12 (CCl\n\n4\n\n+70 mg/kg b.w. rutin).\n",
"Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.\nEffect of Rutin on Indices of hepatotoxicity\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).\nEffect of Rutin on assessment of oxidative stress\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"The authors declare that they have no competing interests.",
"RAK made a significant contribution to conception and design of the study, acquisition and analyses of data and drafting of the manuscript. MRK and SS made contribution in sample collection and design. All the authors read the revised manuscript and approved.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/178/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Drugs and chemicals",
"Animals and treatment",
"Assessment of hepatotoxicity",
"Assessment of oxidative stress",
"DNA damages",
"Statistical analysis",
"Results",
"Body weight, liver weight",
"Lipids profile",
"Genotoxicity studies",
"Indices of hepatotoxicity",
"Assessment of oxidative stress",
"Discussion",
"Conclusion",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl4) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2-4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl4 metabolites and cytokines, which propagate inflammatory response [6]. CCl4 is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7,8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (·CCl3) and peroxy trichloromethyl radicals (·OOCCl3). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl4 exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9,10].\nAn increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11,12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].\nFlavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18,19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20,21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl4-induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity.",
" Drugs and chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.\nReduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.\n Animals and treatment Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.\nAfter 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.\nSix week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.\nAfter 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.\n Assessment of hepatotoxicity Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.\nLiver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.\n Assessment of oxidative stress For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].\nFor determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].\n DNA damages Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.\nHepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.\n Statistical analysis To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.\nTo determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.",
"Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.",
"Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.\nAfter 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.",
"Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.",
"For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].",
"Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.",
"To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.",
" Body weight, liver weight Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).\nEffect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\nTreatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).\nEffect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n Lipids profile Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.\nEffect of Rutin on Lipids profile\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\nAdministration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.\nEffect of Rutin on Lipids profile\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n Genotoxicity studies Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).\nEffect of Rutin on Genotoxicity studies\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n\nProtective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl\n\n4\n\ntreated rats), 9,10 (CCl\n\n4\n\n+50 mg/kg b. w. rutin), 11,12 (CCl\n\n4\n\n+70 mg/kg b.w. rutin).\n\nExposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).\nEffect of Rutin on Genotoxicity studies\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n\nProtective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl\n\n4\n\ntreated rats), 9,10 (CCl\n\n4\n\n+50 mg/kg b. w. rutin), 11,12 (CCl\n\n4\n\n+70 mg/kg b.w. rutin).\n\n Indices of hepatotoxicity Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.\nEffect of Rutin on Indices of hepatotoxicity\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\nAdministration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.\nEffect of Rutin on Indices of hepatotoxicity\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n Assessment of oxidative stress CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).\nEffect of Rutin on assessment of oxidative stress\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\nCCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).\nEffect of Rutin on assessment of oxidative stress\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).\nEffect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.\nEffect of Rutin on Lipids profile\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).\nEffect of Rutin on Genotoxicity studies\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.\n\nProtective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl\n\n4\n\ntreated rats), 9,10 (CCl\n\n4\n\n+50 mg/kg b. w. rutin), 11,12 (CCl\n\n4\n\n+70 mg/kg b.w. rutin).\n",
"Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.\nEffect of Rutin on Indices of hepatotoxicity\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).\nEffect of Rutin on assessment of oxidative stress\nMean ±SE (n=6 number).\n** indicate significance from the control group at P<0.01 probability level.\n++ indicate significance from the CCl4 group at P<0.01 probability level.",
"The fields of dietary modification and chemoprevention show considerable effective approaches against oxidative stress and are the focus of research these days [36]. Various studies have shown that several mutagens and carcinogens cause generation of oxygen-free radicals, which play a major role in the emergence of cancer and other health disturbances [37,38]. The present study revealed that CCl4-induction in rats remarkably increased the level of ALT, AST, ALP and γ-GT. CCl4 causes acute hepatocyte injuries, altered membrane integrity and as a result enzymes in hepatocytes leak out [39]. However, after treatment with rutin, the pathological increases in ALT, AST, ALP and γ-GT were significantly restored. These results indicate that rutin has the ability to protect against CCl4-induced hepatocyte injury, which is in agreement with a previous study [40] that reported the protective consequence of polyphenolic compounds against CCl4-induced liver cirrhosis. Importantly, the increased serum concentrations of triglycerides, total cholesterol and LDL, and the decreased level of HDL, were restored to normal values with rutin co-treatment. This may be explained on the basis that rutin has a strong ability to chelate multivalent metal ions, especially zinc, calcium and iron. Indeed, its ability to chelate minerals has been reported to have some protective effects, such as decreasing iron mediated free radical formation and lowering serum cholesterol, triglycerides and lipid peroxides in experimental animals [41]. Similar findings were reported in another study [42] that investigated the hepatoprotective effects of plant bioactive compounds against CCl4-induced hepatic injury in rats.\nROS formed during the biotransformation process of CCl4 are more reactive and toxic than the parental compound. Biotransformation of CCl4 occurs in the endoplasmic reticulum and the isoenzyme implicated in this process is CYP2E1 [43,44]. Our results showed that the active free radical/intermediate of CCl4 caused a reduction in CYP2E1, which was markedly restored by rutin treatment. Our results showed conformity with previous investigations, which demonstrated that the polyphenolic natural product is responsible for its protective, effects [45,46].\nResults of the present study revealed that exposure of rats to CCl4 resulted in depletion of antioxidant activities. In consonance with our results, Szymonik-Lesiuk et al.[1] reported that CCl4 intoxication leads to changes in antioxidant enzymes and reactive intermediates involved in the bioactivation of CCl4 that may truss to those enzymes to prevent their inactivation. Furthermore, our results correspond with [11,12], and are in agreement with an investigation following CCl4 intoxication [47].\nGlutathione provides a first line of defense and scavenges free radical oxygen species (ROS). The decreased concentration of GSH in liver may be due to NADPH reduction or GSH utilization in the exclusion of peroxides [48]. GSH-dependent enzymes offer a second line of protection as they primarily detoxify noxious byproducts generated by ROS and help to avert dissemination of free radicals [49]. GSH-Px detoxifies peroxides by reacting with GSH and converting it into GSSG, which is reduced to GSH by GSR [50]. Our study revealed that CCl4 treatment in rats markedly changed the activity of antioxidant enzymes, which was reverted by the co-administration of rutin. Thiobarbituric acid reactive substances (TBARS), the final metabolites of peroxidized polyunsaturated fatty acids, are considered as a late biomarker of oxidative stress [51]. In our experiments, major decrease in lipid peroxidation and consequent reduction in TBARS were obtained by treatment with rutin. The increment in lipid peroxidation, as assessed by the elevated levels of TBARS following CCl4 administration, has been well documented [12]. Data of the present study indicated that lipid peroxidation induced by oxidative stress caused DNA damage. TBARS react with the DNA strand to form the M1G adduct, the mutagenic pirimedopurinone adduct of deoxyguanosine [52]. Administration of rutin markedly reduced the DNA damage, which is in close agreement with a previous study [11]. This level of DNA damage decreases the expression of p53 and blocks cells in the G phase of the cell cycle, which gives the cells additional time to repair the DNA damage. However, severe DNA damage may elicit apoptosis [53]. The data revealed that CCl4-induction caused marked reduction in p53. This result may be explained on the basis that CCl4 acts as a tumor promoter through increasing the intracellular concentration of ROS necrosis/regeneration and cell proliferation and/or may be due to mutation of p53. Our results regarding p53 are in agreement with previous studies [54,55].",
"These results demonstrate that administration of rutin may be useful in the treatment and prevention of hepatic stress.",
"The authors declare that they have no competing interests.",
"RAK made a significant contribution to conception and design of the study, acquisition and analyses of data and drafting of the manuscript. MRK and SS made contribution in sample collection and design. All the authors read the revised manuscript and approved.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6882/12/178/prepub\n"
] | [
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"methods",
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null,
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null,
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"results",
null,
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"discussion",
"conclusions",
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] | [
"Hepatotoxicity",
"Rutin",
"p53",
"CYP 2E1",
"Antioxidant enzymes"
] | Background:
Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl4) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2-4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl4 metabolites and cytokines, which propagate inflammatory response [6]. CCl4 is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7,8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (·CCl3) and peroxy trichloromethyl radicals (·OOCCl3). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl4 exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9,10].
An increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11,12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].
Flavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18,19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20,21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl4-induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity.
Methods:
Drugs and chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.
Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.
Animals and treatment Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.
After 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.
Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.
After 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.
Assessment of hepatotoxicity Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.
Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.
Assessment of oxidative stress For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].
For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].
DNA damages Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.
Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.
Statistical analysis To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.
To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.
Drugs and chemicals:
Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, gamma-glutamyl p-nitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2-dithio-bis nitro benzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), rutin, CCl4, flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (PCA) were purchased from Sigma Chemicals Co. USA.
Animals and treatment:
Six week old, 24 Sprague–Dawley male rats (200–210 g) were provided by National Institute of Health Islamabad and were kept in ordinary cages at room temperature of 25±3°C with a 12 h dark/light cycles. They have free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Quaid-i-Azam University Islamabad for animal care and experimentation. To study the hepatoprotective effects of rutin, rats were equally divided into four groups (six rats). Animals of group I were treated with 1 ml/kg bw of saline (0.85%) intragastrically and olive oil (3 ml/kg bw) intraperitoneally twice a week for four weeks. Rats of group II, III and IV were treated with CCl4 (30% in olive oil) at a dose of 3 ml/kg bw intraperitoneally twice a week for four weeks. Animals of group II received only CCl4 treatment. However, animals of group III and IV received rutin at a dose of 50 and 70 mg/kg bw intragastrically, respectively, in addition to CCl4 treatment, twice a week for four weeks.
After 24 h of the last treatment, all the animals were weighted, sacrificed, collected the blood while liver was removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −70 °C for further studies.
Assessment of hepatotoxicity:
Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), lipid profile [total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride] were estimated by using standard AMP diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). CYP 2E1 (cat no: E90988Ra, Uscn Life Science Inc.), oxo8dG and p53 (PUMA ELISA Kit cat no: E91909Ra) concentration was determined with ELISA kit.
Assessment of oxidative stress:
For determination of oxidative stress liver tissue was homogenized in 10 volumes of 100 mmol KH2PO4 buffer containing 1 mmol EDTA (pH 7.4) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below. Protein concentration was determined by using crystalline BSA as standard. CAT and SOD activities are determined with protocol of [27,28] while phase II metabolizing enzyme, including glutathione-S-transferase (GST), glutathione reductase (GSR), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) [29-32] and thiobarbituric acid reactive substances (TBARS) contents, respectively [33].
DNA damages:
Hepatic DNA damages (fragmentation % by DPA assay), DNA ladder assay [34] and number of NORs per cell [35] were determined.
Statistical analysis:
To determine the treatment effects, one-way analysis of variance was carried by computer software SPSS 13.0. Level of significance among the various treatments was determined by LSD at 0.05% and 0.01% level of probability.
Results:
Body weight, liver weight Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).
Effect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).
Effect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Lipids profile Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.
Effect of Rutin on Lipids profile
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.
Effect of Rutin on Lipids profile
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Genotoxicity studies Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).
Effect of Rutin on Genotoxicity studies
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Protective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl
4
treated rats), 9,10 (CCl
4
+50 mg/kg b. w. rutin), 11,12 (CCl
4
+70 mg/kg b.w. rutin).
Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).
Effect of Rutin on Genotoxicity studies
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Protective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl
4
treated rats), 9,10 (CCl
4
+50 mg/kg b. w. rutin), 11,12 (CCl
4
+70 mg/kg b.w. rutin).
Indices of hepatotoxicity Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.
Effect of Rutin on Indices of hepatotoxicity
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.
Effect of Rutin on Indices of hepatotoxicity
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Assessment of oxidative stress CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).
Effect of Rutin on assessment of oxidative stress
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).
Effect of Rutin on assessment of oxidative stress
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Body weight, liver weight:
Treatment of CCl4 caused significant reduction (P<0.01) in body weight while increased the absolute liver and relative liver weight comparatively to control group was significantly (P<0.01) restored with 50 mg/kg bw and 70 mg/kg bw treatment of rutin (Table 1).
Effect of Rutin on absolute liver weight, relative liver weight, and % increase in body weight
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Lipids profile:
Administration of CCl4 increased triglycerides, total cholesterol, LDL while decreased the HDL as shown in Table 2. Concentration of HDL was significantly (P<0.01) increased by rutin whereas concentration of triglycerides, total cholesterol and LDL was appreciably (P<0.01) augmented to compensate the CCl4-induced toxicity.
Effect of Rutin on Lipids profile
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Genotoxicity studies:
Exposure of CCl4 elicited the hepatic DNA damages (% fragmentation) and number of AgNORs/cell. Percent serum level of oxo8dG was increased whereas the % activity level of p53 and CYP 2E1 was decreased in hepatic samples of rat. Treatment of rats with 50 mg/kg bw and 70 mg/kg bw of rutin restored the level of these markers (Table 3). DNA ladder assay showed conformity to the DNA fragmentation assay (Figure 1).
Effect of Rutin on Genotoxicity studies
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Protective effects of rutin on DNA; Lane 1–4 (non treated control), 5–8 (CCl
4
treated rats), 9,10 (CCl
4
+50 mg/kg b. w. rutin), 11,12 (CCl
4
+70 mg/kg b.w. rutin).
Indices of hepatotoxicity:
Administration of CCl4 markedly increased (P<0.01) the activity of liver serum marker enzymes such as AST, ALT, ALP and γ-GT as compared with the control group. Elevations in the secretion of these enzymes were significantly decreased (P<0.01) by 50 mg/kg bw and 70 mg/kg bw of rutin as compared to the CCl4 group are shown in Table 4.
Effect of Rutin on Indices of hepatotoxicity
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Assessment of oxidative stress:
CCl4 treatment in rats significantly decreased (P<0.01) the activity of CAT, SOD, GST, GSH-Px, GSR, GSH while increased TBARS contents in liver samples. The increase of lipid peroxidation caused; reduction in the activities of antioxidant enzymes and glutathione (GSH) contents were markedly attenuated (P<0.01) by administration of 50 mg/kg bw and 70 mg/kg bw of rutin in intoxicated rats (Table 5).
Effect of Rutin on assessment of oxidative stress
Mean ±SE (n=6 number).
** indicate significance from the control group at P<0.01 probability level.
++ indicate significance from the CCl4 group at P<0.01 probability level.
Discussion:
The fields of dietary modification and chemoprevention show considerable effective approaches against oxidative stress and are the focus of research these days [36]. Various studies have shown that several mutagens and carcinogens cause generation of oxygen-free radicals, which play a major role in the emergence of cancer and other health disturbances [37,38]. The present study revealed that CCl4-induction in rats remarkably increased the level of ALT, AST, ALP and γ-GT. CCl4 causes acute hepatocyte injuries, altered membrane integrity and as a result enzymes in hepatocytes leak out [39]. However, after treatment with rutin, the pathological increases in ALT, AST, ALP and γ-GT were significantly restored. These results indicate that rutin has the ability to protect against CCl4-induced hepatocyte injury, which is in agreement with a previous study [40] that reported the protective consequence of polyphenolic compounds against CCl4-induced liver cirrhosis. Importantly, the increased serum concentrations of triglycerides, total cholesterol and LDL, and the decreased level of HDL, were restored to normal values with rutin co-treatment. This may be explained on the basis that rutin has a strong ability to chelate multivalent metal ions, especially zinc, calcium and iron. Indeed, its ability to chelate minerals has been reported to have some protective effects, such as decreasing iron mediated free radical formation and lowering serum cholesterol, triglycerides and lipid peroxides in experimental animals [41]. Similar findings were reported in another study [42] that investigated the hepatoprotective effects of plant bioactive compounds against CCl4-induced hepatic injury in rats.
ROS formed during the biotransformation process of CCl4 are more reactive and toxic than the parental compound. Biotransformation of CCl4 occurs in the endoplasmic reticulum and the isoenzyme implicated in this process is CYP2E1 [43,44]. Our results showed that the active free radical/intermediate of CCl4 caused a reduction in CYP2E1, which was markedly restored by rutin treatment. Our results showed conformity with previous investigations, which demonstrated that the polyphenolic natural product is responsible for its protective, effects [45,46].
Results of the present study revealed that exposure of rats to CCl4 resulted in depletion of antioxidant activities. In consonance with our results, Szymonik-Lesiuk et al.[1] reported that CCl4 intoxication leads to changes in antioxidant enzymes and reactive intermediates involved in the bioactivation of CCl4 that may truss to those enzymes to prevent their inactivation. Furthermore, our results correspond with [11,12], and are in agreement with an investigation following CCl4 intoxication [47].
Glutathione provides a first line of defense and scavenges free radical oxygen species (ROS). The decreased concentration of GSH in liver may be due to NADPH reduction or GSH utilization in the exclusion of peroxides [48]. GSH-dependent enzymes offer a second line of protection as they primarily detoxify noxious byproducts generated by ROS and help to avert dissemination of free radicals [49]. GSH-Px detoxifies peroxides by reacting with GSH and converting it into GSSG, which is reduced to GSH by GSR [50]. Our study revealed that CCl4 treatment in rats markedly changed the activity of antioxidant enzymes, which was reverted by the co-administration of rutin. Thiobarbituric acid reactive substances (TBARS), the final metabolites of peroxidized polyunsaturated fatty acids, are considered as a late biomarker of oxidative stress [51]. In our experiments, major decrease in lipid peroxidation and consequent reduction in TBARS were obtained by treatment with rutin. The increment in lipid peroxidation, as assessed by the elevated levels of TBARS following CCl4 administration, has been well documented [12]. Data of the present study indicated that lipid peroxidation induced by oxidative stress caused DNA damage. TBARS react with the DNA strand to form the M1G adduct, the mutagenic pirimedopurinone adduct of deoxyguanosine [52]. Administration of rutin markedly reduced the DNA damage, which is in close agreement with a previous study [11]. This level of DNA damage decreases the expression of p53 and blocks cells in the G phase of the cell cycle, which gives the cells additional time to repair the DNA damage. However, severe DNA damage may elicit apoptosis [53]. The data revealed that CCl4-induction caused marked reduction in p53. This result may be explained on the basis that CCl4 acts as a tumor promoter through increasing the intracellular concentration of ROS necrosis/regeneration and cell proliferation and/or may be due to mutation of p53. Our results regarding p53 are in agreement with previous studies [54,55].
Conclusion:
These results demonstrate that administration of rutin may be useful in the treatment and prevention of hepatic stress.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
RAK made a significant contribution to conception and design of the study, acquisition and analyses of data and drafting of the manuscript. MRK and SS made contribution in sample collection and design. All the authors read the revised manuscript and approved.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6882/12/178/prepub
| Background:
Rutin is a polyphenolic natural flavonoid which possesses antioxidant and anticancer activity. In the present study the hepatoprotective effect of rutin was evaluated against carbon tetrachloride (CCl₄)-induced liver injuries in rats.
Methods:
24 Sprague-Dawley male rats were equally divided into 4 groups for the assessment of hepatoprotective potential of rutin. Rats of group I (control) received only vehicles; 1 ml/kg bw of saline (0.85%) and olive oil (3 ml/kg) and had free access to food and water. Rats of group II, III and IV were treated with CCl₄ (30% in olive oil, 3 ml/kg bw) via the intraperitoneal route twice a week for four weeks. The rutin at the doses of 50 and 70 mg/kg were administered intragastrically after 48 h of CCl₄ treatment to group III and IV, respectively. Protective effect of rutin on serum enzyme level, lipid profile, activities of antioxidant enzymes and molecular markers were calculated in CCl₄-induced hepatotoxicity in rat.
Results:
Rutin showed significant protection with the depletion of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT) in serum as was raised by the induction of CCl₄. Concentration of serum triglycerides, total cholesterol and low density lipoproteins was increased while high-density lipoprotein was decreased with rutin in a dose dependent manner. Activity level of endogenous liver antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHpx), glutathione-S-transferase (GST) and glutathione reductase (GSR) and glutathione (GSH) contents were increased while lipid peroxidation (TBARS) was decreased dose dependently with rutin. Moreover, increase in DNA fragmentation and oxo8dG damages while decrease in p53 and CYP 2E1 expression induced with CCl₄ was restored with the treatment of rutin.
Conclusions:
From these results, it is suggested that rutin possesses hepatoprotective properties. | Background:
Exposure to toxic chemicals, environmental pollutants and drugs can cause cellular injuries through metabolic activation of reactive oxygen species (ROS) [1]. Carbon tetrachloride (CCl4) has been used extensively to study hepatotoxicity in animal models by initiating lipid peroxidation, thereby causing injuries to kidney, heart, testis and brain [2-4], in addition to liver pathogenesis [5]. Liver is particularly susceptible to oxidative stress due to the direct release of CCl4 metabolites and cytokines, which propagate inflammatory response [6]. CCl4 is one of the xenobiotics that has been reported to induce acute and chronic tissue injuries [7,8] through bioactivation of the phase I cytochrome P450 system to form reactive metabolic trichloromethyl radicals (·CCl3) and peroxy trichloromethyl radicals (·OOCCl3). These free radicals can covalently bind to macromolecules such as proteins, lipids and nucleic acids. The double allylic hydrogen bonds of polyunsaturated fatty acid (PUFA) are susceptible to abstraction by free radicals; CCl4 exposure induces an increase in lipoperoxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [9,10].
An increase in unsaturated fatty acid lipoperoxide and free peroxide radical concentrations [11,12], can induce alterations in the cholesterol profile and decrease in hepatic antioxidant enzymes [13], in addition to induction of oxidative DNA damage including formation of DNA adducts, genetic mutations, strand breakage and chromosomal alterations [14]. These free radicals can cause depletion of CYP2E1 activity [15] and increase in oxo8dG concentration in tissues of experimental animals [16]. DNA fragmentation induces p53 gene expression, blocks cells in the G phase of the cell cycle, and gives additional time for DNA repair, while severe DNA damage triggers apoptosis [17]. It has been reported that CCl4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [11].
Flavonoids are a large group of polyphenolic compounds that play an important role in detoxification of free radicals and are markedly found in fruits, vegetables and medicinal plants [18,19]. Glycosidic flavonoids such as rutin are much more readily absorbed by humans than aglycones [20,21]. Rutin possesses antitumor [22], anti-inflammatory [23] and antimutagenic potential [24], besides myocardial protection [25] and immunomodulating activities [26]. Therefore, the present study was designed to investigate the hepatoprotective effects of rutin against CCl4-induced oxidative stress and its role in alleviation of lipid peroxidation and restoration of p53 and CYP2E1 activity.
Conclusion:
These results demonstrate that administration of rutin may be useful in the treatment and prevention of hepatic stress. | Background:
Rutin is a polyphenolic natural flavonoid which possesses antioxidant and anticancer activity. In the present study the hepatoprotective effect of rutin was evaluated against carbon tetrachloride (CCl₄)-induced liver injuries in rats.
Methods:
24 Sprague-Dawley male rats were equally divided into 4 groups for the assessment of hepatoprotective potential of rutin. Rats of group I (control) received only vehicles; 1 ml/kg bw of saline (0.85%) and olive oil (3 ml/kg) and had free access to food and water. Rats of group II, III and IV were treated with CCl₄ (30% in olive oil, 3 ml/kg bw) via the intraperitoneal route twice a week for four weeks. The rutin at the doses of 50 and 70 mg/kg were administered intragastrically after 48 h of CCl₄ treatment to group III and IV, respectively. Protective effect of rutin on serum enzyme level, lipid profile, activities of antioxidant enzymes and molecular markers were calculated in CCl₄-induced hepatotoxicity in rat.
Results:
Rutin showed significant protection with the depletion of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT) in serum as was raised by the induction of CCl₄. Concentration of serum triglycerides, total cholesterol and low density lipoproteins was increased while high-density lipoprotein was decreased with rutin in a dose dependent manner. Activity level of endogenous liver antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHpx), glutathione-S-transferase (GST) and glutathione reductase (GSR) and glutathione (GSH) contents were increased while lipid peroxidation (TBARS) was decreased dose dependently with rutin. Moreover, increase in DNA fragmentation and oxo8dG damages while decrease in p53 and CYP 2E1 expression induced with CCl₄ was restored with the treatment of rutin.
Conclusions:
From these results, it is suggested that rutin possesses hepatoprotective properties. | 5,603 | 375 | [
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] | [CONTENT] Hepatotoxicity | Rutin | p53 | CYP 2E1 | Antioxidant enzymes [SUMMARY] | [CONTENT] Hepatotoxicity | Rutin | p53 | CYP 2E1 | Antioxidant enzymes [SUMMARY] | [CONTENT] Hepatotoxicity | Rutin | p53 | CYP 2E1 | Antioxidant enzymes [SUMMARY] | [CONTENT] Hepatotoxicity | Rutin | p53 | CYP 2E1 | Antioxidant enzymes [SUMMARY] | [CONTENT] Hepatotoxicity | Rutin | p53 | CYP 2E1 | Antioxidant enzymes [SUMMARY] | [CONTENT] Hepatotoxicity | Rutin | p53 | CYP 2E1 | Antioxidant enzymes [SUMMARY] | [CONTENT] Animals | Antioxidants | Carbon Tetrachloride | Chemical and Drug Induced Liver Injury | Cytochrome P-450 CYP2E1 | DNA Fragmentation | Dose-Response Relationship, Drug | Enzymes | Lipid Peroxidation | Lipids | Liver | Male | Oxidative Stress | Phytotherapy | Plant Extracts | Rats | Rats, Sprague-Dawley | Rutin | Tumor Suppressor Protein p53 [SUMMARY] | [CONTENT] Animals | Antioxidants | Carbon Tetrachloride | Chemical and Drug Induced Liver Injury | Cytochrome P-450 CYP2E1 | DNA Fragmentation | Dose-Response Relationship, Drug | Enzymes | Lipid Peroxidation | Lipids | Liver | Male | Oxidative Stress | Phytotherapy | Plant Extracts | Rats | Rats, Sprague-Dawley | Rutin | Tumor Suppressor Protein p53 [SUMMARY] | [CONTENT] Animals | Antioxidants | Carbon Tetrachloride | Chemical and Drug Induced Liver Injury | Cytochrome P-450 CYP2E1 | DNA Fragmentation | Dose-Response Relationship, Drug | Enzymes | Lipid Peroxidation | Lipids | Liver | Male | Oxidative Stress | Phytotherapy | Plant Extracts | Rats | Rats, Sprague-Dawley | Rutin | Tumor Suppressor Protein p53 [SUMMARY] | [CONTENT] Animals | Antioxidants | Carbon Tetrachloride | Chemical and Drug Induced Liver Injury | Cytochrome P-450 CYP2E1 | DNA Fragmentation | Dose-Response Relationship, Drug | Enzymes | Lipid Peroxidation | Lipids | Liver | Male | Oxidative Stress | Phytotherapy | Plant Extracts | Rats | Rats, Sprague-Dawley | Rutin | Tumor Suppressor Protein p53 [SUMMARY] | [CONTENT] Animals | Antioxidants | Carbon Tetrachloride | Chemical and Drug Induced Liver Injury | Cytochrome P-450 CYP2E1 | DNA Fragmentation | Dose-Response Relationship, Drug | Enzymes | Lipid Peroxidation | Lipids | Liver | Male | Oxidative Stress | Phytotherapy | Plant Extracts | Rats | Rats, Sprague-Dawley | Rutin | Tumor Suppressor Protein p53 [SUMMARY] | [CONTENT] Animals | Antioxidants | Carbon Tetrachloride | Chemical and Drug Induced Liver Injury | Cytochrome P-450 CYP2E1 | DNA Fragmentation | Dose-Response Relationship, Drug | Enzymes | Lipid Peroxidation | Lipids | Liver | Male | Oxidative Stress | Phytotherapy | Plant Extracts | Rats | Rats, Sprague-Dawley | Rutin | Tumor Suppressor Protein p53 [SUMMARY] | [CONTENT] ccl4 induced hepatocyte | ccl4 metabolites cytokines | oxidative stress liver | ccl4 reactive toxic | oxidative stress ccl4 [SUMMARY] | [CONTENT] ccl4 induced hepatocyte | ccl4 metabolites cytokines | oxidative stress liver | ccl4 reactive toxic | oxidative stress ccl4 [SUMMARY] | [CONTENT] ccl4 induced hepatocyte | ccl4 metabolites cytokines | oxidative stress liver | ccl4 reactive toxic | oxidative stress ccl4 [SUMMARY] | [CONTENT] ccl4 induced hepatocyte | ccl4 metabolites cytokines | oxidative stress liver | ccl4 reactive toxic | oxidative stress ccl4 [SUMMARY] | [CONTENT] ccl4 induced hepatocyte | ccl4 metabolites cytokines | oxidative stress liver | ccl4 reactive toxic | oxidative stress ccl4 [SUMMARY] | [CONTENT] ccl4 induced hepatocyte | ccl4 metabolites cytokines | oxidative stress liver | ccl4 reactive toxic | oxidative stress ccl4 [SUMMARY] | [CONTENT] ccl4 | rutin | 01 | group | level | kg | bw | liver | kg bw | significance [SUMMARY] | [CONTENT] ccl4 | rutin | 01 | group | level | kg | bw | liver | kg bw | significance [SUMMARY] | [CONTENT] ccl4 | rutin | 01 | group | level | kg | bw | liver | kg bw | significance [SUMMARY] | [CONTENT] ccl4 | rutin | 01 | group | level | kg | bw | liver | kg bw | significance [SUMMARY] | [CONTENT] ccl4 | rutin | 01 | group | level | kg | bw | liver | kg bw | significance [SUMMARY] | [CONTENT] ccl4 | rutin | 01 | group | level | kg | bw | liver | kg bw | significance [SUMMARY] | [CONTENT] radicals | free | free radicals | dna | ccl4 | injuries | cause | increase | reactive | lipoperoxide free [SUMMARY] | [CONTENT] glutathione | acid | week | determined | animals | week weeks | ml | ml kg | ml kg bw | twice week weeks [SUMMARY] | [CONTENT] 01 | level | group | indicate significance | 01 probability level | group 01 | group 01 probability | group 01 probability level | probability level | 01 probability [SUMMARY] | [CONTENT] results demonstrate administration rutin | demonstrate administration rutin useful | treatment prevention | treatment prevention hepatic | treatment prevention hepatic stress | rutin useful treatment prevention | rutin useful treatment | rutin useful | prevention hepatic stress | prevention [SUMMARY] | [CONTENT] 01 | ccl4 | rutin | level | group | kg | dna | significance | probability | bw [SUMMARY] | [CONTENT] 01 | ccl4 | rutin | level | group | kg | dna | significance | probability | bw [SUMMARY] | [CONTENT] Rutin ||| [SUMMARY] | [CONTENT] 24 | Sprague-Dawley | 4 ||| 1 ml/kg | 0.85% | 3 ml/kg ||| II | III | IV | CCl₄ | 30% | 3 ml/kg | four weeks ||| 50 | 70 | 48 | CCl₄ | III | IV ||| [SUMMARY] | [CONTENT] Rutin | CCl₄. Concentration ||| CAT | SOD | GSR ||| 2E1 | CCl₄ [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] Rutin ||| ||| 24 | Sprague-Dawley | 4 ||| 1 ml/kg | 0.85% | 3 ml/kg ||| II | III | IV | CCl₄ | 30% | 3 ml/kg | four weeks ||| 50 | 70 | 48 | CCl₄ | III | IV ||| ||| ||| Rutin | CCl₄. Concentration ||| CAT | SOD | GSR ||| 2E1 | CCl₄ ||| [SUMMARY] | [CONTENT] Rutin ||| ||| 24 | Sprague-Dawley | 4 ||| 1 ml/kg | 0.85% | 3 ml/kg ||| II | III | IV | CCl₄ | 30% | 3 ml/kg | four weeks ||| 50 | 70 | 48 | CCl₄ | III | IV ||| ||| ||| Rutin | CCl₄. Concentration ||| CAT | SOD | GSR ||| 2E1 | CCl₄ ||| [SUMMARY] |
||||||
Association between Anxious and Depressive Symptomatology and Sexual Activity in Spain: A Cross-Sectional Study during the COVID-19 Quarantine. | 35010405 | Evidence on sexual behaviour and COVID-19 shows a change in sexual habits; however, there is no research on the association between mental health and sexual activity. | INTRODUCTION | A sample of 305 adults filled out an online questionnaire. Sexual activity was assessed with one question. Anxiety and depression symptoms were assessed using the Beck Anxiety Inventory (BAI) and the Beck Depression Inventory (BDI), respectively. To check associations between levels of both anxiety and depressive symptoms (exposure) and weekly prevalence of sexual activity (outcome), we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, chronic physical conditions, chronic psychiatric conditions, physical symptoms, and days of confinement). | METHODS | Higher depression level was associated with lower weekly sexual activity in a dose-response fashion in the three implemented models. Participants with higher levels of depression were associated with significantly lower sexual activity in the fully adjusted model (OR: 0.09, 95% CI 0.01-0.61). Mild anxiety-level participants consistently presented significantly lower ORs for lower sexual activity than their minimal-anxiety category counterparts. Particularly, the fully adjusted model showed the lower values (OR: 0.40, 95% CI 0.19-0.84). | RESULTS | The results of this study support existing evidence stressing the association between mental health and sexual activity in quarantined adults. | CONCLUSION | [
"Adult",
"Anxiety",
"COVID-19",
"Cross-Sectional Studies",
"Depression",
"Humans",
"Quarantine",
"SARS-CoV-2",
"Sexual Behavior",
"Spain"
] | 8751132 | 1. Introduction | As in many other countries, the Spanish Government has been implementing different public health strategies in order to slow the spread and “flatten the curve” of the ongoing human-to-human transmission of the COVID-19. In this regard, after the statement of the outbreak of COVID-19 as a global pandemic by the World Health Organisation and due to the high number of diagnosed cases in Spain, from 15 March until 2 May 2020, a period of total quarantine was established in Spain [1].
The health crisis and the restrictions imposed prompted a drastic situation for everyone with radical changes in daily life [2,3]. Among the different collateral impacts due to the pandemic situation and the public health measures to combat COVID-19 infection, sexual behaviour is one of the main concerns and it is being studied internationally [4,5,6]. This is because sexuality is an important element of human life and, according to The World Health Organisation, sexual health is “a state of physical, emotional, mental and social well-being related to sexuality; not merely the absence of disease, dysfunction or infirmity”. Consequently, a frequent and trouble-free sex life is associated with a plethora of physical and mental health benefits [4].
First studies on sexual behaviour and COVID-19 pointed out that there has been a change in sexual habits; in this regard, the lockdown has affected the sexual activity between cohabitants due to the difficulty of finding a moment of intimacy. As for the stable relationships who were not living together, the lockdown has made the physical contact impossible. Finally, for those looking for occasional sexual relations, the lockdown has created a drastic barrier for meeting a sexual partner [5]. Studies have suggested some changes based on the relationship status and the availability for living together during the implementation of movement control measures during the COVID-19 pandemic [6]. In this regard, there is evidence that all these changes have supposed that the quality of sex and satisfaction has decreased significantly [5,6]. Furthermore, Spanish women have been identified as a target group for promoting healthy regular practices regarding sex during COVID-19 quarantine since they were observed to reduce sexual activity during that period [7].
Scientific evidence shows that sexual inactivity is significantly related to different mental health conditions [8]. Moreover, it has been found that sexual dysfunction might lead to interpersonal conflicts by deteriorating either self-esteem or partner relationships [9]. On the other hand, there is evidence that positive mental health outcomes for males and females are significantly related to successful sexual activity. In this regard, psychological well-being by improving mood, even in depressed and high-anxiety patients [10,11], is positively related to trouble-free sexual activity.
To our knowledge, there is no evidence explaining how mental health affected the sexual activity status of the Spanish population during the quarantine. Therefore, this study aims to explain the association between depression/anxiety and sexual activity during quarantine among this population. | null | null | 3. Results | A total of 305 Spanish confined adults completed the survey. Overall, 85 (27.9%) males and 220 females (72.1%) participated. The age distribution was: 18–44 years (n = 277), 45–74 years (n = 28). Significant differences between participants regarding marital status, employment, depression, anxiety, and sexual activity before the COVID-19 confinement were identified. The rest of the sample characteristics are shown in Table 1.
The association between the level of depression and sexual activity is displayed in Table 2. Higher depression level was associated with lower sexual activity in a dose-response fashion in the three implemented models. Participants with higher levels of depression were associated with significantly lower sexual activity in the fully adjusted model (OR: 0.09, 95% CI 0.01–0.61). Also, Table 3 shows the association between anxiety and sexual activity, in which mild anxiety level was associated with lower sexual activity (OR: 0.40, 95% CI 0.19–0.84; fully adjusted model). | 5. Conclusions | In conclusion, in the present sample of confined Spanish adults, those who had more anxious and depressive symptomatology presented less sexual activity during the quarantine. To our knowledge, this is the first study on mental health and sexual activity in a sample of quarantined adults. In this regard, mental health outcomes and sexual activity seem to be associated, for that reason, future research must focus on inferring the direction of this association. | [
"2. Materials and Methods",
"2.2. Key Measures",
"2.2.1. Sexual Activity (Exposure)",
"2.2.2. Anxiety (Outcome)",
"2.2.3. Depression (Outcome)",
"2.2.4. Covariates",
"2.3. Statistical Analyses"
] | [
" 2.1. Study Design and Participants A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12]. \nSpanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13]. \nA cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12]. \nSpanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13]. \n 2.2. Key Measures 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\nIn the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\n 2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \nAnxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \n 2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\nDepression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\n 2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\nCollected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\n 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\nIn the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\n 2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \nAnxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \n 2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\nDepression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\n 2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\nCollected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\n 2.3. Statistical Analyses The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.\nThe statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.",
" 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\nIn the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\n 2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \nAnxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \n 2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\nDepression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\n 2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\nCollected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).",
"In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”",
"Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. ",
"Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].",
"Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).",
"The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05."
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"1. Introduction",
"2. Materials and Methods",
"2.1. Study Design and Participants",
"2.2. Key Measures",
"2.2.1. Sexual Activity (Exposure)",
"2.2.2. Anxiety (Outcome)",
"2.2.3. Depression (Outcome)",
"2.2.4. Covariates",
"2.3. Statistical Analyses",
"3. Results",
"4. Discussion",
"5. Conclusions"
] | [
"As in many other countries, the Spanish Government has been implementing different public health strategies in order to slow the spread and “flatten the curve” of the ongoing human-to-human transmission of the COVID-19. In this regard, after the statement of the outbreak of COVID-19 as a global pandemic by the World Health Organisation and due to the high number of diagnosed cases in Spain, from 15 March until 2 May 2020, a period of total quarantine was established in Spain [1]. \nThe health crisis and the restrictions imposed prompted a drastic situation for everyone with radical changes in daily life [2,3]. Among the different collateral impacts due to the pandemic situation and the public health measures to combat COVID-19 infection, sexual behaviour is one of the main concerns and it is being studied internationally [4,5,6]. This is because sexuality is an important element of human life and, according to The World Health Organisation, sexual health is “a state of physical, emotional, mental and social well-being related to sexuality; not merely the absence of disease, dysfunction or infirmity”. Consequently, a frequent and trouble-free sex life is associated with a plethora of physical and mental health benefits [4].\nFirst studies on sexual behaviour and COVID-19 pointed out that there has been a change in sexual habits; in this regard, the lockdown has affected the sexual activity between cohabitants due to the difficulty of finding a moment of intimacy. As for the stable relationships who were not living together, the lockdown has made the physical contact impossible. Finally, for those looking for occasional sexual relations, the lockdown has created a drastic barrier for meeting a sexual partner [5]. Studies have suggested some changes based on the relationship status and the availability for living together during the implementation of movement control measures during the COVID-19 pandemic [6]. In this regard, there is evidence that all these changes have supposed that the quality of sex and satisfaction has decreased significantly [5,6]. Furthermore, Spanish women have been identified as a target group for promoting healthy regular practices regarding sex during COVID-19 quarantine since they were observed to reduce sexual activity during that period [7].\nScientific evidence shows that sexual inactivity is significantly related to different mental health conditions [8]. Moreover, it has been found that sexual dysfunction might lead to interpersonal conflicts by deteriorating either self-esteem or partner relationships [9]. On the other hand, there is evidence that positive mental health outcomes for males and females are significantly related to successful sexual activity. In this regard, psychological well-being by improving mood, even in depressed and high-anxiety patients [10,11], is positively related to trouble-free sexual activity.\nTo our knowledge, there is no evidence explaining how mental health affected the sexual activity status of the Spanish population during the quarantine. Therefore, this study aims to explain the association between depression/anxiety and sexual activity during quarantine among this population. ",
" 2.1. Study Design and Participants A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12]. \nSpanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13]. \nA cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12]. \nSpanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13]. \n 2.2. Key Measures 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\nIn the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\n 2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \nAnxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \n 2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\nDepression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\n 2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\nCollected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\n 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\nIn the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\n 2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \nAnxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \n 2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\nDepression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\n 2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\nCollected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\n 2.3. Statistical Analyses The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.\nThe statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.",
"A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12]. \nSpanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13]. ",
" 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\nIn the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”\n 2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \nAnxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. \n 2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\nDepression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].\n 2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).\nCollected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).",
"In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”",
"Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis. ",
"Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].",
"Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).",
"The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.",
"A total of 305 Spanish confined adults completed the survey. Overall, 85 (27.9%) males and 220 females (72.1%) participated. The age distribution was: 18–44 years (n = 277), 45–74 years (n = 28). Significant differences between participants regarding marital status, employment, depression, anxiety, and sexual activity before the COVID-19 confinement were identified. The rest of the sample characteristics are shown in Table 1. \nThe association between the level of depression and sexual activity is displayed in Table 2. Higher depression level was associated with lower sexual activity in a dose-response fashion in the three implemented models. Participants with higher levels of depression were associated with significantly lower sexual activity in the fully adjusted model (OR: 0.09, 95% CI 0.01–0.61). Also, Table 3 shows the association between anxiety and sexual activity, in which mild anxiety level was associated with lower sexual activity (OR: 0.40, 95% CI 0.19–0.84; fully adjusted model). ",
"The main objective of this study was to investigate the association between mental health and sexual activity during quarantine for adults in Spain. In the present sample, comprising 305 Spanish confined adults, we found that both higher anxiety and depressive symptoms had a negative dose-response on the level of sexual activity. \nThe finding that sexual activity is associated with worse mental health outcomes is important and supports and adds to previous evidence [10,11]. Indeed, the present study found that people who have higher levels of depression have less sexual activity during the quarantine. Additionally, we found that the participants who have higher levels of anxiety presented a significantly lower chance to have sexual activity. Taken together, the present findings suggest that better mental health status, having less anxious and depressive symptomatology, is associated with having a more active sexual activity during COVID-19 quarantine. \nMoreover, the COVID-19 pandemic is having an impact on the sexual life of the population [20,21]. There is evidence showing a reduction and different changes in sexual activity due to the fear of how contagious COVID-19 is, as well as the consequences of the lockdown, such as loss of privacy and the incapacity of meeting sexual partners [22,23]. This reduction of sexual activity might lead to a decrement of the level of physical activity, which, in turn, could worsen mental health [24]. In this regard, physical activity was significantly associated with lower perceived anxiety and lower perceived worse mood in a large sample of Spanish confined adults [25]. Similar evidence has been found between depression and anxiety symptoms and general physical activity in a sample of adults aged 18 years and over, residing in UK and social-distancing (i.e., following UK government enforced restrictions that limited movement of people) due to COVID-19 [26].\nOther studies have shown that the COVID-19 pandemic had a wide range of psychological impact on the Spanish population, and this might affect the predisposition and the mood for having personal intercourse [27]. Different studies have shown that the uncertainty of the COVID-19 situation, as well as its development and consequences, have led to worse mental health outcomes such as anxious and depressive symptomology [27,28]. For that reason, the promotion of protective factors for mental health could lead to the development of tailored strategies focusing on physical activity and an active and healthy sexual life during the whole cycle of life, due to the potential relationship of these variables. Another implication, also highlighted in another study, should be the need to focus on enhancing sexual wellness by educating on and supporting sexual activities during the implementation of movement control measures [29].\nThe main strength of this study is that it is the first study reporting the association between mental health and sexual activity in a sample of quarantined adults. Another strength of the present study is the use of two well-known valid and reliable inventories to assess mental health: Beck Anxiety Inventory (BAI) and Beck Depression Inventory (BDI). However, the present findings must be interpreted in light of the study limitations. First, sexual activity was self-reported, potentially introducing self-reporting bias into the findings. Second, due to the method of sampling (convenience sampling), there is the possibility of a selection bias. Third, analyses were cross-sectional, and, thus, it was not possible to determine the direction of the association. Moreover, given the critical and unprecedented situation, we avoided asking too many questions, thus, important information, such as the number of days confined was not collected. Finally, more than 70% of the sample are women, thus, a selection bias regarding gender is plausible. Similarly, because the option of binary gender was not given to the participants, there is still a chance for some misclassification bias. ",
"In conclusion, in the present sample of confined Spanish adults, those who had more anxious and depressive symptomatology presented less sexual activity during the quarantine. To our knowledge, this is the first study on mental health and sexual activity in a sample of quarantined adults. In this regard, mental health outcomes and sexual activity seem to be associated, for that reason, future research must focus on inferring the direction of this association. "
] | [
"intro",
null,
"subjects",
null,
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusions"
] | [
"COVID-19",
"lockdown",
"sexual health",
"mental health",
"anxiety",
"depression"
] | 1. Introduction:
As in many other countries, the Spanish Government has been implementing different public health strategies in order to slow the spread and “flatten the curve” of the ongoing human-to-human transmission of the COVID-19. In this regard, after the statement of the outbreak of COVID-19 as a global pandemic by the World Health Organisation and due to the high number of diagnosed cases in Spain, from 15 March until 2 May 2020, a period of total quarantine was established in Spain [1].
The health crisis and the restrictions imposed prompted a drastic situation for everyone with radical changes in daily life [2,3]. Among the different collateral impacts due to the pandemic situation and the public health measures to combat COVID-19 infection, sexual behaviour is one of the main concerns and it is being studied internationally [4,5,6]. This is because sexuality is an important element of human life and, according to The World Health Organisation, sexual health is “a state of physical, emotional, mental and social well-being related to sexuality; not merely the absence of disease, dysfunction or infirmity”. Consequently, a frequent and trouble-free sex life is associated with a plethora of physical and mental health benefits [4].
First studies on sexual behaviour and COVID-19 pointed out that there has been a change in sexual habits; in this regard, the lockdown has affected the sexual activity between cohabitants due to the difficulty of finding a moment of intimacy. As for the stable relationships who were not living together, the lockdown has made the physical contact impossible. Finally, for those looking for occasional sexual relations, the lockdown has created a drastic barrier for meeting a sexual partner [5]. Studies have suggested some changes based on the relationship status and the availability for living together during the implementation of movement control measures during the COVID-19 pandemic [6]. In this regard, there is evidence that all these changes have supposed that the quality of sex and satisfaction has decreased significantly [5,6]. Furthermore, Spanish women have been identified as a target group for promoting healthy regular practices regarding sex during COVID-19 quarantine since they were observed to reduce sexual activity during that period [7].
Scientific evidence shows that sexual inactivity is significantly related to different mental health conditions [8]. Moreover, it has been found that sexual dysfunction might lead to interpersonal conflicts by deteriorating either self-esteem or partner relationships [9]. On the other hand, there is evidence that positive mental health outcomes for males and females are significantly related to successful sexual activity. In this regard, psychological well-being by improving mood, even in depressed and high-anxiety patients [10,11], is positively related to trouble-free sexual activity.
To our knowledge, there is no evidence explaining how mental health affected the sexual activity status of the Spanish population during the quarantine. Therefore, this study aims to explain the association between depression/anxiety and sexual activity during quarantine among this population.
2. Materials and Methods:
2.1. Study Design and Participants A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12].
Spanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13].
A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12].
Spanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13].
2.2. Key Measures 2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
2.3. Statistical Analyses The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.
The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.
2.1. Study Design and Participants:
A cross-sectional online survey was administered during the COVID-19 quarantine in Spain. This study followed the principles of the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Research in Humans of the University of Valencia (1 April 2020; register code 1278789). The data collection comprised one month (from 1 April 2020 to 1 May 2020). On 9 May 2020, the gradual opening in Spain commenced [12].
Spanish adults aged 18 years and over (i.e., the age range of those who completed the survey 18–74 years) that confirmed to be self-isolated due to the COVID-19 pandemic were eligible to participate. A convenience sample of participants were recruited through social media (e.g., Facebook, Twitter, Whatsapp). They were directed to a data encrypted website where they indicated their consent to participate after reading an information sheet, and confirmed that they were in a quarantine situation. Overall, a participation rate of 42% was reached. The data provided were anonymous and treated accordingly to Spanish law regarding general data protection. This manuscript was written in accordance with the STROBE Statement (Strengthening the Reporting of Observational Studies in Epidemiology) [13].
2.2. Key Measures:
2.2.1. Sexual Activity (Exposure) In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
2.2.2. Anxiety (Outcome) Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
2.2.3. Depression (Outcome) Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
2.2.4. Covariates Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
2.2.1. Sexual Activity (Exposure):
In the participant survey, sexual activity was defined as sexual intercourse, masturbation, petting, or fondling. Participants were asked, “On average, after self-isolating/social-distancing, how many times have you engaged in sexual activity weekly?”
2.2.2. Anxiety (Outcome):
Anxiety symptoms were assessed using the Spanish version of the Beck Anxiety Inventory (BAI) [14], which has shown high consistency (Cronbach alpha coefficient = 0.93) and moderate correlation (r = 0.32) with the Trait-Anger scale of the STAXI 2 [15] The BAI is composed of 21 items. Each item consists of a different anxiety symptom in which the participant scores how he/she felt in relation to that symptom during the last month, on a scale that varies from 0 (Not at all) to 3 (Severely, it bothered me a lot). The overall scores range from 0 to 63. Scores ranging from 0–7 indicate minimal/no anxiety symptoms; 8–15 indicate mild anxiety symptoms; 16–25 indicate moderate anxiety symptoms, and 26–63 indicate severe anxiety symptoms [14]. For this study, the group of severe anxiety was underrepresented with 4 participants, so they were excluded from the analysis.
2.2.3. Depression (Outcome):
Depression symptoms were assessed using the Spanish version of the Beck Depression Inventory (BDI) [16]. This has shown high consistency (Cronbach alpha coefficient = 0.83) [17]. The BDI is composed of 21 items. Each item consists of a series of four statements based on the severity of depression symptoms. The score of each item varies from 0 (minimum score) to 3 maximum score). The overall score of the instrument range from 0 to 63. Scores ranging from 0–13 indicate minimal/no depression; 14–19 indicate mild depressive symptoms; 20–28 indicate moderate depressive symptoms, and 29–63 indicate severe depressive symptoms [16,18].
2.2.4. Covariates:
Collected demographic data were conducted through single-item questions: “What is your gender?” and potential answers comprising “male” or “female”, “What is your Age?”, and potential answers including 10-year age bands, “What is your marital status?”, with potential answers including “single”, “divorced”, “separated”, “widowed” or “married/in a domestic partnership”, “What is your current status?”, and possibilities comprising “employed”, and “not employed”, “What is your average household annual income?”, and possible answers including “<€15,000”, “€15,000–<€25,000”, “€25,000–<€40,000”, “€40,000–<€60,000”, “≤€60,000”. Participants were also asked whether they were residing in the Iberian Peninsula or other Spanish regions outside the Iberian Peninsula (“What part of the country do you live in?”), and possible answers comprising “Peninsula”, “Balearic Islands”, “Canary Islands”, and “Ceuta or Melilla”. Measures of health status included whether respondents were a current consumer of alcohol (“Do you drink alcohol?”), and smoker (“Do you smoke?”), and possible answers for these two questions comprising “yes”, or “no”; “Have you ever been diagnosed by a health professional with: (tick all that apply)”: “hypertension”, “obesity”, “myocardial infarction”, “angina pectoris and other coronary diseases”, “other cardiac diseases”, “varicose veins of lower extremities”, “osteoarthritis”, “chronic neck pain”, “chronic low back pain”, “chronic allergy (excluding allergic asthma)”, “asthma (including allergic asthma)”, “chronic bronchitis”, “emphysema or chronic obstructive pulmonary disease (COPD)”, “type 1 diabetes”, “type 2 diabetes”, “diabetic retinopathy”, “peptic ulcer disease”, “cataract”, “urinary incontinence or urine control problems”, “hypercholesterolemia”, “chronic skin disease”, “chronic constipation”, “liver cirrhosis and other hepatic disorders”, “stroke”, “chronic migraine and other frequent chronic headaches”, “hemorrhoids”, “cancer”, “osteoporosis”, “thyroid disease”, “injury”, and “renal disease”, as well as the option to declare “other psychiatric conditions”. Moreover, participants were asked if they had experienced any physical symptom of COVID-19 during the confinement: “What guidelines are you following for self-isolation?”, and responses comprising the selection of one or more of the following symptoms: “high temperature”, “persistent cough”, “sore throat”, and “runny nose”. Finally, the participants were asked for the number of days they had been confined (“What day of self-isolation are you currently on?”).
2.3. Statistical Analyses:
The statistical analyses were performed with Stata v16.1. (StataCorp, TX, USA) Participants with and without regular sexual activity were compared regarding socioeconomic characteristics, health characteristics, and other specific characteristics concerning COVID-19 confinement using chi-squared tests for categorical variables and t-tests for continuous variables. The cut-off point for age was set at 45 years old since it is a turning point concerning mental health for Spanish men and women [19]. Estimations for effect sizes were conducted using phi coefficient (chi-squared tests with binary categorical variables), Cramer’s V (chi-squared tests with categorical variables with more than 2 categories), and Cohen’s d (t-tests with continuous variables). To check associations between weekly prevalence of sexual activity among different levels of both depression and anxiety, we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, physical conditions, psychiatric conditions, physical symptoms, and days of confinement). There were less than 3.5% of missing values for the variables used in this study, thus, complete case analysis was carried out. The level of statistical significance was set at p < 0.05.
3. Results:
A total of 305 Spanish confined adults completed the survey. Overall, 85 (27.9%) males and 220 females (72.1%) participated. The age distribution was: 18–44 years (n = 277), 45–74 years (n = 28). Significant differences between participants regarding marital status, employment, depression, anxiety, and sexual activity before the COVID-19 confinement were identified. The rest of the sample characteristics are shown in Table 1.
The association between the level of depression and sexual activity is displayed in Table 2. Higher depression level was associated with lower sexual activity in a dose-response fashion in the three implemented models. Participants with higher levels of depression were associated with significantly lower sexual activity in the fully adjusted model (OR: 0.09, 95% CI 0.01–0.61). Also, Table 3 shows the association between anxiety and sexual activity, in which mild anxiety level was associated with lower sexual activity (OR: 0.40, 95% CI 0.19–0.84; fully adjusted model).
4. Discussion:
The main objective of this study was to investigate the association between mental health and sexual activity during quarantine for adults in Spain. In the present sample, comprising 305 Spanish confined adults, we found that both higher anxiety and depressive symptoms had a negative dose-response on the level of sexual activity.
The finding that sexual activity is associated with worse mental health outcomes is important and supports and adds to previous evidence [10,11]. Indeed, the present study found that people who have higher levels of depression have less sexual activity during the quarantine. Additionally, we found that the participants who have higher levels of anxiety presented a significantly lower chance to have sexual activity. Taken together, the present findings suggest that better mental health status, having less anxious and depressive symptomatology, is associated with having a more active sexual activity during COVID-19 quarantine.
Moreover, the COVID-19 pandemic is having an impact on the sexual life of the population [20,21]. There is evidence showing a reduction and different changes in sexual activity due to the fear of how contagious COVID-19 is, as well as the consequences of the lockdown, such as loss of privacy and the incapacity of meeting sexual partners [22,23]. This reduction of sexual activity might lead to a decrement of the level of physical activity, which, in turn, could worsen mental health [24]. In this regard, physical activity was significantly associated with lower perceived anxiety and lower perceived worse mood in a large sample of Spanish confined adults [25]. Similar evidence has been found between depression and anxiety symptoms and general physical activity in a sample of adults aged 18 years and over, residing in UK and social-distancing (i.e., following UK government enforced restrictions that limited movement of people) due to COVID-19 [26].
Other studies have shown that the COVID-19 pandemic had a wide range of psychological impact on the Spanish population, and this might affect the predisposition and the mood for having personal intercourse [27]. Different studies have shown that the uncertainty of the COVID-19 situation, as well as its development and consequences, have led to worse mental health outcomes such as anxious and depressive symptomology [27,28]. For that reason, the promotion of protective factors for mental health could lead to the development of tailored strategies focusing on physical activity and an active and healthy sexual life during the whole cycle of life, due to the potential relationship of these variables. Another implication, also highlighted in another study, should be the need to focus on enhancing sexual wellness by educating on and supporting sexual activities during the implementation of movement control measures [29].
The main strength of this study is that it is the first study reporting the association between mental health and sexual activity in a sample of quarantined adults. Another strength of the present study is the use of two well-known valid and reliable inventories to assess mental health: Beck Anxiety Inventory (BAI) and Beck Depression Inventory (BDI). However, the present findings must be interpreted in light of the study limitations. First, sexual activity was self-reported, potentially introducing self-reporting bias into the findings. Second, due to the method of sampling (convenience sampling), there is the possibility of a selection bias. Third, analyses were cross-sectional, and, thus, it was not possible to determine the direction of the association. Moreover, given the critical and unprecedented situation, we avoided asking too many questions, thus, important information, such as the number of days confined was not collected. Finally, more than 70% of the sample are women, thus, a selection bias regarding gender is plausible. Similarly, because the option of binary gender was not given to the participants, there is still a chance for some misclassification bias.
5. Conclusions:
In conclusion, in the present sample of confined Spanish adults, those who had more anxious and depressive symptomatology presented less sexual activity during the quarantine. To our knowledge, this is the first study on mental health and sexual activity in a sample of quarantined adults. In this regard, mental health outcomes and sexual activity seem to be associated, for that reason, future research must focus on inferring the direction of this association.
| Background:
Evidence on sexual behaviour and COVID-19 shows a change in sexual habits; however, there is no research on the association between mental health and sexual activity.
Methods:
A sample of 305 adults filled out an online questionnaire. Sexual activity was assessed with one question. Anxiety and depression symptoms were assessed using the Beck Anxiety Inventory (BAI) and the Beck Depression Inventory (BDI), respectively. To check associations between levels of both anxiety and depressive symptoms (exposure) and weekly prevalence of sexual activity (outcome), we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, chronic physical conditions, chronic psychiatric conditions, physical symptoms, and days of confinement).
Results:
Higher depression level was associated with lower weekly sexual activity in a dose-response fashion in the three implemented models. Participants with higher levels of depression were associated with significantly lower sexual activity in the fully adjusted model (OR: 0.09, 95% CI 0.01-0.61). Mild anxiety-level participants consistently presented significantly lower ORs for lower sexual activity than their minimal-anxiety category counterparts. Particularly, the fully adjusted model showed the lower values (OR: 0.40, 95% CI 0.19-0.84).
Conclusions:
The results of this study support existing evidence stressing the association between mental health and sexual activity in quarantined adults. | 1. Introduction:
As in many other countries, the Spanish Government has been implementing different public health strategies in order to slow the spread and “flatten the curve” of the ongoing human-to-human transmission of the COVID-19. In this regard, after the statement of the outbreak of COVID-19 as a global pandemic by the World Health Organisation and due to the high number of diagnosed cases in Spain, from 15 March until 2 May 2020, a period of total quarantine was established in Spain [1].
The health crisis and the restrictions imposed prompted a drastic situation for everyone with radical changes in daily life [2,3]. Among the different collateral impacts due to the pandemic situation and the public health measures to combat COVID-19 infection, sexual behaviour is one of the main concerns and it is being studied internationally [4,5,6]. This is because sexuality is an important element of human life and, according to The World Health Organisation, sexual health is “a state of physical, emotional, mental and social well-being related to sexuality; not merely the absence of disease, dysfunction or infirmity”. Consequently, a frequent and trouble-free sex life is associated with a plethora of physical and mental health benefits [4].
First studies on sexual behaviour and COVID-19 pointed out that there has been a change in sexual habits; in this regard, the lockdown has affected the sexual activity between cohabitants due to the difficulty of finding a moment of intimacy. As for the stable relationships who were not living together, the lockdown has made the physical contact impossible. Finally, for those looking for occasional sexual relations, the lockdown has created a drastic barrier for meeting a sexual partner [5]. Studies have suggested some changes based on the relationship status and the availability for living together during the implementation of movement control measures during the COVID-19 pandemic [6]. In this regard, there is evidence that all these changes have supposed that the quality of sex and satisfaction has decreased significantly [5,6]. Furthermore, Spanish women have been identified as a target group for promoting healthy regular practices regarding sex during COVID-19 quarantine since they were observed to reduce sexual activity during that period [7].
Scientific evidence shows that sexual inactivity is significantly related to different mental health conditions [8]. Moreover, it has been found that sexual dysfunction might lead to interpersonal conflicts by deteriorating either self-esteem or partner relationships [9]. On the other hand, there is evidence that positive mental health outcomes for males and females are significantly related to successful sexual activity. In this regard, psychological well-being by improving mood, even in depressed and high-anxiety patients [10,11], is positively related to trouble-free sexual activity.
To our knowledge, there is no evidence explaining how mental health affected the sexual activity status of the Spanish population during the quarantine. Therefore, this study aims to explain the association between depression/anxiety and sexual activity during quarantine among this population.
5. Conclusions:
In conclusion, in the present sample of confined Spanish adults, those who had more anxious and depressive symptomatology presented less sexual activity during the quarantine. To our knowledge, this is the first study on mental health and sexual activity in a sample of quarantined adults. In this regard, mental health outcomes and sexual activity seem to be associated, for that reason, future research must focus on inferring the direction of this association. | Background:
Evidence on sexual behaviour and COVID-19 shows a change in sexual habits; however, there is no research on the association between mental health and sexual activity.
Methods:
A sample of 305 adults filled out an online questionnaire. Sexual activity was assessed with one question. Anxiety and depression symptoms were assessed using the Beck Anxiety Inventory (BAI) and the Beck Depression Inventory (BDI), respectively. To check associations between levels of both anxiety and depressive symptoms (exposure) and weekly prevalence of sexual activity (outcome), we conducted multiple logistic regression adjusted for control variables (marital status, employment, average household annual income, place of living, pre-COVID-19 sexual activity, current smoking, current alcohol consumption, chronic physical conditions, chronic psychiatric conditions, physical symptoms, and days of confinement).
Results:
Higher depression level was associated with lower weekly sexual activity in a dose-response fashion in the three implemented models. Participants with higher levels of depression were associated with significantly lower sexual activity in the fully adjusted model (OR: 0.09, 95% CI 0.01-0.61). Mild anxiety-level participants consistently presented significantly lower ORs for lower sexual activity than their minimal-anxiety category counterparts. Particularly, the fully adjusted model showed the lower values (OR: 0.40, 95% CI 0.19-0.84).
Conclusions:
The results of this study support existing evidence stressing the association between mental health and sexual activity in quarantined adults. | 9,700 | 285 | [
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|||||
Tablet, web-based, or paper questionnaires for measuring anxiety in patients suspected of breast cancer: patients' preferences and quality of collected data. | 25364951 | Electronic applications are increasingly being used in hospitals for numerous purposes. | BACKGROUND | Between October 2012 and June 2013, 136 patients participated in a study on diagnosis-induced stress and anxiety. Patients were asked to fill out questionnaires at six different moments during the diagnostic phase. They were given the opportunity to fill out the questionnaires on paper or electronically (a combination of tablet and Web-based questionnaires). Demographic characteristics and completeness of returned data were compared between groups. | METHODS | Nearly two-thirds of patients (88/136, 64.7%) chose to fill out the questionnaires on paper, and just over a third (48/136, 35.3%) preferred the electronic option. Patients choosing electronic questionnaires were significantly younger (mean 47.3 years vs mean 53.5 in the paper group, P=.01) and higher educated (P=.004). There was significantly more missing information (ie, at least one question not answered) in the paper group during the diagnostic day compared to the electronic group (using a tablet) (28/88 vs 1/48, P<.001). However, in the week after the diagnostic day, missing information was significantly higher in the electronic group (Web-based questionnaires) compared to the paper group (41/48 vs 38/88, P<.001). | RESULTS | Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires resulted in a better response than Web-based questionnaires. | CONCLUSIONS | [
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] | 4259914 | Introduction | With the evolution of modern technology, electronic applications are increasingly being used in hospitals. Web-based applications and touchscreen devices are finding their way into hospitals for numerous purposes. These electronic applications can be useful for research purposes, for collecting patient-reported outcomes, and questionnaires [1-3]. Some of the most important advantages of electronic over paper questionnaires include easy usage and immediate electronic storage of results. The use of electronic applications has been evaluated for informed consenting procedures, assessing quality of life, medical education, interventions, diagnostics, and filling out questionnaires [1,3-11].
Obtaining high response rates without missing information is important for research purposes, as non-responders can bias study results [12]. Response rates have been found to be lower using electronic questionnaires compared to paper questionnaires [13-15]. In order to potentially improve response rates, specific patient subgroups with a preference for electronic questionnaires could be identified. For example, elderly patients may not be as experienced with electronic applications. Aiello et al compared the use of a tablet to paper questionnaires in a mammography clinic. They found that older women (>60 years) had a slightly harder time learning to use the tablet compared to younger patients, but preference towards the tablet was similar in both groups [2].
The aim of our study was to assess differences in demographic characteristics of patients choosing paper versus electronic questionnaires and to evaluate data quality and completeness of data of both approaches. | Methods | Study Context This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.
Between mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.
This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.
Between mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.
Outcome Measures Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.
Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.
Methods for Data Analysis Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.
Overview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.
Screenshot of the electronic questionnaire (measuring moment 2).
Screenshot of the paper questionnaire (measuring moment 2).
Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.
Overview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.
Screenshot of the electronic questionnaire (measuring moment 2).
Screenshot of the paper questionnaire (measuring moment 2). | Results | Demographic Data Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.
The mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).
Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.
The mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).
Outcome Data: Missing Information There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).
In the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.
Demographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).
a
P values are based on valid proportions.
bCalculated by chi-square test.
cCalculated by independent samples t test.
dLevel of education is based on the Dutch educational system
eLow-moderate education includes primary education/low pre-vocational/secondary general education.
fModerate-high education includes secondary vocational/higher general and pre-university education.
gHigh education includes higher vocational education/university.
Differences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).
aIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.
bIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.
There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).
In the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.
Demographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).
a
P values are based on valid proportions.
bCalculated by chi-square test.
cCalculated by independent samples t test.
dLevel of education is based on the Dutch educational system
eLow-moderate education includes primary education/low pre-vocational/secondary general education.
fModerate-high education includes secondary vocational/higher general and pre-university education.
gHigh education includes higher vocational education/university.
Differences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).
aIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.
bIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6. | Conclusions | Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires. | [
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"Conclusions"
] | [
"This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.\nBetween mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.",
"Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.",
"Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.\nOverview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.\nScreenshot of the electronic questionnaire (measuring moment 2).\nScreenshot of the paper questionnaire (measuring moment 2).",
"Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.\nThe mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).",
"There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).\nIn the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.\nDemographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).\n\na\nP values are based on valid proportions.\n\nbCalculated by chi-square test.\n\ncCalculated by independent samples t test.\n\ndLevel of education is based on the Dutch educational system\n\neLow-moderate education includes primary education/low pre-vocational/secondary general education.\n\nfModerate-high education includes secondary vocational/higher general and pre-university education.\n\ngHigh education includes higher vocational education/university.\nDifferences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).\n\naIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.\n\nbIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.",
"The use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.\nA major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.\nPossible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].\nCompleteness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.",
"A possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).",
"Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires."
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Study Context",
"Outcome Measures",
"Methods for Data Analysis",
"Results",
"Demographic Data",
"Outcome Data: Missing Information",
"Discussion",
"Principal Findings",
"Limitations",
"Conclusions"
] | [
"With the evolution of modern technology, electronic applications are increasingly being used in hospitals. Web-based applications and touchscreen devices are finding their way into hospitals for numerous purposes. These electronic applications can be useful for research purposes, for collecting patient-reported outcomes, and questionnaires [1-3]. Some of the most important advantages of electronic over paper questionnaires include easy usage and immediate electronic storage of results. The use of electronic applications has been evaluated for informed consenting procedures, assessing quality of life, medical education, interventions, diagnostics, and filling out questionnaires [1,3-11].\nObtaining high response rates without missing information is important for research purposes, as non-responders can bias study results [12]. Response rates have been found to be lower using electronic questionnaires compared to paper questionnaires [13-15]. In order to potentially improve response rates, specific patient subgroups with a preference for electronic questionnaires could be identified. For example, elderly patients may not be as experienced with electronic applications. Aiello et al compared the use of a tablet to paper questionnaires in a mammography clinic. They found that older women (>60 years) had a slightly harder time learning to use the tablet compared to younger patients, but preference towards the tablet was similar in both groups [2].\nThe aim of our study was to assess differences in demographic characteristics of patients choosing paper versus electronic questionnaires and to evaluate data quality and completeness of data of both approaches.",
" Study Context This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.\nBetween mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.\nThis study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.\nBetween mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.\n Outcome Measures Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.\nOutcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.\n Methods for Data Analysis Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.\nOverview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.\nScreenshot of the electronic questionnaire (measuring moment 2).\nScreenshot of the paper questionnaire (measuring moment 2).\nDemographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.\nOverview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.\nScreenshot of the electronic questionnaire (measuring moment 2).\nScreenshot of the paper questionnaire (measuring moment 2).",
"This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.\nBetween mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.",
"Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.",
"Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.\nOverview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.\nScreenshot of the electronic questionnaire (measuring moment 2).\nScreenshot of the paper questionnaire (measuring moment 2).",
" Demographic Data Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.\nThe mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).\nOf 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.\nThe mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).\n Outcome Data: Missing Information There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).\nIn the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.\nDemographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).\n\na\nP values are based on valid proportions.\n\nbCalculated by chi-square test.\n\ncCalculated by independent samples t test.\n\ndLevel of education is based on the Dutch educational system\n\neLow-moderate education includes primary education/low pre-vocational/secondary general education.\n\nfModerate-high education includes secondary vocational/higher general and pre-university education.\n\ngHigh education includes higher vocational education/university.\nDifferences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).\n\naIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.\n\nbIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.\nThere was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).\nIn the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.\nDemographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).\n\na\nP values are based on valid proportions.\n\nbCalculated by chi-square test.\n\ncCalculated by independent samples t test.\n\ndLevel of education is based on the Dutch educational system\n\neLow-moderate education includes primary education/low pre-vocational/secondary general education.\n\nfModerate-high education includes secondary vocational/higher general and pre-university education.\n\ngHigh education includes higher vocational education/university.\nDifferences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).\n\naIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.\n\nbIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.",
"Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.\nThe mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).",
"There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).\nIn the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.\nDemographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).\n\na\nP values are based on valid proportions.\n\nbCalculated by chi-square test.\n\ncCalculated by independent samples t test.\n\ndLevel of education is based on the Dutch educational system\n\neLow-moderate education includes primary education/low pre-vocational/secondary general education.\n\nfModerate-high education includes secondary vocational/higher general and pre-university education.\n\ngHigh education includes higher vocational education/university.\nDifferences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).\n\naIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.\n\nbIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.",
" Principal Findings The use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.\nA major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.\nPossible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].\nCompleteness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.\nThe use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.\nA major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.\nPossible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].\nCompleteness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.\n Limitations A possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).\nA possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).\n Conclusions Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires.\nYounger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires.",
"The use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.\nA major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.\nPossible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].\nCompleteness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.",
"A possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).",
"Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires."
] | [
"introduction",
"methods",
null,
null,
null,
"results",
null,
null,
"discussion",
null,
null,
null
] | [
"breast cancer",
"electronic questionnaires",
"paper questionnaires",
"quality of collected data"
] | Introduction:
With the evolution of modern technology, electronic applications are increasingly being used in hospitals. Web-based applications and touchscreen devices are finding their way into hospitals for numerous purposes. These electronic applications can be useful for research purposes, for collecting patient-reported outcomes, and questionnaires [1-3]. Some of the most important advantages of electronic over paper questionnaires include easy usage and immediate electronic storage of results. The use of electronic applications has been evaluated for informed consenting procedures, assessing quality of life, medical education, interventions, diagnostics, and filling out questionnaires [1,3-11].
Obtaining high response rates without missing information is important for research purposes, as non-responders can bias study results [12]. Response rates have been found to be lower using electronic questionnaires compared to paper questionnaires [13-15]. In order to potentially improve response rates, specific patient subgroups with a preference for electronic questionnaires could be identified. For example, elderly patients may not be as experienced with electronic applications. Aiello et al compared the use of a tablet to paper questionnaires in a mammography clinic. They found that older women (>60 years) had a slightly harder time learning to use the tablet compared to younger patients, but preference towards the tablet was similar in both groups [2].
The aim of our study was to assess differences in demographic characteristics of patients choosing paper versus electronic questionnaires and to evaluate data quality and completeness of data of both approaches.
Methods:
Study Context This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.
Between mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.
This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.
Between mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.
Outcome Measures Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.
Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.
Methods for Data Analysis Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.
Overview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.
Screenshot of the electronic questionnaire (measuring moment 2).
Screenshot of the paper questionnaire (measuring moment 2).
Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.
Overview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.
Screenshot of the electronic questionnaire (measuring moment 2).
Screenshot of the paper questionnaire (measuring moment 2).
Study Context:
This study was performed in the University Medical Center Utrecht, the Netherlands (approximate caseload of 180 newly diagnosed breast cancer patients per year). In 2011, same-day diagnosis for breast cancer was introduced with the aim to provide a definitive diagnosis within one day in over 80% of patients. Reducing the time of uncertainty about a diagnosis could potentially reduce anxiety and stress. All patients suspected of breast cancer visited the outpatient breast clinic and underwent physical examination, diagnostic imaging (mammography and ultrasound) with a histological biopsy if indicated, and received a final diagnosis at the end of the day after a multidisciplinary meeting.
Between mid-October 2012 and June 2013, all patients referred to the same-day diagnosis out-patient breast clinic were eligible to participate in the study. Approval for this study was obtained from the local ethics committee, and all patients signed written informed consent. All patients were asked to fill out the 6-item State Trait Anxiety Inventory (STAI) [16,17] questionnaire at six different time points (measuring moments) during the diagnostic phase to evaluate levels of stress and anxiety (Figure 1). Patients were given the opportunity to fill out the questionnaires electronically (Figure 2) or on paper (Figure 3). Preference towards paper or electronic questionnaires was measured at baseline. The paper questionnaires were returned by mail. In the electronic scenario, the first three questionnaires (administered on the day of diagnosis in the hospital) were offered by means of tablets (iPad). For the last three electronic questionnaires that were to be filled out at home, we used Web-based (hypertext markup language [HTML]) questionnaires. An email with login information to the questionnaires was sent to participants by email on the diagnostic day. The STAI questionnaire was displayed on one page, and all six questions needed to be answered before the form could be submitted. If a question was left blank, an automated message appeared saying that all questions needed to be answered. Patients were not able to look back at previously completed questionnaires. The tablets were also used for providing information and entertainment. An information app was built to provide information on the diagnostic process, diagnostic procedures, treatment team, and routing during the diagnostic day. Several forms of entertainment were available on the tablet, including digital newspapers, magazines, games, and music. The paper questionnaires were returned by mail in a pre-stamped return envelope.
Outcome Measures:
Outcome measures included differences in demographic characteristics between patients choosing paper or electronic questionnaires and data quality, focusing on age, reason for referral, breast cancer history, level of education, and baseline anxiety. Data quality was assessed by focusing on missing information, defined as a questionnaire containing at least one unanswered question. To assess if a breast cancer diagnosis affected the quality of data, subgroup analysis including only patients with a benign diagnosis was performed.
Methods for Data Analysis:
Demographics, history of breast disease, and diagnostic findings were described as proportions and means with standard deviation. Differences in demographic characteristics, reported anxiety score, and completeness of reported data between the electronic and the paper group were compared by means of chi-square test and independent samples t test, where appropriate. Significant differences were defined as P values of .05 or less. All statistical analyses were performed using SPSS version 20.0.
Overview of the six measuring moments during the diagnostic phase to evaluate level of stress and anxiety.
Screenshot of the electronic questionnaire (measuring moment 2).
Screenshot of the paper questionnaire (measuring moment 2).
Results:
Demographic Data Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.
The mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).
Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.
The mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).
Outcome Data: Missing Information There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).
In the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.
Demographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).
a
P values are based on valid proportions.
bCalculated by chi-square test.
cCalculated by independent samples t test.
dLevel of education is based on the Dutch educational system
eLow-moderate education includes primary education/low pre-vocational/secondary general education.
fModerate-high education includes secondary vocational/higher general and pre-university education.
gHigh education includes higher vocational education/university.
Differences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).
aIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.
bIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.
There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).
In the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.
Demographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).
a
P values are based on valid proportions.
bCalculated by chi-square test.
cCalculated by independent samples t test.
dLevel of education is based on the Dutch educational system
eLow-moderate education includes primary education/low pre-vocational/secondary general education.
fModerate-high education includes secondary vocational/higher general and pre-university education.
gHigh education includes higher vocational education/university.
Differences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).
aIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.
bIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.
Demographic Data:
Of 321 patients referred to our out-patient breast clinic, 136 patients (42.4%) agreed to participate in the study. All patients were offered the choice of paper or electronic questionnaires.
The mean age was 51.3 years (range 18-85 years) and 35.3% (48/136) patients chose to fill out the questionnaires electronically (Table 1). Reason for referral, family history of breast cancer, and breast-related medical history were similar in both groups. Baseline anxiety scores (as measured by the STAI) did not differ between the groups (46.4 in the paper group versus 43.8 in the electronic group, P=.30). Diagnostic imaging findings and proportion of patients undergoing biopsy were similar in both groups. Patients choosing to fill out questionnaires electronically were significantly younger compared to those opting for paper questionnaires (47.3 years vs 53.5, respectively; P=.01) and had a higher level of education (P=.004).
Outcome Data: Missing Information:
There was significantly more missing information (ie, questionnaires containing at least one unanswered question) in the paper group during the diagnostic day (measuring moments 1-3) compared to the electronic group (28/88 vs 1/48, P<.001) (Table 2). In the paper group, this included two patients who did not fill out one or two questions (instead of complete questionnaires not filled out).
In the week after the diagnostic day (measuring moments 4-6), missing information was significantly more prevalent in the electronic group (41/48, 85%) compared to the paper group (38/88, 43%) (P<.001). This included 7 patients in the paper group who left one or two questions unanswered. These differences persisted in subgroup analysis including only patients with a benign diagnosis.
Demographic characteristics of patients undergoing 1-day diagnosis for suspected breast cancer, comparing patients choosing paper questionnaires (n=88) to those choosing electronic questionnaires (n=48).
a
P values are based on valid proportions.
bCalculated by chi-square test.
cCalculated by independent samples t test.
dLevel of education is based on the Dutch educational system
eLow-moderate education includes primary education/low pre-vocational/secondary general education.
fModerate-high education includes secondary vocational/higher general and pre-university education.
gHigh education includes higher vocational education/university.
Differences in proportion of patients with incompletely filled out questionnaires between patients opting for paper questionnaires (n=88) and patients choosing electronic questionnaires (n=48).
aIncludes all patients with at least 1 incomplete questionnaire in measuring moment 1, 2, or 3.
bIncludes all patients with at least 1 incomplete questionnaire in measuring moment 4, 5, or 6.
Discussion:
Principal Findings The use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.
A major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.
Possible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].
Completeness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.
The use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.
A major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.
Possible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].
Completeness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.
Limitations A possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).
A possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).
Conclusions Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires.
Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires.
Principal Findings:
The use of tablets and Web-based questionnaires for collection of patient-reported data has many potential advantages over the use of paper questionnaires. Still, the present study shows that a majority of patients preferred paper over electronic questionnaires. Younger patients and those with a higher level of education were more likely to opt for electronic questionnaires. When using tablets (during the diagnostic day in the hospital), more complete information was collected compared to using paper questionnaires. These data suggests that tablets are superior to paper questionnaires. However, the use of Web-based questionnaires resulted in less complete data collection than paper questionnaires. This might be due to the study design where patients could fill out the electronic questionnaires only on a specific day.
A major advantage of filling out electronic questionnaires is that information is immediately saved. Other advantages of the use of tablets include the possibility of automatically reminding the patient to fill out the questionnaires, and providing information and entertainment. We did not electronically remind patients by email to fill out the questionnaires. Considering the high percentage of incompletely filled out Web-based questionnaires (85%), we would definitively incorporate this in a future study. We did use an automated message when not all questions were answered. This resulted in completely filled out questionnaires in the electronic group, which could possibly lead to more complete data.
Possible drawbacks of using Web-based questionnaires are high non-response rates, impaired reliability and validity, and safety or confidentiality issues [18]. Drawbacks of tablets are the need for upgrades, wireless network unreliability, hardware theft [19], and costs. Fritz et al performed a cost-effectiveness analysis comparing the costs of electronic questionnaires offered on a tablet with paper questionnaires. They found the break-even point to be at 1737 paper sheets per year [1].
Completeness of data collection was very high in the tablet group, with only 1 of the 48 patients not filling out all questions at the first three measuring moments. Missing information was highest in the Web-based group, where many patients (41/48) did not fill out all questions at the last three measuring moments. One likely reason for the high rate of missing information in this group was that patients could fill out the questionnaires only on the correct day (ie, exactly 1, 3, or 7 days after the patient’s visit). Our aim was to measure patients’ anxiety at specific moments in time, and we limited the possibility of filling out the questionnaires to the correct day only. Patients in the paper group, however, were able to fill out the questionnaires at any given time. This led to more missing information in the Web-based group, and results on missing information need to be interpreted with this information in mind. However, limiting patients to one specific moment to fill out the questionnaires might lead to more accurate measurements of patients’ anxiety at that specific moment. For higher response in the Web-based group, automated email reminders could be useful.
Limitations:
A possible limitation of this study was that we included only breast cancer patients and consequently, 98% were female. These results are therefore not generalizable to other populations. There could be reduced missing data in the electronic group when other groups are included in a similar study (eg, men, young adults).
Conclusions:
Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires.
| Background:
Electronic applications are increasingly being used in hospitals for numerous purposes.
Methods:
Between October 2012 and June 2013, 136 patients participated in a study on diagnosis-induced stress and anxiety. Patients were asked to fill out questionnaires at six different moments during the diagnostic phase. They were given the opportunity to fill out the questionnaires on paper or electronically (a combination of tablet and Web-based questionnaires). Demographic characteristics and completeness of returned data were compared between groups.
Results:
Nearly two-thirds of patients (88/136, 64.7%) chose to fill out the questionnaires on paper, and just over a third (48/136, 35.3%) preferred the electronic option. Patients choosing electronic questionnaires were significantly younger (mean 47.3 years vs mean 53.5 in the paper group, P=.01) and higher educated (P=.004). There was significantly more missing information (ie, at least one question not answered) in the paper group during the diagnostic day compared to the electronic group (using a tablet) (28/88 vs 1/48, P<.001). However, in the week after the diagnostic day, missing information was significantly higher in the electronic group (Web-based questionnaires) compared to the paper group (41/48 vs 38/88, P<.001).
Conclusions:
Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires resulted in a better response than Web-based questionnaires. | Introduction:
With the evolution of modern technology, electronic applications are increasingly being used in hospitals. Web-based applications and touchscreen devices are finding their way into hospitals for numerous purposes. These electronic applications can be useful for research purposes, for collecting patient-reported outcomes, and questionnaires [1-3]. Some of the most important advantages of electronic over paper questionnaires include easy usage and immediate electronic storage of results. The use of electronic applications has been evaluated for informed consenting procedures, assessing quality of life, medical education, interventions, diagnostics, and filling out questionnaires [1,3-11].
Obtaining high response rates without missing information is important for research purposes, as non-responders can bias study results [12]. Response rates have been found to be lower using electronic questionnaires compared to paper questionnaires [13-15]. In order to potentially improve response rates, specific patient subgroups with a preference for electronic questionnaires could be identified. For example, elderly patients may not be as experienced with electronic applications. Aiello et al compared the use of a tablet to paper questionnaires in a mammography clinic. They found that older women (>60 years) had a slightly harder time learning to use the tablet compared to younger patients, but preference towards the tablet was similar in both groups [2].
The aim of our study was to assess differences in demographic characteristics of patients choosing paper versus electronic questionnaires and to evaluate data quality and completeness of data of both approaches.
Conclusions:
Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires result in a better response compared to Web-based questionnaires. | Background:
Electronic applications are increasingly being used in hospitals for numerous purposes.
Methods:
Between October 2012 and June 2013, 136 patients participated in a study on diagnosis-induced stress and anxiety. Patients were asked to fill out questionnaires at six different moments during the diagnostic phase. They were given the opportunity to fill out the questionnaires on paper or electronically (a combination of tablet and Web-based questionnaires). Demographic characteristics and completeness of returned data were compared between groups.
Results:
Nearly two-thirds of patients (88/136, 64.7%) chose to fill out the questionnaires on paper, and just over a third (48/136, 35.3%) preferred the electronic option. Patients choosing electronic questionnaires were significantly younger (mean 47.3 years vs mean 53.5 in the paper group, P=.01) and higher educated (P=.004). There was significantly more missing information (ie, at least one question not answered) in the paper group during the diagnostic day compared to the electronic group (using a tablet) (28/88 vs 1/48, P<.001). However, in the week after the diagnostic day, missing information was significantly higher in the electronic group (Web-based questionnaires) compared to the paper group (41/48 vs 38/88, P<.001).
Conclusions:
Younger patients and patients with a higher level of education have a preference towards filling out questionnaires electronically. In the hospital, a tablet is an excellent medium for patients to fill out questionnaires with very little missing information. However, for filling out questionnaires at home, paper questionnaires resulted in a better response than Web-based questionnaires. | 6,066 | 310 | [
467,
84,
124,
177,
338,
580,
60,
64
] | 12 | [
"questionnaires",
"patients",
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"electronic",
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] | [
"electronic questionnaires younger",
"questionnaires electronically significantly",
"choosing electronic questionnaires",
"patients experienced electronic",
"questionnaires electronically hospital"
] | [CONTENT] breast cancer | electronic questionnaires | paper questionnaires | quality of collected data [SUMMARY] | [CONTENT] breast cancer | electronic questionnaires | paper questionnaires | quality of collected data [SUMMARY] | [CONTENT] breast cancer | electronic questionnaires | paper questionnaires | quality of collected data [SUMMARY] | [CONTENT] breast cancer | electronic questionnaires | paper questionnaires | quality of collected data [SUMMARY] | [CONTENT] breast cancer | electronic questionnaires | paper questionnaires | quality of collected data [SUMMARY] | [CONTENT] breast cancer | electronic questionnaires | paper questionnaires | quality of collected data [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Anxiety Disorders | Breast Neoplasms | Data Collection | Female | Humans | Internet | Microcomputers | Middle Aged | Patient Preference | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Anxiety Disorders | Breast Neoplasms | Data Collection | Female | Humans | Internet | Microcomputers | Middle Aged | Patient Preference | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Anxiety Disorders | Breast Neoplasms | Data Collection | Female | Humans | Internet | Microcomputers | Middle Aged | Patient Preference | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Anxiety Disorders | Breast Neoplasms | Data Collection | Female | Humans | Internet | Microcomputers | Middle Aged | Patient Preference | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Anxiety Disorders | Breast Neoplasms | Data Collection | Female | Humans | Internet | Microcomputers | Middle Aged | Patient Preference | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Anxiety Disorders | Breast Neoplasms | Data Collection | Female | Humans | Internet | Microcomputers | Middle Aged | Patient Preference | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] electronic questionnaires younger | questionnaires electronically significantly | choosing electronic questionnaires | patients experienced electronic | questionnaires electronically hospital [SUMMARY] | [CONTENT] electronic questionnaires younger | questionnaires electronically significantly | choosing electronic questionnaires | patients experienced electronic | questionnaires electronically hospital [SUMMARY] | [CONTENT] electronic questionnaires younger | questionnaires electronically significantly | choosing electronic questionnaires | patients experienced electronic | questionnaires electronically hospital [SUMMARY] | [CONTENT] electronic questionnaires younger | questionnaires electronically significantly | choosing electronic questionnaires | patients experienced electronic | questionnaires electronically hospital [SUMMARY] | [CONTENT] electronic questionnaires younger | questionnaires electronically significantly | choosing electronic questionnaires | patients experienced electronic | questionnaires electronically hospital [SUMMARY] | [CONTENT] electronic questionnaires younger | questionnaires electronically significantly | choosing electronic questionnaires | patients experienced electronic | questionnaires electronically hospital [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | fill | breast | data [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | fill | breast | data [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | fill | breast | data [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | fill | breast | data [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | fill | breast | data [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | fill | breast | data [SUMMARY] | [CONTENT] applications | electronic applications | electronic | questionnaires | purposes | response rates | rates | use | response | research [SUMMARY] | [CONTENT] diagnosis | questionnaires | day | diagnostic | patients | breast | anxiety | questionnaire | paper | figure [SUMMARY] | [CONTENT] patients | questionnaires | education | group | 88 | paper | 48 | paper group | vocational | includes [SUMMARY] | [CONTENT] questionnaires | filling | filling questionnaires | patients | electronically hospital | higher level education preference | education preference filling questionnaires | education preference filling | education preference | preference filling questionnaires electronically [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | breast | data | diagnosis [SUMMARY] | [CONTENT] questionnaires | patients | paper | electronic | group | information | day | breast | data | diagnosis [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] Between October 2012 and June 2013 | 136 ||| six ||| ||| [SUMMARY] | [CONTENT] Nearly two-thirds | 88/136 | 64.7% | just over a third | 48/136 | 35.3% ||| 47.3 years | 53.5 | P=.01 ||| at least one | 28/88 | 1/48 ||| the week | 41/48 | 38/88 [SUMMARY] | [CONTENT] ||| ||| [SUMMARY] | [CONTENT] ||| Between October 2012 and June 2013 | 136 ||| six ||| ||| ||| Nearly two-thirds | 88/136 | 64.7% | just over a third | 48/136 | 35.3% ||| 47.3 years | 53.5 | P=.01 ||| at least one | 28/88 | 1/48 ||| the week | 41/48 | 38/88 ||| ||| ||| [SUMMARY] | [CONTENT] ||| Between October 2012 and June 2013 | 136 ||| six ||| ||| ||| Nearly two-thirds | 88/136 | 64.7% | just over a third | 48/136 | 35.3% ||| 47.3 years | 53.5 | P=.01 ||| at least one | 28/88 | 1/48 ||| the week | 41/48 | 38/88 ||| ||| ||| [SUMMARY] |
||||||
Actinic lesions in fishermen's lower lip: clinical, cytopathological and histopathologic analysis. | 20454492 | Actinic cheilitis (AC) is considered to be a pre-malignant lesion or an incipient and superficial form of lip squamous cell carcinoma. It is commonly found in individuals whose occupational activities are related to chronic sun exposure and the definitive diagnosis is performed with biopsy. Although Exfoliative cytology has been used as a screening procedure to evaluate cancer of the oral cavity no studies have proposed the use of exfoliative cytologic analysis to evaluate and diagnose AC. | INTRODUCTION | Smears taken from the vermilion of the lower lips of 125 fishermen and 30 control individuals were subjected to cytologic analysis. | PATIENTS AND METHODS | The harvested cells were sufficient for cytologic analysis in 83.2% of the samples. Sixteen fishermen exhibited prominent lower lip lesions that justified biopsy and histological studies. In total, 4 specimens were malignant (3.2%), and 12 displayed epithelial dysplasia, demonstrating that the prevalence of epithelial dysplasia and malignant lesions was high among the fishermen population. These conditions were strongly associated with infiltration and blurring of the vermilion margin of the lower lip. | RESULTS | The cytologic analysis was not useful for detecting epithelial dysplasia or malignant alterations. | CONCLUSION | [
"Adult",
"Biopsy",
"Brazil",
"Carcinoma, Squamous Cell",
"Cheilitis",
"Chi-Square Distribution",
"Female",
"Humans",
"Lip",
"Lip Neoplasms",
"Male",
"Middle Aged",
"Occupational Exposure",
"Precancerous Conditions",
"Statistics, Nonparametric",
"Sunlight"
] | 2862672 | INTRODUCTION | Actinic cheilitis is considered to be a pre-malignant lesion or an incipient and superficial form of lip squamous cell carcinoma (SCC). It is commonly found in individuals whose occupational activities are related to chronic sun exposure, especially fishermen.1–7 The clinical changes of actinic cheilitis include erythema, keratosis, atrophy, erosion, crusts and fissures. Infiltration and blurring of the vermilion margin are clinical signs that correlate with lower lip neoplasia.8–10
No consensus has been reached in the literature regarding the clinical manifestations and histopathologic aggressiveness of actinic cheilitis (AC), thus histopathologic analyses should be used to confirm this condition.9,10 Exfoliative cytology has been used as a screening procedure to evaluate oral pre-malignant lesions and cancer of the oral cavity.11–14
Considering the well-established association between AC and lower-lip SCC, especially in patients at risk, we herein suggest procedures to increase the diagnostic accuracy for this lesion. Exfoliative cytology is a non-invasive and low-cost examination technique that may be useful for the assessment of lower lip AC and SCC, as it has been used to screen and diagnose pre-malignant oral lesions.15–29 However, few studies have used exfoliative cytology for oral lip lesions. To our knowledge, no studies have proposed the use of exfoliative cytologic analysis to evaluate and diagnose AC.
The aim of this study was to evaluate the clinical, cytologic and histopathologic features of AC in fishermen living in the city of Florianopolis, in the state of Santa Catarina, Brazil. | null | null | Histopathologic Findings | Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies. | null | null | [
"RESULTS"
] | [
"The control group consisted of 28 male and 2 female Caucasians, with a mean age of 50.6±6.4 years. The fishermen group (FG) included 125 Caucasians (121 males and 4 females) with a mean age of 50.58±12.77 years. The sunlight exposure period for the FG was 32.14±12.47 years, with an average of 7.71±1.23 hours/day. In total, 92.8% of the fishermen did not use any type of sunscreen protection. Alcohol consumption was observed in 32% and smoking in 38.4% of the fishermen. Only 2% of fishermen were aware of the malignant potential of their lip lesions.\nThe characteristic clinical manifestations of the actinic-induced alterations were mild in this sample (Table 1). Lower lip biopsies were obtained from 16 fishermen (12.8%) based on the abovementioned clinical criteria.\nThe number of cells in the FG smears was lower than that in the CG, and the number of inconclusive cases (cellular amount = 0) was significantly higher in the FG (p<0.001). Although the cellular amount in the FG smears was lower, the exfoliative cytology displayed more intermediate (p<0.001) and atypical (inflammatory) squamous cells (p=0.027) in the FG than in the CG (Table 2).\n Histopathologic Findings Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.\nHistopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies."
] | [
"results"
] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"RESULTS",
"Histopathologic Findings",
"DISCUSSION"
] | [
"Actinic cheilitis is considered to be a pre-malignant lesion or an incipient and superficial form of lip squamous cell carcinoma (SCC). It is commonly found in individuals whose occupational activities are related to chronic sun exposure, especially fishermen.1–7 The clinical changes of actinic cheilitis include erythema, keratosis, atrophy, erosion, crusts and fissures. Infiltration and blurring of the vermilion margin are clinical signs that correlate with lower lip neoplasia.8–10\nNo consensus has been reached in the literature regarding the clinical manifestations and histopathologic aggressiveness of actinic cheilitis (AC), thus histopathologic analyses should be used to confirm this condition.9,10 Exfoliative cytology has been used as a screening procedure to evaluate oral pre-malignant lesions and cancer of the oral cavity.11–14\nConsidering the well-established association between AC and lower-lip SCC, especially in patients at risk, we herein suggest procedures to increase the diagnostic accuracy for this lesion. Exfoliative cytology is a non-invasive and low-cost examination technique that may be useful for the assessment of lower lip AC and SCC, as it has been used to screen and diagnose pre-malignant oral lesions.15–29 However, few studies have used exfoliative cytology for oral lip lesions. To our knowledge, no studies have proposed the use of exfoliative cytologic analysis to evaluate and diagnose AC.\nThe aim of this study was to evaluate the clinical, cytologic and histopathologic features of AC in fishermen living in the city of Florianopolis, in the state of Santa Catarina, Brazil.",
"The studied sample included 125 fishermen with a history of chronic sun exposure (FG) living in Florianopolis, Santa Catarina, Brazil (latitude 270 35′ 48″ S, longitude 480 32′ 57″ O). The control group (n=30) did not have a history of chronic sun exposure (CG) and included individuals selected from the same geographic region who generally participated in only indoor activities. The study was approved by the Research Ethics Board of the institution. Informed consent was obtained from all patients.\nClinical manifestations including erythema, desquamation, fissure, infiltration, atrophy, ulceration, crust, leukokeratosis and blurring of the vermilion margin were graded according to their severity from 0 (absent) to 4 (intense).9,10\nThe patients in control group (CG) were evaluated for clinical features that could indicate lip epithelial dysplasia or malignancy.\nAll individuals underwent lower-lip exfoliative cytology with a metal spatula.32,33,11 The parched lip areas were humidified with saline solution for 10 minutes and scraped to remove the excess keratin.30,31 Cytologic smears were fixed in a 70% alcohol solution33,14 and stained by Papanicolaou’s method.\nThe cytologic analysis evaluated the following: 1) cellular characteristics, as defined for gynecological smears (Bibbo, 1997; Koss, 1992); 2) oral smear pattern, as proposed by Silva (1997); and 3) the correlation between cytologic characteristics and histopathologic findings in biopsied patients. The number of cells in the smears was scored from absent / inconclusive (0) to intense (3).\nPatients that presented blurring of the vermilion margin, infiltration of the lower lip9 and cytologic results suggestive of dysplasia/malignancy underwent a biopsy and histopathologic analysis.\nThe specimens were obtained using punch or scalpel biopsy. The specimens were fixed in 10% formalin and stained with hematoxylin and eosin. The histopathologic analysis used the cytologic and architectural criteria proposed by the WHO (1978), Pindborg et al. (1997) and Nico et al. (2007). Three independent, blinded pathologists performed the cytologic and histopathologic analyses.\nThe data obtained were compared using the Chi-square, Mann-Whitney and Kruskall-Wallis tests in SPSS version 15.0 (spssinc©). p < 0.05 was considered significant.",
"The control group consisted of 28 male and 2 female Caucasians, with a mean age of 50.6±6.4 years. The fishermen group (FG) included 125 Caucasians (121 males and 4 females) with a mean age of 50.58±12.77 years. The sunlight exposure period for the FG was 32.14±12.47 years, with an average of 7.71±1.23 hours/day. In total, 92.8% of the fishermen did not use any type of sunscreen protection. Alcohol consumption was observed in 32% and smoking in 38.4% of the fishermen. Only 2% of fishermen were aware of the malignant potential of their lip lesions.\nThe characteristic clinical manifestations of the actinic-induced alterations were mild in this sample (Table 1). Lower lip biopsies were obtained from 16 fishermen (12.8%) based on the abovementioned clinical criteria.\nThe number of cells in the FG smears was lower than that in the CG, and the number of inconclusive cases (cellular amount = 0) was significantly higher in the FG (p<0.001). Although the cellular amount in the FG smears was lower, the exfoliative cytology displayed more intermediate (p<0.001) and atypical (inflammatory) squamous cells (p=0.027) in the FG than in the CG (Table 2).\n Histopathologic Findings Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.\nHistopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.",
"Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.",
"The epidemiological profile of the FG concerning smoking and chronic sunlight exposure supports the data in the literature regarding AC. The results obtained in this study were also similar to the first description of AC and other published data.834,35\nIn this study, there was no correlation between age or sunlight exposure time and the histopathologic diagnosis of dysplasia or malignancy. This may reflect the difficulty in predicting the malignant transformation of pre-malignant lesions, even with well-defined etiological factors.36\nAll biopsied fishermen presented dysplastic or malignant alterations, showing that the established criteria for indication of biopsy9 had 100% specificity for dysplasia/malignancy. The presence of pre-malignant lesions has been associated with malignant transformation.41,42\nIn our study, 12 cases showed assorted dysplasia grades at the histopathologic examination. Four (3.2%) of the fishermen presented the histopathologic diagnosis of SCC. This prevalence is slightly higher than that reported for the normal population.33 Lip cancer incidence in Brazil is 2.2/100,000 cases per year. In the southern region of Brazil, the incidence is 1.2/10,000 cases.33\nConsidering the discrepancy between the clinical alterations of AC and the histopathologic findings, particularly when evaluated in a single biopsy, the use of other techniques with improved diagnostic accuracy as well as methods (such as cytologic smears) that could be used as screening tools is very important. In this study, we evaluated exfoliative cytology as an effective option for the early diagnosis of AC, and used smears as a screening tool to determine the best area for biopsy.\nDysplastic alterations occur in the deeper epithelial layers. For this reason, the cytologic diagnosis of keratinized lesions can be difficult. Studies evaluating the quality, sensitivity and specificity of cytologic examinations for oral cancer and pre-malignant lesions usually refer to intra-oral lesions. There are no published cytologic results for lip lesions associated with AC and lower-lip cancer.\nThe sample quality, according to the number of cells obtained from the lower-lip smears of the fishermen, was considered adequate for evaluation (scores 2 and 3) in 83.2% of cases. The few cytologic studies that have addressed lip lesions discussed the difficulty of cell harvesting, due to lip dryness and keratinization. In our study, this difficulty was overcome by first humidifying the lip with saline solution and then using a metal spatula to attain more intense frictional contact with the selected area.11–14\nAlthough recent studies have supported the use of exfoliative cytology for oral pre-malignant and malignant diagnoses, our study clearly shows that such analyses should not be used for strictly morphological diagnosis of AC and lower-lip cancer.11,13,23\nPathological studies of AC and SCC have shown that these lesions can develop from the basal epithelial layer to the lamina propria, or even present alterations throughout all epithelial layers before evolving into fully developed malignant lesions.34\nThe sensitivity and specificity of exfoliative cytology may be enhanced when associated with novel technologies such as tumor markers, p53 identification, cytomorphometry, growth factors, gene expression and others.11–13,15–17,19–24 It is known that AC and SCC present molecular alterations even before their morphological features develop. It is possible that these alterations may be demonstrated in adequate cellular samples (even superficial and intermediate ones). Exfoliative cytology might be a non-invasive tool for initial studies of AC and lip cancer when used with the aforementioned new techniques.\nConsidering the data obtained, we conclude that the studied fishermen presented a high prevalence of actinically induced lesions. These conditions were strongly associated with infiltration and blurring of the vermilion margin of the lower lip. Routine exfoliative cytology was not useful for screening pre-malignant and malignant lower-lip lesions."
] | [
"intro",
"materials|methods",
"results",
"results",
"discussion"
] | [
"Actinic cheilitis",
"Lip carcinoma",
"Pre-malignant lesion",
"Exfoliative cytology"
] | INTRODUCTION:
Actinic cheilitis is considered to be a pre-malignant lesion or an incipient and superficial form of lip squamous cell carcinoma (SCC). It is commonly found in individuals whose occupational activities are related to chronic sun exposure, especially fishermen.1–7 The clinical changes of actinic cheilitis include erythema, keratosis, atrophy, erosion, crusts and fissures. Infiltration and blurring of the vermilion margin are clinical signs that correlate with lower lip neoplasia.8–10
No consensus has been reached in the literature regarding the clinical manifestations and histopathologic aggressiveness of actinic cheilitis (AC), thus histopathologic analyses should be used to confirm this condition.9,10 Exfoliative cytology has been used as a screening procedure to evaluate oral pre-malignant lesions and cancer of the oral cavity.11–14
Considering the well-established association between AC and lower-lip SCC, especially in patients at risk, we herein suggest procedures to increase the diagnostic accuracy for this lesion. Exfoliative cytology is a non-invasive and low-cost examination technique that may be useful for the assessment of lower lip AC and SCC, as it has been used to screen and diagnose pre-malignant oral lesions.15–29 However, few studies have used exfoliative cytology for oral lip lesions. To our knowledge, no studies have proposed the use of exfoliative cytologic analysis to evaluate and diagnose AC.
The aim of this study was to evaluate the clinical, cytologic and histopathologic features of AC in fishermen living in the city of Florianopolis, in the state of Santa Catarina, Brazil.
MATERIALS AND METHODS:
The studied sample included 125 fishermen with a history of chronic sun exposure (FG) living in Florianopolis, Santa Catarina, Brazil (latitude 270 35′ 48″ S, longitude 480 32′ 57″ O). The control group (n=30) did not have a history of chronic sun exposure (CG) and included individuals selected from the same geographic region who generally participated in only indoor activities. The study was approved by the Research Ethics Board of the institution. Informed consent was obtained from all patients.
Clinical manifestations including erythema, desquamation, fissure, infiltration, atrophy, ulceration, crust, leukokeratosis and blurring of the vermilion margin were graded according to their severity from 0 (absent) to 4 (intense).9,10
The patients in control group (CG) were evaluated for clinical features that could indicate lip epithelial dysplasia or malignancy.
All individuals underwent lower-lip exfoliative cytology with a metal spatula.32,33,11 The parched lip areas were humidified with saline solution for 10 minutes and scraped to remove the excess keratin.30,31 Cytologic smears were fixed in a 70% alcohol solution33,14 and stained by Papanicolaou’s method.
The cytologic analysis evaluated the following: 1) cellular characteristics, as defined for gynecological smears (Bibbo, 1997; Koss, 1992); 2) oral smear pattern, as proposed by Silva (1997); and 3) the correlation between cytologic characteristics and histopathologic findings in biopsied patients. The number of cells in the smears was scored from absent / inconclusive (0) to intense (3).
Patients that presented blurring of the vermilion margin, infiltration of the lower lip9 and cytologic results suggestive of dysplasia/malignancy underwent a biopsy and histopathologic analysis.
The specimens were obtained using punch or scalpel biopsy. The specimens were fixed in 10% formalin and stained with hematoxylin and eosin. The histopathologic analysis used the cytologic and architectural criteria proposed by the WHO (1978), Pindborg et al. (1997) and Nico et al. (2007). Three independent, blinded pathologists performed the cytologic and histopathologic analyses.
The data obtained were compared using the Chi-square, Mann-Whitney and Kruskall-Wallis tests in SPSS version 15.0 (spssinc©). p < 0.05 was considered significant.
RESULTS:
The control group consisted of 28 male and 2 female Caucasians, with a mean age of 50.6±6.4 years. The fishermen group (FG) included 125 Caucasians (121 males and 4 females) with a mean age of 50.58±12.77 years. The sunlight exposure period for the FG was 32.14±12.47 years, with an average of 7.71±1.23 hours/day. In total, 92.8% of the fishermen did not use any type of sunscreen protection. Alcohol consumption was observed in 32% and smoking in 38.4% of the fishermen. Only 2% of fishermen were aware of the malignant potential of their lip lesions.
The characteristic clinical manifestations of the actinic-induced alterations were mild in this sample (Table 1). Lower lip biopsies were obtained from 16 fishermen (12.8%) based on the abovementioned clinical criteria.
The number of cells in the FG smears was lower than that in the CG, and the number of inconclusive cases (cellular amount = 0) was significantly higher in the FG (p<0.001). Although the cellular amount in the FG smears was lower, the exfoliative cytology displayed more intermediate (p<0.001) and atypical (inflammatory) squamous cells (p=0.027) in the FG than in the CG (Table 2).
Histopathologic Findings Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.
Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.
Histopathologic Findings:
Histopathologic analysis identified four SCCs (two in situ carcinoma and two superficially invasive SCCs), seven cases of light dysplasia and five cases of mild dysplasia. No correlation was observed between dysplasia or malignancy and age or sunlight exposure time. Table 3 presents the mean scores for each morphological feature observed in the 16 lower-lip biopsies.
DISCUSSION:
The epidemiological profile of the FG concerning smoking and chronic sunlight exposure supports the data in the literature regarding AC. The results obtained in this study were also similar to the first description of AC and other published data.834,35
In this study, there was no correlation between age or sunlight exposure time and the histopathologic diagnosis of dysplasia or malignancy. This may reflect the difficulty in predicting the malignant transformation of pre-malignant lesions, even with well-defined etiological factors.36
All biopsied fishermen presented dysplastic or malignant alterations, showing that the established criteria for indication of biopsy9 had 100% specificity for dysplasia/malignancy. The presence of pre-malignant lesions has been associated with malignant transformation.41,42
In our study, 12 cases showed assorted dysplasia grades at the histopathologic examination. Four (3.2%) of the fishermen presented the histopathologic diagnosis of SCC. This prevalence is slightly higher than that reported for the normal population.33 Lip cancer incidence in Brazil is 2.2/100,000 cases per year. In the southern region of Brazil, the incidence is 1.2/10,000 cases.33
Considering the discrepancy between the clinical alterations of AC and the histopathologic findings, particularly when evaluated in a single biopsy, the use of other techniques with improved diagnostic accuracy as well as methods (such as cytologic smears) that could be used as screening tools is very important. In this study, we evaluated exfoliative cytology as an effective option for the early diagnosis of AC, and used smears as a screening tool to determine the best area for biopsy.
Dysplastic alterations occur in the deeper epithelial layers. For this reason, the cytologic diagnosis of keratinized lesions can be difficult. Studies evaluating the quality, sensitivity and specificity of cytologic examinations for oral cancer and pre-malignant lesions usually refer to intra-oral lesions. There are no published cytologic results for lip lesions associated with AC and lower-lip cancer.
The sample quality, according to the number of cells obtained from the lower-lip smears of the fishermen, was considered adequate for evaluation (scores 2 and 3) in 83.2% of cases. The few cytologic studies that have addressed lip lesions discussed the difficulty of cell harvesting, due to lip dryness and keratinization. In our study, this difficulty was overcome by first humidifying the lip with saline solution and then using a metal spatula to attain more intense frictional contact with the selected area.11–14
Although recent studies have supported the use of exfoliative cytology for oral pre-malignant and malignant diagnoses, our study clearly shows that such analyses should not be used for strictly morphological diagnosis of AC and lower-lip cancer.11,13,23
Pathological studies of AC and SCC have shown that these lesions can develop from the basal epithelial layer to the lamina propria, or even present alterations throughout all epithelial layers before evolving into fully developed malignant lesions.34
The sensitivity and specificity of exfoliative cytology may be enhanced when associated with novel technologies such as tumor markers, p53 identification, cytomorphometry, growth factors, gene expression and others.11–13,15–17,19–24 It is known that AC and SCC present molecular alterations even before their morphological features develop. It is possible that these alterations may be demonstrated in adequate cellular samples (even superficial and intermediate ones). Exfoliative cytology might be a non-invasive tool for initial studies of AC and lip cancer when used with the aforementioned new techniques.
Considering the data obtained, we conclude that the studied fishermen presented a high prevalence of actinically induced lesions. These conditions were strongly associated with infiltration and blurring of the vermilion margin of the lower lip. Routine exfoliative cytology was not useful for screening pre-malignant and malignant lower-lip lesions.
| Background:
Actinic cheilitis (AC) is considered to be a pre-malignant lesion or an incipient and superficial form of lip squamous cell carcinoma. It is commonly found in individuals whose occupational activities are related to chronic sun exposure and the definitive diagnosis is performed with biopsy. Although Exfoliative cytology has been used as a screening procedure to evaluate cancer of the oral cavity no studies have proposed the use of exfoliative cytologic analysis to evaluate and diagnose AC.
Methods:
Smears taken from the vermilion of the lower lips of 125 fishermen and 30 control individuals were subjected to cytologic analysis.
Results:
The harvested cells were sufficient for cytologic analysis in 83.2% of the samples. Sixteen fishermen exhibited prominent lower lip lesions that justified biopsy and histological studies. In total, 4 specimens were malignant (3.2%), and 12 displayed epithelial dysplasia, demonstrating that the prevalence of epithelial dysplasia and malignant lesions was high among the fishermen population. These conditions were strongly associated with infiltration and blurring of the vermilion margin of the lower lip.
Conclusions:
The cytologic analysis was not useful for detecting epithelial dysplasia or malignant alterations. | null | null | 1,829 | 216 | [
365
] | 5 | [
"lip",
"lower",
"histopathologic",
"malignant",
"lesions",
"dysplasia",
"ac",
"lower lip",
"cytologic",
"fishermen"
] | [
"changes actinic cheilitis",
"oral lip lesions",
"actinic cheilitis include",
"lesion exfoliative cytology",
"exfoliative cytology oral"
] | null | null | null | [CONTENT] Actinic cheilitis | Lip carcinoma | Pre-malignant lesion | Exfoliative cytology [SUMMARY] | null | [CONTENT] Actinic cheilitis | Lip carcinoma | Pre-malignant lesion | Exfoliative cytology [SUMMARY] | null | [CONTENT] Actinic cheilitis | Lip carcinoma | Pre-malignant lesion | Exfoliative cytology [SUMMARY] | null | [CONTENT] Adult | Biopsy | Brazil | Carcinoma, Squamous Cell | Cheilitis | Chi-Square Distribution | Female | Humans | Lip | Lip Neoplasms | Male | Middle Aged | Occupational Exposure | Precancerous Conditions | Statistics, Nonparametric | Sunlight [SUMMARY] | null | [CONTENT] Adult | Biopsy | Brazil | Carcinoma, Squamous Cell | Cheilitis | Chi-Square Distribution | Female | Humans | Lip | Lip Neoplasms | Male | Middle Aged | Occupational Exposure | Precancerous Conditions | Statistics, Nonparametric | Sunlight [SUMMARY] | null | [CONTENT] Adult | Biopsy | Brazil | Carcinoma, Squamous Cell | Cheilitis | Chi-Square Distribution | Female | Humans | Lip | Lip Neoplasms | Male | Middle Aged | Occupational Exposure | Precancerous Conditions | Statistics, Nonparametric | Sunlight [SUMMARY] | null | [CONTENT] changes actinic cheilitis | oral lip lesions | actinic cheilitis include | lesion exfoliative cytology | exfoliative cytology oral [SUMMARY] | null | [CONTENT] changes actinic cheilitis | oral lip lesions | actinic cheilitis include | lesion exfoliative cytology | exfoliative cytology oral [SUMMARY] | null | [CONTENT] changes actinic cheilitis | oral lip lesions | actinic cheilitis include | lesion exfoliative cytology | exfoliative cytology oral [SUMMARY] | null | [CONTENT] lip | lower | histopathologic | malignant | lesions | dysplasia | ac | lower lip | cytologic | fishermen [SUMMARY] | null | [CONTENT] lip | lower | histopathologic | malignant | lesions | dysplasia | ac | lower lip | cytologic | fishermen [SUMMARY] | null | [CONTENT] lip | lower | histopathologic | malignant | lesions | dysplasia | ac | lower lip | cytologic | fishermen [SUMMARY] | null | [CONTENT] ac | evaluate | actinic cheilitis | cheilitis | oral | scc | pre | pre malignant | actinic | lip [SUMMARY] | null | [CONTENT] dysplasia | observed | sccs | cases | feature observed | superficially invasive sccs seven | superficially invasive sccs | superficially invasive | dysplasia malignancy age sunlight | dysplasia malignancy age [SUMMARY] | null | [CONTENT] lip | dysplasia | ac | cases | observed | sccs | lower | lesions | malignant | cytologic [SUMMARY] | null | [CONTENT] Actinic | AC ||| ||| AC [SUMMARY] | null | [CONTENT] 83.2% ||| Sixteen ||| 4 | 3.2% | 12 ||| [SUMMARY] | null | [CONTENT] Actinic | AC ||| ||| AC ||| 125 | 30 ||| ||| 83.2% ||| Sixteen ||| 4 | 3.2% | 12 ||| ||| [SUMMARY] | null |
|||
Cervical Human Papillomavirus genotypes in HIV-infected women: a cross-sectional analysis of the VALHIDATE study. | 29707656 | Primary-prevention by prophylactic vaccination against HPV-related cancers and HPV-based screening programs are based on HPV-type distribution in immunocompetent individuals. HIV-infected women are at high risk of invasive HPV-disease sustained by a broader range of HPV-types and have higher multi-type infection rates than immunocompetent hosts. | INTRODUCTION | This is a cross-sectional analysis of High Risk HPV (HR HPV) type distribution in 805 HIV+ women (HIW) compared with a control group of 1402 immunocompetent HIV- women (SPW) enrolled in the VALHIDATE study in order to define HPV type-specific distribution according to cytology. | METHODS | HIW had a 3.8, 3.6, and 2.7 times higher risk of atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) than SPW respectively. HPV-DNA prevalence was 28.4% in HIW and 11.81% in SPW (p<0.0001). The prevalence of infection increased from normal cytology to HSIL both in HIW (from 21.45% to 90.91%) and SPW (from 9.54% to 75%). The OR for women with normal cytology of having a positive HPV-DNA test result of was 2.6 times higher in HIW than in SPW. The cumulative prevalence of HPV-16/18 in HSIL is much lower in HIW (36.4±28.4) than SPW (62.5±33.5). | RESULTS | A higher prevalence of infection and broader HPV type distribution were observed in HIV+ women compared to the general population. More than 60% of HSIL lesions of HIW patients are caused by single or multi-type infections from non-HPV16/18 HPVs. The potential 9v-HPV vaccine coverage could be even higher than that expected for the general population given the wide panel of HPV-types observed in the HSIL of HIV+ women. | CONCLUSIONS | [
"Adult",
"Atypical Squamous Cells of the Cervix",
"Case-Control Studies",
"Cervix Uteri",
"Coinfection",
"Cross-Sectional Studies",
"DNA, Viral",
"Female",
"Genotype",
"HIV Infections",
"Humans",
"Immunocompetence",
"Immunocompromised Host",
"Italy",
"Middle Aged",
"Molecular Epidemiology",
"Odds Ratio",
"Papillomaviridae",
"Papillomavirus Infections",
"Squamous Intraepithelial Lesions of the Cervix",
"Uterine Cervical Neoplasms"
] | 5912788 | Introduction | Thirteen Human Papillomavirus (HPV) genotypes classified as carcinogenic and probably carcinogenic (group 1 and 2A) and six other HPV types with an invasive cervical cancer (ICC)/normal cytology ratio greater than 1.0, classified as possibly carcinogenic genotypes (HPV 26, 30, 67, 69, 73, and 82 – group 2B) are the cause of more than 90% of all ICCs worldwide [1, 2].
Their prevalence varies widely across world regions, but HPV-16 and -18 infections are the most prevalent and carcinogenic all over the world. Apart from the HPV type there are several co-factors that can contribute to invasive evolution [3].
Women affected by HIV/AIDS are at higher risk of invasive disease which is mainly due to the extent of immune-depression [4, 5]. However, the broader range of HPV types sustaining infections and the higher rate of multi-type infections in women living with HIV/AIDS could differently affect HPV type-specific carcinogenicity [6-8]. This is particularly relevant for cervical cancer prevention in immune-compromised hosts: in fact both vaccine primary prevention and HPV-based screening programs are based on the HPV-type distribution in immunocompetent individuals.
Three different HPV vaccines have been approved and licensed by the European Medicines Agency (EMA) and are available in Italy: the 2-valent (Cervarix®, GSK biologicals) vaccine, which prevents infections with High Risk HPV (HR HPV) types 16 and 18; the 4-valent vaccine (Gardasil®, Merck, Sanofi Pasteur MSD) which also targets Low Risk HPV (LR HPV) types 6 and 11 and the 9-valent vaccine (Gardasil9®, Merck, Sanofi Pasteur MSD) which, in addition to the four types of the 4-valent vaccine, also targets five additional HR HPV types (31, 33, 45, 52, and 58). Several studies [9, 10] have indicated that using a 9-valent vaccine could improve the prevention of invasive cervical cancers worldwide from 70% to 90%.
This paper reports a cross-sectional analysis of HPV-type distribution in HIV infected Women (HIW) compared with a control group of immunocompetent HIV-negative women enrolled in the eVALuation and monitoring of HPV infections and relATEd cervical diseases (VALHIDATE) study [11]. The VALHIDATE study [11] was a 5-year (Dec. 2010-Dec.2015) multicenter open prospective cohort study aimed at gaining insight into the molecular epidemiology of HPV infection and cervical diseases in high-risk women in the Lombardy Region, Italy. HIW aged 26-64 years were one of the high-risk cohorts of the study. The control group was composed of women in the same age group attending spontaneous Pap screening-programs (SPW).
The aim of the study was to evaluate HPV type-specific distribution according to cytology among HIV infected women. Moreover, these data will enable us to establish the pre-vaccine type-specific prevalence of HPV–associated diseases in this population in order to evaluate the potential impact of the newly approved 9-valent HPV vaccine. | Methods | STUDY DESIGN With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.
The protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.
A total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.
The Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).
With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.
The protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.
A total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.
The Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).
DNA EXTRACTION, HPV DETECTION AND GENOTYPING DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].
DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].
STATISTICAL ANALYSIS HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).
HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com). | null | null | Discussion and conclusions | Worldwide, 69.4% of invasive cervical cancers are caused by HPV-16/18 infections [15]. In Italy, current estimates indicate that approximately 3.3% of females with normal cytology were infected with these two genotypes and the prevalence increased with disease progression [15]. The data reported in our study closely resemble the figures for SPW, even a slightly lower prevalence of HPV infection was observed in women with normal cytology; a comparable distribution of HPV types in women with normal cytology was found for HIW as well as a relatively lower presence of HPV-16/18 infections in the HSIL lesions compared to the Italian figures. HPV type distribution stratified by cytological results showed a substantial equivalence between women with HIV and SPW in normal cytology: HPV-16 is the most common viral type in both populations, followed by HPV-52, 66, 31, 53 for SPW and by HPV-66, 53, 52, 31 for HIW. Due to the limited number of cases with abnormal cytology observed in this study, it was impossible to identify statistically significant differences of distribution between HIW and SPW. However, although HPV-16 is by far the most common type expressed in HSIL of SPW, there is a greater heterogeneity of genotypes in women with HIV infection and HSIL. Keller et al. [16] highlighted that HIV-infected women with HPV-16 infection and normal Pap test results have a similar pre-cancer risk as those with LSIL and therefore referral for colposcopy is warranted. However the presence of non HPV-16/18 infections was observed in 70% and 16.7% of HSIL in HIW and SPW respectively. McKenzie et al. [17] reported that cervical dysplasia specimens from 23 HIV infected women were infected (55%) by non-16/18 high-risk HPV types. If confirmed by larger cohorts, this finding may have major implications in the screening and triage strategies for women infected with HIV.As described by several Authors [18-19], it was observed that HIV-infected women have a higher prevalence of HPV and multi-type infections than the control group. A high prevalence of rare HPV types was detected in HIW, both in normal cytology and in cervical abnormalities and to date little is known about their carcinogenicity. The IARC classification and the analysis of rare HPV types show a wide variability in the carcinogenic potential of the rare and common HPV types found in normal cytology [1, 2]. In HIV-infected women, variability towards oncogenesis can be further influenced by the simultaneous presence of other cofactors, in particular cell-mediated immunosuppression [5], while the additional risks associated with each HPV type in multi-type infections is difficult to establish. Although no statistically significant differences were found due to lack of data, the description of viral types in the cervical specimens of HIV-infected women and correlation with the severity of cytological lesions can contribute to the mapping of HPV-related pre-invasive or invasive disease and provide a better risk stratification of disease evolution in HIV-infected women. Even in the context of primary prevention through vaccination, the wide distribution of viral types found in high-grade lesions of HIV-infected women demonstrates the need for multivalent vaccines. This means that a primary prophylaxis with a 9v-HPV vaccine could have prevented infections in over 50% of the women included in this study, whether HIV infected or not. The assessment of the impact of HPV vaccines in the study population shows how immunization with a 9v-HPV-vaccine could prevent a significantly higher proportion of viral infections, both in HIV-infected people and in the control population, than 2v- and 4v-HPV vaccines. The ICC preventable fraction in the general population increased by 12-19% when the 9v-HPV vaccine was introduced due to the addition of 7 high risk HPVs to the 2v or 4vHPV vaccines [10, 20, 21] which could be even higher in HIV-infected people considering the wide range of HPV types observed in progressive diseases. | [
"Introduction",
"STUDY DESIGN",
"DNA EXTRACTION, HPV DETECTION AND GENOTYPING",
"STATISTICAL ANALYSIS",
"Results",
"CYTOLOGICAL RESULTS",
"HUMAN PAPILLOMAVIRUS INFECTION BY CYTOLOGICAL STATUS",
"HPV TYPING BY CYTOLOGICAL STATUS",
"INFECTION FROM VACCINE HPV-TYPES IN WOMEN WITH NORMAL CYTOLOGY",
"Discussion and conclusions"
] | [
"Thirteen Human Papillomavirus (HPV) genotypes classified as carcinogenic and probably carcinogenic (group 1 and 2A) and six other HPV types with an invasive cervical cancer (ICC)/normal cytology ratio greater than 1.0, classified as possibly carcinogenic genotypes (HPV 26, 30, 67, 69, 73, and 82 – group 2B) are the cause of more than 90% of all ICCs worldwide [1, 2].\nTheir prevalence varies widely across world regions, but HPV-16 and -18 infections are the most prevalent and carcinogenic all over the world. Apart from the HPV type there are several co-factors that can contribute to invasive evolution [3].\nWomen affected by HIV/AIDS are at higher risk of invasive disease which is mainly due to the extent of immune-depression [4, 5]. However, the broader range of HPV types sustaining infections and the higher rate of multi-type infections in women living with HIV/AIDS could differently affect HPV type-specific carcinogenicity [6-8]. This is particularly relevant for cervical cancer prevention in immune-compromised hosts: in fact both vaccine primary prevention and HPV-based screening programs are based on the HPV-type distribution in immunocompetent individuals.\nThree different HPV vaccines have been approved and licensed by the European Medicines Agency (EMA) and are available in Italy: the 2-valent (Cervarix®, GSK biologicals) vaccine, which prevents infections with High Risk HPV (HR HPV) types 16 and 18; the 4-valent vaccine (Gardasil®, Merck, Sanofi Pasteur MSD) which also targets Low Risk HPV (LR HPV) types 6 and 11 and the 9-valent vaccine (Gardasil9®, Merck, Sanofi Pasteur MSD) which, in addition to the four types of the 4-valent vaccine, also targets five additional HR HPV types (31, 33, 45, 52, and 58). Several studies [9, 10] have indicated that using a 9-valent vaccine could improve the prevention of invasive cervical cancers worldwide from 70% to 90%.\nThis paper reports a cross-sectional analysis of HPV-type distribution in HIV infected Women (HIW) compared with a control group of immunocompetent HIV-negative women enrolled in the eVALuation and monitoring of HPV infections and relATEd cervical diseases (VALHIDATE) study [11]. The VALHIDATE study [11] was a 5-year (Dec. 2010-Dec.2015) multicenter open prospective cohort study aimed at gaining insight into the molecular epidemiology of HPV infection and cervical diseases in high-risk women in the Lombardy Region, Italy. HIW aged 26-64 years were one of the high-risk cohorts of the study. The control group was composed of women in the same age group attending spontaneous Pap screening-programs (SPW).\nThe aim of the study was to evaluate HPV type-specific distribution according to cytology among HIV infected women. Moreover, these data will enable us to establish the pre-vaccine type-specific prevalence of HPV–associated diseases in this population in order to evaluate the potential impact of the newly approved 9-valent HPV vaccine.",
"With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.\nThe protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.\nA total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.\nThe Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).",
"DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].",
"HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).",
" CYTOLOGICAL RESULTS Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).\nCytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).\n HUMAN PAPILLOMAVIRUS INFECTION BY CYTOLOGICAL STATUS HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).\nThe OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.\nDifferent HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).\nHPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).\nThe OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.\nDifferent HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).\n HPV TYPING BY CYTOLOGICAL STATUS Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.\nHPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.\nThe cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).\nMulti-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).\nType-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.\nHPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.\nThe cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).\nMulti-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).\n INFECTION FROM VACCINE HPV-TYPES IN WOMEN WITH NORMAL CYTOLOGY No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).\nNo differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).",
"Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).",
"HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).\nThe OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.\nDifferent HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).",
"Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.\nHPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.\nThe cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).\nMulti-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).",
"No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).",
"Worldwide, 69.4% of invasive cervical cancers are caused by HPV-16/18 infections [15]. In Italy, current estimates indicate that approximately 3.3% of females with normal cytology were infected with these two genotypes and the prevalence increased with disease progression [15]. The data reported in our study closely resemble the figures for SPW, even a slightly lower prevalence of HPV infection was observed in women with normal cytology; a comparable distribution of HPV types in women with normal cytology was found for HIW as well as a relatively lower presence of HPV-16/18 infections in the HSIL lesions compared to the Italian figures. HPV type distribution stratified by cytological results showed a substantial equivalence between women with HIV and SPW in normal cytology: HPV-16 is the most common viral type in both populations, followed by HPV-52, 66, 31, 53 for SPW and by HPV-66, 53, 52, 31 for HIW. Due to the limited number of cases with abnormal cytology observed in this study, it was impossible to identify statistically significant differences of distribution between HIW and SPW. However, although HPV-16 is by far the most common type expressed in HSIL of SPW, there is a greater heterogeneity of genotypes in women with HIV infection and HSIL. Keller et al. [16] highlighted that HIV-infected women with HPV-16 infection and normal Pap test results have a similar pre-cancer risk as those with LSIL and therefore referral for colposcopy is warranted. However the presence of non HPV-16/18 infections was observed in 70% and 16.7% of HSIL in HIW and SPW respectively. McKenzie et al. [17] reported that cervical dysplasia specimens from 23 HIV infected women were infected (55%) by non-16/18 high-risk HPV types. If confirmed by larger cohorts, this finding may have major implications in the screening and triage strategies for women infected with HIV.As described by several Authors [18-19], it was observed that HIV-infected women have a higher prevalence of HPV and multi-type infections than the control group. A high prevalence of rare HPV types was detected in HIW, both in normal cytology and in cervical abnormalities and to date little is known about their carcinogenicity. The IARC classification and the analysis of rare HPV types show a wide variability in the carcinogenic potential of the rare and common HPV types found in normal cytology [1, 2]. In HIV-infected women, variability towards oncogenesis can be further influenced by the simultaneous presence of other cofactors, in particular cell-mediated immunosuppression [5], while the additional risks associated with each HPV type in multi-type infections is difficult to establish. Although no statistically significant differences were found due to lack of data, the description of viral types in the cervical specimens of HIV-infected women and correlation with the severity of cytological lesions can contribute to the mapping of HPV-related pre-invasive or invasive disease and provide a better risk stratification of disease evolution in HIV-infected women. Even in the context of primary prevention through vaccination, the wide distribution of viral types found in high-grade lesions of HIV-infected women demonstrates the need for multivalent vaccines. This means that a primary prophylaxis with a 9v-HPV vaccine could have prevented infections in over 50% of the women included in this study, whether HIV infected or not. The assessment of the impact of HPV vaccines in the study population shows how immunization with a 9v-HPV-vaccine could prevent a significantly higher proportion of viral infections, both in HIV-infected people and in the control population, than 2v- and 4v-HPV vaccines. The ICC preventable fraction in the general population increased by 12-19% when the 9v-HPV vaccine was introduced due to the addition of 7 high risk HPVs to the 2v or 4vHPV vaccines [10, 20, 21] which could be even higher in HIV-infected people considering the wide range of HPV types observed in progressive diseases."
] | [
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] | [
"Introduction",
"Methods",
"STUDY DESIGN",
"DNA EXTRACTION, HPV DETECTION AND GENOTYPING",
"STATISTICAL ANALYSIS",
"Results",
"CYTOLOGICAL RESULTS",
"HUMAN PAPILLOMAVIRUS INFECTION BY CYTOLOGICAL STATUS",
"HPV TYPING BY CYTOLOGICAL STATUS",
"INFECTION FROM VACCINE HPV-TYPES IN WOMEN WITH NORMAL CYTOLOGY",
"Discussion and conclusions"
] | [
"Thirteen Human Papillomavirus (HPV) genotypes classified as carcinogenic and probably carcinogenic (group 1 and 2A) and six other HPV types with an invasive cervical cancer (ICC)/normal cytology ratio greater than 1.0, classified as possibly carcinogenic genotypes (HPV 26, 30, 67, 69, 73, and 82 – group 2B) are the cause of more than 90% of all ICCs worldwide [1, 2].\nTheir prevalence varies widely across world regions, but HPV-16 and -18 infections are the most prevalent and carcinogenic all over the world. Apart from the HPV type there are several co-factors that can contribute to invasive evolution [3].\nWomen affected by HIV/AIDS are at higher risk of invasive disease which is mainly due to the extent of immune-depression [4, 5]. However, the broader range of HPV types sustaining infections and the higher rate of multi-type infections in women living with HIV/AIDS could differently affect HPV type-specific carcinogenicity [6-8]. This is particularly relevant for cervical cancer prevention in immune-compromised hosts: in fact both vaccine primary prevention and HPV-based screening programs are based on the HPV-type distribution in immunocompetent individuals.\nThree different HPV vaccines have been approved and licensed by the European Medicines Agency (EMA) and are available in Italy: the 2-valent (Cervarix®, GSK biologicals) vaccine, which prevents infections with High Risk HPV (HR HPV) types 16 and 18; the 4-valent vaccine (Gardasil®, Merck, Sanofi Pasteur MSD) which also targets Low Risk HPV (LR HPV) types 6 and 11 and the 9-valent vaccine (Gardasil9®, Merck, Sanofi Pasteur MSD) which, in addition to the four types of the 4-valent vaccine, also targets five additional HR HPV types (31, 33, 45, 52, and 58). Several studies [9, 10] have indicated that using a 9-valent vaccine could improve the prevention of invasive cervical cancers worldwide from 70% to 90%.\nThis paper reports a cross-sectional analysis of HPV-type distribution in HIV infected Women (HIW) compared with a control group of immunocompetent HIV-negative women enrolled in the eVALuation and monitoring of HPV infections and relATEd cervical diseases (VALHIDATE) study [11]. The VALHIDATE study [11] was a 5-year (Dec. 2010-Dec.2015) multicenter open prospective cohort study aimed at gaining insight into the molecular epidemiology of HPV infection and cervical diseases in high-risk women in the Lombardy Region, Italy. HIW aged 26-64 years were one of the high-risk cohorts of the study. The control group was composed of women in the same age group attending spontaneous Pap screening-programs (SPW).\nThe aim of the study was to evaluate HPV type-specific distribution according to cytology among HIV infected women. Moreover, these data will enable us to establish the pre-vaccine type-specific prevalence of HPV–associated diseases in this population in order to evaluate the potential impact of the newly approved 9-valent HPV vaccine.",
" STUDY DESIGN With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.\nThe protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.\nA total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.\nThe Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).\nWith the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.\nThe protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.\nA total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.\nThe Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).\n DNA EXTRACTION, HPV DETECTION AND GENOTYPING DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].\nDNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].\n STATISTICAL ANALYSIS HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).\nHPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).",
"With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.\nThe protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.\nA total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.\nThe Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).",
"DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].",
"HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).",
" CYTOLOGICAL RESULTS Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).\nCytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).\n HUMAN PAPILLOMAVIRUS INFECTION BY CYTOLOGICAL STATUS HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).\nThe OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.\nDifferent HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).\nHPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).\nThe OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.\nDifferent HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).\n HPV TYPING BY CYTOLOGICAL STATUS Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.\nHPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.\nThe cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).\nMulti-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).\nType-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.\nHPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.\nThe cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).\nMulti-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).\n INFECTION FROM VACCINE HPV-TYPES IN WOMEN WITH NORMAL CYTOLOGY No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).\nNo differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).",
"Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).",
"HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).\nThe OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.\nDifferent HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).",
"Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.\nHPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.\nThe cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).\nMulti-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).",
"No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).",
"Worldwide, 69.4% of invasive cervical cancers are caused by HPV-16/18 infections [15]. In Italy, current estimates indicate that approximately 3.3% of females with normal cytology were infected with these two genotypes and the prevalence increased with disease progression [15]. The data reported in our study closely resemble the figures for SPW, even a slightly lower prevalence of HPV infection was observed in women with normal cytology; a comparable distribution of HPV types in women with normal cytology was found for HIW as well as a relatively lower presence of HPV-16/18 infections in the HSIL lesions compared to the Italian figures. HPV type distribution stratified by cytological results showed a substantial equivalence between women with HIV and SPW in normal cytology: HPV-16 is the most common viral type in both populations, followed by HPV-52, 66, 31, 53 for SPW and by HPV-66, 53, 52, 31 for HIW. Due to the limited number of cases with abnormal cytology observed in this study, it was impossible to identify statistically significant differences of distribution between HIW and SPW. However, although HPV-16 is by far the most common type expressed in HSIL of SPW, there is a greater heterogeneity of genotypes in women with HIV infection and HSIL. Keller et al. [16] highlighted that HIV-infected women with HPV-16 infection and normal Pap test results have a similar pre-cancer risk as those with LSIL and therefore referral for colposcopy is warranted. However the presence of non HPV-16/18 infections was observed in 70% and 16.7% of HSIL in HIW and SPW respectively. McKenzie et al. [17] reported that cervical dysplasia specimens from 23 HIV infected women were infected (55%) by non-16/18 high-risk HPV types. If confirmed by larger cohorts, this finding may have major implications in the screening and triage strategies for women infected with HIV.As described by several Authors [18-19], it was observed that HIV-infected women have a higher prevalence of HPV and multi-type infections than the control group. A high prevalence of rare HPV types was detected in HIW, both in normal cytology and in cervical abnormalities and to date little is known about their carcinogenicity. The IARC classification and the analysis of rare HPV types show a wide variability in the carcinogenic potential of the rare and common HPV types found in normal cytology [1, 2]. In HIV-infected women, variability towards oncogenesis can be further influenced by the simultaneous presence of other cofactors, in particular cell-mediated immunosuppression [5], while the additional risks associated with each HPV type in multi-type infections is difficult to establish. Although no statistically significant differences were found due to lack of data, the description of viral types in the cervical specimens of HIV-infected women and correlation with the severity of cytological lesions can contribute to the mapping of HPV-related pre-invasive or invasive disease and provide a better risk stratification of disease evolution in HIV-infected women. Even in the context of primary prevention through vaccination, the wide distribution of viral types found in high-grade lesions of HIV-infected women demonstrates the need for multivalent vaccines. This means that a primary prophylaxis with a 9v-HPV vaccine could have prevented infections in over 50% of the women included in this study, whether HIV infected or not. The assessment of the impact of HPV vaccines in the study population shows how immunization with a 9v-HPV-vaccine could prevent a significantly higher proportion of viral infections, both in HIV-infected people and in the control population, than 2v- and 4v-HPV vaccines. The ICC preventable fraction in the general population increased by 12-19% when the 9v-HPV vaccine was introduced due to the addition of 7 high risk HPVs to the 2v or 4vHPV vaccines [10, 20, 21] which could be even higher in HIV-infected people considering the wide range of HPV types observed in progressive diseases."
] | [
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"HIV-infected women",
"HPV types",
"Cervical lesions",
"Molecular Epidemiology"
] | Introduction:
Thirteen Human Papillomavirus (HPV) genotypes classified as carcinogenic and probably carcinogenic (group 1 and 2A) and six other HPV types with an invasive cervical cancer (ICC)/normal cytology ratio greater than 1.0, classified as possibly carcinogenic genotypes (HPV 26, 30, 67, 69, 73, and 82 – group 2B) are the cause of more than 90% of all ICCs worldwide [1, 2].
Their prevalence varies widely across world regions, but HPV-16 and -18 infections are the most prevalent and carcinogenic all over the world. Apart from the HPV type there are several co-factors that can contribute to invasive evolution [3].
Women affected by HIV/AIDS are at higher risk of invasive disease which is mainly due to the extent of immune-depression [4, 5]. However, the broader range of HPV types sustaining infections and the higher rate of multi-type infections in women living with HIV/AIDS could differently affect HPV type-specific carcinogenicity [6-8]. This is particularly relevant for cervical cancer prevention in immune-compromised hosts: in fact both vaccine primary prevention and HPV-based screening programs are based on the HPV-type distribution in immunocompetent individuals.
Three different HPV vaccines have been approved and licensed by the European Medicines Agency (EMA) and are available in Italy: the 2-valent (Cervarix®, GSK biologicals) vaccine, which prevents infections with High Risk HPV (HR HPV) types 16 and 18; the 4-valent vaccine (Gardasil®, Merck, Sanofi Pasteur MSD) which also targets Low Risk HPV (LR HPV) types 6 and 11 and the 9-valent vaccine (Gardasil9®, Merck, Sanofi Pasteur MSD) which, in addition to the four types of the 4-valent vaccine, also targets five additional HR HPV types (31, 33, 45, 52, and 58). Several studies [9, 10] have indicated that using a 9-valent vaccine could improve the prevention of invasive cervical cancers worldwide from 70% to 90%.
This paper reports a cross-sectional analysis of HPV-type distribution in HIV infected Women (HIW) compared with a control group of immunocompetent HIV-negative women enrolled in the eVALuation and monitoring of HPV infections and relATEd cervical diseases (VALHIDATE) study [11]. The VALHIDATE study [11] was a 5-year (Dec. 2010-Dec.2015) multicenter open prospective cohort study aimed at gaining insight into the molecular epidemiology of HPV infection and cervical diseases in high-risk women in the Lombardy Region, Italy. HIW aged 26-64 years were one of the high-risk cohorts of the study. The control group was composed of women in the same age group attending spontaneous Pap screening-programs (SPW).
The aim of the study was to evaluate HPV type-specific distribution according to cytology among HIV infected women. Moreover, these data will enable us to establish the pre-vaccine type-specific prevalence of HPV–associated diseases in this population in order to evaluate the potential impact of the newly approved 9-valent HPV vaccine.
Methods:
STUDY DESIGN With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.
The protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.
A total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.
The Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).
With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.
The protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.
A total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.
The Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).
DNA EXTRACTION, HPV DETECTION AND GENOTYPING DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].
DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].
STATISTICAL ANALYSIS HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).
HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).
STUDY DESIGN:
With the aim of evaluating the baseline HPV type-specific distribution stratified by the cervical cytological results, HIV-infected women (HIW) were compared with the control group (SPW). HIW and SPW cohorts were recruited consecutively for 12 months from 3 Infectious Diseases Units and 4 Gynecology Units of the four general hospitals located in Lombardy participating in the VALHIDATE study [11]. In particular, HIW were recruited from those followed up for HIV infection and SPW from those attending a spontaneous Pap screening program. Exclusion criteria were: history of histologically proven grade II or higher Cervical Intraepithelial Neoplasia (CIN) requiring treatment, pregnancy at the time of enrollment, inability to provide informed consent.
The protocol enrollment was approved by the Sacco Hospital Ethical Committees (Resolution n174/2010, 9 March 2010) and all participants provided written informed consent.
A total of 828 HIW and 1423 SPW were enrolled in the VALHIDATE study. The consenting women underwent basal co-testing with conventional Pap tests and HPV-DNA testing/genotyping. The cervical brush (Cytobrush Plus MedscandW Medical AB) sample collected at the baseline visit was used to perform the conventional Pap smear and then immersed and stored in a PreservCyt solution (ThinPrep® Pap Test, Hologic Italia Srl) to be analyzed for HPV-DNA and HPV genotyping.
The Pap tests were evaluated according to the 2001 Bethesda System terminology [12] by expert cytopathologists from the participating Centers. The cases were classified according to cytology at baseline evaluation as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL).
DNA EXTRACTION, HPV DETECTION AND GENOTYPING:
DNA was extracted with a commercial kit (NucliSENS® EasyMAG®, bioMérieux, Lyon, France) and HPV-DNA was detected through PCR amplification of a 450 bp segment of ORF L1 using the degenerate primer pair ELSI-f and ELSI-r in the central reference laboratory of the University of Milan [11, 13]. HPV genotyping was performed on HPV-DNA positive cervical brushes using the commercially available Inno-LiPA® HPV Genotyping Extra (Innogenetics N.V., Belgium) method in the microbiology laboratories of the participating Centers. This test allows for the identification of 27 HPV types. All of the HPV-DNA positive cervical samples resulted as non-typeable by the Inno-Lipa test (HPV-X) were subjected to the Restriction Fragment Length Polymorphism (RFLP) type analysis which is capable of identifying all types of the High-Risk clade (HR-clade) and Low-Risk (LR) types of the alpha genus according to the 2011 IARC classification [1, 14].
STATISTICAL ANALYSIS:
HPV-DNA prevalence and type-specific HPV prevalence were expressed as crude proportions with corresponding 95% confidence intervals (95%CI) calculated assuming a normal distribution. The data are presented as the median (interquartile range, IQR) and percentages (with 95%CI) as appropriate. Comparisons between groups were made using the Chi-square test or Fisher’s exact test. A P-value less than 0.05 was considered statistically significant (two-tailed test). All of the statistical analyses were performed using GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com).
Results:
CYTOLOGICAL RESULTS Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).
Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).
HUMAN PAPILLOMAVIRUS INFECTION BY CYTOLOGICAL STATUS HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).
The OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.
Different HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).
HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).
The OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.
Different HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).
HPV TYPING BY CYTOLOGICAL STATUS Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.
HPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.
The cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).
Multi-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).
Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.
HPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.
The cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).
Multi-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).
INFECTION FROM VACCINE HPV-TYPES IN WOMEN WITH NORMAL CYTOLOGY No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).
No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).
CYTOLOGICAL RESULTS:
Cytological results are provided for 805 HIW (97.2%) and 1402 SPW (98.5%). Women included in the HIW group had an overall 3.7-fold increased risk of ASCUS, a 3.6-fold increased risk of LSIL and a 2.7-fold increased risk of HSIL than those included in the control SPW group (Tab. I).
HUMAN PAPILLOMAVIRUS INFECTION BY CYTOLOGICAL STATUS:
HPV-DNA prevalence was 28.4% (95%CI 25.32-31.48) among HIW and 11.81% (95%CI 10.14-13.49) among SPW (p < 0.0001). HPV prevalence increased with the progression of the severity of cytological abnormalities from 21.45% to 90.91% in HIW and from 9.54% to 75% in SPW in normal cytology and HSIL respectively (Fig. 1).
The OR for women with normal cytology of having a positive HPV-DNA was 2.6 times higher in HIW (95%CI 2.0-3.3) than in SPW.
Different HPV-DNA prevalences were found for ASCUS and LSIL, while no differences were observed for HSIL lesions between the two groups (Tab. II).
HPV TYPING BY CYTOLOGICAL STATUS:
Type-specific HPV prevalence is reported in Fig 2a: HPV-16 was the most prevalent type which was detected in 4.74% of HIW and in 4.61% of SPW. No significant differences were observed in the patterns of distribution of the HIW and SPW groups, except for the identification of infections sustained by HPV-67 and HPV-34 in HIW, absent in SPW and infections by HPV- 42 and HPV-72 types in SPW, absent in HIW.
HPV typing stratified by cervical cytological results (Fig. 2b) showed very similar patterns in HIW and SPW with normal cytology. There is a lack of data concerning women with abnormal cytological results therefore we were unable to establish a different distribution pattern; however, HPV-16 is by far the most common type of HPV in women with abnormal cytology (any grade SIL, Fig. 2c) in SPW, while HPV-16 prevalence is similar to other high-risk HPV types (HPV-52, HPV-33 and HPV-66) in HIW. Several other HR-HPV types have been reported in HIW patients with cytological abnormalities (Fig 2c-2d). HSIL lesions sustained by non-HPV-16/18 types were 16.7% (1 out of 6) in SPW and 70% (7 out of 10) in HIW.
The cumulative prevalence of the two main oncogenic types (HPV-16/18), broken down by cytological outcome, is lower in LSIL and HSIL among the HIW than in the SPW and the general Italian population (Fig. 3).
Multi-type HPV infections (2 to 9 HPV types) occurred in 54.62% (95%CI 4.68-63.57) and 37.38% (95%CI 28.22-46.55) of HIW and SPW with normal cytology respectively (p 0.011). No differences were observed in the prevalence of multi-type HPV infections in women with cytological lesions (any grade or HSIL) (Table III).
INFECTION FROM VACCINE HPV-TYPES IN WOMEN WITH NORMAL CYTOLOGY:
No differences were observed in the proportion of infections sustained by at least one of the HPV types included in 2v-, 4v-, or 9v-HPV vaccines between HIW and SPW with normal cytology: 20.0% vs 23.62% for 2v-, 26.90% vs 27.56% for 4v-, and 51.7% vs 55.9% for 9v-HPV vaccine respectively. A highly significant difference in potential coverage was observed when we compared the 9v-HPV vaccine with the 2v- and 4v-HPV vaccines in both HIW and in SPW (Fig 4).
Discussion and conclusions:
Worldwide, 69.4% of invasive cervical cancers are caused by HPV-16/18 infections [15]. In Italy, current estimates indicate that approximately 3.3% of females with normal cytology were infected with these two genotypes and the prevalence increased with disease progression [15]. The data reported in our study closely resemble the figures for SPW, even a slightly lower prevalence of HPV infection was observed in women with normal cytology; a comparable distribution of HPV types in women with normal cytology was found for HIW as well as a relatively lower presence of HPV-16/18 infections in the HSIL lesions compared to the Italian figures. HPV type distribution stratified by cytological results showed a substantial equivalence between women with HIV and SPW in normal cytology: HPV-16 is the most common viral type in both populations, followed by HPV-52, 66, 31, 53 for SPW and by HPV-66, 53, 52, 31 for HIW. Due to the limited number of cases with abnormal cytology observed in this study, it was impossible to identify statistically significant differences of distribution between HIW and SPW. However, although HPV-16 is by far the most common type expressed in HSIL of SPW, there is a greater heterogeneity of genotypes in women with HIV infection and HSIL. Keller et al. [16] highlighted that HIV-infected women with HPV-16 infection and normal Pap test results have a similar pre-cancer risk as those with LSIL and therefore referral for colposcopy is warranted. However the presence of non HPV-16/18 infections was observed in 70% and 16.7% of HSIL in HIW and SPW respectively. McKenzie et al. [17] reported that cervical dysplasia specimens from 23 HIV infected women were infected (55%) by non-16/18 high-risk HPV types. If confirmed by larger cohorts, this finding may have major implications in the screening and triage strategies for women infected with HIV.As described by several Authors [18-19], it was observed that HIV-infected women have a higher prevalence of HPV and multi-type infections than the control group. A high prevalence of rare HPV types was detected in HIW, both in normal cytology and in cervical abnormalities and to date little is known about their carcinogenicity. The IARC classification and the analysis of rare HPV types show a wide variability in the carcinogenic potential of the rare and common HPV types found in normal cytology [1, 2]. In HIV-infected women, variability towards oncogenesis can be further influenced by the simultaneous presence of other cofactors, in particular cell-mediated immunosuppression [5], while the additional risks associated with each HPV type in multi-type infections is difficult to establish. Although no statistically significant differences were found due to lack of data, the description of viral types in the cervical specimens of HIV-infected women and correlation with the severity of cytological lesions can contribute to the mapping of HPV-related pre-invasive or invasive disease and provide a better risk stratification of disease evolution in HIV-infected women. Even in the context of primary prevention through vaccination, the wide distribution of viral types found in high-grade lesions of HIV-infected women demonstrates the need for multivalent vaccines. This means that a primary prophylaxis with a 9v-HPV vaccine could have prevented infections in over 50% of the women included in this study, whether HIV infected or not. The assessment of the impact of HPV vaccines in the study population shows how immunization with a 9v-HPV-vaccine could prevent a significantly higher proportion of viral infections, both in HIV-infected people and in the control population, than 2v- and 4v-HPV vaccines. The ICC preventable fraction in the general population increased by 12-19% when the 9v-HPV vaccine was introduced due to the addition of 7 high risk HPVs to the 2v or 4vHPV vaccines [10, 20, 21] which could be even higher in HIV-infected people considering the wide range of HPV types observed in progressive diseases.
| Background:
Primary-prevention by prophylactic vaccination against HPV-related cancers and HPV-based screening programs are based on HPV-type distribution in immunocompetent individuals. HIV-infected women are at high risk of invasive HPV-disease sustained by a broader range of HPV-types and have higher multi-type infection rates than immunocompetent hosts.
Methods:
This is a cross-sectional analysis of High Risk HPV (HR HPV) type distribution in 805 HIV+ women (HIW) compared with a control group of 1402 immunocompetent HIV- women (SPW) enrolled in the VALHIDATE study in order to define HPV type-specific distribution according to cytology.
Results:
HIW had a 3.8, 3.6, and 2.7 times higher risk of atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) than SPW respectively. HPV-DNA prevalence was 28.4% in HIW and 11.81% in SPW (p<0.0001). The prevalence of infection increased from normal cytology to HSIL both in HIW (from 21.45% to 90.91%) and SPW (from 9.54% to 75%). The OR for women with normal cytology of having a positive HPV-DNA test result of was 2.6 times higher in HIW than in SPW. The cumulative prevalence of HPV-16/18 in HSIL is much lower in HIW (36.4±28.4) than SPW (62.5±33.5).
Conclusions:
A higher prevalence of infection and broader HPV type distribution were observed in HIV+ women compared to the general population. More than 60% of HSIL lesions of HIW patients are caused by single or multi-type infections from non-HPV16/18 HPVs. The potential 9v-HPV vaccine coverage could be even higher than that expected for the general population given the wide panel of HPV-types observed in the HSIL of HIV+ women. | Introduction:
Thirteen Human Papillomavirus (HPV) genotypes classified as carcinogenic and probably carcinogenic (group 1 and 2A) and six other HPV types with an invasive cervical cancer (ICC)/normal cytology ratio greater than 1.0, classified as possibly carcinogenic genotypes (HPV 26, 30, 67, 69, 73, and 82 – group 2B) are the cause of more than 90% of all ICCs worldwide [1, 2].
Their prevalence varies widely across world regions, but HPV-16 and -18 infections are the most prevalent and carcinogenic all over the world. Apart from the HPV type there are several co-factors that can contribute to invasive evolution [3].
Women affected by HIV/AIDS are at higher risk of invasive disease which is mainly due to the extent of immune-depression [4, 5]. However, the broader range of HPV types sustaining infections and the higher rate of multi-type infections in women living with HIV/AIDS could differently affect HPV type-specific carcinogenicity [6-8]. This is particularly relevant for cervical cancer prevention in immune-compromised hosts: in fact both vaccine primary prevention and HPV-based screening programs are based on the HPV-type distribution in immunocompetent individuals.
Three different HPV vaccines have been approved and licensed by the European Medicines Agency (EMA) and are available in Italy: the 2-valent (Cervarix®, GSK biologicals) vaccine, which prevents infections with High Risk HPV (HR HPV) types 16 and 18; the 4-valent vaccine (Gardasil®, Merck, Sanofi Pasteur MSD) which also targets Low Risk HPV (LR HPV) types 6 and 11 and the 9-valent vaccine (Gardasil9®, Merck, Sanofi Pasteur MSD) which, in addition to the four types of the 4-valent vaccine, also targets five additional HR HPV types (31, 33, 45, 52, and 58). Several studies [9, 10] have indicated that using a 9-valent vaccine could improve the prevention of invasive cervical cancers worldwide from 70% to 90%.
This paper reports a cross-sectional analysis of HPV-type distribution in HIV infected Women (HIW) compared with a control group of immunocompetent HIV-negative women enrolled in the eVALuation and monitoring of HPV infections and relATEd cervical diseases (VALHIDATE) study [11]. The VALHIDATE study [11] was a 5-year (Dec. 2010-Dec.2015) multicenter open prospective cohort study aimed at gaining insight into the molecular epidemiology of HPV infection and cervical diseases in high-risk women in the Lombardy Region, Italy. HIW aged 26-64 years were one of the high-risk cohorts of the study. The control group was composed of women in the same age group attending spontaneous Pap screening-programs (SPW).
The aim of the study was to evaluate HPV type-specific distribution according to cytology among HIV infected women. Moreover, these data will enable us to establish the pre-vaccine type-specific prevalence of HPV–associated diseases in this population in order to evaluate the potential impact of the newly approved 9-valent HPV vaccine.
Discussion and conclusions:
Worldwide, 69.4% of invasive cervical cancers are caused by HPV-16/18 infections [15]. In Italy, current estimates indicate that approximately 3.3% of females with normal cytology were infected with these two genotypes and the prevalence increased with disease progression [15]. The data reported in our study closely resemble the figures for SPW, even a slightly lower prevalence of HPV infection was observed in women with normal cytology; a comparable distribution of HPV types in women with normal cytology was found for HIW as well as a relatively lower presence of HPV-16/18 infections in the HSIL lesions compared to the Italian figures. HPV type distribution stratified by cytological results showed a substantial equivalence between women with HIV and SPW in normal cytology: HPV-16 is the most common viral type in both populations, followed by HPV-52, 66, 31, 53 for SPW and by HPV-66, 53, 52, 31 for HIW. Due to the limited number of cases with abnormal cytology observed in this study, it was impossible to identify statistically significant differences of distribution between HIW and SPW. However, although HPV-16 is by far the most common type expressed in HSIL of SPW, there is a greater heterogeneity of genotypes in women with HIV infection and HSIL. Keller et al. [16] highlighted that HIV-infected women with HPV-16 infection and normal Pap test results have a similar pre-cancer risk as those with LSIL and therefore referral for colposcopy is warranted. However the presence of non HPV-16/18 infections was observed in 70% and 16.7% of HSIL in HIW and SPW respectively. McKenzie et al. [17] reported that cervical dysplasia specimens from 23 HIV infected women were infected (55%) by non-16/18 high-risk HPV types. If confirmed by larger cohorts, this finding may have major implications in the screening and triage strategies for women infected with HIV.As described by several Authors [18-19], it was observed that HIV-infected women have a higher prevalence of HPV and multi-type infections than the control group. A high prevalence of rare HPV types was detected in HIW, both in normal cytology and in cervical abnormalities and to date little is known about their carcinogenicity. The IARC classification and the analysis of rare HPV types show a wide variability in the carcinogenic potential of the rare and common HPV types found in normal cytology [1, 2]. In HIV-infected women, variability towards oncogenesis can be further influenced by the simultaneous presence of other cofactors, in particular cell-mediated immunosuppression [5], while the additional risks associated with each HPV type in multi-type infections is difficult to establish. Although no statistically significant differences were found due to lack of data, the description of viral types in the cervical specimens of HIV-infected women and correlation with the severity of cytological lesions can contribute to the mapping of HPV-related pre-invasive or invasive disease and provide a better risk stratification of disease evolution in HIV-infected women. Even in the context of primary prevention through vaccination, the wide distribution of viral types found in high-grade lesions of HIV-infected women demonstrates the need for multivalent vaccines. This means that a primary prophylaxis with a 9v-HPV vaccine could have prevented infections in over 50% of the women included in this study, whether HIV infected or not. The assessment of the impact of HPV vaccines in the study population shows how immunization with a 9v-HPV-vaccine could prevent a significantly higher proportion of viral infections, both in HIV-infected people and in the control population, than 2v- and 4v-HPV vaccines. The ICC preventable fraction in the general population increased by 12-19% when the 9v-HPV vaccine was introduced due to the addition of 7 high risk HPVs to the 2v or 4vHPV vaccines [10, 20, 21] which could be even higher in HIV-infected people considering the wide range of HPV types observed in progressive diseases. | Background:
Primary-prevention by prophylactic vaccination against HPV-related cancers and HPV-based screening programs are based on HPV-type distribution in immunocompetent individuals. HIV-infected women are at high risk of invasive HPV-disease sustained by a broader range of HPV-types and have higher multi-type infection rates than immunocompetent hosts.
Methods:
This is a cross-sectional analysis of High Risk HPV (HR HPV) type distribution in 805 HIV+ women (HIW) compared with a control group of 1402 immunocompetent HIV- women (SPW) enrolled in the VALHIDATE study in order to define HPV type-specific distribution according to cytology.
Results:
HIW had a 3.8, 3.6, and 2.7 times higher risk of atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) than SPW respectively. HPV-DNA prevalence was 28.4% in HIW and 11.81% in SPW (p<0.0001). The prevalence of infection increased from normal cytology to HSIL both in HIW (from 21.45% to 90.91%) and SPW (from 9.54% to 75%). The OR for women with normal cytology of having a positive HPV-DNA test result of was 2.6 times higher in HIW than in SPW. The cumulative prevalence of HPV-16/18 in HSIL is much lower in HIW (36.4±28.4) than SPW (62.5±33.5).
Conclusions:
A higher prevalence of infection and broader HPV type distribution were observed in HIV+ women compared to the general population. More than 60% of HSIL lesions of HIW patients are caused by single or multi-type infections from non-HPV16/18 HPVs. The potential 9v-HPV vaccine coverage could be even higher than that expected for the general population given the wide panel of HPV-types observed in the HSIL of HIV+ women. | 5,294 | 355 | [
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Case-Control Studies | Cervix Uteri | Coinfection | Cross-Sectional Studies | DNA, Viral | Female | Genotype | HIV Infections | Humans | Immunocompetence | Immunocompromised Host | Italy | Middle Aged | Molecular Epidemiology | Odds Ratio | Papillomaviridae | Papillomavirus Infections | Squamous Intraepithelial Lesions of the Cervix | Uterine Cervical Neoplasms [SUMMARY] | null | [CONTENT] Adult | Atypical Squamous Cells of the Cervix | Case-Control Studies | Cervix Uteri | Coinfection | Cross-Sectional Studies | DNA, Viral | Female | Genotype | HIV Infections | Humans | Immunocompetence | Immunocompromised Host | Italy | Middle Aged | Molecular Epidemiology | Odds Ratio | Papillomaviridae | Papillomavirus Infections | Squamous Intraepithelial Lesions of the Cervix | Uterine Cervical Neoplasms [SUMMARY] | [CONTENT] Adult | Atypical Squamous Cells of the Cervix | Case-Control Studies | Cervix Uteri | Coinfection | Cross-Sectional Studies | DNA, Viral | Female | Genotype | HIV Infections | Humans | Immunocompetence | Immunocompromised Host | Italy | Middle Aged | Molecular Epidemiology | Odds Ratio | Papillomaviridae | Papillomavirus Infections | Squamous Intraepithelial Lesions of the Cervix | Uterine Cervical Neoplasms [SUMMARY] | [CONTENT] Adult | Atypical Squamous Cells of the Cervix | Case-Control Studies | Cervix Uteri | Coinfection | Cross-Sectional Studies | DNA, Viral | Female | Genotype | HIV Infections | Humans | Immunocompetence | Immunocompromised Host | Italy | Middle Aged | Molecular Epidemiology | Odds Ratio | Papillomaviridae | Papillomavirus Infections | Squamous Intraepithelial Lesions of the Cervix | Uterine Cervical Neoplasms [SUMMARY] | [CONTENT] prevalence hpv associated | infections hpv types | risk hpv types | carcinogenic genotypes hpv | human papillomavirus hpv [SUMMARY] | [CONTENT] prevalence hpv associated | infections hpv types | risk hpv types | carcinogenic genotypes hpv | human papillomavirus hpv [SUMMARY] | null | [CONTENT] prevalence hpv associated | infections hpv types | risk hpv types | carcinogenic genotypes hpv | human papillomavirus hpv [SUMMARY] | [CONTENT] prevalence hpv associated | infections hpv types | risk hpv types | carcinogenic genotypes hpv | human papillomavirus hpv [SUMMARY] | [CONTENT] prevalence hpv associated | infections hpv types | risk hpv types | carcinogenic genotypes hpv | human papillomavirus hpv [SUMMARY] | [CONTENT] hpv | hiw | spw | types | women | type | dna | cytology | cytological | normal [SUMMARY] | [CONTENT] hpv | hiw | spw | types | women | type | dna | cytology | cytological | normal [SUMMARY] | null | [CONTENT] hpv | hiw | spw | types | women | type | dna | cytology | cytological | normal [SUMMARY] | [CONTENT] hpv | hiw | spw | types | women | type | dna | cytology | cytological | normal [SUMMARY] | [CONTENT] hpv | hiw | spw | types | women | type | dna | cytology | cytological | normal [SUMMARY] | [CONTENT] hpv | valent | vaccine | valent vaccine | invasive | carcinogenic | hiv | study | hpv type | women [SUMMARY] | [CONTENT] hpv | dna | test | genotyping | pap | hpv dna | baseline | graphpad | cervical | participating [SUMMARY] | null | [CONTENT] hpv | hiv | infected | hiv infected | 16 | women | infected women | hiv infected women | infections | hpv 16 [SUMMARY] | [CONTENT] hpv | hiw | spw | types | dna | women | 95 | hpv dna | 16 | risk [SUMMARY] | [CONTENT] hpv | hiw | spw | types | dna | women | 95 | hpv dna | 16 | risk [SUMMARY] | [CONTENT] HPV | HPV ||| HPV [SUMMARY] | [CONTENT] High Risk HPV | HPV | 805 | HIV+ | HIW | 1402 | SPW | HPV [SUMMARY] | null | [CONTENT] HPV | HIV+ ||| More than 60% | HSIL | HIW ||| 9v | HPV | HSIL | HIV+ [SUMMARY] | [CONTENT] HPV | HPV ||| HPV ||| High Risk HPV | HPV | 805 | HIV+ | HIW | 1402 | SPW | HPV ||| HIW | 3.8 | 3.6 | 2.7 | LSIL | HSIL | SPW ||| 28.4% | HIW | 11.81% | SPW ||| HSIL | HIW | 21.45% | 90.91% | SPW | 9.54% to 75% ||| 2.6 | HIW | SPW ||| HSIL | HIW | 36.4±28.4 | SPW | 62.5±33.5 ||| HPV | HIV+ ||| More than 60% | HSIL | HIW ||| 9v | HPV | HSIL | HIV+ [SUMMARY] | [CONTENT] HPV | HPV ||| HPV ||| High Risk HPV | HPV | 805 | HIV+ | HIW | 1402 | SPW | HPV ||| HIW | 3.8 | 3.6 | 2.7 | LSIL | HSIL | SPW ||| 28.4% | HIW | 11.81% | SPW ||| HSIL | HIW | 21.45% | 90.91% | SPW | 9.54% to 75% ||| 2.6 | HIW | SPW ||| HSIL | HIW | 36.4±28.4 | SPW | 62.5±33.5 ||| HPV | HIV+ ||| More than 60% | HSIL | HIW ||| 9v | HPV | HSIL | HIV+ [SUMMARY] |
|||||
Physiotherapists' perceptions and experiences of home-based rehabilitation in Libya: a qualitative study. | 35251450 | home-based rehabilitation (HBR) is a rehabilitation model that aims to help people with disabilities to integrate into the community and be independent as much as possible. HBR is a promising alternative to institution-based rehabilitation, in which rehabilitation services are provided at patients' homes. However, challenges and barriers to HBR practice in Libya have never been researched before. This study explores physiotherapists' perceptions of home-based rehabilitation (HBR) in Libya and examines their views and the concerns they face. | INTRODUCTION | eight physiotherapists (2 females, 6 males) with at least two years of work experience in the Libyan physiotherapy community participated in in-depth semi-structured interviews. The interviews were audio-recorded and transcribed verbatim, and the data were analyzed using framework method. | METHODS | three themes emerged from the data, namely: i) access problems, including lack of infrastructure; ii) lack of governmental policies, such as the absence of governmental support (e.g., lack of programs and resources); iii) poor awareness and misconception issues, including that of patients and families. | RESULTS | although all the interviewed physiotherapists described HBR as an essential practice in Libya, they expressed concerns about several factors that hinder its development and may influence the quality of interventions provided in the community. Given the fact that this is the first qualitative study in this field in Libya, there is a need for future research to explore HBR from other perspectives, such as those of policymakers, healthcare planners, or patients and their families and/or caregivers. | CONCLUSION | [
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] | 8856979 | Introduction | Home-based rehabilitation (HBR) is an alternative to traditional institution-based rehabilitation that aims to optimize health-related quality of life through delivering rehabilitation services at patients´ homes by rehabilitation professionals such as physiotherapists [1]. There is growing evidence in the literature that supports HBR over other rehabilitation settings [2,3]. Home-based rehabilitation has been documented as an effective intervention for many patient groups, among whom are stroke survivors [4], patients with total knee replacements [5], and patients with neuromuscular conditions [6], to mention a few. In Libya, rehabilitation services are provided in both the public and private sectors. Often, after discharge from hospitals, patients cannot receive rehabilitation services in private facilities, either due to economic reasons or because they are too frail or disabled to travel there regularly [7]. These individuals end up on long waiting lists for public hospitals, and the inherent delays may cause their conditions to worsen. Therefore, HBR is considered an important and relatively cost-effective way of providing rehabilitation services to the public. The healthcare problem in Libya was exacerbated by the war that followed the revolution in 2011 [8]. As a result, the healthcare system, as with many other governmental organizations, collapsed [9]. In early 2012, Libya requested support from the World Health Organization (WHO) to deal with the crippled healthcare system, as the war resulted in large numbers of injured people, often with permanent disabilities as a result of amputations and spinal cord injuries [10]. The current situation in Libya clearly calls for a more practical approach that can provide more efficient rehabilitation services such as HBR.
Despite the benefits and practicality of HBR as it pertains to Libya, there are still gaps in health policy research, and Tashani [11] suggested that Libya needs more research to inform policy developments. Furthermore, little is known about the perceptions of physiotherapists with respect to HBR for people with disabilities in the community. To date, there have been few studies internationally that have examined the views of physiotherapists who manage HBR. Examples of these studies include Carson et al. [12] and Hall et al. [13]. The interviewees of these studies mentioned several barriers that limit HBR services, including a lack of resources. With regard to Libya, there is a lack of qualitative research in the context of rehabilitation. To the best of our knowledge, this is the first qualitative study focusing specifically on the experiences of physiotherapists who provide HBR in Libya. As rehabilitation services in Libya are provided mainly by physiotherapists, this project was therefore limited to home-based physiotherapy services. Awareness of the perceptions and attitudes of physiotherapists is important, as ingrained beliefs about HBR may restrict its promotion and development. Understanding these views may also improve the therapeutic collaborative relationships between people with disabilities and healthcare providers, in which interns improve the quality of life of home-dwelling patients, relieve the burden on their families, and improve the healthcare system at large. Additionally, this research could provide the foundations necessary to develop and design HBR programs that are efficient and meaningful to the community. To that end, the aim of this research was to explore Libyan physiotherapists´ perceptions, views, and opinions on barriers that potentially influence current HBR practices in Libya. | Methods | Research design: this study was underpinned by phenomenology, as it aimed to achieve an in-depth understanding of reality from physiotherapists´ narratives as related to their experience of HBR services. To that end, qualitative in-depth semi-structured interviews were used.
Participants and recruitment: purposeful sampling was used for the identification of the study´s participants [14]. As stated by Cresswell [15], purposive sampling involves identifying and selecting individuals who are knowledgeable or experienced about a given phenomenon. The sample size was determined by data saturation [16]. When we reached the point where no new information were observed, data collection was terminated. However, to ensure the quality of the data collected, when data saturation was observed, two more interviews were conducted in order to make sure that we did not miss any relevant information. Recruitment took place in the city of Misrata (Libya) from August 4th to December 10th, 2019. The participants had no previous acquaintance with the interviewer. Variation in years of experience in HBR, gender of participant, and number of geographic locations of work experience were considered in order to obtain diversity in the experiences with HBR. The participants were eligible if they: i) were registered physiotherapists in Misrata city; ii) were able to sign the consent form; iii) were working in HBR practice and had at least two years of work experience in that field. Two years of HBR experience was applied to make sure that potential interviewees had enough knowledge and experience to discuss the concerns and challenges of HBR. The researchers initially approached two rehabilitation institutions and asked them to nominate relevant participants. To screen for the inclusion criteria, the researchers had preliminary face-to-face meetings with nominated participants prior to the interviews to confirm their eligibility and interest. Overall, seventeen physiotherapists were invited to a preliminary discussion; and out of them, eight agreed to participate.
Data collection: an interview guide was developed based on a literature review, clinical knowledge, and research experience see Table 1. The interview guide was piloted with professionals who were familiar with the topic, and minor modifications were made for simplicity and clarity. The researchers contacted potential participants via the phone and invited them to a face-to-face meeting. The meetings were scheduled based on the participants´ preferences, and were held in a private office in the participants´ workplaces. In the meeting, the study's purpose was explained, and the consent form was provided. The participants returned their signed consent forms before their appointments for the interviews. Semi-structured, in-depth telephone interviews were conducted at times that were convenient for the participants. With the participants´ permission, all interviews were audio-recorded and lasted between 30 to 45 minutes. The interviews, were in the Arabic language, and the quotations were translated to English by professional translators who were not in the research team by using the forward-backward method [17].
interview guide
Data analysis: the phone interviews were audio-recorded and transcribed verbatim by a professional transcriber. The transcripts were read several times by (AJ and AR) independently to get an overall understanding of meanings of the content. During this process, if clarification were needed, the participants were contacted, and the transcripts amended. The data were analyzed using the Framework Method [18], which involves five iterative steps: familiarization with the primary data, coding, identifying a thematic conceptual framework, applying the analytical framework, and charting data into the framework matrix [18,19]. The data analysis method was selected based on three main criteria: the research question, the nature of the data, and the pragmatics of working together in a multidisciplinary research team. The Framework Method of analysis was chosen because it meets the above criteria [20].
Rigor: to establish the trustworthiness of our data, we followed the recommendations by Shenton [21]. Credibility was achieved by conducting semi-structured in-depth interviews with an experienced qualitative interviewer, and the collected data were validated by employing a peer-debriefing technique [22]. Also, data analysis of the transcripts was done independently by the two authors of this study (Alhadi Mohamed Jahan and Ali Emhemed Rwaiha). The findings were then discussed by the research team and any disagreements were solved using the consensus technique [23]. Furthermore, the transcriptions were sent to the participants for them to confirm and to suggest changes in case of any misunderstandings. To address transferability, we relied on the variability of interviewees´ characteristics, including their work experience, as well as the rich variety of thoughts and quotations collected in the interviews. Moreover, the researchers who conducted this study came from the same community and work environment as those of the participants; this made the process of analyzing and interpreting the data more straightforward. Finally, the researchers attempted to provide sufficient details about the research methodology, context, and data collection in this report; this would enhance the reproducibility and the transferability of this study [24].
Ethics, consent, and permissions: ethical and research governance approvals for this project were obtained from the College of Medical Technology, Al-Tadamon Rehabilitation Centre and Alpha Medical Centre in Misrata, Libya Ethics certificate# EXT-198-2019. The objectives of the study and the voluntary nature of participation were discussed with the participants during face-to-face individual meetings with the researchers. The participants provided the signed consent form, including consent for being audio-recorded, before scheduling the telephone interviews. To ensure confidentiality, codes were used (e.g., PT3, PT5 etc.) instead of the participants´ names. We also did not collect any identifying information such as age or city of origin due to the limited number of physiotherapists who are involved in HBR in Misrata region. | Results | The sample consisted of eight physiotherapists (2 females, 6 males) with experience working with people with disabilities in HBR programs ranging from 4 to 10 years see Table 2. Three main aspects of the challenges facing the physiotherapists in HBR were described from the data and grouped according to three themes: access problems, lack of governmental policies, and poor awareness and misconception issues. These three themes and the corresponding subthemes are presented in the following section.
participant characteristics
Access problems Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”
Lack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”
Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”
Lack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”
Lack of governmental policies Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”
Poor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”
Lack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”
Poor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.
Patients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”
Family members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”
Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”
Poor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”
Lack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”
Poor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.
Patients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”
Family members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.” | Conclusion | Overall, this study provides the first qualitative attempt to explore physiotherapists’ experience with HBR from the point of view of the current challenges in Libya’s healthcare system. Moreover, the aim of this study was to present a useful starting point for the implementation and sustainable management of HBR services by approaching the process from the reality of practice and in relation to the available rehabilitation services. The interviews revealed challenges related to patients’ capabilities as well as access, government policies, and public awareness about the HBR model. By addressing these challenges, we can ensure that HBR services are delivered in a way that contributes to the best possible outcomes. Finally, it is worth noting that the current study explored the experiences of physiotherapists delivering care to a variety of patient populations, and therefore it is imperative to focus future research on addressing HBR challenges from the perspectives of people receiving these services as well as their family members and or caregivers.
What is known about this topic
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
What this study adds
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time. | [
"Access problems",
"Lack of governmental policies",
"\nWhat is known about this topic\n",
"\nWhat this study adds\n"
] | [
"Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”\nLack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”",
"Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”\nPoor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”\nLack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”\nPoor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.\nPatients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”\nFamily members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”",
"\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\nLimited resources is an acknowledged barrier to its application in western societies.\n\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\n\nLimited resources is an acknowledged barrier to its application in western societies.",
"\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.\n\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\n\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time."
] | [
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Results",
"Access problems",
"Lack of governmental policies",
"Discussion",
"Conclusion",
"\nWhat is known about this topic\n",
"\nWhat this study adds\n"
] | [
"Home-based rehabilitation (HBR) is an alternative to traditional institution-based rehabilitation that aims to optimize health-related quality of life through delivering rehabilitation services at patients´ homes by rehabilitation professionals such as physiotherapists [1]. There is growing evidence in the literature that supports HBR over other rehabilitation settings [2,3]. Home-based rehabilitation has been documented as an effective intervention for many patient groups, among whom are stroke survivors [4], patients with total knee replacements [5], and patients with neuromuscular conditions [6], to mention a few. In Libya, rehabilitation services are provided in both the public and private sectors. Often, after discharge from hospitals, patients cannot receive rehabilitation services in private facilities, either due to economic reasons or because they are too frail or disabled to travel there regularly [7]. These individuals end up on long waiting lists for public hospitals, and the inherent delays may cause their conditions to worsen. Therefore, HBR is considered an important and relatively cost-effective way of providing rehabilitation services to the public. The healthcare problem in Libya was exacerbated by the war that followed the revolution in 2011 [8]. As a result, the healthcare system, as with many other governmental organizations, collapsed [9]. In early 2012, Libya requested support from the World Health Organization (WHO) to deal with the crippled healthcare system, as the war resulted in large numbers of injured people, often with permanent disabilities as a result of amputations and spinal cord injuries [10]. The current situation in Libya clearly calls for a more practical approach that can provide more efficient rehabilitation services such as HBR.\nDespite the benefits and practicality of HBR as it pertains to Libya, there are still gaps in health policy research, and Tashani [11] suggested that Libya needs more research to inform policy developments. Furthermore, little is known about the perceptions of physiotherapists with respect to HBR for people with disabilities in the community. To date, there have been few studies internationally that have examined the views of physiotherapists who manage HBR. Examples of these studies include Carson et al. [12] and Hall et al. [13]. The interviewees of these studies mentioned several barriers that limit HBR services, including a lack of resources. With regard to Libya, there is a lack of qualitative research in the context of rehabilitation. To the best of our knowledge, this is the first qualitative study focusing specifically on the experiences of physiotherapists who provide HBR in Libya. As rehabilitation services in Libya are provided mainly by physiotherapists, this project was therefore limited to home-based physiotherapy services. Awareness of the perceptions and attitudes of physiotherapists is important, as ingrained beliefs about HBR may restrict its promotion and development. Understanding these views may also improve the therapeutic collaborative relationships between people with disabilities and healthcare providers, in which interns improve the quality of life of home-dwelling patients, relieve the burden on their families, and improve the healthcare system at large. Additionally, this research could provide the foundations necessary to develop and design HBR programs that are efficient and meaningful to the community. To that end, the aim of this research was to explore Libyan physiotherapists´ perceptions, views, and opinions on barriers that potentially influence current HBR practices in Libya.",
"Research design: this study was underpinned by phenomenology, as it aimed to achieve an in-depth understanding of reality from physiotherapists´ narratives as related to their experience of HBR services. To that end, qualitative in-depth semi-structured interviews were used.\nParticipants and recruitment: purposeful sampling was used for the identification of the study´s participants [14]. As stated by Cresswell [15], purposive sampling involves identifying and selecting individuals who are knowledgeable or experienced about a given phenomenon. The sample size was determined by data saturation [16]. When we reached the point where no new information were observed, data collection was terminated. However, to ensure the quality of the data collected, when data saturation was observed, two more interviews were conducted in order to make sure that we did not miss any relevant information. Recruitment took place in the city of Misrata (Libya) from August 4th to December 10th, 2019. The participants had no previous acquaintance with the interviewer. Variation in years of experience in HBR, gender of participant, and number of geographic locations of work experience were considered in order to obtain diversity in the experiences with HBR. The participants were eligible if they: i) were registered physiotherapists in Misrata city; ii) were able to sign the consent form; iii) were working in HBR practice and had at least two years of work experience in that field. Two years of HBR experience was applied to make sure that potential interviewees had enough knowledge and experience to discuss the concerns and challenges of HBR. The researchers initially approached two rehabilitation institutions and asked them to nominate relevant participants. To screen for the inclusion criteria, the researchers had preliminary face-to-face meetings with nominated participants prior to the interviews to confirm their eligibility and interest. Overall, seventeen physiotherapists were invited to a preliminary discussion; and out of them, eight agreed to participate.\nData collection: an interview guide was developed based on a literature review, clinical knowledge, and research experience see Table 1. The interview guide was piloted with professionals who were familiar with the topic, and minor modifications were made for simplicity and clarity. The researchers contacted potential participants via the phone and invited them to a face-to-face meeting. The meetings were scheduled based on the participants´ preferences, and were held in a private office in the participants´ workplaces. In the meeting, the study's purpose was explained, and the consent form was provided. The participants returned their signed consent forms before their appointments for the interviews. Semi-structured, in-depth telephone interviews were conducted at times that were convenient for the participants. With the participants´ permission, all interviews were audio-recorded and lasted between 30 to 45 minutes. The interviews, were in the Arabic language, and the quotations were translated to English by professional translators who were not in the research team by using the forward-backward method [17].\ninterview guide\nData analysis: the phone interviews were audio-recorded and transcribed verbatim by a professional transcriber. The transcripts were read several times by (AJ and AR) independently to get an overall understanding of meanings of the content. During this process, if clarification were needed, the participants were contacted, and the transcripts amended. The data were analyzed using the Framework Method [18], which involves five iterative steps: familiarization with the primary data, coding, identifying a thematic conceptual framework, applying the analytical framework, and charting data into the framework matrix [18,19]. The data analysis method was selected based on three main criteria: the research question, the nature of the data, and the pragmatics of working together in a multidisciplinary research team. The Framework Method of analysis was chosen because it meets the above criteria [20].\nRigor: to establish the trustworthiness of our data, we followed the recommendations by Shenton [21]. Credibility was achieved by conducting semi-structured in-depth interviews with an experienced qualitative interviewer, and the collected data were validated by employing a peer-debriefing technique [22]. Also, data analysis of the transcripts was done independently by the two authors of this study (Alhadi Mohamed Jahan and Ali Emhemed Rwaiha). The findings were then discussed by the research team and any disagreements were solved using the consensus technique [23]. Furthermore, the transcriptions were sent to the participants for them to confirm and to suggest changes in case of any misunderstandings. To address transferability, we relied on the variability of interviewees´ characteristics, including their work experience, as well as the rich variety of thoughts and quotations collected in the interviews. Moreover, the researchers who conducted this study came from the same community and work environment as those of the participants; this made the process of analyzing and interpreting the data more straightforward. Finally, the researchers attempted to provide sufficient details about the research methodology, context, and data collection in this report; this would enhance the reproducibility and the transferability of this study [24].\nEthics, consent, and permissions: ethical and research governance approvals for this project were obtained from the College of Medical Technology, Al-Tadamon Rehabilitation Centre and Alpha Medical Centre in Misrata, Libya Ethics certificate# EXT-198-2019. The objectives of the study and the voluntary nature of participation were discussed with the participants during face-to-face individual meetings with the researchers. The participants provided the signed consent form, including consent for being audio-recorded, before scheduling the telephone interviews. To ensure confidentiality, codes were used (e.g., PT3, PT5 etc.) instead of the participants´ names. We also did not collect any identifying information such as age or city of origin due to the limited number of physiotherapists who are involved in HBR in Misrata region.",
"The sample consisted of eight physiotherapists (2 females, 6 males) with experience working with people with disabilities in HBR programs ranging from 4 to 10 years see Table 2. Three main aspects of the challenges facing the physiotherapists in HBR were described from the data and grouped according to three themes: access problems, lack of governmental policies, and poor awareness and misconception issues. These three themes and the corresponding subthemes are presented in the following section.\nparticipant characteristics\n Access problems Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”\nLack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”\nPatients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”\nLack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”\n Lack of governmental policies Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”\nPoor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”\nLack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”\nPoor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.\nPatients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”\nFamily members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”\nAbsence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”\nPoor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”\nLack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”\nPoor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.\nPatients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”\nFamily members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”",
"Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”\nLack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”",
"Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”\nPoor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”\nLack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”\nPoor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.\nPatients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”\nFamily members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”",
"This research aimed to explore Libyan physiotherapists´ experiences and views about barriers to HBR services and to discuss how these insights may influence current HBR practices in Libya. The views of the interviewees were grouped into three main themes and seven subthemes. These views may help clinicians and policymakers in Libya and beyond to implement effective strategies that accommodate essential evidence-based practices. While many of these practices have been reported in the literature, our interviewees highlighted novel insights specific to developing countries like Libya. Evidently, this qualitative study is the first of its kind done in Libya that addresses the challenges and barriers in HBR. Our interviewees agreed that HBR is of utmost importance for the health care system in Libya today, as a large number of people have need for it, especially due to the ongoing war that started in 2011 following the revolution [8]. The challenges in the healthcare system are exacerbated by the closures of healthcare institutions due to violent attacks or due to lack of staff and equipment, as documented by the World Health Organization [25]. Therefore, as expected, the findings from the current study highlight several challenges that make HBR delivery in Libya complicated at this time. All the participants agreed that accessibility and patients' capabilities are the most critical factors influencing HBR delivery. By “patients' capabilities”, we mean the physical and cognitive capabilities of home-dwelling patients to fulfil and adhere to rehabilitation treatment plans. As our interviewees recognized, patients´ physical and cognitive abilities determine the success of HBR, because patients with complex medical conditions, such as stroke survivors, those with head and spinal cord injuries, or with other neurological conditions may find it hard to follow treatment plans effectively. Patients' cognitive and physical functioning may determine the success or failure of HBR; for example, patients with communication problems, multimorbidity, or frailness may pose challenges to physiotherapists when treating these populations [26,27]. In line with this theme, a recent qualitative study by Hall et al. (2017) suggested similar challenges for physiotherapists when treating patients with dementia [13], and similar experiences have been reported by physiotherapists in patients with neurological and palliative problems [12].\nIn the context of Libya, home-dwelling patients most likely have advanced medical conditions (e.g. cancer) or permanent disabilities (e.g., spinal cord injuries). Given the fact that there are no health insurance plans that cover important equipment, such as mobility assistive devices for instance, patients may end up bedridden for months and even years, and physiotherapists face multiple problems related to decreased mobility, which of course requires a lot of work and effort. Adding to the problem, patients sometimes experience cognitive or behavioral problems in addition to physical impairment which further hinder the provision of HBR services. Consistent with our findings, research has documented that physical and cognitive impairments make healthcare services for patients with such impairments more complicated [28,29]. Patients with physical and cognitive impairments pose extra challenges for their physiotherapists because they require more supervision, often experience behavioral problems, are unlikely to express gratitude, and are most likely to experience stress and depression [30]. Physical and cognitive impairments can also put extra pressure on physiotherapists to ensure their patients´ safety. Mental burdens are relatively high in the Libyan community as reported in literature [31]. These burdens pose several risks to patient safety in the home setting, such as environmental hazards, risk of falling, and patient education problems, to mention a few. Evidently there is a large research gap in Libya, as we do not know the prevalence of physical and mental burdens among patients who receive healthcare services at their homes, and we encourage further research in this regard. Therefore, assessing safety risks as well as access options, and determining clear inclusion and exclusion criteria when moving to HBR is warranted.\nIn order to meet the HBR goal, it was essential to first understand the barriers and challenges that face physiotherapists in Libya with regard to HBR delivery. Conceptualizing the HBR programs in Libya in a holistic way (i.e. thinking outside the box) might be necessary at this challenging time as the healthcare system struggles to address the basic medical needs of the community [32]. Therefore, it is imperative to plan HBR services as an “integrated community program” that benefits from a wide array of available community resources [1,33,34]. Thus, it is essential to involve community partners, such as non-governmental organizations, in HBR service delivery. This suggestion was mentioned by six out of the eight interviewees as a potential facilitator for HBR in Libya in the coming months or years. Another level of cooperation that was mentioned by our interviewees is networking with other healthcare professionals at the intermediate level. At this level, good communication with healthcare professionals in both private and public healthcare institutions could benefit not only the quality of service delivery, but also the development of HBR programs. Based on the participants´ views, this can be achieved by offering referral services when needed and providing training and technical supervision to rehabilitation personnel. All the aforementioned dimensions of professional networking have been discussed in the literature [33,35,36]. This view was further supported by a recent qualitative study in the United Kingdom. In that study, the researchers used a qualitative interview process that included eleven physiotherapists involved in delivering HBR in palliative care, and highlighted lack of cooperation with other healthcare professionals as a barrier to the provision of HBR to palliative patients [12].\nPoor awareness and misconception is another theme that emerged from the interviews with our participants. The interviewees agreed that the levels of awareness and attitudes among patients and their families play an instrumental role in HBR delivery. Some interviewees also connected levels of family awareness with treatment outcomes, as they noted better treatment outcomes when the patient and their family were aware of HBR´s goals and services. However, the interviewees did not hide their concerns about the difficulties in the implementation of the HBR model to urban regions due to misconceptions over the definition of HBR among the diverse cultural and ethnic groups in Libya. A few limitations of this study should be noted. First, the researchers used phone interviews to conduct the study, making it impossible to record non-verbal signals, which might affect the quality of the collected data. However, to ensure the accuracy of the collected data, transcriptions were sent to participants to confirm the transcriptions´ content. Second, all the participants were physiotherapists, so the findings of the current study stemmed solely from their points of view without considering patients´ or families´ opinions, which might affect the generalizability of the results. Also, the gender distribution of the interviewees was not equal, as we interviewed two females and six males. This gender imbalance is due to the fact that the majority of physiotherapists who provide HBR in Libya are males. Only a few females provide HBR services, and most of the females who were invited declined to participate in this study.",
"Overall, this study provides the first qualitative attempt to explore physiotherapists’ experience with HBR from the point of view of the current challenges in Libya’s healthcare system. Moreover, the aim of this study was to present a useful starting point for the implementation and sustainable management of HBR services by approaching the process from the reality of practice and in relation to the available rehabilitation services. The interviews revealed challenges related to patients’ capabilities as well as access, government policies, and public awareness about the HBR model. By addressing these challenges, we can ensure that HBR services are delivered in a way that contributes to the best possible outcomes. Finally, it is worth noting that the current study explored the experiences of physiotherapists delivering care to a variety of patient populations, and therefore it is imperative to focus future research on addressing HBR challenges from the perspectives of people receiving these services as well as their family members and or caregivers.\n \nWhat is known about this topic\n \n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\nLimited resources is an acknowledged barrier to its application in western societies.\n\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\n\nLimited resources is an acknowledged barrier to its application in western societies.\n\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\nLimited resources is an acknowledged barrier to its application in western societies.\n\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\n\nLimited resources is an acknowledged barrier to its application in western societies.\n \nWhat this study adds\n \n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.\n\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\n\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.\n\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.\n\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\n\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.",
"\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\nLimited resources is an acknowledged barrier to its application in western societies.\n\n\nHome-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;\n\n\nHome-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;\n\nLimited resources is an acknowledged barrier to its application in western societies.",
"\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.\n\n\nPublic's poor awareness and misconception about the HBR model is a big challenge in Libya;\n\n\nImproved documentations and communications between healthcare institutions can enhance the applicability of HBR;\n\nFuture research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time."
] | [
"intro",
"methods",
"results",
null,
null,
"discussion",
"conclusions",
null,
null
] | [
"Home-based rehabilitation",
"experiences",
"physiotherapy",
"barriers",
"qualitative",
"Libya"
] | Introduction:
Home-based rehabilitation (HBR) is an alternative to traditional institution-based rehabilitation that aims to optimize health-related quality of life through delivering rehabilitation services at patients´ homes by rehabilitation professionals such as physiotherapists [1]. There is growing evidence in the literature that supports HBR over other rehabilitation settings [2,3]. Home-based rehabilitation has been documented as an effective intervention for many patient groups, among whom are stroke survivors [4], patients with total knee replacements [5], and patients with neuromuscular conditions [6], to mention a few. In Libya, rehabilitation services are provided in both the public and private sectors. Often, after discharge from hospitals, patients cannot receive rehabilitation services in private facilities, either due to economic reasons or because they are too frail or disabled to travel there regularly [7]. These individuals end up on long waiting lists for public hospitals, and the inherent delays may cause their conditions to worsen. Therefore, HBR is considered an important and relatively cost-effective way of providing rehabilitation services to the public. The healthcare problem in Libya was exacerbated by the war that followed the revolution in 2011 [8]. As a result, the healthcare system, as with many other governmental organizations, collapsed [9]. In early 2012, Libya requested support from the World Health Organization (WHO) to deal with the crippled healthcare system, as the war resulted in large numbers of injured people, often with permanent disabilities as a result of amputations and spinal cord injuries [10]. The current situation in Libya clearly calls for a more practical approach that can provide more efficient rehabilitation services such as HBR.
Despite the benefits and practicality of HBR as it pertains to Libya, there are still gaps in health policy research, and Tashani [11] suggested that Libya needs more research to inform policy developments. Furthermore, little is known about the perceptions of physiotherapists with respect to HBR for people with disabilities in the community. To date, there have been few studies internationally that have examined the views of physiotherapists who manage HBR. Examples of these studies include Carson et al. [12] and Hall et al. [13]. The interviewees of these studies mentioned several barriers that limit HBR services, including a lack of resources. With regard to Libya, there is a lack of qualitative research in the context of rehabilitation. To the best of our knowledge, this is the first qualitative study focusing specifically on the experiences of physiotherapists who provide HBR in Libya. As rehabilitation services in Libya are provided mainly by physiotherapists, this project was therefore limited to home-based physiotherapy services. Awareness of the perceptions and attitudes of physiotherapists is important, as ingrained beliefs about HBR may restrict its promotion and development. Understanding these views may also improve the therapeutic collaborative relationships between people with disabilities and healthcare providers, in which interns improve the quality of life of home-dwelling patients, relieve the burden on their families, and improve the healthcare system at large. Additionally, this research could provide the foundations necessary to develop and design HBR programs that are efficient and meaningful to the community. To that end, the aim of this research was to explore Libyan physiotherapists´ perceptions, views, and opinions on barriers that potentially influence current HBR practices in Libya.
Methods:
Research design: this study was underpinned by phenomenology, as it aimed to achieve an in-depth understanding of reality from physiotherapists´ narratives as related to their experience of HBR services. To that end, qualitative in-depth semi-structured interviews were used.
Participants and recruitment: purposeful sampling was used for the identification of the study´s participants [14]. As stated by Cresswell [15], purposive sampling involves identifying and selecting individuals who are knowledgeable or experienced about a given phenomenon. The sample size was determined by data saturation [16]. When we reached the point where no new information were observed, data collection was terminated. However, to ensure the quality of the data collected, when data saturation was observed, two more interviews were conducted in order to make sure that we did not miss any relevant information. Recruitment took place in the city of Misrata (Libya) from August 4th to December 10th, 2019. The participants had no previous acquaintance with the interviewer. Variation in years of experience in HBR, gender of participant, and number of geographic locations of work experience were considered in order to obtain diversity in the experiences with HBR. The participants were eligible if they: i) were registered physiotherapists in Misrata city; ii) were able to sign the consent form; iii) were working in HBR practice and had at least two years of work experience in that field. Two years of HBR experience was applied to make sure that potential interviewees had enough knowledge and experience to discuss the concerns and challenges of HBR. The researchers initially approached two rehabilitation institutions and asked them to nominate relevant participants. To screen for the inclusion criteria, the researchers had preliminary face-to-face meetings with nominated participants prior to the interviews to confirm their eligibility and interest. Overall, seventeen physiotherapists were invited to a preliminary discussion; and out of them, eight agreed to participate.
Data collection: an interview guide was developed based on a literature review, clinical knowledge, and research experience see Table 1. The interview guide was piloted with professionals who were familiar with the topic, and minor modifications were made for simplicity and clarity. The researchers contacted potential participants via the phone and invited them to a face-to-face meeting. The meetings were scheduled based on the participants´ preferences, and were held in a private office in the participants´ workplaces. In the meeting, the study's purpose was explained, and the consent form was provided. The participants returned their signed consent forms before their appointments for the interviews. Semi-structured, in-depth telephone interviews were conducted at times that were convenient for the participants. With the participants´ permission, all interviews were audio-recorded and lasted between 30 to 45 minutes. The interviews, were in the Arabic language, and the quotations were translated to English by professional translators who were not in the research team by using the forward-backward method [17].
interview guide
Data analysis: the phone interviews were audio-recorded and transcribed verbatim by a professional transcriber. The transcripts were read several times by (AJ and AR) independently to get an overall understanding of meanings of the content. During this process, if clarification were needed, the participants were contacted, and the transcripts amended. The data were analyzed using the Framework Method [18], which involves five iterative steps: familiarization with the primary data, coding, identifying a thematic conceptual framework, applying the analytical framework, and charting data into the framework matrix [18,19]. The data analysis method was selected based on three main criteria: the research question, the nature of the data, and the pragmatics of working together in a multidisciplinary research team. The Framework Method of analysis was chosen because it meets the above criteria [20].
Rigor: to establish the trustworthiness of our data, we followed the recommendations by Shenton [21]. Credibility was achieved by conducting semi-structured in-depth interviews with an experienced qualitative interviewer, and the collected data were validated by employing a peer-debriefing technique [22]. Also, data analysis of the transcripts was done independently by the two authors of this study (Alhadi Mohamed Jahan and Ali Emhemed Rwaiha). The findings were then discussed by the research team and any disagreements were solved using the consensus technique [23]. Furthermore, the transcriptions were sent to the participants for them to confirm and to suggest changes in case of any misunderstandings. To address transferability, we relied on the variability of interviewees´ characteristics, including their work experience, as well as the rich variety of thoughts and quotations collected in the interviews. Moreover, the researchers who conducted this study came from the same community and work environment as those of the participants; this made the process of analyzing and interpreting the data more straightforward. Finally, the researchers attempted to provide sufficient details about the research methodology, context, and data collection in this report; this would enhance the reproducibility and the transferability of this study [24].
Ethics, consent, and permissions: ethical and research governance approvals for this project were obtained from the College of Medical Technology, Al-Tadamon Rehabilitation Centre and Alpha Medical Centre in Misrata, Libya Ethics certificate# EXT-198-2019. The objectives of the study and the voluntary nature of participation were discussed with the participants during face-to-face individual meetings with the researchers. The participants provided the signed consent form, including consent for being audio-recorded, before scheduling the telephone interviews. To ensure confidentiality, codes were used (e.g., PT3, PT5 etc.) instead of the participants´ names. We also did not collect any identifying information such as age or city of origin due to the limited number of physiotherapists who are involved in HBR in Misrata region.
Results:
The sample consisted of eight physiotherapists (2 females, 6 males) with experience working with people with disabilities in HBR programs ranging from 4 to 10 years see Table 2. Three main aspects of the challenges facing the physiotherapists in HBR were described from the data and grouped according to three themes: access problems, lack of governmental policies, and poor awareness and misconception issues. These three themes and the corresponding subthemes are presented in the following section.
participant characteristics
Access problems Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”
Lack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”
Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”
Lack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”
Lack of governmental policies Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”
Poor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”
Lack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”
Poor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.
Patients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”
Family members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”
Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”
Poor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”
Lack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”
Poor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.
Patients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”
Family members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”
Access problems:
Patients´ capability: patients´ capability was the most common subtheme reported by the participants. The participants agreed that a patient's physical capacity to do tasks as recommended by their physiotherapist is considered an essential factor that controls the delivery of HBR. PT6: “Most patients with mobility limitations need to use a wheelchair or other device to help them to move, so if they do not have any assistive devices at home, physiotherapy will not be as effective as it is supposed to be, especially in the first few weeks after injury.” Any patient who is experiencing physical impairment, is bedridden, or has limited mobility is a potential candidate for HBR. However, patients who are cognitively or intellectually challenged or are medically fragile due to a chronic condition which results in a prolonged dependency on medical care for which daily skilled nursing intervention is medically necessary, renders HBR services next to impossible. PT8: “Patients with complicated medical conditions cannot have HBR sessions at all. This also applies to elders with cognitive problems who require a caregiver available at all times to support the physiotherapist during the treatment session.”
Lack of infrastructure: some participants thought that a lack of public transport was a barrier to HBR applications, as this restricts the ability of physiotherapists to be involved in HBR. Also, poor transportation further limits the ability of physiotherapists of low socio-economic levels who do not own vehicles to access patients´ homes to provide treatment. PT2: “you know, in Libya; we do not have public transports like buses. Therefore, you have to rely on your private car or a taxi, which are not always available.” Furthermore, participants talked about other barriers like poor road conditions, especially in the winter months. For example, in rural areas, the roads and infrastructure are deficient or lacking; therefore, lack of access to patients who live in such areas is an obstacle to the implementation of HBR. PT3: “The unpaved roads prevent physiotherapists from accessing their patients at home easily you know, in the winter months, roads are literally blocked due to accumulated rainwater, maybe because of poor drainage systems, I really do not know.”
Lack of governmental policies:
Absence of government support: from the participants´ point of view, the absence of government support for HBR programs has an adverse effect on service delivery. Since patients often require equipment, the absence of funds from the government affects the progress of HBR. PT4: “The government should support the patients and provide them with assistive devices or other expensive in-home devices that facilitate treatment sessions... ”
Poor medical management: limited documentation of patient information and poor communication between healthcare institutions was a significant barrier mentioned by all the interviewees. From the participants´ point of view, physiotherapists need to get access to a patient´s information beforehand in order to get a good picture of their condition. Equally important, awareness of other environmental factors is essential for planning a treatment program that accommodates important ethical, psychological, and social considerations. PT5: “The policymakers in the healthcare system need to develop programs for better documentation and communication between healthcare providers in order to help physiotherapist to provide HBR services efficiently...”
Lack of collaboration and teamwork: a few interviewees mentioned the role of collaboration with other healthcare professionals, as some patients need more than just physiotherapy. For example, patients may sometimes be frail seniors who need neurologists, psychologists, or social workers in addition to physiotherapists or occupational therapists. Therefore, working as a team during the physiotherapy process could positively influence the rehabilitation outcomes. PT1: “ ...legislation by the Ministry of Health and the Ministry of Social Works for implementing HBR lacks important details about teamwork...” Moreover, participants stressed the importance of multidisciplinary teams in providing care for those living at home: PT7 : “ ...Home-based rehabilitation programs should include a team of healthcare professionals such as physicians, psychologists, and occupational therapists in order to achieve better outcomes...”
Poor awareness and misconception issues: poor awareness of HBR and how it differs from institution-based rehabilitation can cause several problems. According to most of the participants, awareness about HBR services plays an essential role in the effective and smooth delivery of such services. The participants highlighted two components of poor awareness and misconception that should be considered: patients´ and family members´. Most of the participants agreed that a lack of patient and family knowledge regarding home visits provided by physiotherapists could interrupt the treatment plan, as many questions and misconceptions may arise after the beginning of the program. In other words, the greater the awareness, the better the delivery of HBR services.
Patients´ poor awareness and misconceptions: a few participants voiced that patients often thought that having treatment sessions at their home was not appropriate for them. PT3: “ ...some patients think that physiotherapy Also, when patients believe that their disability is a result of the aging process and that physiotherapists cannot make it any better, their misconception complicates the treatment plan. PT6: “...elderly patients believe that they experience mobility limitations because they are old and therefore, physiotherapy will not help them. ”Furthermore, patients sometimes believed that their homes were very personal spaces, and they did not want any “strangers” there. PT4: “...some patients say that their home is a private place and they refuse any strangers wanting to come into their home, and if you did, they look at you as being unwelcome it the situation is uncomfortable for everyone. ”Moreover, participants repeatedly mentioned that patients often request physiotherapists PT1: “Some patients stated that they would prefer to see a physiotherapist they were already familiar with and they trusted to deliver HBR.” One participant highlighted the importance of patient education in promoting awareness and perceptions about HBR services. PT3: “I have been working for years in home-based rehabilitation, and none of my patients have ever had any educational training about their condition or about HBR.”
Family members´ poor awareness and misconceptions: most home-based patients receive some level of care from their family members, but this informal care can be significantly interrupted in scope, duration, and intensity due to misperceptions about HBR. Often, informal caregivers think that HBR can be substituted with care provided by family members. PT2: “Family members sometimes stop providing support for patients when HBR starts they think that physiotherapy visits are enough to cure their loved ones.” Likewise, participant PT1 said: “There is a general belief among family members that physiotherapists are healthcare professionals who can treat the patients alone, without any help from the families, which is wrong we physiotherapists need to work together with family members to achieve meaningful outcomes.”
Discussion:
This research aimed to explore Libyan physiotherapists´ experiences and views about barriers to HBR services and to discuss how these insights may influence current HBR practices in Libya. The views of the interviewees were grouped into three main themes and seven subthemes. These views may help clinicians and policymakers in Libya and beyond to implement effective strategies that accommodate essential evidence-based practices. While many of these practices have been reported in the literature, our interviewees highlighted novel insights specific to developing countries like Libya. Evidently, this qualitative study is the first of its kind done in Libya that addresses the challenges and barriers in HBR. Our interviewees agreed that HBR is of utmost importance for the health care system in Libya today, as a large number of people have need for it, especially due to the ongoing war that started in 2011 following the revolution [8]. The challenges in the healthcare system are exacerbated by the closures of healthcare institutions due to violent attacks or due to lack of staff and equipment, as documented by the World Health Organization [25]. Therefore, as expected, the findings from the current study highlight several challenges that make HBR delivery in Libya complicated at this time. All the participants agreed that accessibility and patients' capabilities are the most critical factors influencing HBR delivery. By “patients' capabilities”, we mean the physical and cognitive capabilities of home-dwelling patients to fulfil and adhere to rehabilitation treatment plans. As our interviewees recognized, patients´ physical and cognitive abilities determine the success of HBR, because patients with complex medical conditions, such as stroke survivors, those with head and spinal cord injuries, or with other neurological conditions may find it hard to follow treatment plans effectively. Patients' cognitive and physical functioning may determine the success or failure of HBR; for example, patients with communication problems, multimorbidity, or frailness may pose challenges to physiotherapists when treating these populations [26,27]. In line with this theme, a recent qualitative study by Hall et al. (2017) suggested similar challenges for physiotherapists when treating patients with dementia [13], and similar experiences have been reported by physiotherapists in patients with neurological and palliative problems [12].
In the context of Libya, home-dwelling patients most likely have advanced medical conditions (e.g. cancer) or permanent disabilities (e.g., spinal cord injuries). Given the fact that there are no health insurance plans that cover important equipment, such as mobility assistive devices for instance, patients may end up bedridden for months and even years, and physiotherapists face multiple problems related to decreased mobility, which of course requires a lot of work and effort. Adding to the problem, patients sometimes experience cognitive or behavioral problems in addition to physical impairment which further hinder the provision of HBR services. Consistent with our findings, research has documented that physical and cognitive impairments make healthcare services for patients with such impairments more complicated [28,29]. Patients with physical and cognitive impairments pose extra challenges for their physiotherapists because they require more supervision, often experience behavioral problems, are unlikely to express gratitude, and are most likely to experience stress and depression [30]. Physical and cognitive impairments can also put extra pressure on physiotherapists to ensure their patients´ safety. Mental burdens are relatively high in the Libyan community as reported in literature [31]. These burdens pose several risks to patient safety in the home setting, such as environmental hazards, risk of falling, and patient education problems, to mention a few. Evidently there is a large research gap in Libya, as we do not know the prevalence of physical and mental burdens among patients who receive healthcare services at their homes, and we encourage further research in this regard. Therefore, assessing safety risks as well as access options, and determining clear inclusion and exclusion criteria when moving to HBR is warranted.
In order to meet the HBR goal, it was essential to first understand the barriers and challenges that face physiotherapists in Libya with regard to HBR delivery. Conceptualizing the HBR programs in Libya in a holistic way (i.e. thinking outside the box) might be necessary at this challenging time as the healthcare system struggles to address the basic medical needs of the community [32]. Therefore, it is imperative to plan HBR services as an “integrated community program” that benefits from a wide array of available community resources [1,33,34]. Thus, it is essential to involve community partners, such as non-governmental organizations, in HBR service delivery. This suggestion was mentioned by six out of the eight interviewees as a potential facilitator for HBR in Libya in the coming months or years. Another level of cooperation that was mentioned by our interviewees is networking with other healthcare professionals at the intermediate level. At this level, good communication with healthcare professionals in both private and public healthcare institutions could benefit not only the quality of service delivery, but also the development of HBR programs. Based on the participants´ views, this can be achieved by offering referral services when needed and providing training and technical supervision to rehabilitation personnel. All the aforementioned dimensions of professional networking have been discussed in the literature [33,35,36]. This view was further supported by a recent qualitative study in the United Kingdom. In that study, the researchers used a qualitative interview process that included eleven physiotherapists involved in delivering HBR in palliative care, and highlighted lack of cooperation with other healthcare professionals as a barrier to the provision of HBR to palliative patients [12].
Poor awareness and misconception is another theme that emerged from the interviews with our participants. The interviewees agreed that the levels of awareness and attitudes among patients and their families play an instrumental role in HBR delivery. Some interviewees also connected levels of family awareness with treatment outcomes, as they noted better treatment outcomes when the patient and their family were aware of HBR´s goals and services. However, the interviewees did not hide their concerns about the difficulties in the implementation of the HBR model to urban regions due to misconceptions over the definition of HBR among the diverse cultural and ethnic groups in Libya. A few limitations of this study should be noted. First, the researchers used phone interviews to conduct the study, making it impossible to record non-verbal signals, which might affect the quality of the collected data. However, to ensure the accuracy of the collected data, transcriptions were sent to participants to confirm the transcriptions´ content. Second, all the participants were physiotherapists, so the findings of the current study stemmed solely from their points of view without considering patients´ or families´ opinions, which might affect the generalizability of the results. Also, the gender distribution of the interviewees was not equal, as we interviewed two females and six males. This gender imbalance is due to the fact that the majority of physiotherapists who provide HBR in Libya are males. Only a few females provide HBR services, and most of the females who were invited declined to participate in this study.
Conclusion:
Overall, this study provides the first qualitative attempt to explore physiotherapists’ experience with HBR from the point of view of the current challenges in Libya’s healthcare system. Moreover, the aim of this study was to present a useful starting point for the implementation and sustainable management of HBR services by approaching the process from the reality of practice and in relation to the available rehabilitation services. The interviews revealed challenges related to patients’ capabilities as well as access, government policies, and public awareness about the HBR model. By addressing these challenges, we can ensure that HBR services are delivered in a way that contributes to the best possible outcomes. Finally, it is worth noting that the current study explored the experiences of physiotherapists delivering care to a variety of patient populations, and therefore it is imperative to focus future research on addressing HBR challenges from the perspectives of people receiving these services as well as their family members and or caregivers.
What is known about this topic
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
What this study adds
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
What is known about this topic
:
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
What this study adds
:
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
| Background:
home-based rehabilitation (HBR) is a rehabilitation model that aims to help people with disabilities to integrate into the community and be independent as much as possible. HBR is a promising alternative to institution-based rehabilitation, in which rehabilitation services are provided at patients' homes. However, challenges and barriers to HBR practice in Libya have never been researched before. This study explores physiotherapists' perceptions of home-based rehabilitation (HBR) in Libya and examines their views and the concerns they face.
Methods:
eight physiotherapists (2 females, 6 males) with at least two years of work experience in the Libyan physiotherapy community participated in in-depth semi-structured interviews. The interviews were audio-recorded and transcribed verbatim, and the data were analyzed using framework method.
Results:
three themes emerged from the data, namely: i) access problems, including lack of infrastructure; ii) lack of governmental policies, such as the absence of governmental support (e.g., lack of programs and resources); iii) poor awareness and misconception issues, including that of patients and families.
Conclusions:
although all the interviewed physiotherapists described HBR as an essential practice in Libya, they expressed concerns about several factors that hinder its development and may influence the quality of interventions provided in the community. Given the fact that this is the first qualitative study in this field in Libya, there is a need for future research to explore HBR from other perspectives, such as those of policymakers, healthcare planners, or patients and their families and/or caregivers. | Introduction:
Home-based rehabilitation (HBR) is an alternative to traditional institution-based rehabilitation that aims to optimize health-related quality of life through delivering rehabilitation services at patients´ homes by rehabilitation professionals such as physiotherapists [1]. There is growing evidence in the literature that supports HBR over other rehabilitation settings [2,3]. Home-based rehabilitation has been documented as an effective intervention for many patient groups, among whom are stroke survivors [4], patients with total knee replacements [5], and patients with neuromuscular conditions [6], to mention a few. In Libya, rehabilitation services are provided in both the public and private sectors. Often, after discharge from hospitals, patients cannot receive rehabilitation services in private facilities, either due to economic reasons or because they are too frail or disabled to travel there regularly [7]. These individuals end up on long waiting lists for public hospitals, and the inherent delays may cause their conditions to worsen. Therefore, HBR is considered an important and relatively cost-effective way of providing rehabilitation services to the public. The healthcare problem in Libya was exacerbated by the war that followed the revolution in 2011 [8]. As a result, the healthcare system, as with many other governmental organizations, collapsed [9]. In early 2012, Libya requested support from the World Health Organization (WHO) to deal with the crippled healthcare system, as the war resulted in large numbers of injured people, often with permanent disabilities as a result of amputations and spinal cord injuries [10]. The current situation in Libya clearly calls for a more practical approach that can provide more efficient rehabilitation services such as HBR.
Despite the benefits and practicality of HBR as it pertains to Libya, there are still gaps in health policy research, and Tashani [11] suggested that Libya needs more research to inform policy developments. Furthermore, little is known about the perceptions of physiotherapists with respect to HBR for people with disabilities in the community. To date, there have been few studies internationally that have examined the views of physiotherapists who manage HBR. Examples of these studies include Carson et al. [12] and Hall et al. [13]. The interviewees of these studies mentioned several barriers that limit HBR services, including a lack of resources. With regard to Libya, there is a lack of qualitative research in the context of rehabilitation. To the best of our knowledge, this is the first qualitative study focusing specifically on the experiences of physiotherapists who provide HBR in Libya. As rehabilitation services in Libya are provided mainly by physiotherapists, this project was therefore limited to home-based physiotherapy services. Awareness of the perceptions and attitudes of physiotherapists is important, as ingrained beliefs about HBR may restrict its promotion and development. Understanding these views may also improve the therapeutic collaborative relationships between people with disabilities and healthcare providers, in which interns improve the quality of life of home-dwelling patients, relieve the burden on their families, and improve the healthcare system at large. Additionally, this research could provide the foundations necessary to develop and design HBR programs that are efficient and meaningful to the community. To that end, the aim of this research was to explore Libyan physiotherapists´ perceptions, views, and opinions on barriers that potentially influence current HBR practices in Libya.
Conclusion:
Overall, this study provides the first qualitative attempt to explore physiotherapists’ experience with HBR from the point of view of the current challenges in Libya’s healthcare system. Moreover, the aim of this study was to present a useful starting point for the implementation and sustainable management of HBR services by approaching the process from the reality of practice and in relation to the available rehabilitation services. The interviews revealed challenges related to patients’ capabilities as well as access, government policies, and public awareness about the HBR model. By addressing these challenges, we can ensure that HBR services are delivered in a way that contributes to the best possible outcomes. Finally, it is worth noting that the current study explored the experiences of physiotherapists delivering care to a variety of patient populations, and therefore it is imperative to focus future research on addressing HBR challenges from the perspectives of people receiving these services as well as their family members and or caregivers.
What is known about this topic
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
Home-based rehabilitation is an effective rehabilitation model that can relieve the stress on the healthcare system by providing services at patients’ homes;
Home-based rehabilitation can benefit a large population, including stroke survivors, patients with spinal cord injuries, and the elderly, to mention a few;
Limited resources is an acknowledged barrier to its application in western societies.
What this study adds
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time.
Public's poor awareness and misconception about the HBR model is a big challenge in Libya;
Improved documentations and communications between healthcare institutions can enhance the applicability of HBR;
Future research should focus on the community-dwelling patients as data on the prevalence of medical, psychological, and social burdens among such populations in Libya are not available at this time. | Background:
home-based rehabilitation (HBR) is a rehabilitation model that aims to help people with disabilities to integrate into the community and be independent as much as possible. HBR is a promising alternative to institution-based rehabilitation, in which rehabilitation services are provided at patients' homes. However, challenges and barriers to HBR practice in Libya have never been researched before. This study explores physiotherapists' perceptions of home-based rehabilitation (HBR) in Libya and examines their views and the concerns they face.
Methods:
eight physiotherapists (2 females, 6 males) with at least two years of work experience in the Libyan physiotherapy community participated in in-depth semi-structured interviews. The interviews were audio-recorded and transcribed verbatim, and the data were analyzed using framework method.
Results:
three themes emerged from the data, namely: i) access problems, including lack of infrastructure; ii) lack of governmental policies, such as the absence of governmental support (e.g., lack of programs and resources); iii) poor awareness and misconception issues, including that of patients and families.
Conclusions:
although all the interviewed physiotherapists described HBR as an essential practice in Libya, they expressed concerns about several factors that hinder its development and may influence the quality of interventions provided in the community. Given the fact that this is the first qualitative study in this field in Libya, there is a need for future research to explore HBR from other perspectives, such as those of policymakers, healthcare planners, or patients and their families and/or caregivers. | 8,083 | 302 | [
415,
864,
142,
138
] | 9 | [
"hbr",
"patients",
"participants",
"physiotherapists",
"home",
"rehabilitation",
"services",
"healthcare",
"libya",
"awareness"
] | [
"efficient rehabilitation services",
"providing rehabilitation services",
"based rehabilitation hbr",
"mention libya rehabilitation",
"hbr libya rehabilitation"
] | [CONTENT] Home-based rehabilitation | experiences | physiotherapy | barriers | qualitative | Libya [SUMMARY] | [CONTENT] Home-based rehabilitation | experiences | physiotherapy | barriers | qualitative | Libya [SUMMARY] | [CONTENT] Home-based rehabilitation | experiences | physiotherapy | barriers | qualitative | Libya [SUMMARY] | [CONTENT] Home-based rehabilitation | experiences | physiotherapy | barriers | qualitative | Libya [SUMMARY] | [CONTENT] Home-based rehabilitation | experiences | physiotherapy | barriers | qualitative | Libya [SUMMARY] | [CONTENT] Home-based rehabilitation | experiences | physiotherapy | barriers | qualitative | Libya [SUMMARY] | [CONTENT] Caregivers | Female | Humans | Libya | Male | Physical Therapists | Physical Therapy Modalities | Qualitative Research [SUMMARY] | [CONTENT] Caregivers | Female | Humans | Libya | Male | Physical Therapists | Physical Therapy Modalities | Qualitative Research [SUMMARY] | [CONTENT] Caregivers | Female | Humans | Libya | Male | Physical Therapists | Physical Therapy Modalities | Qualitative Research [SUMMARY] | [CONTENT] Caregivers | Female | Humans | Libya | Male | Physical Therapists | Physical Therapy Modalities | Qualitative Research [SUMMARY] | [CONTENT] Caregivers | Female | Humans | Libya | Male | Physical Therapists | Physical Therapy Modalities | Qualitative Research [SUMMARY] | [CONTENT] Caregivers | Female | Humans | Libya | Male | Physical Therapists | Physical Therapy Modalities | Qualitative Research [SUMMARY] | [CONTENT] efficient rehabilitation services | providing rehabilitation services | based rehabilitation hbr | mention libya rehabilitation | hbr libya rehabilitation [SUMMARY] | [CONTENT] efficient rehabilitation services | providing rehabilitation services | based rehabilitation hbr | mention libya rehabilitation | hbr libya rehabilitation [SUMMARY] | [CONTENT] efficient rehabilitation services | providing rehabilitation services | based rehabilitation hbr | mention libya rehabilitation | hbr libya rehabilitation [SUMMARY] | [CONTENT] efficient rehabilitation services | providing rehabilitation services | based rehabilitation hbr | mention libya rehabilitation | hbr libya rehabilitation [SUMMARY] | [CONTENT] efficient rehabilitation services | providing rehabilitation services | based rehabilitation hbr | mention libya rehabilitation | hbr libya rehabilitation [SUMMARY] | [CONTENT] efficient rehabilitation services | providing rehabilitation services | based rehabilitation hbr | mention libya rehabilitation | hbr libya rehabilitation [SUMMARY] | [CONTENT] hbr | patients | participants | physiotherapists | home | rehabilitation | services | healthcare | libya | awareness [SUMMARY] | [CONTENT] hbr | patients | participants | physiotherapists | home | rehabilitation | services | healthcare | libya | awareness [SUMMARY] | [CONTENT] hbr | patients | participants | physiotherapists | home | rehabilitation | services | healthcare | libya | awareness [SUMMARY] | [CONTENT] hbr | patients | participants | physiotherapists | home | rehabilitation | services | healthcare | libya | awareness [SUMMARY] | [CONTENT] hbr | patients | participants | physiotherapists | home | rehabilitation | services | healthcare | libya | awareness [SUMMARY] | [CONTENT] hbr | patients | participants | physiotherapists | home | rehabilitation | services | healthcare | libya | awareness [SUMMARY] | [CONTENT] rehabilitation | hbr | rehabilitation services | libya | services | physiotherapists | improve | studies | research | views [SUMMARY] | [CONTENT] participants | data | interviews | consent | face | researchers | framework | research | experience | study [SUMMARY] | [CONTENT] patients | hbr | participants | poor | family | home | physiotherapists | family members | members | awareness [SUMMARY] | [CONTENT] rehabilitation | model | patients | hbr | based rehabilitation | home based rehabilitation | home based | future research | future | focus [SUMMARY] | [CONTENT] hbr | patients | participants | rehabilitation | home | physiotherapists | libya | healthcare | services | poor [SUMMARY] | [CONTENT] hbr | patients | participants | rehabilitation | home | physiotherapists | libya | healthcare | services | poor [SUMMARY] | [CONTENT] HBR ||| HBR ||| HBR | Libya ||| HBR | Libya [SUMMARY] | [CONTENT] eight | 2 | 6 | at least two years | Libyan ||| [SUMMARY] | [CONTENT] three [SUMMARY] | [CONTENT] HBR | Libya ||| first | Libya | HBR [SUMMARY] | [CONTENT] HBR ||| HBR ||| HBR | Libya ||| HBR | Libya ||| eight | 2 | 6 | at least two years | Libyan ||| ||| three ||| HBR | Libya ||| first | Libya | HBR [SUMMARY] | [CONTENT] HBR ||| HBR ||| HBR | Libya ||| HBR | Libya ||| eight | 2 | 6 | at least two years | Libyan ||| ||| three ||| HBR | Libya ||| first | Libya | HBR [SUMMARY] |
||||||
Comparative Effectiveness of Electrical Stimulation and Aerobic Exercise in the Management of Erectile Dysfunction: A Randomized Clinical Trial. | 33883841 | Electrical stimulation and aerobic exercise have been indicated to be beneficial in the management of erectile dysfunction individually. However, there is a scarcity of evidence comparing the two treatment approaches. This study investigated the effects of Electrical Stimulation (ES) compared with Eerobic Exercise (AE) in the management of individuals with Erectile Dysfunction (ED). | BACKGROUND | This study was a single-blind parallel randomized clinical trial. Thirty (30) patients diagnosed with ED (Mean age of 39.17 ± 6.21 years) were recruited and randomized into two groups, A and B with 15 participants in each group. Group A received ES while Group B received AE. International Index of Erectile Function (IIEF-5) was used to assess the sexual functions of the participants at baseline and after 6 weeks of intervention. Within-group and between-group differences were analyzed using dependent and independent t-tests respectively. | METHODS | The result indicated a significant difference between groups A and B [20.83 (1.83) Vs 14.33 (2.07), p=0.001] after 6 weeks of intervention. However, the mean effect was significantly higher in the ES group than in the AE group. | RESULTS | The finding of this study indicated that ES is more effective than AE in the management of individuals with ED.Trial Registration: Pan African Clinical Trial Registry (PACTR201906776769795). | CONCLUSION | [
"Adult",
"Double-Blind Method",
"Electric Stimulation",
"Erectile Dysfunction",
"Exercise",
"Humans",
"Male",
"Middle Aged",
"Single-Blind Method",
"Treatment Outcome"
] | 8047238 | Introduction | The term male Erectile Dysfunction (ED) refers to a recurring and persistent condition where a man is unable to achieve or maintain an erection and complete sexual intercourse (1). ED is very common, and its prevalence as well as severity increases with age (2). A review of published studies found that estimates for the global prevalence of erectile dysfunction vary widely, ranging from 3% to 76.5%. In Nigeria, a recent study reported a high prevalence of ED (66.4%) among 378 male adults, with most of the participants having poor health-seeking behavior due to associated stigmatization (4).
Therapeutic options for men with ED are mainly administration of phosphodiesterase type 5 inhibitors, intracavernous injections of vasoactive agents (for example, prostaglandin El, papaverine/phentolamine, or triple-drug), intraurethral administration of prostaglandin El, and administration of centrally acting drugs (5,6). However, all of these methods treat the patient's problem temporarily, and patients are not cured of impotence. Patients typically remain dependent on these treatments for the remainder of their sexually active lives and for these reason, alternative effective treatment options are needed (6).
ED is usually of multifactorial origin ranging from physiologic, organic, psychogenic and endocrine factors (7). However, it has been recognized that the major cause of ED is atherosclerosis affecting the pelvic vasculature (1). It is known that atherosclerotic lesions prevent blood flow into cavernosal tissues resulting in ED (8). Although vascular factors predominate in the etiology of ED, conditions associated with reduced nerve and endothelium function, such as ageing, hypertension, smoking, hypercholesterolemia and diabetes, also alter the balance between contraction and relaxation factors (9). Almost any disease that may affect erectile function such as benign prostatic hyperplasia, prostate cancer, depression, and infectious diseases among others, alter the nervous, vascular and hormonal systems (10).
Considering the multiple causes of ED and the subsequent vascular or nervous affectation, it would be helpful to determine the most effective treatment between exercise and electrical stimulation of nerves. There has already been a report of the inverse relationship between physical activity levels and biomarkers of inflammation in both healthy individuals and subjects with cardiovascular conditions (11). Accordingly, recent studies have indicated the role of aerobic exercise in the management of erectile dysfunction (12–16). Aerobic exercise has been revealed to strengthen the cardiovascular system by making the heart stronger and the lungs more efficient (17). A stronger heart delivers more blood to the body with fewer beats, which also lowers blood pressure. Efficient lungs can transfer more oxygen into the bloodstream with each breath (17). Nevertheless, there is the need for a reliable alternative to aerobic exercise in case of contraindications such as electrocardiography changes, myocardial infarction, congestive heart failure, unstable angina, complete heart block, and uncontrolled hypertension. Moreover, exercise may also not be very effective if nervous affectation is implicated in the pathophysiology of individual's ED.
Recent studies have demonstrated the effectiveness of penile electrical stimulation in the management of ED (11,18–20). Also reported was a randomized control trial to find out the efficacy of magnetic stimulation of the cavernous nerve for the treatment of erectile dysfunction, which proved to be effective in producing increased intercorporal pressure and penile tumescence and rigidity (21). By applying electrical stimulation with appropriate stimulation parameters, the insufficiency of the carvenosal-smooth muscle can be treated (22). The muscle will be strengthened and the resulting increase of muscular mass and physical strength can lead to an improvement of the functioning of venous occlusion mechanism and enable the required filling of the corpus carvenosal bodies with the blood (22).
Considering the contraindications of electrical stimulation such as impaired sensation, insufficient blood flow and presence of a metallic implant, and also the aforementioned contraindications of exercise, in addition to multifactorial causes of ED, it will be important to determine the relative efficacy of aerobic exercise in comparison to electrical stimulation in the management of this condition. This will help in providing the patients with the most cost-effective and relatively safe choice among the two options according to their conditions and need. Furthermore, an extensive literature search indicated scarcity of studies that compared electrical stimulation with aerobic exercise in the management of individuals with ED. It was hypothesized that there will be no significant difference between electrical stimulation and aerobic exercise in the management of individuals with erectile dysfunction. | null | null | Results | Thirty (30) patients with ED participated in this study with the age range of 25 to 65 years (mean of 39.17 ± 6.21 years). Details of the demographic and clinical parameters of the study participants are presented in Table 1.
Demographic and Clinical Parameters of the Participants at Baseline
N = Number of participants; SD = Standard Deviation; Group A= Electrical stimulation; Group B= Aerobic exercise; BMI= body mass index; IIEF-5=International Index of Erectile Function
The within-group differences are presented in Table 2. The result indicated that the comparison of mean IIEF-5 scores before (11.17±1.72) and after (20.83±1.83) electrical stimulation (Group A) revealed a significant difference (p<0.05). Similarly, there was a significant difference (p<0.05) between patients' IIEF-5 means scores before (10.67±1.63) and after (14.33±2.07) aerobic exercise (Group B). In both the two groups, the post-intervention IIEF-5 scores had the highest means.
Comparison of Pre- and Post-intervention IIEF-5 Scores for within Group Differences
Significance at p-value<0.05
Group A= Electrical stimulation; Group B= Aerobic exercise; IIEF-5=International Index of Erectile Function
The between-group difference was presented in Table 3. The result indicated that there was a significant difference (p<0.05) when post-intervention mean IIEF-5 score of group A (20.83±1.83) was compared with that of group B (14.33±2.07). This also indicated that Group A was better than Group B in terms of treatment effect. | null | null | [] | [] | [] | [
"Introduction",
"Material and Methods",
"Results",
"Discussion"
] | [
"The term male Erectile Dysfunction (ED) refers to a recurring and persistent condition where a man is unable to achieve or maintain an erection and complete sexual intercourse (1). ED is very common, and its prevalence as well as severity increases with age (2). A review of published studies found that estimates for the global prevalence of erectile dysfunction vary widely, ranging from 3% to 76.5%. In Nigeria, a recent study reported a high prevalence of ED (66.4%) among 378 male adults, with most of the participants having poor health-seeking behavior due to associated stigmatization (4).\nTherapeutic options for men with ED are mainly administration of phosphodiesterase type 5 inhibitors, intracavernous injections of vasoactive agents (for example, prostaglandin El, papaverine/phentolamine, or triple-drug), intraurethral administration of prostaglandin El, and administration of centrally acting drugs (5,6). However, all of these methods treat the patient's problem temporarily, and patients are not cured of impotence. Patients typically remain dependent on these treatments for the remainder of their sexually active lives and for these reason, alternative effective treatment options are needed (6).\nED is usually of multifactorial origin ranging from physiologic, organic, psychogenic and endocrine factors (7). However, it has been recognized that the major cause of ED is atherosclerosis affecting the pelvic vasculature (1). It is known that atherosclerotic lesions prevent blood flow into cavernosal tissues resulting in ED (8). Although vascular factors predominate in the etiology of ED, conditions associated with reduced nerve and endothelium function, such as ageing, hypertension, smoking, hypercholesterolemia and diabetes, also alter the balance between contraction and relaxation factors (9). Almost any disease that may affect erectile function such as benign prostatic hyperplasia, prostate cancer, depression, and infectious diseases among others, alter the nervous, vascular and hormonal systems (10).\nConsidering the multiple causes of ED and the subsequent vascular or nervous affectation, it would be helpful to determine the most effective treatment between exercise and electrical stimulation of nerves. There has already been a report of the inverse relationship between physical activity levels and biomarkers of inflammation in both healthy individuals and subjects with cardiovascular conditions (11). Accordingly, recent studies have indicated the role of aerobic exercise in the management of erectile dysfunction (12–16). Aerobic exercise has been revealed to strengthen the cardiovascular system by making the heart stronger and the lungs more efficient (17). A stronger heart delivers more blood to the body with fewer beats, which also lowers blood pressure. Efficient lungs can transfer more oxygen into the bloodstream with each breath (17). Nevertheless, there is the need for a reliable alternative to aerobic exercise in case of contraindications such as electrocardiography changes, myocardial infarction, congestive heart failure, unstable angina, complete heart block, and uncontrolled hypertension. Moreover, exercise may also not be very effective if nervous affectation is implicated in the pathophysiology of individual's ED.\nRecent studies have demonstrated the effectiveness of penile electrical stimulation in the management of ED (11,18–20). Also reported was a randomized control trial to find out the efficacy of magnetic stimulation of the cavernous nerve for the treatment of erectile dysfunction, which proved to be effective in producing increased intercorporal pressure and penile tumescence and rigidity (21). By applying electrical stimulation with appropriate stimulation parameters, the insufficiency of the carvenosal-smooth muscle can be treated (22). The muscle will be strengthened and the resulting increase of muscular mass and physical strength can lead to an improvement of the functioning of venous occlusion mechanism and enable the required filling of the corpus carvenosal bodies with the blood (22).\nConsidering the contraindications of electrical stimulation such as impaired sensation, insufficient blood flow and presence of a metallic implant, and also the aforementioned contraindications of exercise, in addition to multifactorial causes of ED, it will be important to determine the relative efficacy of aerobic exercise in comparison to electrical stimulation in the management of this condition. This will help in providing the patients with the most cost-effective and relatively safe choice among the two options according to their conditions and need. Furthermore, an extensive literature search indicated scarcity of studies that compared electrical stimulation with aerobic exercise in the management of individuals with ED. It was hypothesized that there will be no significant difference between electrical stimulation and aerobic exercise in the management of individuals with erectile dysfunction.",
"Study design and ethics: This study was a single-blind parallel randomized clinical trial (the research participants blinded to randomization). Because of the nature of the study, investigators were not blinded; however, outcome assessors and data analysts were blinded. The study has been registered with the Pan African Clinical Trial Registry (PACTR201906776769795). Any change in the study protocol was updated in the clinical trial registry. By the Declaration of Helsinki, ethical approval was sought and obtained from the Health Research and Ethics Committee of Lagos University Teaching Hospital (LUTH), Idiaraba, Lagos State, Nigeria (Registration Number: ADM/DCST/HREC/2132).\nSample size and study population: The sample size was calculated using the G*Power version 3.9.1. The Effect Size (ES) used for calculating the sample size was obtained from the previous study (19) using the International Index of Erectile Function (IIEF-5) primary outcome. The probability level (α), the power (p) and the Effect Size (ES) used for the calculation were then set at 0.05, 0.95 and 3.3 respectively which yielded a sample size of 4 participants per group (total sample size was 8) using independent ttest for between-group analysis. The sample size was increased to 30 to obtain better treatment effects.\nThe targeted population included patients diagnosed with ED and attending urology and Physiotherapy Outpatient Clinics of LUTH. All the patients were said to have been diagnosed more than six months before the commencement of the study as stipulated in the 5-Items version of the International Index of Erectile Function (IIEF-5).\nEligibility criteria: Fourty five (45) male participants, between the ages of 25 to 65 were recruited. The inclusion criteria included participants who have stable medical conditions and diagnosed with ED due to; 1) Neurogenic causes—10(22.2%), 2) Venous occlusion or arterial insufficiency—11 (24.4%), 3)Psychogenic causes—8(17.8%), and 3) Electric shock—1(2.2%).\nThe exclusion criteria included participants without stable medical conditions and diagnosed with ED due to; 1) Hormonal causes―5(11.1%), 2) Diabetes and renal diseases―10(22.2%). Other exclusion criteria included; participants with priapism, cardiac pacemaker, history of psychiatry or psychological disorders, penile skin lesion/ulcers, or uncontrolled high blood pressure. Additionally, patients on other oral medications were given 2 weeks washout period. In the end, 30 patients were included in the study.\nRole of funding source: This study did not receive any funding and no organization played any role in the design, conduct, or reporting of this study.\nData collection instruments: The instruments used during this study were treadmill machine (an exercise machine for running or walking while staying in one place), Electrical stimulator machine, and IIEF-5. The electrical stimulator is a device that uses electric current to stimulate the nerves for therapeutic purposes. The current is normally produced when the machine is connected to the source of electricity. It has different modes of treatment which include faradic and galvanic currents, and two electrodes (active and inactive). The electrodes are normally applied to the skin surface of the affected area.\nThe IIEF-5 is an instrument used to determine the presence and extent of ED (23). This Questionnaire consists of only five questions and each IIEF-5 item is scored on a five-point ordinal scale. A response of 1 indicates the least sexual function, whereas a response of 5 indicates the highest sexual function. The highest possible cumulative score for the IIEF-5 is 25, while the least score is 1. A score above 21 was considered as a normal erectile function and a score at or below this value was considered as ED. Overall, according to this scale, ED was classified into four categories: severe (1–7), moderate (8–11), moderate to mild (12–16), mild (17–21), and no ED (22–25) (23).\nOutcomes assessment: The IIEF-5 questionnaires were given to the participants (self-administered) to assess the level of their ED before the commencement of the treatment. This was repeated at 6 weeks post-intervention. For those subjects that could not read the questionnaire, research assistants interpreted it to them.\nRandomization and concealment: Eligible participants who provide informed consent were randomized into one of two treatment groups; Electrical Stimulation (ES) or Aerobic Exercise (AE). A randomization timeline was prepared by a research assistant who did not have communication with any participant throughout the trial and was unaware of the recruitment, screening, assessment, enrolment or treatment process. The randomization series was created by the use of SAS 9.4 statistical software (Cary, NC, USA) with the participants likely to be assigned to a group with an equal chance of allocation. See Figure 1 for the study flow chart.\nExperimental flow chart\nIntervention procedures: The participants were randomly assigned to two experimental groups namely; group A and B, using sealed envelopes, with each group having fifteen (15) participants. Participants in Group A received electrical stimulation, while those in group B received aerobic exercise. The full intervention procedures were described below:\nGroup A (Electrical Stimulation): The participants were positioned in supine lying and the areas to be treated were adequately exposed. The treatment was carried out by the use of an electronic muscle stimulator (α-wave healthtronic, BM-1006, serial no: C2018-6016, Cybermed Inc. 1618 Valdorast., Davis, CA 95618, USA), using the Erect-fit Stimulation System with two monopolar self-adhesive electrodes. The active electrode was circularly placed on the penis and the inactive electrode at the central-sacral region of the spine (the origin of pelvic plexus). The proper arrangement was made so that the current could be transferred to the inner muscular system, and the intensity was increased to the maximum tolerance level of the participants. The stimulation was carried out for 30 minutes using the following parameters: frequency of 5 Hz, a pulse width of 150µs, and contraction time of 3 seconds (11). Two treatment sessions were given per week for 6 weeks.\nGroup B (Aerobic Exercise)-Treadmill: Roger black treadmill machine, with serial number A0013863; and power of AC220V+10% 50/60 Hz and input of 900V was used for this study. It has a menu which consists of buttons for the following parameters: distance, pulse, speed, time, calories, and start menu. The exercise protocol was based on the guidelines by the American College of Sports Medicine in terms of improving peak Vo2 (55% – 90% of maximal heart rate V02 or 40%–85% of heart rate reserve; 3 – 5 days, 20 – 30 minutes per session) (24). Two treatment sessions were given for 30 minutes each per week for 6 weeks.\nData analysis: Descriptive statistics of means and standard deviations were used to summarize the demographic and clinical parameters of the participants. Shapiro-Wilk test was used to determine the normality of the data. Following normal distributions, the data were statistically analyzed using dependent and independent t-tests to find out the difference between the pre- and post-intervention scores of the IIEF-5 within and between the two groups respectively. Independent t-test was also used to compare participants at baseline. All the statistical analysis was performed using Statistical Package for Social Science- SPSS (Windows Version 20.0) at an alpha level of 0.05.\nData sharing statement: All participants data collected during the trial will be shared after de-identification. The study protocol, statistical analysis plan, and informed consent form will also be shared. All this information will be shared via a link to be provided immediately after publication so that the data will be available to anyone who wishes to assess it. All data and publication records will equally be updated in the Pan African Clinical Trial registry.",
"Thirty (30) patients with ED participated in this study with the age range of 25 to 65 years (mean of 39.17 ± 6.21 years). Details of the demographic and clinical parameters of the study participants are presented in Table 1.\nDemographic and Clinical Parameters of the Participants at Baseline\nN = Number of participants; SD = Standard Deviation; Group A= Electrical stimulation; Group B= Aerobic exercise; BMI= body mass index; IIEF-5=International Index of Erectile Function\nThe within-group differences are presented in Table 2. The result indicated that the comparison of mean IIEF-5 scores before (11.17±1.72) and after (20.83±1.83) electrical stimulation (Group A) revealed a significant difference (p<0.05). Similarly, there was a significant difference (p<0.05) between patients' IIEF-5 means scores before (10.67±1.63) and after (14.33±2.07) aerobic exercise (Group B). In both the two groups, the post-intervention IIEF-5 scores had the highest means.\nComparison of Pre- and Post-intervention IIEF-5 Scores for within Group Differences\nSignificance at p-value<0.05\nGroup A= Electrical stimulation; Group B= Aerobic exercise; IIEF-5=International Index of Erectile Function\nThe between-group difference was presented in Table 3. The result indicated that there was a significant difference (p<0.05) when post-intervention mean IIEF-5 score of group A (20.83±1.83) was compared with that of group B (14.33±2.07). This also indicated that Group A was better than Group B in terms of treatment effect.",
"The findings of this study indicated that both electrical stimulation and aerobic exercise are effective non-invasive treatment options for ED. On direct comparison, however, electrical stimulation proved to be of superior effectiveness. All the important characteristics of the participants such as BMI, age, the duration of ED and IIEF-5 scores, which could have affected the findings of this study, were similar at baseline. This validated the outcomes of the study as being solely due to the effect of the interventions.\nConcerning the effect of aerobic exercise on ED, our finding has corroborated with those of the previous studies (12–16). The physiological basis of the therapeutic effect of aerobic exercise on ED in this study could be attributed to both acute and long-term changes in the blood vessel walls (12). The immediate vascular relaxation following physical activity is due to body warming effects; decrease in nerve activities; local production of certain chemicals such as lactic acid, and nitric oxide (NO), and changes in certain hormones and their receptors (25). The repetitive physical activity-induced increased blood flow and vascular shear stress lead to substantial remodeling of the vascular system (26). This adaptive response alters the endothelium by increasing the expression of nitric oxide synthase mRNA; resulting in increased synthesis of NO and improved endothelial function (27). Although most of the adaptive changes are limited to the working muscles, the endothelial functional improvement seems to be the entire body response to exercise (26). For example, Belardinelli et al. (28) reported a significant improvement in flow-mediated dilation of brachial artery in the group of patients with ED subjected to 8-weeks aerobic exercise compared to controls.\nAnother possible physiological basis of our finding was the improvement in the testosterone concentration in the body. Even though testosterone level has been reported to rise after resistant training (29), a recent randomized controlled trial involving animal models revealed that the normal plasma level of testosterone was completely restored in a group of high-fat diet (HFD) fed rabbits with metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction, following a 12-week treadmill training compared to controls (30). Analysis of penile gene expression indicates that physical activity increases genes related to a smooth muscle phenotype along with NO formation and signaling (30). Since erectile tissue function is finely regulated by a healthy smooth muscle tissue whose relaxant or contractile mechanisms are dependent on NO signaling and formation (31), the improvement in ED seen in our participants may not be unrelated to this these mechanisms. In addition, considering that NO signaling within the penis is also androgen-dependent (32), it is possible to conceive that the aforementioned findings in our participants are related to the restoration of normal androgen levels induced by physical exercise. Thus, we believe that the improvement in erectile function seen in our patients following treadmill exercise may be attributed to the increase in both NO synthesis and testosterone levels.\nOn the other hand, the present study reported significant improvement in erectile function following electrical stimulation. This is consistent with the findings of previous studies. For instance, Carboni et al. (19) reported improvement in erectile function in individuals with ED following 4-weeks of functional electrical stimulations. A similar finding has been reported by Kayigil et al. (33) and Van Kampen et al. (18), who concluded that electrical stimulation plays an important role in the rehabilitation of ED. It will be pertinent to note that electrical stimulation is also effective in the management of neurogenic ED. For example, Fayiz et al. (11) conducted a study to determine the effectiveness of transcutaneous electrical stimulation in the rehabilitation of patients with post-prostatectomy ED. They reported significant improvement in erectile function of the patients compared to controls. A similar finding has been reported by Derouet et al. (34). These were supportive of our findings considering the fact that many studies excluded this group of patients from their research envisaging that electrical stimulation will not affect neurogenic ED. Nevertheless, the possible physiologic rationale behind the positive effect of electrical stimulation in the current study could be the already established ability of this modality to induce regeneration of cavernous sinusoidal endothelium smooth muscle, with increased NO release (35,36). It was further hypothesized that the increased penile strength and the resulting gain in muscular mass will improve the venous occlusion mechanism leading to required filling of corpus cavernosal bodies with blood (20,21). Concerning the positive effect of electrical stimulation on neurogenic ED, it may be possible that the electric currents have spread to the deeply situated cavernosal nerves. This is possible since a very low stimulating frequency of 5Hz was used in the study and the inactive electrode was placed on the sacral region of the spine, the origin of pelvic plexus. This may constitute part of the reasons for the effectiveness of our intervention. An animal study has demonstrated the ability of electrical stimulation to rescue erectile function after cavernosal nerve injury by inducing nerve regeneration through pericyte (PC)-derived exosome (37). Pericytes are bionanoparticles that are distributed in the erectile tissue and play important roles in the regulation of penile erection, including promoting angiogenesis and neurogenesis through interacting with endothelial cells. Even though all the participants in the current study have intact cavernous nerve, the electric current from our intervention might have led to an increase in intracavernous pressure (38).\nThe most important finding of the current study is the superiority of electrical stimulation over aerobic exercise in the management of ED, which did not conform to our initial hypothesis. This finding could be attributed to the multifactorial nature of the etiologies of ED. Based on its causes, ED has been classified into vasculogenic, neurogenic, hormonal, or psychogenic. Vasculogenic ED is the one that presents with vascular endothelial dysfunction, though ED is generally considered to be an early indicator of cerebrovascular disease. Aerobic exercise seems to be more effective in the rehabilitation of vasculogenic ED due to its ability to restore endothelial function. Moreover, the long-term adaptive changes such as an increase in the expression of nitric oxide synthase occur only with chronic exercise. In the present study, almost half (46.6%) of the participants were diagnosed with vasculogenic ED and might have derived more benefit from exercise intervention than the remaining participants. Contrary to this, electrical stimulation can provide the local and more immediate effect to all forms of ED through its effect on cavernosal smooth muscle and nerve.\nIn conclusion, this study indicated that electrical stimulation has been proven to be more effective than aerobic exercise in the treatment of individuals with erectile dysfunction. This study was conducted on a few participants (30) diagnosed with ED due to the lack of funding for the research. Furthermore, long-term follow-up was not conducted because of time constraint. Future studies may, therefore, include large sample sizes and conduct sufficient follow-ups to see the long-term effects of the treatments."
] | [
"intro",
"materials|methodss",
"results",
"discussion"
] | [
"Erectile dysfunction",
"Electrical stimulation",
"Aerobic exercise",
"International Index of Erectile Function"
] | Introduction:
The term male Erectile Dysfunction (ED) refers to a recurring and persistent condition where a man is unable to achieve or maintain an erection and complete sexual intercourse (1). ED is very common, and its prevalence as well as severity increases with age (2). A review of published studies found that estimates for the global prevalence of erectile dysfunction vary widely, ranging from 3% to 76.5%. In Nigeria, a recent study reported a high prevalence of ED (66.4%) among 378 male adults, with most of the participants having poor health-seeking behavior due to associated stigmatization (4).
Therapeutic options for men with ED are mainly administration of phosphodiesterase type 5 inhibitors, intracavernous injections of vasoactive agents (for example, prostaglandin El, papaverine/phentolamine, or triple-drug), intraurethral administration of prostaglandin El, and administration of centrally acting drugs (5,6). However, all of these methods treat the patient's problem temporarily, and patients are not cured of impotence. Patients typically remain dependent on these treatments for the remainder of their sexually active lives and for these reason, alternative effective treatment options are needed (6).
ED is usually of multifactorial origin ranging from physiologic, organic, psychogenic and endocrine factors (7). However, it has been recognized that the major cause of ED is atherosclerosis affecting the pelvic vasculature (1). It is known that atherosclerotic lesions prevent blood flow into cavernosal tissues resulting in ED (8). Although vascular factors predominate in the etiology of ED, conditions associated with reduced nerve and endothelium function, such as ageing, hypertension, smoking, hypercholesterolemia and diabetes, also alter the balance between contraction and relaxation factors (9). Almost any disease that may affect erectile function such as benign prostatic hyperplasia, prostate cancer, depression, and infectious diseases among others, alter the nervous, vascular and hormonal systems (10).
Considering the multiple causes of ED and the subsequent vascular or nervous affectation, it would be helpful to determine the most effective treatment between exercise and electrical stimulation of nerves. There has already been a report of the inverse relationship between physical activity levels and biomarkers of inflammation in both healthy individuals and subjects with cardiovascular conditions (11). Accordingly, recent studies have indicated the role of aerobic exercise in the management of erectile dysfunction (12–16). Aerobic exercise has been revealed to strengthen the cardiovascular system by making the heart stronger and the lungs more efficient (17). A stronger heart delivers more blood to the body with fewer beats, which also lowers blood pressure. Efficient lungs can transfer more oxygen into the bloodstream with each breath (17). Nevertheless, there is the need for a reliable alternative to aerobic exercise in case of contraindications such as electrocardiography changes, myocardial infarction, congestive heart failure, unstable angina, complete heart block, and uncontrolled hypertension. Moreover, exercise may also not be very effective if nervous affectation is implicated in the pathophysiology of individual's ED.
Recent studies have demonstrated the effectiveness of penile electrical stimulation in the management of ED (11,18–20). Also reported was a randomized control trial to find out the efficacy of magnetic stimulation of the cavernous nerve for the treatment of erectile dysfunction, which proved to be effective in producing increased intercorporal pressure and penile tumescence and rigidity (21). By applying electrical stimulation with appropriate stimulation parameters, the insufficiency of the carvenosal-smooth muscle can be treated (22). The muscle will be strengthened and the resulting increase of muscular mass and physical strength can lead to an improvement of the functioning of venous occlusion mechanism and enable the required filling of the corpus carvenosal bodies with the blood (22).
Considering the contraindications of electrical stimulation such as impaired sensation, insufficient blood flow and presence of a metallic implant, and also the aforementioned contraindications of exercise, in addition to multifactorial causes of ED, it will be important to determine the relative efficacy of aerobic exercise in comparison to electrical stimulation in the management of this condition. This will help in providing the patients with the most cost-effective and relatively safe choice among the two options according to their conditions and need. Furthermore, an extensive literature search indicated scarcity of studies that compared electrical stimulation with aerobic exercise in the management of individuals with ED. It was hypothesized that there will be no significant difference between electrical stimulation and aerobic exercise in the management of individuals with erectile dysfunction.
Material and Methods:
Study design and ethics: This study was a single-blind parallel randomized clinical trial (the research participants blinded to randomization). Because of the nature of the study, investigators were not blinded; however, outcome assessors and data analysts were blinded. The study has been registered with the Pan African Clinical Trial Registry (PACTR201906776769795). Any change in the study protocol was updated in the clinical trial registry. By the Declaration of Helsinki, ethical approval was sought and obtained from the Health Research and Ethics Committee of Lagos University Teaching Hospital (LUTH), Idiaraba, Lagos State, Nigeria (Registration Number: ADM/DCST/HREC/2132).
Sample size and study population: The sample size was calculated using the G*Power version 3.9.1. The Effect Size (ES) used for calculating the sample size was obtained from the previous study (19) using the International Index of Erectile Function (IIEF-5) primary outcome. The probability level (α), the power (p) and the Effect Size (ES) used for the calculation were then set at 0.05, 0.95 and 3.3 respectively which yielded a sample size of 4 participants per group (total sample size was 8) using independent ttest for between-group analysis. The sample size was increased to 30 to obtain better treatment effects.
The targeted population included patients diagnosed with ED and attending urology and Physiotherapy Outpatient Clinics of LUTH. All the patients were said to have been diagnosed more than six months before the commencement of the study as stipulated in the 5-Items version of the International Index of Erectile Function (IIEF-5).
Eligibility criteria: Fourty five (45) male participants, between the ages of 25 to 65 were recruited. The inclusion criteria included participants who have stable medical conditions and diagnosed with ED due to; 1) Neurogenic causes—10(22.2%), 2) Venous occlusion or arterial insufficiency—11 (24.4%), 3)Psychogenic causes—8(17.8%), and 3) Electric shock—1(2.2%).
The exclusion criteria included participants without stable medical conditions and diagnosed with ED due to; 1) Hormonal causes―5(11.1%), 2) Diabetes and renal diseases―10(22.2%). Other exclusion criteria included; participants with priapism, cardiac pacemaker, history of psychiatry or psychological disorders, penile skin lesion/ulcers, or uncontrolled high blood pressure. Additionally, patients on other oral medications were given 2 weeks washout period. In the end, 30 patients were included in the study.
Role of funding source: This study did not receive any funding and no organization played any role in the design, conduct, or reporting of this study.
Data collection instruments: The instruments used during this study were treadmill machine (an exercise machine for running or walking while staying in one place), Electrical stimulator machine, and IIEF-5. The electrical stimulator is a device that uses electric current to stimulate the nerves for therapeutic purposes. The current is normally produced when the machine is connected to the source of electricity. It has different modes of treatment which include faradic and galvanic currents, and two electrodes (active and inactive). The electrodes are normally applied to the skin surface of the affected area.
The IIEF-5 is an instrument used to determine the presence and extent of ED (23). This Questionnaire consists of only five questions and each IIEF-5 item is scored on a five-point ordinal scale. A response of 1 indicates the least sexual function, whereas a response of 5 indicates the highest sexual function. The highest possible cumulative score for the IIEF-5 is 25, while the least score is 1. A score above 21 was considered as a normal erectile function and a score at or below this value was considered as ED. Overall, according to this scale, ED was classified into four categories: severe (1–7), moderate (8–11), moderate to mild (12–16), mild (17–21), and no ED (22–25) (23).
Outcomes assessment: The IIEF-5 questionnaires were given to the participants (self-administered) to assess the level of their ED before the commencement of the treatment. This was repeated at 6 weeks post-intervention. For those subjects that could not read the questionnaire, research assistants interpreted it to them.
Randomization and concealment: Eligible participants who provide informed consent were randomized into one of two treatment groups; Electrical Stimulation (ES) or Aerobic Exercise (AE). A randomization timeline was prepared by a research assistant who did not have communication with any participant throughout the trial and was unaware of the recruitment, screening, assessment, enrolment or treatment process. The randomization series was created by the use of SAS 9.4 statistical software (Cary, NC, USA) with the participants likely to be assigned to a group with an equal chance of allocation. See Figure 1 for the study flow chart.
Experimental flow chart
Intervention procedures: The participants were randomly assigned to two experimental groups namely; group A and B, using sealed envelopes, with each group having fifteen (15) participants. Participants in Group A received electrical stimulation, while those in group B received aerobic exercise. The full intervention procedures were described below:
Group A (Electrical Stimulation): The participants were positioned in supine lying and the areas to be treated were adequately exposed. The treatment was carried out by the use of an electronic muscle stimulator (α-wave healthtronic, BM-1006, serial no: C2018-6016, Cybermed Inc. 1618 Valdorast., Davis, CA 95618, USA), using the Erect-fit Stimulation System with two monopolar self-adhesive electrodes. The active electrode was circularly placed on the penis and the inactive electrode at the central-sacral region of the spine (the origin of pelvic plexus). The proper arrangement was made so that the current could be transferred to the inner muscular system, and the intensity was increased to the maximum tolerance level of the participants. The stimulation was carried out for 30 minutes using the following parameters: frequency of 5 Hz, a pulse width of 150µs, and contraction time of 3 seconds (11). Two treatment sessions were given per week for 6 weeks.
Group B (Aerobic Exercise)-Treadmill: Roger black treadmill machine, with serial number A0013863; and power of AC220V+10% 50/60 Hz and input of 900V was used for this study. It has a menu which consists of buttons for the following parameters: distance, pulse, speed, time, calories, and start menu. The exercise protocol was based on the guidelines by the American College of Sports Medicine in terms of improving peak Vo2 (55% – 90% of maximal heart rate V02 or 40%–85% of heart rate reserve; 3 – 5 days, 20 – 30 minutes per session) (24). Two treatment sessions were given for 30 minutes each per week for 6 weeks.
Data analysis: Descriptive statistics of means and standard deviations were used to summarize the demographic and clinical parameters of the participants. Shapiro-Wilk test was used to determine the normality of the data. Following normal distributions, the data were statistically analyzed using dependent and independent t-tests to find out the difference between the pre- and post-intervention scores of the IIEF-5 within and between the two groups respectively. Independent t-test was also used to compare participants at baseline. All the statistical analysis was performed using Statistical Package for Social Science- SPSS (Windows Version 20.0) at an alpha level of 0.05.
Data sharing statement: All participants data collected during the trial will be shared after de-identification. The study protocol, statistical analysis plan, and informed consent form will also be shared. All this information will be shared via a link to be provided immediately after publication so that the data will be available to anyone who wishes to assess it. All data and publication records will equally be updated in the Pan African Clinical Trial registry.
Results:
Thirty (30) patients with ED participated in this study with the age range of 25 to 65 years (mean of 39.17 ± 6.21 years). Details of the demographic and clinical parameters of the study participants are presented in Table 1.
Demographic and Clinical Parameters of the Participants at Baseline
N = Number of participants; SD = Standard Deviation; Group A= Electrical stimulation; Group B= Aerobic exercise; BMI= body mass index; IIEF-5=International Index of Erectile Function
The within-group differences are presented in Table 2. The result indicated that the comparison of mean IIEF-5 scores before (11.17±1.72) and after (20.83±1.83) electrical stimulation (Group A) revealed a significant difference (p<0.05). Similarly, there was a significant difference (p<0.05) between patients' IIEF-5 means scores before (10.67±1.63) and after (14.33±2.07) aerobic exercise (Group B). In both the two groups, the post-intervention IIEF-5 scores had the highest means.
Comparison of Pre- and Post-intervention IIEF-5 Scores for within Group Differences
Significance at p-value<0.05
Group A= Electrical stimulation; Group B= Aerobic exercise; IIEF-5=International Index of Erectile Function
The between-group difference was presented in Table 3. The result indicated that there was a significant difference (p<0.05) when post-intervention mean IIEF-5 score of group A (20.83±1.83) was compared with that of group B (14.33±2.07). This also indicated that Group A was better than Group B in terms of treatment effect.
Discussion:
The findings of this study indicated that both electrical stimulation and aerobic exercise are effective non-invasive treatment options for ED. On direct comparison, however, electrical stimulation proved to be of superior effectiveness. All the important characteristics of the participants such as BMI, age, the duration of ED and IIEF-5 scores, which could have affected the findings of this study, were similar at baseline. This validated the outcomes of the study as being solely due to the effect of the interventions.
Concerning the effect of aerobic exercise on ED, our finding has corroborated with those of the previous studies (12–16). The physiological basis of the therapeutic effect of aerobic exercise on ED in this study could be attributed to both acute and long-term changes in the blood vessel walls (12). The immediate vascular relaxation following physical activity is due to body warming effects; decrease in nerve activities; local production of certain chemicals such as lactic acid, and nitric oxide (NO), and changes in certain hormones and their receptors (25). The repetitive physical activity-induced increased blood flow and vascular shear stress lead to substantial remodeling of the vascular system (26). This adaptive response alters the endothelium by increasing the expression of nitric oxide synthase mRNA; resulting in increased synthesis of NO and improved endothelial function (27). Although most of the adaptive changes are limited to the working muscles, the endothelial functional improvement seems to be the entire body response to exercise (26). For example, Belardinelli et al. (28) reported a significant improvement in flow-mediated dilation of brachial artery in the group of patients with ED subjected to 8-weeks aerobic exercise compared to controls.
Another possible physiological basis of our finding was the improvement in the testosterone concentration in the body. Even though testosterone level has been reported to rise after resistant training (29), a recent randomized controlled trial involving animal models revealed that the normal plasma level of testosterone was completely restored in a group of high-fat diet (HFD) fed rabbits with metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction, following a 12-week treadmill training compared to controls (30). Analysis of penile gene expression indicates that physical activity increases genes related to a smooth muscle phenotype along with NO formation and signaling (30). Since erectile tissue function is finely regulated by a healthy smooth muscle tissue whose relaxant or contractile mechanisms are dependent on NO signaling and formation (31), the improvement in ED seen in our participants may not be unrelated to this these mechanisms. In addition, considering that NO signaling within the penis is also androgen-dependent (32), it is possible to conceive that the aforementioned findings in our participants are related to the restoration of normal androgen levels induced by physical exercise. Thus, we believe that the improvement in erectile function seen in our patients following treadmill exercise may be attributed to the increase in both NO synthesis and testosterone levels.
On the other hand, the present study reported significant improvement in erectile function following electrical stimulation. This is consistent with the findings of previous studies. For instance, Carboni et al. (19) reported improvement in erectile function in individuals with ED following 4-weeks of functional electrical stimulations. A similar finding has been reported by Kayigil et al. (33) and Van Kampen et al. (18), who concluded that electrical stimulation plays an important role in the rehabilitation of ED. It will be pertinent to note that electrical stimulation is also effective in the management of neurogenic ED. For example, Fayiz et al. (11) conducted a study to determine the effectiveness of transcutaneous electrical stimulation in the rehabilitation of patients with post-prostatectomy ED. They reported significant improvement in erectile function of the patients compared to controls. A similar finding has been reported by Derouet et al. (34). These were supportive of our findings considering the fact that many studies excluded this group of patients from their research envisaging that electrical stimulation will not affect neurogenic ED. Nevertheless, the possible physiologic rationale behind the positive effect of electrical stimulation in the current study could be the already established ability of this modality to induce regeneration of cavernous sinusoidal endothelium smooth muscle, with increased NO release (35,36). It was further hypothesized that the increased penile strength and the resulting gain in muscular mass will improve the venous occlusion mechanism leading to required filling of corpus cavernosal bodies with blood (20,21). Concerning the positive effect of electrical stimulation on neurogenic ED, it may be possible that the electric currents have spread to the deeply situated cavernosal nerves. This is possible since a very low stimulating frequency of 5Hz was used in the study and the inactive electrode was placed on the sacral region of the spine, the origin of pelvic plexus. This may constitute part of the reasons for the effectiveness of our intervention. An animal study has demonstrated the ability of electrical stimulation to rescue erectile function after cavernosal nerve injury by inducing nerve regeneration through pericyte (PC)-derived exosome (37). Pericytes are bionanoparticles that are distributed in the erectile tissue and play important roles in the regulation of penile erection, including promoting angiogenesis and neurogenesis through interacting with endothelial cells. Even though all the participants in the current study have intact cavernous nerve, the electric current from our intervention might have led to an increase in intracavernous pressure (38).
The most important finding of the current study is the superiority of electrical stimulation over aerobic exercise in the management of ED, which did not conform to our initial hypothesis. This finding could be attributed to the multifactorial nature of the etiologies of ED. Based on its causes, ED has been classified into vasculogenic, neurogenic, hormonal, or psychogenic. Vasculogenic ED is the one that presents with vascular endothelial dysfunction, though ED is generally considered to be an early indicator of cerebrovascular disease. Aerobic exercise seems to be more effective in the rehabilitation of vasculogenic ED due to its ability to restore endothelial function. Moreover, the long-term adaptive changes such as an increase in the expression of nitric oxide synthase occur only with chronic exercise. In the present study, almost half (46.6%) of the participants were diagnosed with vasculogenic ED and might have derived more benefit from exercise intervention than the remaining participants. Contrary to this, electrical stimulation can provide the local and more immediate effect to all forms of ED through its effect on cavernosal smooth muscle and nerve.
In conclusion, this study indicated that electrical stimulation has been proven to be more effective than aerobic exercise in the treatment of individuals with erectile dysfunction. This study was conducted on a few participants (30) diagnosed with ED due to the lack of funding for the research. Furthermore, long-term follow-up was not conducted because of time constraint. Future studies may, therefore, include large sample sizes and conduct sufficient follow-ups to see the long-term effects of the treatments.
| Background:
Electrical stimulation and aerobic exercise have been indicated to be beneficial in the management of erectile dysfunction individually. However, there is a scarcity of evidence comparing the two treatment approaches. This study investigated the effects of Electrical Stimulation (ES) compared with Eerobic Exercise (AE) in the management of individuals with Erectile Dysfunction (ED).
Methods:
This study was a single-blind parallel randomized clinical trial. Thirty (30) patients diagnosed with ED (Mean age of 39.17 ± 6.21 years) were recruited and randomized into two groups, A and B with 15 participants in each group. Group A received ES while Group B received AE. International Index of Erectile Function (IIEF-5) was used to assess the sexual functions of the participants at baseline and after 6 weeks of intervention. Within-group and between-group differences were analyzed using dependent and independent t-tests respectively.
Results:
The result indicated a significant difference between groups A and B [20.83 (1.83) Vs 14.33 (2.07), p=0.001] after 6 weeks of intervention. However, the mean effect was significantly higher in the ES group than in the AE group.
Conclusions:
The finding of this study indicated that ES is more effective than AE in the management of individuals with ED.Trial Registration: Pan African Clinical Trial Registry (PACTR201906776769795). | null | null | 3,978 | 260 | [] | 4 | [
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|||
Comparative cost-effectiveness analyses of cardiovascular magnetic resonance and coronary angiography combined with fractional flow reserve for the diagnosis of coronary artery disease. | 24461028 | According to recent guidelines, patients with coronary artery disease (CAD) should undergo revascularization if significant myocardial ischemia is present. Both, cardiovascular magnetic resonance (CMR) and fractional flow reserve (FFR) allow for a reliable ischemia assessment and in combination with anatomical information provided by invasive coronary angiography (CXA), such a work-up sets the basis for a decision to revascularize or not. The cost-effectiveness ratio of these two strategies is compared. | BACKGROUND | Strategy 1) CMR to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA), Strategy 2) CXA followed by FFR in angiographically positive stenoses (CXA + FFR). The costs, evaluated from the third party payer perspective in Switzerland, Germany, the United Kingdom (UK), and the United States (US), included public prices of the different outpatient procedures and costs induced by procedural complications and by diagnostic errors. The effectiveness criterion was the correct identification of hemodynamically significant coronary lesion(s) (= significant CAD) complemented by full anatomical information. Test performances were derived from the published literature. Cost-effectiveness ratios for both strategies were compared for hypothetical cohorts with different pretest likelihood of significant CAD. | METHODS | CMR + CXA and CXA + FFR were equally cost-effective at a pretest likelihood of CAD of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the US with costs of CHF 5'794, € 1'517, £ 2'680, and $ 2'179 per patient correctly diagnosed. Below these thresholds, CMR + CXA showed lower costs per patient correctly diagnosed than CXA + FFR. | RESULTS | The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help to optimize resource utilization in the diagnosis of CAD. | CONCLUSIONS | [
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] | 4015639 | Background | In many countries, cardiovascular diseases are the leading cause of morbidity and also of loss of quality of life. In particular, coronary artery disease (CAD) constitutes a major public health problem. In Europe the total costs of CAD and stroke were estimated at 49 billion Euros in the year 2008 [1] and for the United States these costs were estimated as high as 156 billion dollars [2]. It is well established that patients with no evidence of myocardial ischemia have low cardiac events rates, even when invasive coronary angiography (CXA) demonstrates lesions of intermediate severity [3,4]. In addition, patients without ischemia can be treated safely with medical therapy [5,6] thereby reducing the total costs of patient management [7]. On the other hand, randomized trials e.g. in diabetic patients demonstrated a survival benefit of patients with ischemia being treated by revascularization versus medical treatment alone [8]. Accordingly, recent guidelines recommend to revascularize patients if a relevant burden of myocardial ischemia is present (i.e. proximal vessel(s)) with a positive fractional flow reserve (FFR) and/or >10% of myocardium ischemic [9] or ≥2 ischemic segments in cardiovascular magnetic resonance (CMR) perfusion examinations [10].
Nevertheless, neither vessel anatomy nor presence or absence of ischemia is the factor that will exclusively decide on revascularizations. Symptoms, co-morbidities and other factors have to be taken into account before a treatment decision is made. Thus, the current analysis was undertaken to assess the cost-effectiveness to acquire information (significant ischemia and full anatomical information) relevant for decision making, but did not include the costs for all information needed to manage patients with CAD. Also, we do not know whether a large ischemia burden is directly related to adverse effects, whether it represents a marker of higher risk for occlusion of a severe stenosis that causes ischemia, or whether more severe ischaemia is simply a marker of more extensive atherosclerosis and more vulnerable plaques that go along with a worse outcome. Large trials such as ISCHEMIA and others [11] will hopefully improve our knowledge in a near future on how to treat ischemic patients. Despite this current lack of a detailed understanding of the underlying mechanisms that link ischemia to outcome, current guidelines recommend an ischemia-based approach for decision making in patients with CAD. Therefore, the aim of the current study was to assess the cost-effectiveness of two diagnostic strategies that are ischemia-based and provide both full anatomical and functional evaluation of CAD, which are the basis for a revascularization procedure.
A variety of new imaging techniques allow for such a combined anatomical and functional assessment of CAD, and as a result, the selection of the optimum test becomes more and more challenging. The choice of cardiovascular imaging techniques should consider both, their clinical benefits for the patients as well as the costs and cost-effectiveness compared to others. Invasive coronary angiography (CXA) remains the reference for the morphological assessment of CAD and it is often used in daily practice as a first line test, e.g. in patients with a positive treadmill test. While this strategy is not recommended by current guidelines, the advent of the FFR measurement to assess the hemodynamic significance of coronary artery stenoses [12] may even increase the attractivity to use invasive CXA as a first line test, as it can be easily combined with FFR in case of intermediate stenoses. Also, the FFR results were highly predictive for patient outcome [13,14] and the combination of CXA with FFR was more cost-effective than a CXA-only approach for the treatment of CAD [15]. Accordingly, recent guidelines recommend using FFR to correctly identify lesions that should undergo percutaneous coronary interventions (PCI) [9]. However, the invasive nature and radiation exposure of CXA and FFR limit their usefulness in a screening process [16]. Considering the fact that CXA is still extensively used in many industrialized countries as an early step in the work-up of suspected CAD [17,18], and further considering that FFR is recommended in recent guidelines, the combination of CXA + FFR was one diagnostic strategy to be assessed in the current study with respect to its cost-effectiveness.
As an alternative to the FFR measurement, perfusion CMR has emerged as a robust non-invasive technique for the evaluation of myocardial ischemia [19-22]. Furthermore, recent studies demonstrated the excellent prognosis of patients with known or suspected CAD, when perfusion-CMR was normal [23-25]. Accordingly, in the present study, the cost-effectiveness of a combination of CMR + CXA was compared with that of a CXA + FFR strategy.
In the current economic context, the health care systems have to be economically sustainable while preserving high quality medical standards. Consequently, in the following study we estimated the costs of the two different strategies relative to their effectiveness to 1) correctly diagnose the presence of relevant ischemia (= significant CAD) and 2) to yield full anatomical information of the coronary vasculature in case of ischemia. In particular, the cost-effectiveness of the two strategies was compared when applied to patient populations with varying CAD pre-test probabilities. Strategy 1 consists of a CMR examination to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA). This strategy yields complete information on myocardial ischemia and coronary anatomy. Strategy 2 consists of a CXA in all patients followed by a FFR test in patients with intermediate stenoses on CXA (CXA + FFR). Finally, the cost-effectiveness ratios of the two strategies were calculated for the health care systems in Switzerland, Germany, the United Kingdom, and the United States. | Methods | Using a mathematical model, we compared the cost-effectiveness of 2 algorithms for diagnosing the presence of hemodynamically significant coronary lesion(s) (= significant CAD) for hypothetical patient cohorts characterized by different pretest likelihood of CAD (Pisch): 1) A perfusion CMR to assess myocardial ischemia before referring positive patients to CXA and 2) A CXA combined with an FFR in patients with angiographically positive stenoses (see also Figure 1).
Decision tree for CAD diagnosis including strategy 1 and strategy 2 used to design the model.
Model characteristics The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.
The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.
Cost-effectiveness analysis Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
Definition of costs and calculations of the costs per effect The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].
The total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.
The cost-effectiveness ratios were calculated as follows:
Cost
-
effectiveness
Ratio
=
Direct
cost
+
subsequent
costs
effectiveness
Calculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.
The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].
The total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.
The cost-effectiveness ratios were calculated as follows:
Cost
-
effectiveness
Ratio
=
Direct
cost
+
subsequent
costs
effectiveness
Calculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.
Evaluation of the costs in each country The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.
The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.
Sensitivity analysis Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).
Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section). | Results | Effect of the pretest likelihood of significant CAD on effectiveness and on costs of the two strategies Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.
Example for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P
isch.
)
Figure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).
Costs per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P
isch
) for both strategies.
Results for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).
When CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.
Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.
Example for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P
isch.
)
Figure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).
Costs per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P
isch
) for both strategies.
Results for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).
When CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.
Comparison of the cost per effect and of cost-effectiveness for the two strategies Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).
By assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.
Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).
By assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.
Sensitivity analyses Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.
Sensitivity analysis: Switzerland.
Sensitivity analysis: Germany.
Sensitivity analysis: The United Kingdom.
Sensitivity analysis: The United States.
Changing the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.
Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.
Sensitivity analysis: Switzerland.
Sensitivity analysis: Germany.
Sensitivity analysis: The United Kingdom.
Sensitivity analysis: The United States.
Changing the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems. | Conclusions | With a focus on the latest imaging techniques to detect ischemia, this study shows to what extent the cost-effectiveness of two strategies to diagnose hemodynamically significant coronary lesions, i.e. significant CAD, depends on the prevalence of the disease. The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help the decision-making with regard to resource utilization. | [
"Background",
"Model characteristics",
"Cost-effectiveness analysis",
"Definition of effectiveness",
"Definition of costs and calculations of the costs per effect",
"Evaluation of the costs in each country",
"Sensitivity analysis",
"Effect of the pretest likelihood of significant CAD on effectiveness and on costs of the two strategies",
"Comparison of the cost per effect and of cost-effectiveness for the two strategies",
"Sensitivity analyses",
"CAD prevalence for optimum cost-effectiveness of various strategies and current utilization of resources",
"Testing for ischemia by invasive vs non-invasive techniques",
"Limitations",
"Abbreviations",
"Competing interests",
"Authors' contributions"
] | [
"In many countries, cardiovascular diseases are the leading cause of morbidity and also of loss of quality of life. In particular, coronary artery disease (CAD) constitutes a major public health problem. In Europe the total costs of CAD and stroke were estimated at 49 billion Euros in the year 2008 [1] and for the United States these costs were estimated as high as 156 billion dollars [2]. It is well established that patients with no evidence of myocardial ischemia have low cardiac events rates, even when invasive coronary angiography (CXA) demonstrates lesions of intermediate severity [3,4]. In addition, patients without ischemia can be treated safely with medical therapy [5,6] thereby reducing the total costs of patient management [7]. On the other hand, randomized trials e.g. in diabetic patients demonstrated a survival benefit of patients with ischemia being treated by revascularization versus medical treatment alone [8]. Accordingly, recent guidelines recommend to revascularize patients if a relevant burden of myocardial ischemia is present (i.e. proximal vessel(s)) with a positive fractional flow reserve (FFR) and/or >10% of myocardium ischemic [9] or ≥2 ischemic segments in cardiovascular magnetic resonance (CMR) perfusion examinations [10].\nNevertheless, neither vessel anatomy nor presence or absence of ischemia is the factor that will exclusively decide on revascularizations. Symptoms, co-morbidities and other factors have to be taken into account before a treatment decision is made. Thus, the current analysis was undertaken to assess the cost-effectiveness to acquire information (significant ischemia and full anatomical information) relevant for decision making, but did not include the costs for all information needed to manage patients with CAD. Also, we do not know whether a large ischemia burden is directly related to adverse effects, whether it represents a marker of higher risk for occlusion of a severe stenosis that causes ischemia, or whether more severe ischaemia is simply a marker of more extensive atherosclerosis and more vulnerable plaques that go along with a worse outcome. Large trials such as ISCHEMIA and others [11] will hopefully improve our knowledge in a near future on how to treat ischemic patients. Despite this current lack of a detailed understanding of the underlying mechanisms that link ischemia to outcome, current guidelines recommend an ischemia-based approach for decision making in patients with CAD. Therefore, the aim of the current study was to assess the cost-effectiveness of two diagnostic strategies that are ischemia-based and provide both full anatomical and functional evaluation of CAD, which are the basis for a revascularization procedure.\nA variety of new imaging techniques allow for such a combined anatomical and functional assessment of CAD, and as a result, the selection of the optimum test becomes more and more challenging. The choice of cardiovascular imaging techniques should consider both, their clinical benefits for the patients as well as the costs and cost-effectiveness compared to others. Invasive coronary angiography (CXA) remains the reference for the morphological assessment of CAD and it is often used in daily practice as a first line test, e.g. in patients with a positive treadmill test. While this strategy is not recommended by current guidelines, the advent of the FFR measurement to assess the hemodynamic significance of coronary artery stenoses [12] may even increase the attractivity to use invasive CXA as a first line test, as it can be easily combined with FFR in case of intermediate stenoses. Also, the FFR results were highly predictive for patient outcome [13,14] and the combination of CXA with FFR was more cost-effective than a CXA-only approach for the treatment of CAD [15]. Accordingly, recent guidelines recommend using FFR to correctly identify lesions that should undergo percutaneous coronary interventions (PCI) [9]. However, the invasive nature and radiation exposure of CXA and FFR limit their usefulness in a screening process [16]. Considering the fact that CXA is still extensively used in many industrialized countries as an early step in the work-up of suspected CAD [17,18], and further considering that FFR is recommended in recent guidelines, the combination of CXA + FFR was one diagnostic strategy to be assessed in the current study with respect to its cost-effectiveness.\nAs an alternative to the FFR measurement, perfusion CMR has emerged as a robust non-invasive technique for the evaluation of myocardial ischemia [19-22]. Furthermore, recent studies demonstrated the excellent prognosis of patients with known or suspected CAD, when perfusion-CMR was normal [23-25]. Accordingly, in the present study, the cost-effectiveness of a combination of CMR + CXA was compared with that of a CXA + FFR strategy.\nIn the current economic context, the health care systems have to be economically sustainable while preserving high quality medical standards. Consequently, in the following study we estimated the costs of the two different strategies relative to their effectiveness to 1) correctly diagnose the presence of relevant ischemia (= significant CAD) and 2) to yield full anatomical information of the coronary vasculature in case of ischemia. In particular, the cost-effectiveness of the two strategies was compared when applied to patient populations with varying CAD pre-test probabilities. Strategy 1 consists of a CMR examination to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA). This strategy yields complete information on myocardial ischemia and coronary anatomy. Strategy 2 consists of a CXA in all patients followed by a FFR test in patients with intermediate stenoses on CXA (CXA + FFR). Finally, the cost-effectiveness ratios of the two strategies were calculated for the health care systems in Switzerland, Germany, the United Kingdom, and the United States.",
"The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.",
" Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).\nIn the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).",
"In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).",
"The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].\nThe total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.\nThe cost-effectiveness ratios were calculated as follows: \n\n\n\n\n\n\n\nCost\n-\neffectiveness\n\n\nRatio\n\n\n\n=\n\n\n\n\n\nDirect\n\n\ncost\n\n\n\n+\n\n\n\nsubsequent\n\n\ncosts\n\n\n\n\neffectiveness\n\n\n\n\n\nCalculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.",
"The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.",
"Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).",
"Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.\n\nExample for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P\n\nisch.\n\n)\n\nFigure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).\n\nCosts per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P\n\nisch\n\n) for both strategies.\n\nResults for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).\nWhen CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.",
"Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).\nBy assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.",
"Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.\nSensitivity analysis: Switzerland.\nSensitivity analysis: Germany.\nSensitivity analysis: The United Kingdom.\nSensitivity analysis: The United States.\nChanging the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.",
"Current clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.\nA cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.",
"For the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].\nAt a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.",
"In the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.\nFinally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.",
"CAD: Coronary artery disease; CMR: Cardiovascular magnetic resonance; CXA: Invasive x-ray coronary angiography; FFR: Fractional flow reserve; PCI: Percutaneous coronary interventions.",
"The authors declare that they have no competing interests.",
"KM is responsible for the conception and design of the cost-effectiveness analysis; she performed the cost-effectiveness analysis, participated to the data collection and drafted the manuscript. DF participated to data collection and was involved in the calculations of the cost-effectiveness analysis. CP provided advices on the cost-effectiveness analysis. GP provided precisions on how the German health care system works and actively participated in the acquisition of required data in this context. SP provided information on how the UK health care system works and participated in the data acquisition in this context. AW provided explanations on how the US health care system works and participated in the data acquisition in this context. JBW provided explanations on how the Swiss health care system works. JS is responsible for the design of the study, participated to data collection and was involved in the interpretation of the results and drafting the manuscript; he critically revised its intellectual content. In addition, all authors provided helpful comments and relevant suggestions to improve the manuscript and its intellectual content; all authors read and approved the final manuscript."
] | [
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] | [
"Background",
"Methods",
"Model characteristics",
"Cost-effectiveness analysis",
"Definition of effectiveness",
"Definition of costs and calculations of the costs per effect",
"Evaluation of the costs in each country",
"Sensitivity analysis",
"Results",
"Effect of the pretest likelihood of significant CAD on effectiveness and on costs of the two strategies",
"Comparison of the cost per effect and of cost-effectiveness for the two strategies",
"Sensitivity analyses",
"Discussion",
"CAD prevalence for optimum cost-effectiveness of various strategies and current utilization of resources",
"Testing for ischemia by invasive vs non-invasive techniques",
"Limitations",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors' contributions",
"Supplementary Material"
] | [
"In many countries, cardiovascular diseases are the leading cause of morbidity and also of loss of quality of life. In particular, coronary artery disease (CAD) constitutes a major public health problem. In Europe the total costs of CAD and stroke were estimated at 49 billion Euros in the year 2008 [1] and for the United States these costs were estimated as high as 156 billion dollars [2]. It is well established that patients with no evidence of myocardial ischemia have low cardiac events rates, even when invasive coronary angiography (CXA) demonstrates lesions of intermediate severity [3,4]. In addition, patients without ischemia can be treated safely with medical therapy [5,6] thereby reducing the total costs of patient management [7]. On the other hand, randomized trials e.g. in diabetic patients demonstrated a survival benefit of patients with ischemia being treated by revascularization versus medical treatment alone [8]. Accordingly, recent guidelines recommend to revascularize patients if a relevant burden of myocardial ischemia is present (i.e. proximal vessel(s)) with a positive fractional flow reserve (FFR) and/or >10% of myocardium ischemic [9] or ≥2 ischemic segments in cardiovascular magnetic resonance (CMR) perfusion examinations [10].\nNevertheless, neither vessel anatomy nor presence or absence of ischemia is the factor that will exclusively decide on revascularizations. Symptoms, co-morbidities and other factors have to be taken into account before a treatment decision is made. Thus, the current analysis was undertaken to assess the cost-effectiveness to acquire information (significant ischemia and full anatomical information) relevant for decision making, but did not include the costs for all information needed to manage patients with CAD. Also, we do not know whether a large ischemia burden is directly related to adverse effects, whether it represents a marker of higher risk for occlusion of a severe stenosis that causes ischemia, or whether more severe ischaemia is simply a marker of more extensive atherosclerosis and more vulnerable plaques that go along with a worse outcome. Large trials such as ISCHEMIA and others [11] will hopefully improve our knowledge in a near future on how to treat ischemic patients. Despite this current lack of a detailed understanding of the underlying mechanisms that link ischemia to outcome, current guidelines recommend an ischemia-based approach for decision making in patients with CAD. Therefore, the aim of the current study was to assess the cost-effectiveness of two diagnostic strategies that are ischemia-based and provide both full anatomical and functional evaluation of CAD, which are the basis for a revascularization procedure.\nA variety of new imaging techniques allow for such a combined anatomical and functional assessment of CAD, and as a result, the selection of the optimum test becomes more and more challenging. The choice of cardiovascular imaging techniques should consider both, their clinical benefits for the patients as well as the costs and cost-effectiveness compared to others. Invasive coronary angiography (CXA) remains the reference for the morphological assessment of CAD and it is often used in daily practice as a first line test, e.g. in patients with a positive treadmill test. While this strategy is not recommended by current guidelines, the advent of the FFR measurement to assess the hemodynamic significance of coronary artery stenoses [12] may even increase the attractivity to use invasive CXA as a first line test, as it can be easily combined with FFR in case of intermediate stenoses. Also, the FFR results were highly predictive for patient outcome [13,14] and the combination of CXA with FFR was more cost-effective than a CXA-only approach for the treatment of CAD [15]. Accordingly, recent guidelines recommend using FFR to correctly identify lesions that should undergo percutaneous coronary interventions (PCI) [9]. However, the invasive nature and radiation exposure of CXA and FFR limit their usefulness in a screening process [16]. Considering the fact that CXA is still extensively used in many industrialized countries as an early step in the work-up of suspected CAD [17,18], and further considering that FFR is recommended in recent guidelines, the combination of CXA + FFR was one diagnostic strategy to be assessed in the current study with respect to its cost-effectiveness.\nAs an alternative to the FFR measurement, perfusion CMR has emerged as a robust non-invasive technique for the evaluation of myocardial ischemia [19-22]. Furthermore, recent studies demonstrated the excellent prognosis of patients with known or suspected CAD, when perfusion-CMR was normal [23-25]. Accordingly, in the present study, the cost-effectiveness of a combination of CMR + CXA was compared with that of a CXA + FFR strategy.\nIn the current economic context, the health care systems have to be economically sustainable while preserving high quality medical standards. Consequently, in the following study we estimated the costs of the two different strategies relative to their effectiveness to 1) correctly diagnose the presence of relevant ischemia (= significant CAD) and 2) to yield full anatomical information of the coronary vasculature in case of ischemia. In particular, the cost-effectiveness of the two strategies was compared when applied to patient populations with varying CAD pre-test probabilities. Strategy 1 consists of a CMR examination to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA). This strategy yields complete information on myocardial ischemia and coronary anatomy. Strategy 2 consists of a CXA in all patients followed by a FFR test in patients with intermediate stenoses on CXA (CXA + FFR). Finally, the cost-effectiveness ratios of the two strategies were calculated for the health care systems in Switzerland, Germany, the United Kingdom, and the United States.",
"Using a mathematical model, we compared the cost-effectiveness of 2 algorithms for diagnosing the presence of hemodynamically significant coronary lesion(s) (= significant CAD) for hypothetical patient cohorts characterized by different pretest likelihood of CAD (Pisch): 1) A perfusion CMR to assess myocardial ischemia before referring positive patients to CXA and 2) A CXA combined with an FFR in patients with angiographically positive stenoses (see also Figure 1).\nDecision tree for CAD diagnosis including strategy 1 and strategy 2 used to design the model.\n Model characteristics The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.\nThe model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.\n Cost-effectiveness analysis Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).\nIn the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).\n Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).\nIn the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).\n Definition of costs and calculations of the costs per effect The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].\nThe total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.\nThe cost-effectiveness ratios were calculated as follows: \n\n\n\n\n\n\n\nCost\n-\neffectiveness\n\n\nRatio\n\n\n\n=\n\n\n\n\n\nDirect\n\n\ncost\n\n\n\n+\n\n\n\nsubsequent\n\n\ncosts\n\n\n\n\neffectiveness\n\n\n\n\n\nCalculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.\nThe costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].\nThe total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.\nThe cost-effectiveness ratios were calculated as follows: \n\n\n\n\n\n\n\nCost\n-\neffectiveness\n\n\nRatio\n\n\n\n=\n\n\n\n\n\nDirect\n\n\ncost\n\n\n\n+\n\n\n\nsubsequent\n\n\ncosts\n\n\n\n\neffectiveness\n\n\n\n\n\nCalculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.\n Evaluation of the costs in each country The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.\nThe analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.\n Sensitivity analysis Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).\nDue to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).",
"The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.",
" Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).\nIn the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).",
"In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.\nTest performance parameters used in the effectiveness calculations\nIn this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.\nWhen performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).\nTo appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).",
"The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].\nThe total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.\nThe cost-effectiveness ratios were calculated as follows: \n\n\n\n\n\n\n\nCost\n-\neffectiveness\n\n\nRatio\n\n\n\n=\n\n\n\n\n\nDirect\n\n\ncost\n\n\n\n+\n\n\n\nsubsequent\n\n\ncosts\n\n\n\n\neffectiveness\n\n\n\n\n\nCalculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.",
"The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.",
"Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).",
" Effect of the pretest likelihood of significant CAD on effectiveness and on costs of the two strategies Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.\n\nExample for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P\n\nisch.\n\n)\n\nFigure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).\n\nCosts per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P\n\nisch\n\n) for both strategies.\n\nResults for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).\nWhen CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.\nFigure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.\n\nExample for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P\n\nisch.\n\n)\n\nFigure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).\n\nCosts per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P\n\nisch\n\n) for both strategies.\n\nResults for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).\nWhen CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.\n Comparison of the cost per effect and of cost-effectiveness for the two strategies Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).\nBy assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.\nFigure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).\nBy assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.\n Sensitivity analyses Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.\nSensitivity analysis: Switzerland.\nSensitivity analysis: Germany.\nSensitivity analysis: The United Kingdom.\nSensitivity analysis: The United States.\nChanging the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.\nFollowing a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.\nSensitivity analysis: Switzerland.\nSensitivity analysis: Germany.\nSensitivity analysis: The United Kingdom.\nSensitivity analysis: The United States.\nChanging the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.",
"Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.\n\nExample for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P\n\nisch.\n\n)\n\nFigure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).\n\nCosts per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P\n\nisch\n\n) for both strategies.\n\nResults for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).\nWhen CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.",
"Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).\nBy assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.",
"Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.\nSensitivity analysis: Switzerland.\nSensitivity analysis: Germany.\nSensitivity analysis: The United Kingdom.\nSensitivity analysis: The United States.\nChanging the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.",
"The main findings of this study can be summarized as follows: 1) In all four health care systems analyzed, the cost effectiveness ratio decreases hyperbolically for both diagnostic strategies with an increasing prevalence of hemodynamically significant coronary lesions, i.e. with an increasing prevalence of significant CAD. 2) The increase in the cost-effectiveness for strategy 2, i.e. CXA + FFR, with increasing prevalence of significant CAD is more pronounced than that of the primarily non-invasive CMR + CXA strategy, implying that there is a threshold value of CAD prevalence where strategy 2 becomes more cost-effective than strategy 1. and 3) The crossing point indicating an equal cost-effectiveness for the 2 strategies varied for the 4 countries examined. In Switzerland, strategy 1, i.e. CMR + CXA, is more cost-effective than strategy 2 below a CAD prevalence of 62%. In the German, UK, and US health care systems, a higher cost-effectiveness for CMR + CXA is given for a CAD prevalence below 65%, 83%, and 82%, respectively.\n CAD prevalence for optimum cost-effectiveness of various strategies and current utilization of resources Current clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.\nA cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.\nCurrent clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.\nA cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.\n Testing for ischemia by invasive vs non-invasive techniques For the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].\nAt a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.\nFor the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].\nAt a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.\n Limitations In the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.\nFinally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.\nIn the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.\nFinally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.",
"Current clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.\nA cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.",
"For the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].\nAt a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.",
"In the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.\nFinally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.",
"With a focus on the latest imaging techniques to detect ischemia, this study shows to what extent the cost-effectiveness of two strategies to diagnose hemodynamically significant coronary lesions, i.e. significant CAD, depends on the prevalence of the disease. The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help the decision-making with regard to resource utilization.",
"CAD: Coronary artery disease; CMR: Cardiovascular magnetic resonance; CXA: Invasive x-ray coronary angiography; FFR: Fractional flow reserve; PCI: Percutaneous coronary interventions.",
"The authors declare that they have no competing interests.",
"KM is responsible for the conception and design of the cost-effectiveness analysis; she performed the cost-effectiveness analysis, participated to the data collection and drafted the manuscript. DF participated to data collection and was involved in the calculations of the cost-effectiveness analysis. CP provided advices on the cost-effectiveness analysis. GP provided precisions on how the German health care system works and actively participated in the acquisition of required data in this context. SP provided information on how the UK health care system works and participated in the data acquisition in this context. AW provided explanations on how the US health care system works and participated in the data acquisition in this context. JBW provided explanations on how the Swiss health care system works. JS is responsible for the design of the study, participated to data collection and was involved in the interpretation of the results and drafting the manuscript; he critically revised its intellectual content. In addition, all authors provided helpful comments and relevant suggestions to improve the manuscript and its intellectual content; all authors read and approved the final manuscript.",
"Section A1. The relationship FFR-Stenosis. Section A2. Equations to estimate the costs and the effectiveness for each strategy.\nClick here for file\nBrief description on how the costs were derived for each country.\nClick here for file"
] | [
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"results",
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] | [
"Cost-effectiveness analysis",
"Coronary artery disease",
"Cardiovascular magnetic resonance",
"Coronary angiography",
"Fractional flow reserve",
"Decision making"
] | Background:
In many countries, cardiovascular diseases are the leading cause of morbidity and also of loss of quality of life. In particular, coronary artery disease (CAD) constitutes a major public health problem. In Europe the total costs of CAD and stroke were estimated at 49 billion Euros in the year 2008 [1] and for the United States these costs were estimated as high as 156 billion dollars [2]. It is well established that patients with no evidence of myocardial ischemia have low cardiac events rates, even when invasive coronary angiography (CXA) demonstrates lesions of intermediate severity [3,4]. In addition, patients without ischemia can be treated safely with medical therapy [5,6] thereby reducing the total costs of patient management [7]. On the other hand, randomized trials e.g. in diabetic patients demonstrated a survival benefit of patients with ischemia being treated by revascularization versus medical treatment alone [8]. Accordingly, recent guidelines recommend to revascularize patients if a relevant burden of myocardial ischemia is present (i.e. proximal vessel(s)) with a positive fractional flow reserve (FFR) and/or >10% of myocardium ischemic [9] or ≥2 ischemic segments in cardiovascular magnetic resonance (CMR) perfusion examinations [10].
Nevertheless, neither vessel anatomy nor presence or absence of ischemia is the factor that will exclusively decide on revascularizations. Symptoms, co-morbidities and other factors have to be taken into account before a treatment decision is made. Thus, the current analysis was undertaken to assess the cost-effectiveness to acquire information (significant ischemia and full anatomical information) relevant for decision making, but did not include the costs for all information needed to manage patients with CAD. Also, we do not know whether a large ischemia burden is directly related to adverse effects, whether it represents a marker of higher risk for occlusion of a severe stenosis that causes ischemia, or whether more severe ischaemia is simply a marker of more extensive atherosclerosis and more vulnerable plaques that go along with a worse outcome. Large trials such as ISCHEMIA and others [11] will hopefully improve our knowledge in a near future on how to treat ischemic patients. Despite this current lack of a detailed understanding of the underlying mechanisms that link ischemia to outcome, current guidelines recommend an ischemia-based approach for decision making in patients with CAD. Therefore, the aim of the current study was to assess the cost-effectiveness of two diagnostic strategies that are ischemia-based and provide both full anatomical and functional evaluation of CAD, which are the basis for a revascularization procedure.
A variety of new imaging techniques allow for such a combined anatomical and functional assessment of CAD, and as a result, the selection of the optimum test becomes more and more challenging. The choice of cardiovascular imaging techniques should consider both, their clinical benefits for the patients as well as the costs and cost-effectiveness compared to others. Invasive coronary angiography (CXA) remains the reference for the morphological assessment of CAD and it is often used in daily practice as a first line test, e.g. in patients with a positive treadmill test. While this strategy is not recommended by current guidelines, the advent of the FFR measurement to assess the hemodynamic significance of coronary artery stenoses [12] may even increase the attractivity to use invasive CXA as a first line test, as it can be easily combined with FFR in case of intermediate stenoses. Also, the FFR results were highly predictive for patient outcome [13,14] and the combination of CXA with FFR was more cost-effective than a CXA-only approach for the treatment of CAD [15]. Accordingly, recent guidelines recommend using FFR to correctly identify lesions that should undergo percutaneous coronary interventions (PCI) [9]. However, the invasive nature and radiation exposure of CXA and FFR limit their usefulness in a screening process [16]. Considering the fact that CXA is still extensively used in many industrialized countries as an early step in the work-up of suspected CAD [17,18], and further considering that FFR is recommended in recent guidelines, the combination of CXA + FFR was one diagnostic strategy to be assessed in the current study with respect to its cost-effectiveness.
As an alternative to the FFR measurement, perfusion CMR has emerged as a robust non-invasive technique for the evaluation of myocardial ischemia [19-22]. Furthermore, recent studies demonstrated the excellent prognosis of patients with known or suspected CAD, when perfusion-CMR was normal [23-25]. Accordingly, in the present study, the cost-effectiveness of a combination of CMR + CXA was compared with that of a CXA + FFR strategy.
In the current economic context, the health care systems have to be economically sustainable while preserving high quality medical standards. Consequently, in the following study we estimated the costs of the two different strategies relative to their effectiveness to 1) correctly diagnose the presence of relevant ischemia (= significant CAD) and 2) to yield full anatomical information of the coronary vasculature in case of ischemia. In particular, the cost-effectiveness of the two strategies was compared when applied to patient populations with varying CAD pre-test probabilities. Strategy 1 consists of a CMR examination to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA). This strategy yields complete information on myocardial ischemia and coronary anatomy. Strategy 2 consists of a CXA in all patients followed by a FFR test in patients with intermediate stenoses on CXA (CXA + FFR). Finally, the cost-effectiveness ratios of the two strategies were calculated for the health care systems in Switzerland, Germany, the United Kingdom, and the United States.
Methods:
Using a mathematical model, we compared the cost-effectiveness of 2 algorithms for diagnosing the presence of hemodynamically significant coronary lesion(s) (= significant CAD) for hypothetical patient cohorts characterized by different pretest likelihood of CAD (Pisch): 1) A perfusion CMR to assess myocardial ischemia before referring positive patients to CXA and 2) A CXA combined with an FFR in patients with angiographically positive stenoses (see also Figure 1).
Decision tree for CAD diagnosis including strategy 1 and strategy 2 used to design the model.
Model characteristics The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.
The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.
Cost-effectiveness analysis Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
Definition of costs and calculations of the costs per effect The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].
The total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.
The cost-effectiveness ratios were calculated as follows:
Cost
-
effectiveness
Ratio
=
Direct
cost
+
subsequent
costs
effectiveness
Calculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.
The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].
The total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.
The cost-effectiveness ratios were calculated as follows:
Cost
-
effectiveness
Ratio
=
Direct
cost
+
subsequent
costs
effectiveness
Calculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.
Evaluation of the costs in each country The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.
The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.
Sensitivity analysis Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).
Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).
Model characteristics:
The model is based on Bayes’ theorem and consequently assesses cost-effectiveness ratios of strategies in hypothetical patient cohorts with different pretest likelihoods of disease [26]. The mathematical model was initially suggested by Paterson and co-workers [27] and was later on applied by others [28-30]. The simulation approach has the advantage to allow the evaluation of diagnostic algorithms for patients with different pretest likelihoods of CAD regardless of currently accepted and applied clinical strategies to detect CAD. In order to determine the pretest likelihood of CAD in patients, the same testing procedures would precede both strategies, i.e. CMR + CXA and CXA + FFR, implying the same costs for both strategies. Therefore, these “upstream” costs need not to be considered in the model. Similarly, once a treatment decision is made, based on either diagnostic strategy the same treatment costs will occur and therefore, these “downstream” costs were not considered either in the model. No ethics approval was obtained for this study as it is based on simulation model calculations and therefore no patients data from our institution were required. Calculations were performed with Microsoft Office Excel 2007 software.
Cost-effectiveness analysis:
Definition of effectiveness In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
Definition of effectiveness:
In the present study, the criterion of effectiveness is the ability to accurately identify those patients with one or more hemodynamically significant coronary lesion(s) (=significant CAD), combined with the complete anatomical information on the coronary arteries. These patients with a relevant ischemia burden are the primary candidates for revascularizations according the most recent guidelines [10]. This ischemia burden is defined in the newest guidelines as a positive FFR of proximal coronary vessels [9,10] or by ≥2 segments with ischemia on perfusion-CMR [10]. The effectiveness criterion for strategy 1 is achieved by a positive perfusion-CMR study (≥2 segments ischemic, see also Table 1), which is complemented by a complete anatomical information provided by a CXA examination in patients positive for ischemia. For strategy 2, the effectiveness criterion is achieved by the detection of a stenosis ≥50% in CXA combined with an FFR ≤0.75 (= significant CAD). By assumption, invasive CXA and FFR were the reference tests with an assumed 100% diagnostic accuracy (Table 1). For the calculation of hemodynamically significant lesions by the CMR + CXA strategy, per-patient sensitivities (SnCMR = 0.88) and specificities (SpCMR = 0.90) were considered as determined by Rieber et al. who compared ischemia on CMR (i.e. ≥2 segment positive) versus FFR ≤0.75 as the reference for ischemia [31]. Cost-effectiveness is defined as the costs per effect which is calculated as the ratio between the total costs and the number of patients correctly diagnosed as having one or more hemodynamically significant coronary lesion(s) (true positive). Also, the costs of complications in patients with a false negative diagnosis are included in the cost-effectiveness ratio.
Test performance parameters used in the effectiveness calculations
In this study, the objective of the analysis was to compare cost-effectiveness ratios from the third party payer perspective and not to assess the general impact of CAD detection on the society welfare.
When performing cost-effectiveness analysis, a wide variety of factors and parameters related to the costs and the performances of the tests have to be considered. The model must be able to take into account the costs associated with false-positive results (i.e. costs of unnecessary diagnostic tests or treatments) as well as the costs associated with false negative results (i.e. costs of complications because of inappropriate management of the disease). To this end, data from the published literature on the performances of tests (sensitivities, specificities and rate of non-diagnostic examinations) and the complication rates were used (Table 1).
To appropriately model strategy 2 (CXA + FFR), the portion of patients with diameter stenoses ≥50% on CXA having ischemia in FFR must be known. In other words, the relationship between the probability of stenoses ≥50% on CXA (Psten) and the probability of having ischemia on FFR (Pisch) must be known. In order to assess this relationship, we used published data from 5 recent articles (see Additional file 1: Section A1 for details).
Definition of costs and calculations of the costs per effect:
The costs of a diagnostic strategy consist of first-line test costs and subsequent costs. The first-line test costs are the fees (Ft) for the CMR and CXA tests. Subsequent costs were costs of additional tests (i.e. in case of non-diagnostic CMR or unnecessary diagnostic tests in case of false-positive results), costs of major complications induced by the diagnostic procedure or resulting from mis-diagnosis of a patient (e.g. as false negative patients are at risk to have complications per 10-years follow-up because of inappropriate management of the disease). Due to the non-invasive nature of CMR and recent results showing that no severe complications occurred in >17,000 CMR examinations (i.e. 7 mild reactions in >7,200 stress CMR examinations) in the EuroCMR registry [33] and in large multicenter trials [19], we assumed that CMR is not associated with direct procedure-related major complications. As an anaphylactic shock is extremely rare and may occur after administration of both MR- and CXA-related contrast media, this complication was not considered in the analysis. We assumed that a potential complication associated with either a diagnostic CXA or an untreated hemodynamically significant lesion (i.e. false negatives) is a myocardial infarction (MI). Costs for this complication (hereafter C) included medical costs associated with the complications and accounted for a PCI, a hospital stay of one week, and a rehabilitation period of 4 weeks. The risk of developing malignancies induced by radiation exposure was not incorporated into the model. Future complications-related costs were discounted annually at a rate of 3% [34].
The total costs of a diagnostic algorithm were calculated as the sum of direct costs and subsequent costs multiplied by the respective number of patients. The cost-effectiveness ratios were calculated for patient cohorts with different pretest likelihoods ranging from 10% to 100%.
The cost-effectiveness ratios were calculated as follows:
Cost
-
effectiveness
Ratio
=
Direct
cost
+
subsequent
costs
effectiveness
Calculations of the direct and subsequent costs and the detailed equations are presented in the Additional file 1: Section A2.
Evaluation of the costs in each country:
The analyses were conducted from the third party payer perspective in 4 countries. We used 2012 and 2013 costs data in Swiss Francs (CHF) for Switzerland, in Euros (€) for Germany, in Pounds (£) for the United Kingdom (UK), and in American Dollars (US $) for the United States. The Additional file 2 provides a brief description on how the costs were derived for each country.
Sensitivity analysis:
Due to the uncertainty of the data used and the numerous assumptions (parameter values) made in these calculations, a sensitivity analysis was performed to test the robustness of the model. Thus, the model was re-run with 1) changes in the costs of the tests and of the complications, 2) changes in the rates of complications associated with CXA, 3) changes in the accuracy of the CMR test, and 4) change in the threshold of FFR to detect ischemia (<0.75 vs <0.80; regarding the method used refer to Additional file 1). In order to understand the impact of each parameter in the model they were changed one by one in the repeated calculations (for details, see the figures related to the sensitivity analyses in the Results section).
Results:
Effect of the pretest likelihood of significant CAD on effectiveness and on costs of the two strategies Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.
Example for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P
isch.
)
Figure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).
Costs per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P
isch
) for both strategies.
Results for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).
When CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.
Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.
Example for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P
isch.
)
Figure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).
Costs per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P
isch
) for both strategies.
Results for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).
When CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.
Comparison of the cost per effect and of cost-effectiveness for the two strategies Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).
By assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.
Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).
By assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.
Sensitivity analyses Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.
Sensitivity analysis: Switzerland.
Sensitivity analysis: Germany.
Sensitivity analysis: The United Kingdom.
Sensitivity analysis: The United States.
Changing the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.
Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.
Sensitivity analysis: Switzerland.
Sensitivity analysis: Germany.
Sensitivity analysis: The United Kingdom.
Sensitivity analysis: The United States.
Changing the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.
Effect of the pretest likelihood of significant CAD on effectiveness and on costs of the two strategies:
Figure 2 shows the effect of the pretest likelihood of significant CAD on effectiveness. The proportion of patients with CAD for whom a correct diagnosis is made by the CMR-based strategy depends on its sensitivity, specificity, and the rate of non-diagnostic CMR examinations (Additional file 1: Section A2). As CXA and FFR are assumed to be the reference with 100% accuracy, its advantage compared to CMR increases as Pisch increases. We derived that the difference between the 2 strategies slightly decreases with an increase of the rate of patients with non-diagnostic CMR tests (NDx). In the model, the NDx patients after CMR are oriented to strategy 2 in order to achieve 100% accuracy in these cross-over patients.
Example for the Swiss health care system: Proportion of patients with suspected CAD correctly diagnosed (CAD Dx) by the CMR + CXA and CXA + FFR strategies in relation to pre-test likelihood of significant CAD (P
isch.
)
Figure 3 shows the effect of pretest likelihood of significant CAD on cost (example for the Swiss health care system). The cost per patient tested increases with increasing pre-test likelihood of significant CAD for both strategies. The costs for CXA + FFR slightly increase as the need for FFR increases with increasing prevalence of significant CAD. For the CMR + CXA strategy the costs increase steeply with increasing pre-test likelihood of significant CAD, since patients positive for ischemia on CMR have to undergo CXA. The two strategies are equally costly for a prevalence of significant CAD of 0.87 (Figure 4). The value for such a crossing point, within the range of Pisch (0 – 1.0) depends on the relative costs of the tests and the accuracy of the CMR test (NDx and SnCMR and SpCMR) (see formulas of costs).
Costs per patient (Pt) tested in relation to the pre-test likelihood of significant CAD (=P
isch
) for both strategies.
Results for outpatient procedures performed in the 4 countries. Costs per effect (= cost-effectiveness) for both strategies in relation to the prevalence of significant CAD (=Pisch).
When CXA is considered as inpatient test, the cost per patient tested with strategy 1 (CMR + CXA) is lower than the cost per patient tested with strategy 2 (CXA + FFR) at any level of pre-test-likelihood of CAD.
Comparison of the cost per effect and of cost-effectiveness for the two strategies:
Figure 4 shows the cost per effect, i.e. the cost per patient correctly diagnosed for significant CAD at various levels of CAD prevalence in the 4 countries. We observe that the cost per effect decreases hyperbolically for both strategies as the pretest likelihood increases. The hyperbolic relationship between the prevalence of significant CAD and the costs per patient correctly diagnosed shows the high cost per effect in the patient population with low prevalence of significant CAD (= low Pisch values). The costs per effect at low values of Pisch are higher for strategy 2 (CX + FFR) than for strategy 1 (CMR + CXA).
By assuming that all tests are outpatient tests, both strategies are equally cost-effective at a pretest likelihood of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the United States with costs of CHF 5,794, € 1,517, £ 2,680, and $ 2,179 per patient correctly diagnosed, respectively. Below this threshold, CMR + CXA shows lower costs per patient correctly diagnosed than CXA + FFR. When the CXA test is performed as an inpatient examination, the crossing point of the two curves shifts towards the right to a prevalence of significant CAD of 77% with costs of CHF 6,819 in Switzerland, to 90% with costs of € 2′847 in Germany, to 93% with costs of £ 4,633 in the UK, and to 94% with costs of $ 3,849 in the US.
Sensitivity analyses:
Following a reduction of the sensitivity of the CMR examination by 10% the crossing point shifted to the left by 16, 20, 14, and 30 percentage points for the Swiss, the German, the UK, and the US health care systems, respectively. An increase of the CMR sensitivity by 10% shifted the crossing point to the right by 15, 19, and 12 percentage points for the Swiss, the German, and the UK health care systems, respectively. There is no crossing point for Pisch <1 for the US health care system (Figures 5, 6, 7 and 8), i.e. the costs of the CMR strategy are lower than those of the CXA strategy in the US system irrespectively of the pre-test likelihood of CAD.
Sensitivity analysis: Switzerland.
Sensitivity analysis: Germany.
Sensitivity analysis: The United Kingdom.
Sensitivity analysis: The United States.
Changing the specificity of CMR had a minor influence on the crossing point for all health care systems assessed. This was also the case for the other variables tested in the sensitivity analysis except the complications rate. The sensitivity analysis shows that the rate for complications caused by mis-diagnosed CAD, i.e. a lack of detecting CAD, is associated with relevant cost-effectiveness changes at least in the US system. If the rate of complications in false-negative patients by CMR is doubled the crossing point is shifted to the left by 10, 12, 6, and 23 percentage points in the Swiss, German, UK, and US systems, respectively.An increase of the FFR threshold to <0.80 did not substantially influence the cost-effectiveness results as shown in (Figures 5, 6, 7 and 8). The crossing point shifted to the left by 2 or 3 percentage points for the 4 health care systems.
Discussion:
The main findings of this study can be summarized as follows: 1) In all four health care systems analyzed, the cost effectiveness ratio decreases hyperbolically for both diagnostic strategies with an increasing prevalence of hemodynamically significant coronary lesions, i.e. with an increasing prevalence of significant CAD. 2) The increase in the cost-effectiveness for strategy 2, i.e. CXA + FFR, with increasing prevalence of significant CAD is more pronounced than that of the primarily non-invasive CMR + CXA strategy, implying that there is a threshold value of CAD prevalence where strategy 2 becomes more cost-effective than strategy 1. and 3) The crossing point indicating an equal cost-effectiveness for the 2 strategies varied for the 4 countries examined. In Switzerland, strategy 1, i.e. CMR + CXA, is more cost-effective than strategy 2 below a CAD prevalence of 62%. In the German, UK, and US health care systems, a higher cost-effectiveness for CMR + CXA is given for a CAD prevalence below 65%, 83%, and 82%, respectively.
CAD prevalence for optimum cost-effectiveness of various strategies and current utilization of resources Current clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.
A cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.
Current clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.
A cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.
Testing for ischemia by invasive vs non-invasive techniques For the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].
At a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.
For the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].
At a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.
Limitations In the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.
Finally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.
In the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.
Finally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.
CAD prevalence for optimum cost-effectiveness of various strategies and current utilization of resources:
Current clinical practice guidelines recommend proceeding to PCI only, if relevant myocardial ischemia in symptomatic patients is present [9,10]. Therefore, it appears reasonable to incorporate FFR testing or perfusion CMR for ischemia assessment into models that assess the cost-effectiveness of various strategies suggested for a CAD work-up. Moreover, an FFR-guided PCI approach was shown to be more cost-effective than a simple anatomy-guided, i.e. CXA-based PCI approach [7]. The current results show for all four health care systems assessed, that the pre-test likelihood of CAD is a major factor that influences the cost-effectiveness of a CAD work-up. This is in line with the fact that benefits from diagnostic tests depend on its performance but also on the prevalence of the disease within the evaluated population [35]. According to the current analyses a non-invasive CMR-guided approach is cost-effective for patients with an intermediate pre-test likelihood of disease, which is in line with most guidelines defining intermediate pre-test probabilities as 20-80%. Interestingly, in the US 62% of elective CXA examinations performed in a large sample of approximately 400,000 patients were found to be negative for CAD (stenoses <50% diameter reduction) [18], indicating that in the US the pre-test probability of CAD in daily routine is as low as ~38% which is substantially lower than the calculated threshold of 82%. Similarly, in the UK more than 58% of CXA examinations did not result in PCI or CABG procedures [36] indicating that currently, the pre-test likelihood of significant CAD of patients referred for CXA of 42% is substantially lower than 83% calculated for the UK. In the German health care system, the optimum pre-test likelihood of CAD for a CMR-based strategy is below 65%. However, in 2008, only 43% of patients after CXA were revascularized [17], indicating that an invasive approach was applied in a patient population with a relatively low CAD prevalence of approximately 43%. Finally, in Switzerland, the theoretical threshold for a directly invasive strategy is at 62% CAD prevalence. The portion of normal CXA studies ranged between 55% to 66% over the last 3 years [37], which translates into an approximated pre-test likelihood of significant CAD of 34-45% in Switzerland, thus, again still lower than the prevalence for a calculated optimal cost-effectiveness.
A cost analysis was recently performed based on the data of the German sample of the European CMR registry [38]. Cost savings of 50% were calculated between a CMR strategy and an outpatient invasive CXA strategy which is in line with the fact, that the pre-test probability of CAD in this cohort was 27%, i.e. well below the 65% level. The cost savings for this cohort reported in 2012 would be even higher considering that for these calculations costs for FFR were not yet included. Recently, a cost-effectiveness analysis for UK was performed based on the CE-MARC data [39]. For the prevalence of CAD of 39% in CE-MARC [22], the diagnostic strategy based on CMR (preceded or not by a treadmill test) followed by CXA in case of ischemia on CMR was the most cost-effective strategy of all tested. This finding is well in line with the current calculations which suggest cost-effectiveness for the MR-based approach below a CAD prevalence of 83%. In this context, it should be noted, that in the current study the threshold in favor of a CMR-guided work-up compared cost-effectiveness versus an outpatient CXA + FFR strategy. If inpatient CXA is included into the model, the crossing point shifts towards 77% for Switzerland, and is >90% for Germany, the UK and the US. This indicates that the inpatient CXA + FFR procedures can only compete with non-invasive CMR + CXA for very high rates of CAD prevalence. This result is of even greater importance if we note that in-patient CXA is performed in approximately 67% [40,41], 40% [36], and 88% [42], in the US, the UK, and the German system, respectively.
Testing for ischemia by invasive vs non-invasive techniques:
For the current analyses, the FFR technique was assumed to represent the gold standard. The assumption appears justified in the light of a rapidly increasing number of studies confirming the high prognostic value of FFR-guided PCI [5,6,12-14].
At a first glance, a SnCMR of 88% for ischemia detection (and of SnCMR = 80% in the sensitivity analysis) may appear relatively high. However, similar and even higher performances were reported with SnCMR/SpCMR of 82%/94% [43] and SnCMR/SpCMR of 91%/94% [44]. Importantly, it should be taken into account that these SnCMR for ischemia detection compare with an ischemia test, i.e. FFR. When lower sensitivities of CMR for ischemia detection are reported, they typically compare perfusion-CMR with coronary anatomy. FFR is generally accepted as a useful tool to guide treatment in CAD patients, as it discriminates patients at risk for complications (FFR-positive) versus those with minimal risk (FFR-negative). In FFR-positive patients, complication rates (death and non-fatal MI) were 3.2%-11.1%/y versus 0.7-7.3%/y in FFR-negative patients [5,6]. In a recent registry-based FFR study, MI in the FFR non-ischemia group was ~1%/y vs ~1.9%/y in the ischemia group [12]. For perfusion CMR, similar discriminative power was observed in approximately 1,700 patients of the EuroCMR registry, with complications rates of 2.7%/y in CMR-positive patients, i.e. in patients with ischemia, versus 0.7%/y in CMR-negative patients [24]. Thus, with this evidence of a similar discriminative power for CMR and FFR, the assumption of FFR being the gold standard, and thus, classifying CMR results differing from FFR as incorrect, might induce a bias towards an underestimation of the cost-effectiveness of the CMR + CXA strategy.
Limitations:
In the four countries, the unit costs for the cardiac examinations fed into the model may vary between different geographical regions and therefore, the results are representative for the entire health care systems under study, but not for smaller geographical regions. In the current model, no treatment costs were included. Correct absence of disease was not directly included in the criterion of effectiveness but the effect was captured indirectly through the costs of complications induced by false negative results. The model does not take into account intangible costs associated with cardiac death. This is because of the third-party-payer-perspective study design. For the US context, we decided to use costs for the material and a reimbursement of the physician to represent FFR costs similar to the approach used by Fearon et al. [7] as the current US reimbursement system does not consider costs for infrastructure nor material. The sensitivity analyses showed a rather moderate effect of prices for FFR on the cost-effectiveness shifting the crossing point by ±6, ±8, ±8, and ±11 percent points for the Swiss, the German, the UK, and the US system, respectively.
Finally, the modeling approach used here implies that some simplifications are built in and the decision process to revascularize or not is reduced to the presence or absence of hemodynamically significant stenoses. It does therefore not consider the clinical background of the patient, which is always important to guide treatment. Accordingly, the presented results might be helpful for trials planning whereas the use of the presented model with real CMR and FFR data acquired in ongoing trials [11] would most likely yield more relevant results.
Conclusions:
With a focus on the latest imaging techniques to detect ischemia, this study shows to what extent the cost-effectiveness of two strategies to diagnose hemodynamically significant coronary lesions, i.e. significant CAD, depends on the prevalence of the disease. The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help the decision-making with regard to resource utilization.
Abbreviations:
CAD: Coronary artery disease; CMR: Cardiovascular magnetic resonance; CXA: Invasive x-ray coronary angiography; FFR: Fractional flow reserve; PCI: Percutaneous coronary interventions.
Competing interests:
The authors declare that they have no competing interests.
Authors' contributions:
KM is responsible for the conception and design of the cost-effectiveness analysis; she performed the cost-effectiveness analysis, participated to the data collection and drafted the manuscript. DF participated to data collection and was involved in the calculations of the cost-effectiveness analysis. CP provided advices on the cost-effectiveness analysis. GP provided precisions on how the German health care system works and actively participated in the acquisition of required data in this context. SP provided information on how the UK health care system works and participated in the data acquisition in this context. AW provided explanations on how the US health care system works and participated in the data acquisition in this context. JBW provided explanations on how the Swiss health care system works. JS is responsible for the design of the study, participated to data collection and was involved in the interpretation of the results and drafting the manuscript; he critically revised its intellectual content. In addition, all authors provided helpful comments and relevant suggestions to improve the manuscript and its intellectual content; all authors read and approved the final manuscript.
Supplementary Material:
Section A1. The relationship FFR-Stenosis. Section A2. Equations to estimate the costs and the effectiveness for each strategy.
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Brief description on how the costs were derived for each country.
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| Background:
According to recent guidelines, patients with coronary artery disease (CAD) should undergo revascularization if significant myocardial ischemia is present. Both, cardiovascular magnetic resonance (CMR) and fractional flow reserve (FFR) allow for a reliable ischemia assessment and in combination with anatomical information provided by invasive coronary angiography (CXA), such a work-up sets the basis for a decision to revascularize or not. The cost-effectiveness ratio of these two strategies is compared.
Methods:
Strategy 1) CMR to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA), Strategy 2) CXA followed by FFR in angiographically positive stenoses (CXA + FFR). The costs, evaluated from the third party payer perspective in Switzerland, Germany, the United Kingdom (UK), and the United States (US), included public prices of the different outpatient procedures and costs induced by procedural complications and by diagnostic errors. The effectiveness criterion was the correct identification of hemodynamically significant coronary lesion(s) (= significant CAD) complemented by full anatomical information. Test performances were derived from the published literature. Cost-effectiveness ratios for both strategies were compared for hypothetical cohorts with different pretest likelihood of significant CAD.
Results:
CMR + CXA and CXA + FFR were equally cost-effective at a pretest likelihood of CAD of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the US with costs of CHF 5'794, € 1'517, £ 2'680, and $ 2'179 per patient correctly diagnosed. Below these thresholds, CMR + CXA showed lower costs per patient correctly diagnosed than CXA + FFR.
Conclusions:
The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help to optimize resource utilization in the diagnosis of CAD. | Background:
In many countries, cardiovascular diseases are the leading cause of morbidity and also of loss of quality of life. In particular, coronary artery disease (CAD) constitutes a major public health problem. In Europe the total costs of CAD and stroke were estimated at 49 billion Euros in the year 2008 [1] and for the United States these costs were estimated as high as 156 billion dollars [2]. It is well established that patients with no evidence of myocardial ischemia have low cardiac events rates, even when invasive coronary angiography (CXA) demonstrates lesions of intermediate severity [3,4]. In addition, patients without ischemia can be treated safely with medical therapy [5,6] thereby reducing the total costs of patient management [7]. On the other hand, randomized trials e.g. in diabetic patients demonstrated a survival benefit of patients with ischemia being treated by revascularization versus medical treatment alone [8]. Accordingly, recent guidelines recommend to revascularize patients if a relevant burden of myocardial ischemia is present (i.e. proximal vessel(s)) with a positive fractional flow reserve (FFR) and/or >10% of myocardium ischemic [9] or ≥2 ischemic segments in cardiovascular magnetic resonance (CMR) perfusion examinations [10].
Nevertheless, neither vessel anatomy nor presence or absence of ischemia is the factor that will exclusively decide on revascularizations. Symptoms, co-morbidities and other factors have to be taken into account before a treatment decision is made. Thus, the current analysis was undertaken to assess the cost-effectiveness to acquire information (significant ischemia and full anatomical information) relevant for decision making, but did not include the costs for all information needed to manage patients with CAD. Also, we do not know whether a large ischemia burden is directly related to adverse effects, whether it represents a marker of higher risk for occlusion of a severe stenosis that causes ischemia, or whether more severe ischaemia is simply a marker of more extensive atherosclerosis and more vulnerable plaques that go along with a worse outcome. Large trials such as ISCHEMIA and others [11] will hopefully improve our knowledge in a near future on how to treat ischemic patients. Despite this current lack of a detailed understanding of the underlying mechanisms that link ischemia to outcome, current guidelines recommend an ischemia-based approach for decision making in patients with CAD. Therefore, the aim of the current study was to assess the cost-effectiveness of two diagnostic strategies that are ischemia-based and provide both full anatomical and functional evaluation of CAD, which are the basis for a revascularization procedure.
A variety of new imaging techniques allow for such a combined anatomical and functional assessment of CAD, and as a result, the selection of the optimum test becomes more and more challenging. The choice of cardiovascular imaging techniques should consider both, their clinical benefits for the patients as well as the costs and cost-effectiveness compared to others. Invasive coronary angiography (CXA) remains the reference for the morphological assessment of CAD and it is often used in daily practice as a first line test, e.g. in patients with a positive treadmill test. While this strategy is not recommended by current guidelines, the advent of the FFR measurement to assess the hemodynamic significance of coronary artery stenoses [12] may even increase the attractivity to use invasive CXA as a first line test, as it can be easily combined with FFR in case of intermediate stenoses. Also, the FFR results were highly predictive for patient outcome [13,14] and the combination of CXA with FFR was more cost-effective than a CXA-only approach for the treatment of CAD [15]. Accordingly, recent guidelines recommend using FFR to correctly identify lesions that should undergo percutaneous coronary interventions (PCI) [9]. However, the invasive nature and radiation exposure of CXA and FFR limit their usefulness in a screening process [16]. Considering the fact that CXA is still extensively used in many industrialized countries as an early step in the work-up of suspected CAD [17,18], and further considering that FFR is recommended in recent guidelines, the combination of CXA + FFR was one diagnostic strategy to be assessed in the current study with respect to its cost-effectiveness.
As an alternative to the FFR measurement, perfusion CMR has emerged as a robust non-invasive technique for the evaluation of myocardial ischemia [19-22]. Furthermore, recent studies demonstrated the excellent prognosis of patients with known or suspected CAD, when perfusion-CMR was normal [23-25]. Accordingly, in the present study, the cost-effectiveness of a combination of CMR + CXA was compared with that of a CXA + FFR strategy.
In the current economic context, the health care systems have to be economically sustainable while preserving high quality medical standards. Consequently, in the following study we estimated the costs of the two different strategies relative to their effectiveness to 1) correctly diagnose the presence of relevant ischemia (= significant CAD) and 2) to yield full anatomical information of the coronary vasculature in case of ischemia. In particular, the cost-effectiveness of the two strategies was compared when applied to patient populations with varying CAD pre-test probabilities. Strategy 1 consists of a CMR examination to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA). This strategy yields complete information on myocardial ischemia and coronary anatomy. Strategy 2 consists of a CXA in all patients followed by a FFR test in patients with intermediate stenoses on CXA (CXA + FFR). Finally, the cost-effectiveness ratios of the two strategies were calculated for the health care systems in Switzerland, Germany, the United Kingdom, and the United States.
Conclusions:
With a focus on the latest imaging techniques to detect ischemia, this study shows to what extent the cost-effectiveness of two strategies to diagnose hemodynamically significant coronary lesions, i.e. significant CAD, depends on the prevalence of the disease. The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help the decision-making with regard to resource utilization. | Background:
According to recent guidelines, patients with coronary artery disease (CAD) should undergo revascularization if significant myocardial ischemia is present. Both, cardiovascular magnetic resonance (CMR) and fractional flow reserve (FFR) allow for a reliable ischemia assessment and in combination with anatomical information provided by invasive coronary angiography (CXA), such a work-up sets the basis for a decision to revascularize or not. The cost-effectiveness ratio of these two strategies is compared.
Methods:
Strategy 1) CMR to assess ischemia followed by CXA in ischemia-positive patients (CMR + CXA), Strategy 2) CXA followed by FFR in angiographically positive stenoses (CXA + FFR). The costs, evaluated from the third party payer perspective in Switzerland, Germany, the United Kingdom (UK), and the United States (US), included public prices of the different outpatient procedures and costs induced by procedural complications and by diagnostic errors. The effectiveness criterion was the correct identification of hemodynamically significant coronary lesion(s) (= significant CAD) complemented by full anatomical information. Test performances were derived from the published literature. Cost-effectiveness ratios for both strategies were compared for hypothetical cohorts with different pretest likelihood of significant CAD.
Results:
CMR + CXA and CXA + FFR were equally cost-effective at a pretest likelihood of CAD of 62% in Switzerland, 65% in Germany, 83% in the UK, and 82% in the US with costs of CHF 5'794, € 1'517, £ 2'680, and $ 2'179 per patient correctly diagnosed. Below these thresholds, CMR + CXA showed lower costs per patient correctly diagnosed than CXA + FFR.
Conclusions:
The CMR + CXA strategy is more cost-effective than CXA + FFR below a CAD prevalence of 62%, 65%, 83%, and 82% for the Swiss, the German, the UK, and the US health care systems, respectively. These findings may help to optimize resource utilization in the diagnosis of CAD. | 16,613 | 393 | [
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"ischemia treated revascularization"
] | [CONTENT] Cost-effectiveness analysis | Coronary artery disease | Cardiovascular magnetic resonance | Coronary angiography | Fractional flow reserve | Decision making [SUMMARY] | [CONTENT] Cost-effectiveness analysis | Coronary artery disease | Cardiovascular magnetic resonance | Coronary angiography | Fractional flow reserve | Decision making [SUMMARY] | [CONTENT] Cost-effectiveness analysis | Coronary artery disease | Cardiovascular magnetic resonance | Coronary angiography | Fractional flow reserve | Decision making [SUMMARY] | [CONTENT] Cost-effectiveness analysis | Coronary artery disease | Cardiovascular magnetic resonance | Coronary angiography | Fractional flow reserve | Decision making [SUMMARY] | [CONTENT] Cost-effectiveness analysis | Coronary artery disease | Cardiovascular magnetic resonance | Coronary angiography | Fractional flow reserve | Decision making [SUMMARY] | [CONTENT] Cost-effectiveness analysis | Coronary artery disease | Cardiovascular magnetic resonance | Coronary angiography | Fractional flow reserve | Decision making [SUMMARY] | [CONTENT] Comparative Effectiveness Research | Coronary Angiography | Coronary Stenosis | Coronary Vessels | Cost Savings | Cost-Benefit Analysis | Europe | Fractional Flow Reserve, Myocardial | Health Care Costs | Hemodynamics | Humans | Likelihood Functions | Magnetic Resonance Imaging | Models, Economic | Myocardial Revascularization | Patient Selection | Predictive Value of Tests | Prognosis | Reproducibility of Results | Severity of Illness Index | United States [SUMMARY] | [CONTENT] Comparative Effectiveness Research | Coronary Angiography | Coronary Stenosis | Coronary Vessels | Cost Savings | Cost-Benefit Analysis | Europe | Fractional Flow Reserve, Myocardial | Health Care Costs | Hemodynamics | Humans | Likelihood Functions | Magnetic Resonance Imaging | Models, Economic | Myocardial Revascularization | Patient Selection | Predictive Value of Tests | Prognosis | Reproducibility of Results | Severity of Illness Index | United States [SUMMARY] | [CONTENT] Comparative Effectiveness Research | Coronary Angiography | Coronary Stenosis | Coronary Vessels | Cost Savings | Cost-Benefit Analysis | Europe | Fractional Flow Reserve, Myocardial | Health Care Costs | Hemodynamics | Humans | Likelihood Functions | Magnetic Resonance Imaging | Models, Economic | Myocardial Revascularization | Patient Selection | Predictive Value of Tests | Prognosis | Reproducibility of Results | Severity of Illness Index | United States [SUMMARY] | [CONTENT] Comparative Effectiveness Research | Coronary Angiography | Coronary Stenosis | Coronary Vessels | Cost Savings | Cost-Benefit Analysis | Europe | Fractional Flow Reserve, Myocardial | Health Care Costs | Hemodynamics | Humans | Likelihood Functions | Magnetic Resonance Imaging | Models, Economic | Myocardial Revascularization | Patient Selection | Predictive Value of Tests | Prognosis | Reproducibility of Results | Severity of Illness Index | United States [SUMMARY] | [CONTENT] Comparative Effectiveness Research | Coronary Angiography | Coronary Stenosis | Coronary Vessels | Cost Savings | Cost-Benefit Analysis | Europe | Fractional Flow Reserve, Myocardial | Health Care Costs | Hemodynamics | Humans | Likelihood Functions | Magnetic Resonance Imaging | Models, Economic | Myocardial Revascularization | Patient Selection | Predictive Value of Tests | Prognosis | Reproducibility of Results | Severity of Illness Index | United States [SUMMARY] | [CONTENT] Comparative Effectiveness Research | Coronary Angiography | Coronary Stenosis | Coronary Vessels | Cost Savings | Cost-Benefit Analysis | Europe | Fractional Flow Reserve, Myocardial | Health Care Costs | Hemodynamics | Humans | Likelihood Functions | Magnetic Resonance Imaging | Models, Economic | Myocardial Revascularization | Patient Selection | Predictive Value of Tests | Prognosis | Reproducibility of Results | Severity of Illness Index | United States [SUMMARY] | [CONTENT] ischemia cmr cost | ischemia significant cad | ischemia strategy effectiveness | myocardial ischemia coronary | ischemia treated revascularization [SUMMARY] | [CONTENT] ischemia cmr cost | ischemia significant cad | ischemia strategy effectiveness | myocardial ischemia coronary | ischemia treated revascularization [SUMMARY] | [CONTENT] ischemia cmr cost | ischemia significant cad | ischemia strategy effectiveness | myocardial ischemia coronary | ischemia treated revascularization [SUMMARY] | [CONTENT] ischemia cmr cost | ischemia significant cad | ischemia strategy effectiveness | myocardial ischemia coronary | ischemia treated revascularization [SUMMARY] | [CONTENT] ischemia cmr cost | ischemia significant cad | ischemia strategy effectiveness | myocardial ischemia coronary | ischemia treated revascularization [SUMMARY] | [CONTENT] ischemia cmr cost | ischemia significant cad | ischemia strategy effectiveness | myocardial ischemia coronary | ischemia treated revascularization [SUMMARY] | [CONTENT] costs | cxa | cmr | cad | ffr | cost | effectiveness | patients | ischemia | strategy [SUMMARY] | [CONTENT] costs | cxa | cmr | cad | ffr | cost | effectiveness | patients | ischemia | strategy [SUMMARY] | [CONTENT] costs | cxa | cmr | cad | ffr | cost | effectiveness | patients | ischemia | strategy [SUMMARY] | [CONTENT] costs | cxa | cmr | cad | ffr | cost | effectiveness | patients | ischemia | strategy [SUMMARY] | [CONTENT] costs | cxa | cmr | cad | ffr | cost | effectiveness | patients | ischemia | strategy [SUMMARY] | [CONTENT] costs | cxa | cmr | cad | ffr | cost | effectiveness | patients | ischemia | strategy [SUMMARY] | [CONTENT] ischemia | patients | cxa | cad | current | ffr | coronary | information | guidelines | estimated [SUMMARY] | [CONTENT] costs | effectiveness | ischemia | patients | cxa | positive | diagnostic | model | tests | complications [SUMMARY] | [CONTENT] cad | significant cad | significant | cmr | likelihood | costs | sensitivity | cxa | cost | likelihood significant [SUMMARY] | [CONTENT] prevalence | detect ischemia study shows | cmr cxa strategy cost | 83 82 swiss german | extent cost effectiveness | extent cost | techniques detect ischemia study | techniques detect ischemia | techniques detect | extent [SUMMARY] | [CONTENT] costs | cad | cxa | cmr | ffr | patients | cost | ischemia | effectiveness | significant [SUMMARY] | [CONTENT] costs | cad | cxa | cmr | ffr | patients | cost | ischemia | effectiveness | significant [SUMMARY] | [CONTENT] CAD ||| CMR | CXA ||| two [SUMMARY] | [CONTENT] 1 | CMR | CXA | CMR | Strategy 2 | CXA | FFR | CXA ||| third | Switzerland | Germany | the United Kingdom | UK | the United States | US ||| CAD ||| ||| CAD [SUMMARY] | [CONTENT] CMR | CXA + FFR | CAD | 62% | Switzerland | 65% | Germany | 83% | UK | 82% | US | CHF 5'794 | 1'517 | 2'680 | 2'179 ||| CMR + CXA | CXA [SUMMARY] | [CONTENT] CMR | CXA | CXA | CAD | 62% | 65% | 83% | 82% | Swiss | German | UK | US ||| CAD [SUMMARY] | [CONTENT] ||| CAD ||| CMR | CXA ||| two ||| CMR | CXA | CMR | Strategy 2 | CXA | FFR | CXA ||| third | Switzerland | Germany | the United Kingdom | UK | the United States | US ||| CAD ||| ||| CAD ||| CMR | CXA + FFR | CAD | 62% | Switzerland | 65% | Germany | 83% | UK | 82% | US | CHF 5'794 | 1'517 | 2'680 | 2'179 ||| CMR + CXA | CXA ||| CMR | CXA | CXA | CAD | 62% | 65% | 83% | 82% | Swiss | German | UK | US ||| CAD [SUMMARY] | [CONTENT] ||| CAD ||| CMR | CXA ||| two ||| CMR | CXA | CMR | Strategy 2 | CXA | FFR | CXA ||| third | Switzerland | Germany | the United Kingdom | UK | the United States | US ||| CAD ||| ||| CAD ||| CMR | CXA + FFR | CAD | 62% | Switzerland | 65% | Germany | 83% | UK | 82% | US | CHF 5'794 | 1'517 | 2'680 | 2'179 ||| CMR + CXA | CXA ||| CMR | CXA | CXA | CAD | 62% | 65% | 83% | 82% | Swiss | German | UK | US ||| CAD [SUMMARY] |
||||||
Effects of postoperative radiotherapy on cardiovascular-pulmonary disease mortality in patients with stage IIIA-N2 resected NSCLC: analysis of the SEER database. | 34544464 | The role of postoperative radiotherapy (PORT) in cardiovascular-pulmonary disease mortality in patients with stage IIIA-N2 resected non-small cell lung cancer (NSCLC) remains uncertain. The purpose of this population-based analysis was to explore the effect of PORT on cardiovascular-pulmonary disease mortality in these patients. | BACKGROUND | Patients aged ≥ 18 years with stage IIIA-N2 resected NSCLC were identified in the Surveillance, Epidemiology, and End Results (SEER) database from 2004 to 2015 and were grouped according to the use of PORT. Propensity score matching (PSM) was used to account for differences in baseline characteristics between the Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression was used to run univariate and multivariate analyses to evaluate risk factors. | METHODS | A total of 3981 patients were included in the study population. Among them, 1446 patients received PORT, and 2535 did not. A total of 1380 patients remained in each group after PSM, and the baseline characteristics were not significantly different between the two groups. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (HR 1.07, 95% CI 0.77-1.48, p = 0.703). Multivariate analysis indicated that PORT had no significant impact on increased risk, with an HR of 1.18 (p = 0.377). | RESULTS | No significant differences between the PORT and Non-PORT groups were found in cardiovascular-pulmonary-specific modalities in this study. Further studies are required to validate these results. This study highlights the importance of long-term surveillance for NSCLC patients. | CONCLUSIONS | [
"Adult",
"Aged",
"Aged, 80 and over",
"Carcinoma, Non-Small-Cell Lung",
"Cardiovascular Diseases",
"Combined Modality Therapy",
"Humans",
"Lung Diseases",
"Lung Neoplasms",
"Middle Aged",
"Neoplasm Staging",
"Risk Factors",
"SEER Program",
"Young Adult"
] | 8453996 | Background | Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancer cases [1, 2]. Early-stage NSCLC is best managed with complete surgical resection [3]. Despite curative-intent surgical resection, tumor recurrence and metastasis are major causes of death for patients with locally advanced NSCLC [4–6]. Therefore, surgery plus multidisciplinary sequential therapy continues to be the backbone of treatment with curative intent among patients with stage IIIA resected NSCLC [5, 7–9].
Previous studies have shown that postoperative radiotherapy (PORT) in patients with stage IIIA-N2 NSCLC reduces the risk of local recurrence and thus is an appealing means of improving outcomes in NSCLC patients [10, 11], but whether PORT can bring overall survival (OS) benefits to those patients remains controversial [9–12]. Several retrospective studies and meta-analyses have shown the survival benefits of PORT [5, 10, 11, 13–15]. However, recent multi-institutional randomized phase III trials (Lung ART and PORT-C) indicated that PORT failed to improve disease‐free survival and OS [9, 12]. In the Lung ART study, the incidence of grade 3–5 late cardiopulmonary toxicity was 20% versus 7.7%, and the cardiopulmonary specific mortality was 16.2% versus 2% in the PORT versus Non‐PORT cohort, respectively [9]. The survival benefit may be counterbalanced by radiotherapy (RT)‐induced cardiopulmonary-specific death [9].
Thoracic RT increases the risk of cardiovascular-pulmonary disease during or after therapy, such as ischemic heart disease, arterial disease, pericardial disease, vascular and metabolic issues, conduction disorders, pneumonitis and pulmonary fibrosis, chronic pulmonary insufficiency, and cor pulmonale, and resulting in increased mortality [6, 16–20]. Radiation-associated cardiovascular-pulmonary events and deaths have been thoroughly documented in long-term survivors of breast cancer and Hodgkin’s lymphoma [18, 21–24]. However, the data regarding RT‐associated cardiovascular-pulmonary specific death in patients with NSCLC are limited [6]. Currently, there are no large datasets available with PORT and cardiovascular-pulmonary specific mortality in patients with stage IIIA-N2 NSCLC.
Therefore, we conducted a propensity-matched retrospective study to investigate the effect of PORT on cardiovascular-pulmonary related death in patients with resected stage IIIA-N2 NSCLC using the Surveillance, Epidemiology, and End Results (SEER) database. | null | null | Results | Patient characteristics A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.
Table 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM
Non-PORT
(N = 2535)
PORT
(N = 1446)
P-value
Non-PORT
(N = 1380)
PORT
(N = 1380)
P-value
Age, years (range)
67 (22–90)64 (19–88)
< 0.001*
65 (22–89)65 (28–88)0.674
Sex
0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)
Race
0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)
Year of diagnosis
< 0.001*
0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)
Laterality
0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)
Primary site
0.021*
0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)
Tumor size, mm (range)
35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584
Histologic type
0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)
Grade
0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)
Surgical procedure
< 0.001*
0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)
T stage (sixth edition)
0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)
LN examined (range)
11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595
Metastatic LN
< 0.001*
0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)
Positive LN ratio (%)
< 0.001*
0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)
Chemotherapy
< 0.001*
0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)
Median FU, months (range)
27 (1–154)28 (1–154)
0.010*
29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM
LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.
Table 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM
Non-PORT
(N = 2535)
PORT
(N = 1446)
P-value
Non-PORT
(N = 1380)
PORT
(N = 1380)
P-value
Age, years (range)
67 (22–90)64 (19–88)
< 0.001*
65 (22–89)65 (28–88)0.674
Sex
0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)
Race
0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)
Year of diagnosis
< 0.001*
0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)
Laterality
0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)
Primary site
0.021*
0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)
Tumor size, mm (range)
35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584
Histologic type
0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)
Grade
0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)
Surgical procedure
< 0.001*
0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)
T stage (sixth edition)
0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)
LN examined (range)
11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595
Metastatic LN
< 0.001*
0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)
Positive LN ratio (%)
< 0.001*
0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)
Chemotherapy
< 0.001*
0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)
Median FU, months (range)
27 (1–154)28 (1–154)
0.010*
29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM
LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
Cumulative incidence of cardiovascular-pulmonary disease-related death A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.
Fig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
A total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).
A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.
Fig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
A total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).
Risk factors for cardiovascular-pulmonary diseases death Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.
Table 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name
HR
95% CI for HR
p-value
Univariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.601.082.38
0.020*
Sex (Male vs. Female)1.651.182.32
0.004*
Race
BlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111
Year of diagnosis
2004–2007Reference2008–20110.670.460.98
0.037*
2012–20150.310.190.48
< 0.001*
Laterality (Left vs. Right)1.180.851.640.330
Primary site
Main bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696
Tumor size, mm ( < = 40 vs. > 40)1.090.771.540.628
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78
< 0.001*
Others1.030.591.790.919
Grade
Well differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771
Surgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374
T stage (sixth edition)
T1ReferenceT20.810.571.150.244T30.710.351.440.342
Chemotherapy (No vs. Yes)2.041.353.08
0.001*
Metastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395
Positive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204
PORT (Yes vs. No)1.070.771.480.703
Multivariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.360.872.140.179
Sex (Male vs. Female)1.571.062.32
0.026*
Year of diagnosis
2004–2007Reference2008–20110.600.400.91
0.015*
2012–20150.290.180.49
< 0.001
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54
0.010*
Others0.840.461.530.569
Chemotherapy (No vs. Yes)1.410.882.250.154
PORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold
Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model
Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.
HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold
Fig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.
Table 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name
HR
95% CI for HR
p-value
Univariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.601.082.38
0.020*
Sex (Male vs. Female)1.651.182.32
0.004*
Race
BlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111
Year of diagnosis
2004–2007Reference2008–20110.670.460.98
0.037*
2012–20150.310.190.48
< 0.001*
Laterality (Left vs. Right)1.180.851.640.330
Primary site
Main bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696
Tumor size, mm ( < = 40 vs. > 40)1.090.771.540.628
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78
< 0.001*
Others1.030.591.790.919
Grade
Well differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771
Surgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374
T stage (sixth edition)
T1ReferenceT20.810.571.150.244T30.710.351.440.342
Chemotherapy (No vs. Yes)2.041.353.08
0.001*
Metastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395
Positive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204
PORT (Yes vs. No)1.070.771.480.703
Multivariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.360.872.140.179
Sex (Male vs. Female)1.571.062.32
0.026*
Year of diagnosis
2004–2007Reference2008–20110.600.400.91
0.015*
2012–20150.290.180.49
< 0.001
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54
0.010*
Others0.840.461.530.569
Chemotherapy (No vs. Yes)1.410.882.250.154
PORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold
Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model
Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.
HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold
Fig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy | Conclusions | In conclusion, this study shows the cumulative incidence of cardiovascular-pulmonary mortality by PORT use in stage IIIA-N2 NSCLC patients after complete resection and identifies potential prognostic factors for cardiovascular-pulmonary-specific death. Moreover, no significant differences were found in cardiovascular-pulmonary‐related modalities between the PORT and Non‐PORT groups in this study. Further studies are needed to assess these results, and more detailed information on risk factors should be examined in future work. This study highlights the importance of long-term surveillance for NSCLC patients. | [
"Background",
"Patients and methods",
"Data sources",
"Study population and definition",
"Statistical analysis",
"Patient characteristics",
"Cumulative incidence of cardiovascular-pulmonary disease-related death",
"Risk factors for cardiovascular-pulmonary diseases death"
] | [
"Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancer cases [1, 2]. Early-stage NSCLC is best managed with complete surgical resection [3]. Despite curative-intent surgical resection, tumor recurrence and metastasis are major causes of death for patients with locally advanced NSCLC [4–6]. Therefore, surgery plus multidisciplinary sequential therapy continues to be the backbone of treatment with curative intent among patients with stage IIIA resected NSCLC [5, 7–9].\nPrevious studies have shown that postoperative radiotherapy (PORT) in patients with stage IIIA-N2 NSCLC reduces the risk of local recurrence and thus is an appealing means of improving outcomes in NSCLC patients [10, 11], but whether PORT can bring overall survival (OS) benefits to those patients remains controversial [9–12]. Several retrospective studies and meta-analyses have shown the survival benefits of PORT [5, 10, 11, 13–15]. However, recent multi-institutional randomized phase III trials (Lung ART and PORT-C) indicated that PORT failed to improve disease‐free survival and OS [9, 12]. In the Lung ART study, the incidence of grade 3–5 late cardiopulmonary toxicity was 20% versus 7.7%, and the cardiopulmonary specific mortality was 16.2% versus 2% in the PORT versus Non‐PORT cohort, respectively [9]. The survival benefit may be counterbalanced by radiotherapy (RT)‐induced cardiopulmonary-specific death [9].\nThoracic RT increases the risk of cardiovascular-pulmonary disease during or after therapy, such as ischemic heart disease, arterial disease, pericardial disease, vascular and metabolic issues, conduction disorders, pneumonitis and pulmonary fibrosis, chronic pulmonary insufficiency, and cor pulmonale, and resulting in increased mortality [6, 16–20]. Radiation-associated cardiovascular-pulmonary events and deaths have been thoroughly documented in long-term survivors of breast cancer and Hodgkin’s lymphoma [18, 21–24]. However, the data regarding RT‐associated cardiovascular-pulmonary specific death in patients with NSCLC are limited [6]. Currently, there are no large datasets available with PORT and cardiovascular-pulmonary specific mortality in patients with stage IIIA-N2 NSCLC.\nTherefore, we conducted a propensity-matched retrospective study to investigate the effect of PORT on cardiovascular-pulmonary related death in patients with resected stage IIIA-N2 NSCLC using the Surveillance, Epidemiology, and End Results (SEER) database.",
"Data sources The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.\nThe data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.\nStudy population and definition We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.\n\nFig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\n\nFlow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\nVariables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.\nFollow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].\nWe extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.\n\nFig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\n\nFlow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\nVariables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.\nFollow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].\nStatistical analysis Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.\nData are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.",
"The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.",
"We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.\n\nFig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\n\nFlow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\nVariables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.\nFollow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].",
"Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.",
"A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.\n\nTable 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM\nNon-PORT\n\n(N = 2535)\n\nPORT\n\n(N = 1446)\nP-value\n\nNon-PORT \n\n(N = 1380)\n\nPORT \n\n(N = 1380)\nP-value\n\nAge, years (range)\n67 (22–90)64 (19–88)\n< 0.001*\n65 (22–89)65 (28–88)0.674\nSex\n0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)\nRace\n0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)\nYear of diagnosis\n\n< 0.001*\n0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)\nLaterality\n0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)\nPrimary site\n\n0.021*\n0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)\nTumor size, mm (range)\n35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584\nHistologic type\n0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)\nGrade\n0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)\nSurgical procedure\n\n< 0.001*\n0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)\nT stage (sixth edition)\n0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)\nLN examined (range)\n11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595\nMetastatic LN\n\n< 0.001*\n0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)\nPositive LN ratio (%)\n\n< 0.001*\n0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)\nChemotherapy\n\n< 0.001*\n0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)\nMedian FU, months (range)\n27 (1–154)28 (1–154)\n0.010*\n29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)\n\nThe baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM\nLN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)",
"A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.\n\nFig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\n\nCumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\nA total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).",
"Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.\n\nTable 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name\n\nHR\n\n95% CI for HR\np-value\n\n\nUnivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.601.082.38\n0.020*\n\nSex (Male vs. Female)1.651.182.32\n0.004*\n\nRace\nBlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111\nYear of diagnosis\n2004–2007Reference2008–20110.670.460.98\n0.037*\n2012–20150.310.190.48\n< 0.001*\n\nLaterality (Left vs. Right)1.180.851.640.330\nPrimary site\nMain bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696\nTumor size, mm ( < = 40 vs. > 40)1.090.771.540.628\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78\n< 0.001*\nOthers1.030.591.790.919\nGrade\nWell differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771\nSurgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374\nT stage (sixth edition)\nT1ReferenceT20.810.571.150.244T30.710.351.440.342\nChemotherapy (No vs. Yes)2.041.353.08\n0.001*\n\nMetastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395\nPositive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204\nPORT (Yes vs. No)1.070.771.480.703\nMultivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.360.872.140.179\nSex (Male vs. Female)1.571.062.32\n0.026*\n\nYear of diagnosis\n2004–2007Reference2008–20110.600.400.91\n0.015*\n2012–20150.290.180.49\n< 0.001\n\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54\n0.010*\nOthers0.840.461.530.569\nChemotherapy (No vs. Yes)1.410.882.250.154\nPORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold\n\nUnivariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model\nModel adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.\nHR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold\n\nFig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy\n\nSubgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy"
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Patients and methods",
"Data sources",
"Study population and definition",
"Statistical analysis",
"Results",
"Patient characteristics",
"Cumulative incidence of cardiovascular-pulmonary disease-related death",
"Risk factors for cardiovascular-pulmonary diseases death",
"Discussion",
"Conclusions"
] | [
"Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancer cases [1, 2]. Early-stage NSCLC is best managed with complete surgical resection [3]. Despite curative-intent surgical resection, tumor recurrence and metastasis are major causes of death for patients with locally advanced NSCLC [4–6]. Therefore, surgery plus multidisciplinary sequential therapy continues to be the backbone of treatment with curative intent among patients with stage IIIA resected NSCLC [5, 7–9].\nPrevious studies have shown that postoperative radiotherapy (PORT) in patients with stage IIIA-N2 NSCLC reduces the risk of local recurrence and thus is an appealing means of improving outcomes in NSCLC patients [10, 11], but whether PORT can bring overall survival (OS) benefits to those patients remains controversial [9–12]. Several retrospective studies and meta-analyses have shown the survival benefits of PORT [5, 10, 11, 13–15]. However, recent multi-institutional randomized phase III trials (Lung ART and PORT-C) indicated that PORT failed to improve disease‐free survival and OS [9, 12]. In the Lung ART study, the incidence of grade 3–5 late cardiopulmonary toxicity was 20% versus 7.7%, and the cardiopulmonary specific mortality was 16.2% versus 2% in the PORT versus Non‐PORT cohort, respectively [9]. The survival benefit may be counterbalanced by radiotherapy (RT)‐induced cardiopulmonary-specific death [9].\nThoracic RT increases the risk of cardiovascular-pulmonary disease during or after therapy, such as ischemic heart disease, arterial disease, pericardial disease, vascular and metabolic issues, conduction disorders, pneumonitis and pulmonary fibrosis, chronic pulmonary insufficiency, and cor pulmonale, and resulting in increased mortality [6, 16–20]. Radiation-associated cardiovascular-pulmonary events and deaths have been thoroughly documented in long-term survivors of breast cancer and Hodgkin’s lymphoma [18, 21–24]. However, the data regarding RT‐associated cardiovascular-pulmonary specific death in patients with NSCLC are limited [6]. Currently, there are no large datasets available with PORT and cardiovascular-pulmonary specific mortality in patients with stage IIIA-N2 NSCLC.\nTherefore, we conducted a propensity-matched retrospective study to investigate the effect of PORT on cardiovascular-pulmonary related death in patients with resected stage IIIA-N2 NSCLC using the Surveillance, Epidemiology, and End Results (SEER) database.",
"Data sources The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.\nThe data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.\nStudy population and definition We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.\n\nFig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\n\nFlow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\nVariables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.\nFollow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].\nWe extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.\n\nFig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\n\nFlow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\nVariables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.\nFollow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].\nStatistical analysis Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.\nData are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.",
"The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.",
"We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.\n\nFig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\n\nFlow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy\nVariables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.\nFollow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].",
"Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.",
"Patient characteristics A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.\n\nTable 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM\nNon-PORT\n\n(N = 2535)\n\nPORT\n\n(N = 1446)\nP-value\n\nNon-PORT \n\n(N = 1380)\n\nPORT \n\n(N = 1380)\nP-value\n\nAge, years (range)\n67 (22–90)64 (19–88)\n< 0.001*\n65 (22–89)65 (28–88)0.674\nSex\n0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)\nRace\n0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)\nYear of diagnosis\n\n< 0.001*\n0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)\nLaterality\n0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)\nPrimary site\n\n0.021*\n0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)\nTumor size, mm (range)\n35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584\nHistologic type\n0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)\nGrade\n0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)\nSurgical procedure\n\n< 0.001*\n0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)\nT stage (sixth edition)\n0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)\nLN examined (range)\n11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595\nMetastatic LN\n\n< 0.001*\n0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)\nPositive LN ratio (%)\n\n< 0.001*\n0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)\nChemotherapy\n\n< 0.001*\n0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)\nMedian FU, months (range)\n27 (1–154)28 (1–154)\n0.010*\n29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)\n\nThe baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM\nLN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)\nA total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.\n\nTable 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM\nNon-PORT\n\n(N = 2535)\n\nPORT\n\n(N = 1446)\nP-value\n\nNon-PORT \n\n(N = 1380)\n\nPORT \n\n(N = 1380)\nP-value\n\nAge, years (range)\n67 (22–90)64 (19–88)\n< 0.001*\n65 (22–89)65 (28–88)0.674\nSex\n0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)\nRace\n0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)\nYear of diagnosis\n\n< 0.001*\n0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)\nLaterality\n0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)\nPrimary site\n\n0.021*\n0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)\nTumor size, mm (range)\n35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584\nHistologic type\n0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)\nGrade\n0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)\nSurgical procedure\n\n< 0.001*\n0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)\nT stage (sixth edition)\n0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)\nLN examined (range)\n11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595\nMetastatic LN\n\n< 0.001*\n0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)\nPositive LN ratio (%)\n\n< 0.001*\n0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)\nChemotherapy\n\n< 0.001*\n0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)\nMedian FU, months (range)\n27 (1–154)28 (1–154)\n0.010*\n29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)\n\nThe baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM\nLN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)\nCumulative incidence of cardiovascular-pulmonary disease-related death A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.\n\nFig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\n\nCumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\nA total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).\nA total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.\n\nFig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\n\nCumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\nA total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).\nRisk factors for cardiovascular-pulmonary diseases death Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.\n\nTable 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name\n\nHR\n\n95% CI for HR\np-value\n\n\nUnivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.601.082.38\n0.020*\n\nSex (Male vs. Female)1.651.182.32\n0.004*\n\nRace\nBlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111\nYear of diagnosis\n2004–2007Reference2008–20110.670.460.98\n0.037*\n2012–20150.310.190.48\n< 0.001*\n\nLaterality (Left vs. Right)1.180.851.640.330\nPrimary site\nMain bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696\nTumor size, mm ( < = 40 vs. > 40)1.090.771.540.628\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78\n< 0.001*\nOthers1.030.591.790.919\nGrade\nWell differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771\nSurgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374\nT stage (sixth edition)\nT1ReferenceT20.810.571.150.244T30.710.351.440.342\nChemotherapy (No vs. Yes)2.041.353.08\n0.001*\n\nMetastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395\nPositive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204\nPORT (Yes vs. No)1.070.771.480.703\nMultivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.360.872.140.179\nSex (Male vs. Female)1.571.062.32\n0.026*\n\nYear of diagnosis\n2004–2007Reference2008–20110.600.400.91\n0.015*\n2012–20150.290.180.49\n< 0.001\n\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54\n0.010*\nOthers0.840.461.530.569\nChemotherapy (No vs. Yes)1.410.882.250.154\nPORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold\n\nUnivariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model\nModel adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.\nHR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold\n\nFig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy\n\nSubgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy\nUnivariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.\n\nTable 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name\n\nHR\n\n95% CI for HR\np-value\n\n\nUnivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.601.082.38\n0.020*\n\nSex (Male vs. Female)1.651.182.32\n0.004*\n\nRace\nBlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111\nYear of diagnosis\n2004–2007Reference2008–20110.670.460.98\n0.037*\n2012–20150.310.190.48\n< 0.001*\n\nLaterality (Left vs. Right)1.180.851.640.330\nPrimary site\nMain bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696\nTumor size, mm ( < = 40 vs. > 40)1.090.771.540.628\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78\n< 0.001*\nOthers1.030.591.790.919\nGrade\nWell differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771\nSurgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374\nT stage (sixth edition)\nT1ReferenceT20.810.571.150.244T30.710.351.440.342\nChemotherapy (No vs. Yes)2.041.353.08\n0.001*\n\nMetastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395\nPositive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204\nPORT (Yes vs. No)1.070.771.480.703\nMultivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.360.872.140.179\nSex (Male vs. Female)1.571.062.32\n0.026*\n\nYear of diagnosis\n2004–2007Reference2008–20110.600.400.91\n0.015*\n2012–20150.290.180.49\n< 0.001\n\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54\n0.010*\nOthers0.840.461.530.569\nChemotherapy (No vs. Yes)1.410.882.250.154\nPORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold\n\nUnivariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model\nModel adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.\nHR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold\n\nFig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy\n\nSubgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy",
"A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.\n\nTable 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM\nNon-PORT\n\n(N = 2535)\n\nPORT\n\n(N = 1446)\nP-value\n\nNon-PORT \n\n(N = 1380)\n\nPORT \n\n(N = 1380)\nP-value\n\nAge, years (range)\n67 (22–90)64 (19–88)\n< 0.001*\n65 (22–89)65 (28–88)0.674\nSex\n0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)\nRace\n0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)\nYear of diagnosis\n\n< 0.001*\n0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)\nLaterality\n0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)\nPrimary site\n\n0.021*\n0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)\nTumor size, mm (range)\n35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584\nHistologic type\n0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)\nGrade\n0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)\nSurgical procedure\n\n< 0.001*\n0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)\nT stage (sixth edition)\n0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)\nLN examined (range)\n11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595\nMetastatic LN\n\n< 0.001*\n0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)\nPositive LN ratio (%)\n\n< 0.001*\n0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)\nChemotherapy\n\n< 0.001*\n0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)\nMedian FU, months (range)\n27 (1–154)28 (1–154)\n0.010*\n29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)\n\nThe baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM\nLN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)",
"A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.\n\nFig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\n\nCumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy\nA total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).",
"Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.\n\nTable 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name\n\nHR\n\n95% CI for HR\np-value\n\n\nUnivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.601.082.38\n0.020*\n\nSex (Male vs. Female)1.651.182.32\n0.004*\n\nRace\nBlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111\nYear of diagnosis\n2004–2007Reference2008–20110.670.460.98\n0.037*\n2012–20150.310.190.48\n< 0.001*\n\nLaterality (Left vs. Right)1.180.851.640.330\nPrimary site\nMain bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696\nTumor size, mm ( < = 40 vs. > 40)1.090.771.540.628\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78\n< 0.001*\nOthers1.030.591.790.919\nGrade\nWell differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771\nSurgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374\nT stage (sixth edition)\nT1ReferenceT20.810.571.150.244T30.710.351.440.342\nChemotherapy (No vs. Yes)2.041.353.08\n0.001*\n\nMetastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395\nPositive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204\nPORT (Yes vs. No)1.070.771.480.703\nMultivariate analysis (N = 2760)\n\nAge, years ( > = 60 vs. < 60)1.360.872.140.179\nSex (Male vs. Female)1.571.062.32\n0.026*\n\nYear of diagnosis\n2004–2007Reference2008–20110.600.400.91\n0.015*\n2012–20150.290.180.49\n< 0.001\n\nHistologic type\nAdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54\n0.010*\nOthers0.840.461.530.569\nChemotherapy (No vs. Yes)1.410.882.250.154\nPORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold\n\nUnivariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model\nModel adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.\nHR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy\n*P < 0.05 was considered significant and marked in bold\n\nFig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy\n\nSubgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy",
"Cardiovascular-pulmonary disease is the leading nonrecurrence cause of death in NSCLC patients [6]. The cardiovascular system and lungs are among the organs that are most severely affected by RT injury, resulting in increased morbidity and mortality [6, 16, 18]. The mechanisms of RT-related cardiovascular disease include endothelial dysfunction, altered vascular tone, hemostatic imbalance, and inflammatory activation [16]. Previous preclinical research found that radiation triggers lung injury by initiating a cascade of inflammatory reactions, with capillary leaks and alveolar and interstitial exudate, which later organize into collagen [18, 19]. Damage can even occur at least 10 years after RT [28].\nThe present work provides important insights into the risk of cardiovascular-pulmonary disease mortality by PORT in patients with stage IIIA-N2 resected NSCLC based on the SEER database. Our analysis of data from 2760 patients after PSM demonstrated that PORT among those patients was not associated with a higher risk of cardiovascular-pulmonary disease-related death (HR = 1.18, 95% CI 0.82–1.71, p = 0.377). The same conclusions were obtained in the subgroup analyses. Moreover, the multivariate analysis identified several risk factors for cardiovascular-pulmonary disease-related death, including male sex, earlier year of diagnosis, and squamous cell carcinoma pathologic type.\nThese appeared as unexpected findings. This study failed to show a difference in cardiovascular-pulmonary disease mortality, perhaps due to the follow-up not being long enough for outcomes to occur. The median durations of follow-up were 29 months in the Non-PORT group and 28.5 months in the PORT group, respectively. Cardiac-pulmonary toxicity following radiotherapy has been observed in long-term breast cancer and Hodgkin lymphoma survivors, with a typical latency period of more than a decade and increased incidence at younger age at treatment [18, 21, 24]. A previous study evaluating breast cancer after RT showed that the risk of major cardiovascular disease events started within the first 5 years after treatment and continued into the third decade of follow-up [29]. These malignancies portend a more favorable prognosis, while lung cancer is usually associated with poor prognosis and is conversely the leading cause of cancer-related death in the world [1]. In the phase III Lung ART trial [9], the 3-year OS rates were 66.5% in the PORT arm and 68.5% in the observation arm, respectively. In this study, NSCLC was the leading cause of death, with a cumulative incidence rate of 67.37%. It is possible that there was insufficient time for the development of cardiovascular-pulmonary disease in patients with stage IIIA-N2 NSCLC.\nThe definitive role of PORT in cardiopulmonary toxicity in pIIIA-N2 NSCLC remains controversial. In the recent PORT-C trial [12], no RT-induced grade 4 or 5 adverse events were observed. A total of 97 deaths (26.6%) occurred; among the 8 noncancer-related deaths, only 3 (3.1%) were due to cardiopulmonary disease [12]. The findings of the Lung ART study differed from the previous abovementioned study [9], and cardiopulmonary-specific mortality was observed in 2 (2.0%) patients in the Non-PORT cohort and 16 (16.2%) patients in the PORT cohort, respectively. The main reasons for these inconsistent results lie in the discrepancy of radiotherapy techniques and dose restrictions to the heart and lung. Studies have confirmed that patients treated with intensity-modulated radiation therapy (IMRT) for locally advanced NSCLC had lower rates of severe pneumonitis and cardiac doses than those treated with three-dimensional conformal external beam radiation therapy (3D-CRT) [30, 31]. The majority of patients received IMRT (89.3%) in the PORT-C study and 3D-CRT (89%) in the Lung ART study, respectively. The planned delivered dose was 54 Gy/27-30f in the LUNG ART trial. In reality, however, the maximum irradiation dose reached 70 Gy. The lung V20 was limited to less than 31% in patients after lobectomy and 22% after pneumonectomy, heart V30 less than 35% [9]. Another explanation for the low rate of cardiopulmonary-specific mortality in the PORT-C study is the markedly tighter dose restrictions for normal healthy tissues, especially the lungs and heart [12]. More effective modern RT techniques might have attenuated this risk.\nAlthough our study showed no association of PORT with an increased risk of cardiovascular-pulmonary death in patients with stage IIIA-N2 NSCLC, the long-term safety of PORT for those patients remains uncertain. Combined with the results from the two randomized phase III trials [9, 12], new and modern RT techniques such as the use of IMRT are expected to avoid organs at risk and thereby diminish toxicities [30, 31]. Future studies exploring the long-term effects of modern RT on cardiovascular-pulmonary disease morbidity and mortality in NSCLC are required.\nDespite the meaningful insights into radiation-induced cardiovascular-pulmonary disease mortality in NSCLC patients, we acknowledge several limitations. First, this study was based on the SEER database with potential hidden biases. The adoption of PSM in this study balances baseline patient characteristics between groups. Second, the SEER database lacks related information on pre-existing cardiovascular risk factors and cardiovascular-pulmonary diseases, which might influence subsequent mortality. Finally, type of chemotherapy and other treatment modalities (such as immune checkpoint inhibitors) were not available in the SEER database, which is closely associated with cardiopulmonary toxicity.",
"In conclusion, this study shows the cumulative incidence of cardiovascular-pulmonary mortality by PORT use in stage IIIA-N2 NSCLC patients after complete resection and identifies potential prognostic factors for cardiovascular-pulmonary-specific death. Moreover, no significant differences were found in cardiovascular-pulmonary‐related modalities between the PORT and Non‐PORT groups in this study. Further studies are needed to assess these results, and more detailed information on risk factors should be examined in future work. This study highlights the importance of long-term surveillance for NSCLC patients."
] | [
null,
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusion"
] | [
"Postoperative radiotherapy",
"Non-small cell lung cancer",
"Stage IIIA-N2",
"Cardiovascular-pulmonary disease mortality",
"SEER",
"Propensity score matching"
] | Background:
Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancer cases [1, 2]. Early-stage NSCLC is best managed with complete surgical resection [3]. Despite curative-intent surgical resection, tumor recurrence and metastasis are major causes of death for patients with locally advanced NSCLC [4–6]. Therefore, surgery plus multidisciplinary sequential therapy continues to be the backbone of treatment with curative intent among patients with stage IIIA resected NSCLC [5, 7–9].
Previous studies have shown that postoperative radiotherapy (PORT) in patients with stage IIIA-N2 NSCLC reduces the risk of local recurrence and thus is an appealing means of improving outcomes in NSCLC patients [10, 11], but whether PORT can bring overall survival (OS) benefits to those patients remains controversial [9–12]. Several retrospective studies and meta-analyses have shown the survival benefits of PORT [5, 10, 11, 13–15]. However, recent multi-institutional randomized phase III trials (Lung ART and PORT-C) indicated that PORT failed to improve disease‐free survival and OS [9, 12]. In the Lung ART study, the incidence of grade 3–5 late cardiopulmonary toxicity was 20% versus 7.7%, and the cardiopulmonary specific mortality was 16.2% versus 2% in the PORT versus Non‐PORT cohort, respectively [9]. The survival benefit may be counterbalanced by radiotherapy (RT)‐induced cardiopulmonary-specific death [9].
Thoracic RT increases the risk of cardiovascular-pulmonary disease during or after therapy, such as ischemic heart disease, arterial disease, pericardial disease, vascular and metabolic issues, conduction disorders, pneumonitis and pulmonary fibrosis, chronic pulmonary insufficiency, and cor pulmonale, and resulting in increased mortality [6, 16–20]. Radiation-associated cardiovascular-pulmonary events and deaths have been thoroughly documented in long-term survivors of breast cancer and Hodgkin’s lymphoma [18, 21–24]. However, the data regarding RT‐associated cardiovascular-pulmonary specific death in patients with NSCLC are limited [6]. Currently, there are no large datasets available with PORT and cardiovascular-pulmonary specific mortality in patients with stage IIIA-N2 NSCLC.
Therefore, we conducted a propensity-matched retrospective study to investigate the effect of PORT on cardiovascular-pulmonary related death in patients with resected stage IIIA-N2 NSCLC using the Surveillance, Epidemiology, and End Results (SEER) database.
Patients and methods:
Data sources The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.
The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.
Study population and definition We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.
Fig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy
Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy
Variables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.
Follow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].
We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.
Fig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy
Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy
Variables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.
Follow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].
Statistical analysis Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.
Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.
Data sources:
The data were downloaded from the SEER database using SEER∗STAT software (version 8.3.9). The SEER program of the National Cancer Institute in the United States collects data from 18 population-based registered cancer institutes, covering approximately 28% of cancer cases in the United States.
Study population and definition:
We extracted the data of patients with NSCLC registered from 2004 to 2015. The eligibility criteria included the following: (1) age older than 18 years; (2) pathologically confirmed NSCLC (histologic types were selected as adenocarcinoma [codes: 8140, 8250–8255, 8260, 8310, 8323, 8333, 8480, 8481, 8490, 8550, 8570, 8574], squamous cell carcinoma [codes: 8052, 8070–8074, 8083, 8084], and other NSCLC [codes: 8012, 8013, 8022, 8031–8033, 8035, 8046, 8050, 8082, 8123, 8200, 8201, 8430, 8560, 8980]; (3) diagnosis of stage IIIA-N2 NSCLC according to AJCC 6th Edition; (4) one primary malignant lung tumor only (C34.x); (5) previous lobectomy or pneumonectomy (SEER Surgery of Primary Site Codes range were 30–48 [lobectomy] and 55–70 [pneumonectomy]); (6) complete follow-ups and causes of death; and (7) complete record of RT information (received PORT or did not receive any RT). Major exclusion criteria included patients with incomplete registration information required by this study and those who died within 1 month. Patients were divided into Non-PORT and PORT groups according to whether they underwent PORT. Details of the patient selection process are shown in Fig. 1.
Fig. 1Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy
Flow chart of the screened patients. NSCLC non-small cell lung cancer, SEER Surveillance, Epidemiology, and End Results, AJCC American Joint Committee on cancer, LN lymph nodes, PORT postoperative radiotherapy
Variables extracted from the SEER database included age, sex, race, year of diagnosis, laterality (right and left), primary site, tumor size, histology code and behavior, pathologic grade, surgical procedure (lobectomy and pneumonectomy), T stage, the number of lymph nodes (LN) examined, the number of positive LN, radiation sequence with surgery, chemotherapy, survival months, vital status recode, cause of death, and SEER cause-specific death classification. The LN ratio was defined as the ratio of the number of positive LN to the number of examined LN.
Follow-up time was defined as the period between the initial diagnosis of lung cancer and events defined as death, the last follow-up, or the end of follow-up time (December 31, 2017), whichever came first. Cardiovascular-pulmonary disease-related death was calculated using coded causes of death from cardiovascular diseases (including heart diseases, hypertension without heart disease, cerebrovascular diseases, atherosclerosis, aortic aneurysm/dissection, or other diseases of arteries, arterioles, and capillaries) or pulmonary diseases (including chronic obstructive pulmonary disease and allied cond, or pneumonia and influenza) [22, 25, 26].
Statistical analysis:
Data are given as median (range) or n (%). Clinicopathological characteristics were compared between groups using Fisher’s exact test for categorical variables and the two-sample t-test or Mann-Whitney U-test for continuous variables, as appropriate. Propensity score matching (PSM) with 0.01 matching tolerance was used to balance baseline characteristics between Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression (Fine and Gray method) was used to run univariate and multivariate analyses to evaluate risk factors, considering noncardiovascular-pulmonary death as a competing event [27]. Differences were considered statistically significant at p values < 0.05. R software packages (http://www.R-project.org, The R Foundation) and Empower Stats software (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data.
Results:
Patient characteristics A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.
Table 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM
Non-PORT
(N = 2535)
PORT
(N = 1446)
P-value
Non-PORT
(N = 1380)
PORT
(N = 1380)
P-value
Age, years (range)
67 (22–90)64 (19–88)
< 0.001*
65 (22–89)65 (28–88)0.674
Sex
0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)
Race
0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)
Year of diagnosis
< 0.001*
0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)
Laterality
0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)
Primary site
0.021*
0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)
Tumor size, mm (range)
35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584
Histologic type
0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)
Grade
0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)
Surgical procedure
< 0.001*
0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)
T stage (sixth edition)
0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)
LN examined (range)
11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595
Metastatic LN
< 0.001*
0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)
Positive LN ratio (%)
< 0.001*
0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)
Chemotherapy
< 0.001*
0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)
Median FU, months (range)
27 (1–154)28 (1–154)
0.010*
29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM
LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.
Table 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM
Non-PORT
(N = 2535)
PORT
(N = 1446)
P-value
Non-PORT
(N = 1380)
PORT
(N = 1380)
P-value
Age, years (range)
67 (22–90)64 (19–88)
< 0.001*
65 (22–89)65 (28–88)0.674
Sex
0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)
Race
0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)
Year of diagnosis
< 0.001*
0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)
Laterality
0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)
Primary site
0.021*
0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)
Tumor size, mm (range)
35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584
Histologic type
0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)
Grade
0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)
Surgical procedure
< 0.001*
0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)
T stage (sixth edition)
0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)
LN examined (range)
11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595
Metastatic LN
< 0.001*
0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)
Positive LN ratio (%)
< 0.001*
0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)
Chemotherapy
< 0.001*
0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)
Median FU, months (range)
27 (1–154)28 (1–154)
0.010*
29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM
LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
Cumulative incidence of cardiovascular-pulmonary disease-related death A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.
Fig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
A total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).
A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.
Fig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
A total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).
Risk factors for cardiovascular-pulmonary diseases death Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.
Table 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name
HR
95% CI for HR
p-value
Univariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.601.082.38
0.020*
Sex (Male vs. Female)1.651.182.32
0.004*
Race
BlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111
Year of diagnosis
2004–2007Reference2008–20110.670.460.98
0.037*
2012–20150.310.190.48
< 0.001*
Laterality (Left vs. Right)1.180.851.640.330
Primary site
Main bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696
Tumor size, mm ( < = 40 vs. > 40)1.090.771.540.628
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78
< 0.001*
Others1.030.591.790.919
Grade
Well differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771
Surgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374
T stage (sixth edition)
T1ReferenceT20.810.571.150.244T30.710.351.440.342
Chemotherapy (No vs. Yes)2.041.353.08
0.001*
Metastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395
Positive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204
PORT (Yes vs. No)1.070.771.480.703
Multivariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.360.872.140.179
Sex (Male vs. Female)1.571.062.32
0.026*
Year of diagnosis
2004–2007Reference2008–20110.600.400.91
0.015*
2012–20150.290.180.49
< 0.001
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54
0.010*
Others0.840.461.530.569
Chemotherapy (No vs. Yes)1.410.882.250.154
PORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold
Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model
Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.
HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold
Fig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.
Table 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name
HR
95% CI for HR
p-value
Univariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.601.082.38
0.020*
Sex (Male vs. Female)1.651.182.32
0.004*
Race
BlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111
Year of diagnosis
2004–2007Reference2008–20110.670.460.98
0.037*
2012–20150.310.190.48
< 0.001*
Laterality (Left vs. Right)1.180.851.640.330
Primary site
Main bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696
Tumor size, mm ( < = 40 vs. > 40)1.090.771.540.628
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78
< 0.001*
Others1.030.591.790.919
Grade
Well differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771
Surgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374
T stage (sixth edition)
T1ReferenceT20.810.571.150.244T30.710.351.440.342
Chemotherapy (No vs. Yes)2.041.353.08
0.001*
Metastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395
Positive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204
PORT (Yes vs. No)1.070.771.480.703
Multivariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.360.872.140.179
Sex (Male vs. Female)1.571.062.32
0.026*
Year of diagnosis
2004–2007Reference2008–20110.600.400.91
0.015*
2012–20150.290.180.49
< 0.001
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54
0.010*
Others0.840.461.530.569
Chemotherapy (No vs. Yes)1.410.882.250.154
PORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold
Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model
Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.
HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold
Fig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Patient characteristics:
A total of 3981 patients fulfilled our inclusion criteria and were included in the study population. A flow chart of the selection process is shown in Fig. 1. Among them, 1446 (36.32%) patients received PORT. The proportions of patients receiving PORT differed by age, year of diagnosis, primary site, surgical procedure, metastatic LN, positive LN ratio, receipt of chemotherapy, and follow-up time (Table 1). No significant differences in sex, race, laterality, tumor size, histology, grade, T stage, or LN examined were seen between those who received PORT and those who did not. Using PSM at a ratio of 1:1, 1380 patients remained in each group. There were no significant differences in the clinicopathological patient characteristics between the Non-PORT and PORT groups after PSM, as shown in Table 1.
Table 1The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSMClinical parametersBefore PSMAfter PSM
Non-PORT
(N = 2535)
PORT
(N = 1446)
P-value
Non-PORT
(N = 1380)
PORT
(N = 1380)
P-value
Age, years (range)
67 (22–90)64 (19–88)
< 0.001*
65 (22–89)65 (28–88)0.674
Sex
0.9420.469Male1253 (49.43%)713 (49.31%)669 (48.48%)688 (49.86%)Female1282 (50.57%)733 (50.69%)711 (51.52%)692 (50.14%)
Race
0.5850.591Black256 (10.10%)141 (9.75%)152 (11.01%)136 (9.86%)White2051 (80.91%)1161 (80.29%)1091 (79.06%)1109 (80.36%)Others or unknown228 (8.99%)144 (9.96%)137 (9.93%)135 (9.78%)
Year of diagnosis
< 0.001*
0.3602004–20091370 (54.04%)645 (44.61%)653 (47.32%)629 (45.58%)2010–20151165 (45.96%)801 (55.39%)727 (52.68%)751 (54.42%)
Laterality
0.1540.378Right1373 (54.16%)817 (56.50%)760 (55.07%)783 (56.74%)Left1162 (45.84%)629 (43.50%)620 (44.93%)597 (43.26%)
Primary site
0.021*
0.766Main bronchus30 (1.18%)16 (1.11%)18 (1.30%)15 (1.09%)Upper lobe1415 (55.82%)869 (60.10%)793 (57.46%)822 (59.57%)Middle lobe113 (4.46%)74 (5.12%)67 (4.86%)71 (5.14%)Lower lobe,911 (35.94%)463 (32.02%)476 (34.49%)448 (32.46%)Overlapping lesion of lung66 (2.60%)24 (1.66%)26 (1.88%)24 (1.74%)
Tumor size, mm (range)
35 (1–195)35 (5–180)0.32535 (1–190)34 (5–180)0.584
Histologic type
0.1360.876Adenocarcinoma1622 (63.98%)970 (67.08%)920 (66.67%)912 (66.09%)Squamous cell carcinoma597 (23.55%)307 (21.23%)292 (21.16%)303 (21.96%)Others316 (12.47%)169 (11.69%)168 (12.17%)165 (11.96%)
Grade
0.6040.855Well differentiated126 (4.97%)58 (4.01%)64 (4.64%)55 (3.99%)Moderately differentiated1062 (41.89%)603 (41.70%)586 (42.46%)574 (41.59%)Poorly differentiated1132 (44.65%)655 (45.30%)611 (44.28%)625 (45.29%)Undifferentiated; anaplastic64 (2.52%)34 (2.35%)34 (2.46%)33 (2.39%)Unknown151 (5.96%)96 (6.64%)85 (6.16%)93 (6.74%)
Surgical procedure
< 0.001*
0.239Lobectomy2229 (87.93%)1324 (91.56%)1242 (90.00%)1260 (91.30%)Pneumonectomy306 (12.07%)122 (8.44%)138 (10.00%)120 (8.70%)
T stage (sixth edition)
0.0750.122T1697 (27.50%)411 (28.42%)391 (28.33%)391 (28.33%)T21667 (65.76%)912 (63.07%)899 (65.14%)871 (63.12%)T3171 (6.75%)123 (8.51%)90 (6.52%)118 (8.55%)
LN examined (range)
11 (1–90)11 (1–79)0.81211 (1–90)11 (1–79)0.595
Metastatic LN
< 0.001*
0.644> 4630 (24.85%)450 (31.12%)392 (28.41%)403 (29.20%)<=41905 (75.15%)996 (68.88%)988 (71.59%)977 (70.80%)
Positive LN ratio (%)
< 0.001*
0.386> 50488 (19.25%)376 (26.00%)301 (21.81%)320 (23.19%)<=502047 (80.75%)1070 (74.00%)1079 (78.19%)1060 (76.81%)
Chemotherapy
< 0.001*
0.673No1028 (40.55%)151 (10.44%)158 (11.45%)151 (10.94%)Yes1507 (59.45%)1295 (89.56%)1222 (88.55%)1229 (89.06%)
Median FU, months (range)
27 (1–154)28 (1–154)
0.010*
29 (1–154)28.5 (1–154)0.852LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
The baseline clinical characteristics of enrolled patients with stage IIIA-N2 NSCLC before and after PSM
LN, lymph node; FU, follow-up time; PSM, propensity score-matching; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold. Data represent as median (range) or n (%)
Cumulative incidence of cardiovascular-pulmonary disease-related death:
A total of 1708 patients (61.88%) succumbed to primary NSCLC, cardiovascular-pulmonary disease, or deaths due to other causes in the period of 2004 to 2015. The cumulative incidence curve for all causes of death is shown in Fig. 2A. Primary NSCLC remained the leading cause of death for our cohort after PSM, followed by cardiovascular-pulmonary diseases, with cumulative incidence rates of 67.37 and 10.28%, respectively.
Fig. 2Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
Cumulative incidence curves: (A) for cancer-, cardiovascular-pulmonary- and other cause-specific mortality; (B) for cardiovascular-pulmonary disease-related death by PORT use. PORT postoperative radiotherapy
A total of 143 patients (5.18%) died of cardiovascular-pulmonary diseases. Among them, 70 and 73 patients died of cardiovascular-pulmonary causes in the Non-PORT and PORT groups, respectively. The cumulative incidence curve for cardiovascular-pulmonary death is shown in Fig. 2B. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (hazard ratio [HR] 1.07, 95% confidence interval [CI] 0.77–1.48, p = 0.703).
Risk factors for cardiovascular-pulmonary diseases death:
Univariate Fine-Gray hazard model analysis revealed that cardiovascular-pulmonary disease-related mortality was significantly associated with age ≥ 60 (p = 0.020), male sex (p = 0.004), year of diagnosis between 2004 and 2007, squamous cell carcinoma (p < 0.001), and no receipt of chemotherapy (p = 0.001) (Table 2). Multivariate analysis showed that male sex (p = 0.026), year of diagnosis between 2004 and 2007, and squamous cell carcinoma pathologic type (p = 0.010) were independent risk factors for cardiovascular-pulmonary disease-related death (Table 2). We analyzed the impact of PORT on cardiovascular-pulmonary specific mortality, and the univariate and multivariate results suggested that PORT had no significant impact on increased risk (univariate: HR = 1.07, 95% CI 0.77–1.48, p = 0.703; multivariate: HR = 1.18, 95% CI 0.82–1.71, p = 0.377) (Table 2). Furthermore, subgroup analyses revealed similar results to the primary analyses, and patients treated with PORT were not associated with an increased risk of cardiovascular-pulmonary specific mortality compared to those who were not treated with PORT. The HRs and 95% CIs of the different subgroups are listed in Fig. 3.
Table 2Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard modelVariable name
HR
95% CI for HR
p-value
Univariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.601.082.38
0.020*
Sex (Male vs. Female)1.651.182.32
0.004*
Race
BlackReferenceWhite1.150.662.000.618Others or unknown0.460.171.200.111
Year of diagnosis
2004–2007Reference2008–20110.670.460.98
0.037*
2012–20150.310.190.48
< 0.001*
Laterality (Left vs. Right)1.180.851.640.330
Primary site
Main bronchusReferenceUpper lobe0.850.213.480.820Middle lobe0.470.092.590.385Lower lobe,0.960.233.960.950Overlapping lesion of lung0.680.094.850.696
Tumor size, mm ( < = 40 vs. > 40)1.090.771.540.628
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.931.342.78
< 0.001*
Others1.030.591.790.919
Grade
Well differentiatedReferenceModerately differentiated0.870.372.030.750Poorly differentiated1.130.492.600.778Undifferentiated; anaplastic1.160.324.110.823Unknown0.850.292.470.771
Surgical procedure (Pneumonectomy vs. Lobectomy)1.260.752.120.374
T stage (sixth edition)
T1ReferenceT20.810.571.150.244T30.710.351.440.342
Chemotherapy (No vs. Yes)2.041.353.08
0.001*
Metastatic lymph node ( < = 4 vs. > 4)1.180.811.700.395
Positive lymph node ratio (%) ( < = 50 vs. > 50)0.790.541.140.204
PORT (Yes vs. No)1.070.771.480.703
Multivariate analysis (N = 2760)
Age, years ( > = 60 vs. < 60)1.360.872.140.179
Sex (Male vs. Female)1.571.062.32
0.026*
Year of diagnosis
2004–2007Reference2008–20110.600.400.91
0.015*
2012–20150.290.180.49
< 0.001
Histologic type
AdenocarcinomaReferenceSquamous cell carcinoma1.701.132.54
0.010*
Others0.840.461.530.569
Chemotherapy (No vs. Yes)1.410.882.250.154
PORT (Yes vs. No)1.180.821.710.377Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy*P < 0.05 was considered significant and marked in bold
Univariate and multivariate analyses of cardiovascular-pulmonary disease-related mortality using a Fine-Gray hazard model
Model adjusted for multivariate analysis: Age, Year of diagnosis, Sex, Histologic type, Chemotherapy, PORT.
HR, hazard ratio; CI, confidence interval; PORT, postoperative radiotherapy
*P < 0.05 was considered significant and marked in bold
Fig. 3Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Subgroup analyses of cardiovascular-pulmonary disease death by PORT use. HR hazard ratio, CI confidence interval, PORT postoperative radiotherapy
Discussion:
Cardiovascular-pulmonary disease is the leading nonrecurrence cause of death in NSCLC patients [6]. The cardiovascular system and lungs are among the organs that are most severely affected by RT injury, resulting in increased morbidity and mortality [6, 16, 18]. The mechanisms of RT-related cardiovascular disease include endothelial dysfunction, altered vascular tone, hemostatic imbalance, and inflammatory activation [16]. Previous preclinical research found that radiation triggers lung injury by initiating a cascade of inflammatory reactions, with capillary leaks and alveolar and interstitial exudate, which later organize into collagen [18, 19]. Damage can even occur at least 10 years after RT [28].
The present work provides important insights into the risk of cardiovascular-pulmonary disease mortality by PORT in patients with stage IIIA-N2 resected NSCLC based on the SEER database. Our analysis of data from 2760 patients after PSM demonstrated that PORT among those patients was not associated with a higher risk of cardiovascular-pulmonary disease-related death (HR = 1.18, 95% CI 0.82–1.71, p = 0.377). The same conclusions were obtained in the subgroup analyses. Moreover, the multivariate analysis identified several risk factors for cardiovascular-pulmonary disease-related death, including male sex, earlier year of diagnosis, and squamous cell carcinoma pathologic type.
These appeared as unexpected findings. This study failed to show a difference in cardiovascular-pulmonary disease mortality, perhaps due to the follow-up not being long enough for outcomes to occur. The median durations of follow-up were 29 months in the Non-PORT group and 28.5 months in the PORT group, respectively. Cardiac-pulmonary toxicity following radiotherapy has been observed in long-term breast cancer and Hodgkin lymphoma survivors, with a typical latency period of more than a decade and increased incidence at younger age at treatment [18, 21, 24]. A previous study evaluating breast cancer after RT showed that the risk of major cardiovascular disease events started within the first 5 years after treatment and continued into the third decade of follow-up [29]. These malignancies portend a more favorable prognosis, while lung cancer is usually associated with poor prognosis and is conversely the leading cause of cancer-related death in the world [1]. In the phase III Lung ART trial [9], the 3-year OS rates were 66.5% in the PORT arm and 68.5% in the observation arm, respectively. In this study, NSCLC was the leading cause of death, with a cumulative incidence rate of 67.37%. It is possible that there was insufficient time for the development of cardiovascular-pulmonary disease in patients with stage IIIA-N2 NSCLC.
The definitive role of PORT in cardiopulmonary toxicity in pIIIA-N2 NSCLC remains controversial. In the recent PORT-C trial [12], no RT-induced grade 4 or 5 adverse events were observed. A total of 97 deaths (26.6%) occurred; among the 8 noncancer-related deaths, only 3 (3.1%) were due to cardiopulmonary disease [12]. The findings of the Lung ART study differed from the previous abovementioned study [9], and cardiopulmonary-specific mortality was observed in 2 (2.0%) patients in the Non-PORT cohort and 16 (16.2%) patients in the PORT cohort, respectively. The main reasons for these inconsistent results lie in the discrepancy of radiotherapy techniques and dose restrictions to the heart and lung. Studies have confirmed that patients treated with intensity-modulated radiation therapy (IMRT) for locally advanced NSCLC had lower rates of severe pneumonitis and cardiac doses than those treated with three-dimensional conformal external beam radiation therapy (3D-CRT) [30, 31]. The majority of patients received IMRT (89.3%) in the PORT-C study and 3D-CRT (89%) in the Lung ART study, respectively. The planned delivered dose was 54 Gy/27-30f in the LUNG ART trial. In reality, however, the maximum irradiation dose reached 70 Gy. The lung V20 was limited to less than 31% in patients after lobectomy and 22% after pneumonectomy, heart V30 less than 35% [9]. Another explanation for the low rate of cardiopulmonary-specific mortality in the PORT-C study is the markedly tighter dose restrictions for normal healthy tissues, especially the lungs and heart [12]. More effective modern RT techniques might have attenuated this risk.
Although our study showed no association of PORT with an increased risk of cardiovascular-pulmonary death in patients with stage IIIA-N2 NSCLC, the long-term safety of PORT for those patients remains uncertain. Combined with the results from the two randomized phase III trials [9, 12], new and modern RT techniques such as the use of IMRT are expected to avoid organs at risk and thereby diminish toxicities [30, 31]. Future studies exploring the long-term effects of modern RT on cardiovascular-pulmonary disease morbidity and mortality in NSCLC are required.
Despite the meaningful insights into radiation-induced cardiovascular-pulmonary disease mortality in NSCLC patients, we acknowledge several limitations. First, this study was based on the SEER database with potential hidden biases. The adoption of PSM in this study balances baseline patient characteristics between groups. Second, the SEER database lacks related information on pre-existing cardiovascular risk factors and cardiovascular-pulmonary diseases, which might influence subsequent mortality. Finally, type of chemotherapy and other treatment modalities (such as immune checkpoint inhibitors) were not available in the SEER database, which is closely associated with cardiopulmonary toxicity.
Conclusions:
In conclusion, this study shows the cumulative incidence of cardiovascular-pulmonary mortality by PORT use in stage IIIA-N2 NSCLC patients after complete resection and identifies potential prognostic factors for cardiovascular-pulmonary-specific death. Moreover, no significant differences were found in cardiovascular-pulmonary‐related modalities between the PORT and Non‐PORT groups in this study. Further studies are needed to assess these results, and more detailed information on risk factors should be examined in future work. This study highlights the importance of long-term surveillance for NSCLC patients.
| Background:
The role of postoperative radiotherapy (PORT) in cardiovascular-pulmonary disease mortality in patients with stage IIIA-N2 resected non-small cell lung cancer (NSCLC) remains uncertain. The purpose of this population-based analysis was to explore the effect of PORT on cardiovascular-pulmonary disease mortality in these patients.
Methods:
Patients aged ≥ 18 years with stage IIIA-N2 resected NSCLC were identified in the Surveillance, Epidemiology, and End Results (SEER) database from 2004 to 2015 and were grouped according to the use of PORT. Propensity score matching (PSM) was used to account for differences in baseline characteristics between the Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression was used to run univariate and multivariate analyses to evaluate risk factors.
Results:
A total of 3981 patients were included in the study population. Among them, 1446 patients received PORT, and 2535 did not. A total of 1380 patients remained in each group after PSM, and the baseline characteristics were not significantly different between the two groups. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (HR 1.07, 95% CI 0.77-1.48, p = 0.703). Multivariate analysis indicated that PORT had no significant impact on increased risk, with an HR of 1.18 (p = 0.377).
Conclusions:
No significant differences between the PORT and Non-PORT groups were found in cardiovascular-pulmonary-specific modalities in this study. Further studies are required to validate these results. This study highlights the importance of long-term surveillance for NSCLC patients. | Background:
Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancer cases [1, 2]. Early-stage NSCLC is best managed with complete surgical resection [3]. Despite curative-intent surgical resection, tumor recurrence and metastasis are major causes of death for patients with locally advanced NSCLC [4–6]. Therefore, surgery plus multidisciplinary sequential therapy continues to be the backbone of treatment with curative intent among patients with stage IIIA resected NSCLC [5, 7–9].
Previous studies have shown that postoperative radiotherapy (PORT) in patients with stage IIIA-N2 NSCLC reduces the risk of local recurrence and thus is an appealing means of improving outcomes in NSCLC patients [10, 11], but whether PORT can bring overall survival (OS) benefits to those patients remains controversial [9–12]. Several retrospective studies and meta-analyses have shown the survival benefits of PORT [5, 10, 11, 13–15]. However, recent multi-institutional randomized phase III trials (Lung ART and PORT-C) indicated that PORT failed to improve disease‐free survival and OS [9, 12]. In the Lung ART study, the incidence of grade 3–5 late cardiopulmonary toxicity was 20% versus 7.7%, and the cardiopulmonary specific mortality was 16.2% versus 2% in the PORT versus Non‐PORT cohort, respectively [9]. The survival benefit may be counterbalanced by radiotherapy (RT)‐induced cardiopulmonary-specific death [9].
Thoracic RT increases the risk of cardiovascular-pulmonary disease during or after therapy, such as ischemic heart disease, arterial disease, pericardial disease, vascular and metabolic issues, conduction disorders, pneumonitis and pulmonary fibrosis, chronic pulmonary insufficiency, and cor pulmonale, and resulting in increased mortality [6, 16–20]. Radiation-associated cardiovascular-pulmonary events and deaths have been thoroughly documented in long-term survivors of breast cancer and Hodgkin’s lymphoma [18, 21–24]. However, the data regarding RT‐associated cardiovascular-pulmonary specific death in patients with NSCLC are limited [6]. Currently, there are no large datasets available with PORT and cardiovascular-pulmonary specific mortality in patients with stage IIIA-N2 NSCLC.
Therefore, we conducted a propensity-matched retrospective study to investigate the effect of PORT on cardiovascular-pulmonary related death in patients with resected stage IIIA-N2 NSCLC using the Surveillance, Epidemiology, and End Results (SEER) database.
Conclusions:
In conclusion, this study shows the cumulative incidence of cardiovascular-pulmonary mortality by PORT use in stage IIIA-N2 NSCLC patients after complete resection and identifies potential prognostic factors for cardiovascular-pulmonary-specific death. Moreover, no significant differences were found in cardiovascular-pulmonary‐related modalities between the PORT and Non‐PORT groups in this study. Further studies are needed to assess these results, and more detailed information on risk factors should be examined in future work. This study highlights the importance of long-term surveillance for NSCLC patients. | Background:
The role of postoperative radiotherapy (PORT) in cardiovascular-pulmonary disease mortality in patients with stage IIIA-N2 resected non-small cell lung cancer (NSCLC) remains uncertain. The purpose of this population-based analysis was to explore the effect of PORT on cardiovascular-pulmonary disease mortality in these patients.
Methods:
Patients aged ≥ 18 years with stage IIIA-N2 resected NSCLC were identified in the Surveillance, Epidemiology, and End Results (SEER) database from 2004 to 2015 and were grouped according to the use of PORT. Propensity score matching (PSM) was used to account for differences in baseline characteristics between the Non-PORT and PORT groups. The cumulative risk for cardiovascular-pulmonary disease death was estimated using the cumulative incidence curve. Competing risk regression was used to run univariate and multivariate analyses to evaluate risk factors.
Results:
A total of 3981 patients were included in the study population. Among them, 1446 patients received PORT, and 2535 did not. A total of 1380 patients remained in each group after PSM, and the baseline characteristics were not significantly different between the two groups. The cumulative incidence of cardiovascular-pulmonary mortality was 10.93% in the Non-PORT group compared with 9.85% in the PORT group. There was no significant difference in the cumulative risk between the two groups (HR 1.07, 95% CI 0.77-1.48, p = 0.703). Multivariate analysis indicated that PORT had no significant impact on increased risk, with an HR of 1.18 (p = 0.377).
Conclusions:
No significant differences between the PORT and Non-PORT groups were found in cardiovascular-pulmonary-specific modalities in this study. Further studies are required to validate these results. This study highlights the importance of long-term surveillance for NSCLC patients. | 9,694 | 354 | [
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] | null | [CONTENT] Postoperative radiotherapy | Non-small cell lung cancer | Stage IIIA-N2 | Cardiovascular-pulmonary disease mortality | SEER | Propensity score matching [SUMMARY] | null | [CONTENT] Postoperative radiotherapy | Non-small cell lung cancer | Stage IIIA-N2 | Cardiovascular-pulmonary disease mortality | SEER | Propensity score matching [SUMMARY] | [CONTENT] Postoperative radiotherapy | Non-small cell lung cancer | Stage IIIA-N2 | Cardiovascular-pulmonary disease mortality | SEER | Propensity score matching [SUMMARY] | [CONTENT] Postoperative radiotherapy | Non-small cell lung cancer | Stage IIIA-N2 | Cardiovascular-pulmonary disease mortality | SEER | Propensity score matching [SUMMARY] | [CONTENT] Postoperative radiotherapy | Non-small cell lung cancer | Stage IIIA-N2 | Cardiovascular-pulmonary disease mortality | SEER | Propensity score matching [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Non-Small-Cell Lung | Cardiovascular Diseases | Combined Modality Therapy | Humans | Lung Diseases | Lung Neoplasms | Middle Aged | Neoplasm Staging | Risk Factors | SEER Program | Young Adult [SUMMARY] | null | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Non-Small-Cell Lung | Cardiovascular Diseases | Combined Modality Therapy | Humans | Lung Diseases | Lung Neoplasms | Middle Aged | Neoplasm Staging | Risk Factors | SEER Program | Young Adult [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Non-Small-Cell Lung | Cardiovascular Diseases | Combined Modality Therapy | Humans | Lung Diseases | Lung Neoplasms | Middle Aged | Neoplasm Staging | Risk Factors | SEER Program | Young Adult [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Non-Small-Cell Lung | Cardiovascular Diseases | Combined Modality Therapy | Humans | Lung Diseases | Lung Neoplasms | Middle Aged | Neoplasm Staging | Risk Factors | SEER Program | Young Adult [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma, Non-Small-Cell Lung | Cardiovascular Diseases | Combined Modality Therapy | Humans | Lung Diseases | Lung Neoplasms | Middle Aged | Neoplasm Staging | Risk Factors | SEER Program | Young Adult [SUMMARY] | [CONTENT] port postoperative radiotherapy | postoperative radiotherapy cumulative | surgery chemotherapy survival | lung cancer nsclc | favorable prognosis lung [SUMMARY] | null | [CONTENT] port postoperative radiotherapy | postoperative radiotherapy cumulative | surgery chemotherapy survival | lung cancer nsclc | favorable prognosis lung [SUMMARY] | [CONTENT] port postoperative radiotherapy | postoperative radiotherapy cumulative | surgery chemotherapy survival | lung cancer nsclc | favorable prognosis lung [SUMMARY] | [CONTENT] port postoperative radiotherapy | postoperative radiotherapy cumulative | surgery chemotherapy survival | lung cancer nsclc | favorable prognosis lung [SUMMARY] | [CONTENT] port postoperative radiotherapy | postoperative radiotherapy cumulative | surgery chemotherapy survival | lung cancer nsclc | favorable prognosis lung [SUMMARY] | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | disease | nsclc | pulmonary disease | cardiovascular pulmonary disease [SUMMARY] | null | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | disease | nsclc | pulmonary disease | cardiovascular pulmonary disease [SUMMARY] | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | disease | nsclc | pulmonary disease | cardiovascular pulmonary disease [SUMMARY] | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | disease | nsclc | pulmonary disease | cardiovascular pulmonary disease [SUMMARY] | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | disease | nsclc | pulmonary disease | cardiovascular pulmonary disease [SUMMARY] | [CONTENT] nsclc | port | patients | lung | survival | versus | pulmonary | cardiopulmonary | death patients | cancer [SUMMARY] | null | [CONTENT] port | vs | 001 | cardiovascular | pulmonary | cardiovascular pulmonary | hr | 50 | hazard | 11 [SUMMARY] | [CONTENT] study | nsclc patients | cardiovascular pulmonary | pulmonary | cardiovascular | port | factors | differences found cardiovascular | prognostic factors cardiovascular | prognostic factors [SUMMARY] | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | nsclc | cancer | seer | disease [SUMMARY] | [CONTENT] port | pulmonary | cardiovascular | cardiovascular pulmonary | patients | death | nsclc | cancer | seer | disease [SUMMARY] | [CONTENT] NSCLC ||| [SUMMARY] | null | [CONTENT] 3981 ||| 1446 | 2535 ||| 1380 | PSM | two ||| 10.93% | 9.85% ||| two | 1.07 | 95% | CI | 0.703 ||| 1.18 | 0.377 [SUMMARY] | [CONTENT] ||| ||| NSCLC [SUMMARY] | [CONTENT] NSCLC ||| ||| ≥ | 18 years | NSCLC | the Surveillance, Epidemiology | End Results | SEER | 2004 to 2015 ||| ||| ||| ||| ||| 3981 ||| 1446 | 2535 ||| 1380 | PSM | two ||| 10.93% | 9.85% ||| two | 1.07 | 95% | CI | 0.703 ||| 1.18 | 0.377 ||| ||| ||| NSCLC [SUMMARY] | [CONTENT] NSCLC ||| ||| ≥ | 18 years | NSCLC | the Surveillance, Epidemiology | End Results | SEER | 2004 to 2015 ||| ||| ||| ||| ||| 3981 ||| 1446 | 2535 ||| 1380 | PSM | two ||| 10.93% | 9.85% ||| two | 1.07 | 95% | CI | 0.703 ||| 1.18 | 0.377 ||| ||| ||| NSCLC [SUMMARY] |
|||||
Objective Verification of Acute Tinnitus and Validation of Efficacy of Systemic Steroids in Rats. | 32242342 | This study was performed to identify acute tinnitus and evaluate the efficacy of steroids for noise-induced acute tinnitus by measuring the gap-prepulse inhibition of the acoustic startle (GPIAS) value in an animal model. | BACKGROUND | Nineteen rats (the noise group [n = 7] and the noise + dexamethasone [DEX] group [n = 12]) were exposed to narrow-band noise centered at 16 kHz from a sound generator for 4 hours. The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure. Auditory brainstem response and GPIAS value were measured just prior to, and 1 day after noise exposure and on days 1 and 10 days after completing steroid administration. The changes in cochlear structure were evaluated by histological analysis. | METHODS | The threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and no differences in threshold shift were observed between the two groups in each frequency except for 32 kHz 1 day after steroid injection. The mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly higher than that in the noise group (16.4% ± 18.8%) 10 days after intraperitoneal steroid administration (P = 0.017). There were no pathological changes associated with noise trauma in the two groups as determined on hematoxylin and eosin and immunohistochemical staining. | RESULTS | An acute tinnitus model with minimal structural changes by noise exposure was set up, and used to verify tinnitus objectively by measuring the GPIAS value. Steroid therapy for control of tinnitus was validated in this animal model. | CONCLUSION | [
"Acoustics",
"Acute Disease",
"Animals",
"Dexamethasone",
"Disease Models, Animal",
"Evoked Potentials, Auditory, Brain Stem",
"Glucocorticoids",
"Male",
"Noise",
"Rats",
"Tinnitus"
] | 7131900 | INTRODUCTION | Tinnitus, defined as perception of sound in the absence of corresponding external auditory stimuli, has a reported prevalence of 10%–15% in adults1 and about 20% of adults with tinnitus require clinical intervention.2 The most widely accepted theory regarding the central origin of hyperexcitability in tinnitus is an imbalance between excitation and inhibition. After acute insult, such as acoustic trauma or exposure to ototoxic drugs, the spontaneous activity increases in the auditory nerve, inferior colliculus, and auditory cortex.34 The dorsal cochlear nucleus, inferior colliculus, and primary auditory cortex neurons are hyperexcitable during the chronic phase.56
Brain mechanisms have been suggested to be involved in the development of tinnitus, and a number of animal models have been developed to test these hypotheses by measuring tinnitus objectively. Tinnitus was reported to be induced in rodents treated with sodium salicylate, and was associated with the alterations of neural activity in the central auditory system.7 Additional animal models have been reported, including induction of tinnitus by intense sound exposure.89
A behavioral model based on the acoustic startle reflex test was recently adopted and has been widely used for objective measurement of tinnitus.10 Briefly, acoustic startle can be evoked by noise and the magnitude of this startle can be suppressed by a gap of silence in the background sound that precedes the noise. If tinnitus is present, the sound of tinnitus will fill the gap and reduce the degree to which it suppresses the acoustic startle response.11 The ratio between the magnitude of the startle stimulus presented alone (no-gap trial) and trials in which a gap precedes startle stimulus (gap trials) is calculated as the gap-prepulse inhibition of the acoustic startle (GPIAS) ratio12 and reflects gap detection. Reduced inhibition by gaps embedded in specific background noise frequencies is assumed to reflect tinnitus frequency.13
It was reported that damage to cochlear hair cells was essential for the pathogenesis of tinnitus,14 indicating that cochlear lesions result in elevation of hearing thresholds and initiation of tinnitus. Therefore, the early stage of tinnitus could be controlled by appropriate therapy, such as steroid administration, if the cochlear damage remains reversible. The effects of steroids include immunosuppression, antiinflammation, and sodium reabsorption. Intratympanic steroid administration was reported to be equivalent to high-dose oral prednisone therapy as primary therapy in the treatment of inner ear disorders.15
This study was designed to identify acute tinnitus and to evaluate the efficacy of steroid administration for noise-induced acute tinnitus by measuring the GPIAS value in an animal model. | METHODS | Subjects A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).
DEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.
A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).
DEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.
Noise exposure Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).
Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).
GPIAS Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).
GPIAS = gap-prepulse inhibition of the acoustic startle.
GPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.
Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).
GPIAS = gap-prepulse inhibition of the acoustic startle.
GPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.
Systemic steroid administration The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.
The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.
Auditory brainstem response (ABR) ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.
ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.
Histologic analysis The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.
The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.
Immunohistochemistry The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).
H&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.
The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).
H&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.
Data analysis All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.
All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.
Ethics statement All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).
All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005). | RESULTS | ABR ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).
DEX = dexamethasone.
*P < 0.05.
ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).
DEX = dexamethasone.
*P < 0.05.
Tinnitus development and GPIAS During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).
GPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.
*P < 0.05.
During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).
GPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.
*P < 0.05.
Histology Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.
Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.
Immunohistochemistry Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).
Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6). | null | null | [
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"Systemic steroid administration",
"Auditory brainstem response (ABR)",
"Histologic analysis",
"Immunohistochemistry",
"Data analysis",
"Ethics statement",
"ABR",
"Tinnitus development and GPIAS",
"Histology",
"Immunohistochemistry"
] | [
"A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).\nDEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.",
"Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).",
"Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).\nGPIAS = gap-prepulse inhibition of the acoustic startle.\nGPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.",
"The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.",
"ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.",
"The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.",
"The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).\nH&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.",
"All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.",
"All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).",
"ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).\nDEX = dexamethasone.\n*P < 0.05.",
"During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).\nGPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.\n*P < 0.05.",
"Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.",
"Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6)."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"Subjects",
"Noise exposure",
"GPIAS",
"Systemic steroid administration",
"Auditory brainstem response (ABR)",
"Histologic analysis",
"Immunohistochemistry",
"Data analysis",
"Ethics statement",
"RESULTS",
"ABR",
"Tinnitus development and GPIAS",
"Histology",
"Immunohistochemistry",
"DISCUSSION"
] | [
"Tinnitus, defined as perception of sound in the absence of corresponding external auditory stimuli, has a reported prevalence of 10%–15% in adults1 and about 20% of adults with tinnitus require clinical intervention.2 The most widely accepted theory regarding the central origin of hyperexcitability in tinnitus is an imbalance between excitation and inhibition. After acute insult, such as acoustic trauma or exposure to ototoxic drugs, the spontaneous activity increases in the auditory nerve, inferior colliculus, and auditory cortex.34 The dorsal cochlear nucleus, inferior colliculus, and primary auditory cortex neurons are hyperexcitable during the chronic phase.56\nBrain mechanisms have been suggested to be involved in the development of tinnitus, and a number of animal models have been developed to test these hypotheses by measuring tinnitus objectively. Tinnitus was reported to be induced in rodents treated with sodium salicylate, and was associated with the alterations of neural activity in the central auditory system.7 Additional animal models have been reported, including induction of tinnitus by intense sound exposure.89\nA behavioral model based on the acoustic startle reflex test was recently adopted and has been widely used for objective measurement of tinnitus.10 Briefly, acoustic startle can be evoked by noise and the magnitude of this startle can be suppressed by a gap of silence in the background sound that precedes the noise. If tinnitus is present, the sound of tinnitus will fill the gap and reduce the degree to which it suppresses the acoustic startle response.11 The ratio between the magnitude of the startle stimulus presented alone (no-gap trial) and trials in which a gap precedes startle stimulus (gap trials) is calculated as the gap-prepulse inhibition of the acoustic startle (GPIAS) ratio12 and reflects gap detection. Reduced inhibition by gaps embedded in specific background noise frequencies is assumed to reflect tinnitus frequency.13\nIt was reported that damage to cochlear hair cells was essential for the pathogenesis of tinnitus,14 indicating that cochlear lesions result in elevation of hearing thresholds and initiation of tinnitus. Therefore, the early stage of tinnitus could be controlled by appropriate therapy, such as steroid administration, if the cochlear damage remains reversible. The effects of steroids include immunosuppression, antiinflammation, and sodium reabsorption. Intratympanic steroid administration was reported to be equivalent to high-dose oral prednisone therapy as primary therapy in the treatment of inner ear disorders.15\nThis study was designed to identify acute tinnitus and to evaluate the efficacy of steroid administration for noise-induced acute tinnitus by measuring the GPIAS value in an animal model.",
" Subjects A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).\nDEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.\nA total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).\nDEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.\n Noise exposure Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).\nRats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).\n GPIAS Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).\nGPIAS = gap-prepulse inhibition of the acoustic startle.\nGPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.\nEach rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).\nGPIAS = gap-prepulse inhibition of the acoustic startle.\nGPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.\n Systemic steroid administration The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.\nThe noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.\n Auditory brainstem response (ABR) ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.\nABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.\n Histologic analysis The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.\nThe extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.\n Immunohistochemistry The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).\nH&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.\nThe sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).\nH&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.\n Data analysis All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.\nAll data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.\n Ethics statement All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).\nAll of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).",
"A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).\nDEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.",
"Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).",
"Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).\nGPIAS = gap-prepulse inhibition of the acoustic startle.\nGPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.",
"The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.",
"ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.",
"The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.",
"The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).\nH&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.",
"All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.",
"All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).",
" ABR ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).\nDEX = dexamethasone.\n*P < 0.05.\nABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).\nDEX = dexamethasone.\n*P < 0.05.\n Tinnitus development and GPIAS During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).\nGPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.\n*P < 0.05.\nDuring baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).\nGPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.\n*P < 0.05.\n Histology Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.\nHistological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.\n Immunohistochemistry Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).\nImmunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).",
"ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).\nDEX = dexamethasone.\n*P < 0.05.",
"During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).\nGPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.\n*P < 0.05.",
"Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.",
"Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).",
"Various treatments have been used to control tinnitus, including tinnitus retraining therapy, medication, hearing aids, and transcranial magnetic stimulation. However, there have been few reports showing significant and long-lasting improvement of tinnitus.1718\nPrevious studies supported that damage to cochlear hair cells was essential for the pathogenesis of tinnitus.14 Therefore, in the initial stages of tinnitus, the period of reversibility of cochlear lesions, it was considered that acute tinnitus could be eliminated by appropriate therapy. As steroid receptors were identified in the inner ear,19 DEX has been used as an initial medication option for inner ear diseases and its effects on acute tinnitus have been investigated.202122 The efficacy of DEX used in clinic for the treatment of acute tinnitus was also supported by this animal study.\nThe present study was performed to evaluate objectively the effects of steroid administration on acute tinnitus in an animal model by measuring GPIAS. Since its introduction by Turner et al.,12 GPIAS has been widely used to investigate the mechanisms underlying tinnitus in animal models. The advantage of this method for tinnitus screening in rodents is that it does not require training and, instead, uses reflex modification procedures. The GPIAS system is based on the startle response, which is elicited by loud sounds. We checked the hearing level of the rats because hearing loss is known to affect the reliability of GPIAS by attenuating the behavioral response to loud sound perception. To measure the GPIAS value, it is essential to maintain the acceptable hearing level after noise exposure. Acoustic trauma is most commonly used to induce tinnitus in animal models. Galazyuk and Hébert10 summarized the tinnitus protocol of animal model. Sound levels vary from 94 dB SPL to 125 dB SPL. The most common parameters for tinnitus induction include narrow band noise (10, 12, 16, and 17 kHz) presented during 1–2 hours. Based on the study by Galazyuk and Hébert10 we attempted to create noise exposure conditions that caused tinnitus while preserving hearing as much as possible. The mean hearing thresholds of both groups were lower than the background noise (60 dB SPL) and startle stimulus (105 dB SPL) in the GPIAS measurement protocol. Tinnitus perception was shown to be significantly reduced in the noise + DEX group at 10 days after intraperitoneal steroid administration compared to the noise group by measuring the GPIAS value, which means tinnitus intensity was weakened by steroid. Acute insult such as noise trauma causes cochlear damage by reactive oxygen species accumulation23 and cochlear ischemia,24 which could initiate the tinnitus without hearing impairment. The mechanism of action of steroid on tinnitus is related with the decrease in inflammation due to immune system dysfunction in the inner ear, suppression of irritability or sensitivity due to damage of cochlear hair cells, and increased inner ear vascularity.25 These effects of steroid may contribute to tinnitus awareness control in rats with noise trauma.\nWe performed histological analysis of the noise and noise + DEX groups to compare the differences according to GPIAS value. No pathological changes were observed, such as the loss of hair cells and supporting cells, associated with noise trauma in the noise group and the noise + DEX group. The animal model in this study did not represent a model of tinnitus with normal auditory thresholds as hearing thresholds of 16 kHz increased to 40 dB HL. Sanchez et al.26 reported that only 7.4% of tinnitus patients showed normal pure tone thresholds. These findings suggested that acute tinnitus could be induced without structural damage to the cochlea and the animal tinnitus model with normal hearing category might be developed by adjusting the noise exposure condition.\nA recent study showed that immediate and extensive loss of synapses between IHC and ANF can be caused by titrated noise exposure, which is designated as “synaptopathy”.27 We performed immunohistochemical staining to investigate the synapses of IHC in the present study. Although both the noise and noise + DEX groups showed similar patterns of staining for CtBP2, a presynaptic marker protein associated with synaptic ribbons, it should be considered that this finding could be rather preliminary using a small number of samples and ribbon counting in cochlear whole mount specimens and measurement of ABR wave I amplitude should be performed to confirm synaptopathy.\nThe clinical results regarding the effects of steroid administration on tinnitus control are controversial.2228 In this study, tinnitus control using steroid administration was validated in a model of acute tinnitus caused by acoustic trauma, supporting the positive effect of steroids.29 Steroids may eliminate the reversible inflammatory changes in the cochlea at the initial stages of tinnitus.\nThere are several factors to be considered. The effect of intratympanic DEX injection, commonly used in clinical practice, on acute tinnitus should be verified. Ribbon counting data in cochlear whole mount would be helpful. Additionally, comparison with the experiment using salicylate-induced tinnitus model might be significant.\nIn conclusion, we have developed a model of acute tinnitus with minimal structural change associated with noise exposure and objectively verified tinnitus by measuring the GPIAS value. Steroid therapy for tinnitus control was validated in this animal model. Additional trials for other tinnitus models associated with ototoxicity and sudden hearing loss could be required."
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"methods",
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] | [
"Tinnitus",
"GPIAS",
"Auditory Brainstem Response",
"Dexamethasone",
"Noise-Induced Hearing Loss"
] | INTRODUCTION:
Tinnitus, defined as perception of sound in the absence of corresponding external auditory stimuli, has a reported prevalence of 10%–15% in adults1 and about 20% of adults with tinnitus require clinical intervention.2 The most widely accepted theory regarding the central origin of hyperexcitability in tinnitus is an imbalance between excitation and inhibition. After acute insult, such as acoustic trauma or exposure to ototoxic drugs, the spontaneous activity increases in the auditory nerve, inferior colliculus, and auditory cortex.34 The dorsal cochlear nucleus, inferior colliculus, and primary auditory cortex neurons are hyperexcitable during the chronic phase.56
Brain mechanisms have been suggested to be involved in the development of tinnitus, and a number of animal models have been developed to test these hypotheses by measuring tinnitus objectively. Tinnitus was reported to be induced in rodents treated with sodium salicylate, and was associated with the alterations of neural activity in the central auditory system.7 Additional animal models have been reported, including induction of tinnitus by intense sound exposure.89
A behavioral model based on the acoustic startle reflex test was recently adopted and has been widely used for objective measurement of tinnitus.10 Briefly, acoustic startle can be evoked by noise and the magnitude of this startle can be suppressed by a gap of silence in the background sound that precedes the noise. If tinnitus is present, the sound of tinnitus will fill the gap and reduce the degree to which it suppresses the acoustic startle response.11 The ratio between the magnitude of the startle stimulus presented alone (no-gap trial) and trials in which a gap precedes startle stimulus (gap trials) is calculated as the gap-prepulse inhibition of the acoustic startle (GPIAS) ratio12 and reflects gap detection. Reduced inhibition by gaps embedded in specific background noise frequencies is assumed to reflect tinnitus frequency.13
It was reported that damage to cochlear hair cells was essential for the pathogenesis of tinnitus,14 indicating that cochlear lesions result in elevation of hearing thresholds and initiation of tinnitus. Therefore, the early stage of tinnitus could be controlled by appropriate therapy, such as steroid administration, if the cochlear damage remains reversible. The effects of steroids include immunosuppression, antiinflammation, and sodium reabsorption. Intratympanic steroid administration was reported to be equivalent to high-dose oral prednisone therapy as primary therapy in the treatment of inner ear disorders.15
This study was designed to identify acute tinnitus and to evaluate the efficacy of steroid administration for noise-induced acute tinnitus by measuring the GPIAS value in an animal model.
METHODS:
Subjects A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).
DEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.
A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).
DEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.
Noise exposure Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).
Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).
GPIAS Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).
GPIAS = gap-prepulse inhibition of the acoustic startle.
GPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.
Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).
GPIAS = gap-prepulse inhibition of the acoustic startle.
GPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.
Systemic steroid administration The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.
The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.
Auditory brainstem response (ABR) ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.
ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.
Histologic analysis The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.
The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.
Immunohistochemistry The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).
H&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.
The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).
H&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.
Data analysis All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.
All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.
Ethics statement All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).
All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).
Subjects:
A total of 19 male Sprague-Dawley (SD) rats (8 weeks old, 250–300 g) were used in this study. All SD rats were maintained under conditions of controlled temperature (22°C ± 2°C), humidity (70%–75%), and light/dark cycle (lights on at 8:00, lights off at 20:00). Rats were divided into two groups: the noise group (n = 7) and the noise + dexamethasone (DEX) group (n = 12) (Fig. 1).
DEX = dexamethasone, GPIAS = gap-prepulse inhibition of the acoustic startle, ABR = auditory brainstem response.
Noise exposure:
Rats were exposed for 4 hours to narrow-band noise centered at 16 kHz from a sound generator (Sine Random Generator Type 1027; Brüel & Kjær, Copenhagen, Denmark), amplified (R300 Plus Amplifier; Inter-M, Seoul, Korea) to 112 dB SPL, and played through a speaker (#84234xx; Electro-Voice, Burnsville, MN, USA).
GPIAS:
Each rat was placed in an acoustically transparent unconstrained type enclosure in an anechoic chamber (startle reflex chamber). The anechoic chamber had acoustic open cell foam walls and the unconstrained type enclosure rested on an accelerometer, which measured the startle response. All studies were conducted in a soundproof chamber (Fig. 2).
GPIAS = gap-prepulse inhibition of the acoustic startle.
GPIAS sessions were composed of 30 gap trials and 30 no-gap trials, which were presented in random pairs. Trials were separated by conditional random interstimulus interval. This system measures the animal startle response only when an animal is in the stable status by sensing the animal's movements using an accelerometer in real-time to minimize motion artifacts. A 2-minute acclimation period was allowed at the beginning of each session. Each gap trial was composed of 60-dB SPL continuous narrow band background noise centered at 16 kHz, and a startle stimulus (single broadband noise burst, 110 dB SPL, 20 ms length) preceded by a long gap of 50 ms ending 100 ms before the onset of the startle stimulus. In the no-gap trials, the background sound was continuous with no silent period preceding the startle stimulus. GPIAS was measured in all rats just prior to and 1 day after noise exposure and 1 and 10 days after completing steroid administration.
Systemic steroid administration:
The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure.
Auditory brainstem response (ABR):
ABR was recorded in all rats just prior to, and 1 day after, noise exposure and 1 and 10 days after completing steroid administration. Each rat was anesthetized with Zoletil 50 (Virbac Laboratories, Carros, France) 0.1 cc/100 g and Rompun 2% (Bayer Korea, Ansan, Korea) 0.02 cc/100 g by intraperitoneal injection for auditory evaluation. ABR was recorded in a soundproof and electrically shielded room. Auditory evaluation was tested by measuring ABR with a Biosig 32 system (Tucker - Davis Technologies, Gainesville, FL, USA). Recording needle electrodes were inserted subcutaneously into the vertex (+) of the scalp and the postauricular area (−). Ground needle electrodes were inserted into the neck contralateral to the recording electrodes. The stimuli were tone bursts 5 ms in duration with rise/fall times of 1 ms. The sound frequencies delivered were 16 and 32 kHz to the left ear at a rate of 11.1/s. Responses were averaged over at least 512 repetitions of stimuli, which were decreased by 5 dB at subthreshold levels. The thresholds were defined using visual analysis by two investigators blinded to the noise + DEX group.
Histologic analysis:
The extracted cochleae were placed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight and decalcified in Calci-Clear Rapid (National Diagnostics, Charlotte, NC, USA) for 3 days. After decalcification, the tissues were embedded in paraffin blocks, and sections 5 μm thick were cut along the cochlear axis and stained with hematoxylin and eosin (H&E). Hair cells and spiral ganglion in the cochlea were observed.
Immunohistochemistry:
The sections were rehydrated. After washing with PBS containing 0.05% Triton X-100 (Wash buffer), blocking with 1% bovine serum albumin for 1 hour, and washing with Wash buffer, the sections were incubated with anti-C-terminal binding protein 2 antibody (CtbBP2, 1:300 dilution; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Myosin VIIa antibody (MYO7A, 1:300 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing in Wash buffer, the sections were incubated with fluorescein isothiocyanate - conjugated secondary antibodies (1:300 dilution in PBS containing 0.2% Triton X - 100 [PBST]; Jackson ImmunoResearch, West Grove, PA, USA) and Cy™3 - conjugated secondary antibodies (1:600 dilution in PBST; Jackson ImmunoResearch) for 1 hour. After washing with Wash buffer three times for 5 minutes each time, the sections were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were examined using a Zeiss LSM510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).
H&E staining and immunohistochemical staining of the normal cochlea of SD rats (8 weeks old, 250–300 g) were performed for comparison with the noise and noise + steroid groups.
Data analysis:
All data are presented as means ± standard error. The sample size was small in each group, so the values of hearing threshold and GPIAS were analyzed using the Mann-Whitney U test. In all analyses, P < 0.05 was taken to indicate statistical significance.
Ethics statement:
All of the animals were managed in compliance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and all of the experiments were performed in accordance with the Guidelines of the Internal Animal Care and Use Committee of Ajou University Hospital Biomedical Research Institute (protocol No. 2018-0005).
RESULTS:
ABR ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).
DEX = dexamethasone.
*P < 0.05.
ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).
DEX = dexamethasone.
*P < 0.05.
Tinnitus development and GPIAS During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).
GPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.
*P < 0.05.
During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).
GPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.
*P < 0.05.
Histology Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.
Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.
Immunohistochemistry Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).
Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).
ABR:
ABR was measured in all rats before noise exposure and 1 day after exposure to determine whether noise trauma could induce auditory threshold shift. Baseline threshold values in both groups were under 15 dB SPL at 8, 16, and 32 kHz. The threshold shift after noise exposure was not different between the two groups at 8, 16, and 32 kHz. Threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and showed no differences between the two groups in each frequency, except 32 kHz at 1 day after steroid injection (Fig. 3).
DEX = dexamethasone.
*P < 0.05.
Tinnitus development and GPIAS:
During baseline sessions, rats showed a mean GPIAS of 51.2% ± 13.4% in the noise group and 46.6% ± 14.1% in the noise + DEX group (P = 0.484). The mean GPIAS values did not show significant differences between the two groups at 1 day after noise exposure or 1 day after intraperitoneal steroid administration (P = 0.650 and P = 0.289). In contrast, the mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly greater than that in the noise group (16.4% ± 18.8%) at 10 days after intraperitoneal steroid administration (P = 0.017) (Fig. 4).
GPIAS = gap-prepulse inhibition of the acoustic startle, DEX = dexamethasone.
*P < 0.05.
Histology:
Histological analyses of the two groups were performed after functional evaluation. Fig. 5 shows the histological findings for the organ of Corti in the basal, middle, and apical turns. There were no pathological findings associated with noise trauma, such as loss of hair cells and supporting cells, in the two groups.
Immunohistochemistry:
Immunostained cochlear sections, imaged by confocal microscopy, were assessed to quantify hair cells, cochlear neurons, and synaptic structures providing the communication conduits. Auditory nerve fibers (ANF) terminate in a single synapse on a single inner hair cell (IHC).16 Thus, the ribbon count provides an accurate metric of the number of ANFs contacting each IHC. The mean counts of ribbon synapse were similar in the noise group and noise + DEX group (Fig. 6).
DISCUSSION:
Various treatments have been used to control tinnitus, including tinnitus retraining therapy, medication, hearing aids, and transcranial magnetic stimulation. However, there have been few reports showing significant and long-lasting improvement of tinnitus.1718
Previous studies supported that damage to cochlear hair cells was essential for the pathogenesis of tinnitus.14 Therefore, in the initial stages of tinnitus, the period of reversibility of cochlear lesions, it was considered that acute tinnitus could be eliminated by appropriate therapy. As steroid receptors were identified in the inner ear,19 DEX has been used as an initial medication option for inner ear diseases and its effects on acute tinnitus have been investigated.202122 The efficacy of DEX used in clinic for the treatment of acute tinnitus was also supported by this animal study.
The present study was performed to evaluate objectively the effects of steroid administration on acute tinnitus in an animal model by measuring GPIAS. Since its introduction by Turner et al.,12 GPIAS has been widely used to investigate the mechanisms underlying tinnitus in animal models. The advantage of this method for tinnitus screening in rodents is that it does not require training and, instead, uses reflex modification procedures. The GPIAS system is based on the startle response, which is elicited by loud sounds. We checked the hearing level of the rats because hearing loss is known to affect the reliability of GPIAS by attenuating the behavioral response to loud sound perception. To measure the GPIAS value, it is essential to maintain the acceptable hearing level after noise exposure. Acoustic trauma is most commonly used to induce tinnitus in animal models. Galazyuk and Hébert10 summarized the tinnitus protocol of animal model. Sound levels vary from 94 dB SPL to 125 dB SPL. The most common parameters for tinnitus induction include narrow band noise (10, 12, 16, and 17 kHz) presented during 1–2 hours. Based on the study by Galazyuk and Hébert10 we attempted to create noise exposure conditions that caused tinnitus while preserving hearing as much as possible. The mean hearing thresholds of both groups were lower than the background noise (60 dB SPL) and startle stimulus (105 dB SPL) in the GPIAS measurement protocol. Tinnitus perception was shown to be significantly reduced in the noise + DEX group at 10 days after intraperitoneal steroid administration compared to the noise group by measuring the GPIAS value, which means tinnitus intensity was weakened by steroid. Acute insult such as noise trauma causes cochlear damage by reactive oxygen species accumulation23 and cochlear ischemia,24 which could initiate the tinnitus without hearing impairment. The mechanism of action of steroid on tinnitus is related with the decrease in inflammation due to immune system dysfunction in the inner ear, suppression of irritability or sensitivity due to damage of cochlear hair cells, and increased inner ear vascularity.25 These effects of steroid may contribute to tinnitus awareness control in rats with noise trauma.
We performed histological analysis of the noise and noise + DEX groups to compare the differences according to GPIAS value. No pathological changes were observed, such as the loss of hair cells and supporting cells, associated with noise trauma in the noise group and the noise + DEX group. The animal model in this study did not represent a model of tinnitus with normal auditory thresholds as hearing thresholds of 16 kHz increased to 40 dB HL. Sanchez et al.26 reported that only 7.4% of tinnitus patients showed normal pure tone thresholds. These findings suggested that acute tinnitus could be induced without structural damage to the cochlea and the animal tinnitus model with normal hearing category might be developed by adjusting the noise exposure condition.
A recent study showed that immediate and extensive loss of synapses between IHC and ANF can be caused by titrated noise exposure, which is designated as “synaptopathy”.27 We performed immunohistochemical staining to investigate the synapses of IHC in the present study. Although both the noise and noise + DEX groups showed similar patterns of staining for CtBP2, a presynaptic marker protein associated with synaptic ribbons, it should be considered that this finding could be rather preliminary using a small number of samples and ribbon counting in cochlear whole mount specimens and measurement of ABR wave I amplitude should be performed to confirm synaptopathy.
The clinical results regarding the effects of steroid administration on tinnitus control are controversial.2228 In this study, tinnitus control using steroid administration was validated in a model of acute tinnitus caused by acoustic trauma, supporting the positive effect of steroids.29 Steroids may eliminate the reversible inflammatory changes in the cochlea at the initial stages of tinnitus.
There are several factors to be considered. The effect of intratympanic DEX injection, commonly used in clinical practice, on acute tinnitus should be verified. Ribbon counting data in cochlear whole mount would be helpful. Additionally, comparison with the experiment using salicylate-induced tinnitus model might be significant.
In conclusion, we have developed a model of acute tinnitus with minimal structural change associated with noise exposure and objectively verified tinnitus by measuring the GPIAS value. Steroid therapy for tinnitus control was validated in this animal model. Additional trials for other tinnitus models associated with ototoxicity and sudden hearing loss could be required.
| Background:
This study was performed to identify acute tinnitus and evaluate the efficacy of steroids for noise-induced acute tinnitus by measuring the gap-prepulse inhibition of the acoustic startle (GPIAS) value in an animal model.
Methods:
Nineteen rats (the noise group [n = 7] and the noise + dexamethasone [DEX] group [n = 12]) were exposed to narrow-band noise centered at 16 kHz from a sound generator for 4 hours. The noise + DEX group received intraperitoneal steroid administration daily for 5 days (1.5 mg/kg/day) after completing noise exposure. Auditory brainstem response and GPIAS value were measured just prior to, and 1 day after noise exposure and on days 1 and 10 days after completing steroid administration. The changes in cochlear structure were evaluated by histological analysis.
Results:
The threshold shift was checked 1 and 10 days after intraperitoneal steroid injection, and no differences in threshold shift were observed between the two groups in each frequency except for 32 kHz 1 day after steroid injection. The mean GPIAS value in the noise + DEX group (36.4% ± 14.1%) was significantly higher than that in the noise group (16.4% ± 18.8%) 10 days after intraperitoneal steroid administration (P = 0.017). There were no pathological changes associated with noise trauma in the two groups as determined on hematoxylin and eosin and immunohistochemical staining.
Conclusions:
An acute tinnitus model with minimal structural changes by noise exposure was set up, and used to verify tinnitus objectively by measuring the GPIAS value. Steroid therapy for control of tinnitus was validated in this animal model. | null | null | 6,221 | 314 | [
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] | null | null | [CONTENT] Tinnitus | GPIAS | Auditory Brainstem Response | Dexamethasone | Noise-Induced Hearing Loss [SUMMARY] | [CONTENT] Tinnitus | GPIAS | Auditory Brainstem Response | Dexamethasone | Noise-Induced Hearing Loss [SUMMARY] | [CONTENT] Tinnitus | GPIAS | Auditory Brainstem Response | Dexamethasone | Noise-Induced Hearing Loss [SUMMARY] | null | [CONTENT] Tinnitus | GPIAS | Auditory Brainstem Response | Dexamethasone | Noise-Induced Hearing Loss [SUMMARY] | null | [CONTENT] Acoustics | Acute Disease | Animals | Dexamethasone | Disease Models, Animal | Evoked Potentials, Auditory, Brain Stem | Glucocorticoids | Male | Noise | Rats | Tinnitus [SUMMARY] | [CONTENT] Acoustics | Acute Disease | Animals | Dexamethasone | Disease Models, Animal | Evoked Potentials, Auditory, Brain Stem | Glucocorticoids | Male | Noise | Rats | Tinnitus [SUMMARY] | [CONTENT] Acoustics | Acute Disease | Animals | Dexamethasone | Disease Models, Animal | Evoked Potentials, Auditory, Brain Stem | Glucocorticoids | Male | Noise | Rats | Tinnitus [SUMMARY] | null | [CONTENT] Acoustics | Acute Disease | Animals | Dexamethasone | Disease Models, Animal | Evoked Potentials, Auditory, Brain Stem | Glucocorticoids | Male | Noise | Rats | Tinnitus [SUMMARY] | null | [CONTENT] effects acute tinnitus | hyperexcitability tinnitus | cochlea animal tinnitus | tinnitus preserving hearing | tinnitus caused acoustic [SUMMARY] | [CONTENT] effects acute tinnitus | hyperexcitability tinnitus | cochlea animal tinnitus | tinnitus preserving hearing | tinnitus caused acoustic [SUMMARY] | [CONTENT] effects acute tinnitus | hyperexcitability tinnitus | cochlea animal tinnitus | tinnitus preserving hearing | tinnitus caused acoustic [SUMMARY] | null | [CONTENT] effects acute tinnitus | hyperexcitability tinnitus | cochlea animal tinnitus | tinnitus preserving hearing | tinnitus caused acoustic [SUMMARY] | null | [CONTENT] noise | tinnitus | gpias | steroid | group | startle | dex | gap | exposure | rats [SUMMARY] | [CONTENT] noise | tinnitus | gpias | steroid | group | startle | dex | gap | exposure | rats [SUMMARY] | [CONTENT] noise | tinnitus | gpias | steroid | group | startle | dex | gap | exposure | rats [SUMMARY] | null | [CONTENT] noise | tinnitus | gpias | steroid | group | startle | dex | gap | exposure | rats [SUMMARY] | null | [CONTENT] tinnitus | reported | gap | startle | acoustic | auditory | acute | therapy | acoustic startle | cochlear [SUMMARY] | [CONTENT] noise | usa | startle | gap | sections | 300 | ms | 100 | wash | chamber [SUMMARY] | [CONTENT] noise | groups | group | mean | threshold | shift | threshold shift | mean gpias | dex | gpias [SUMMARY] | null | [CONTENT] noise | tinnitus | group | gpias | dex | startle | groups | steroid | gap | exposure [SUMMARY] | null | [CONTENT] acoustic | GPIAS [SUMMARY] | [CONTENT] 7 ||| 12 | 16 | 4 hours ||| 5 days | 1.5 mg/kg/day ||| GPIAS | 1 day | days 1 and 10 days ||| [SUMMARY] | [CONTENT] 1 and 10 days | two | 32 | 1 day ||| GPIAS | 36.4% | 14.1% | 16.4% | 18.8% | 10 days | 0.017 ||| two | hematoxylin [SUMMARY] | null | [CONTENT] acoustic | GPIAS ||| 7 ||| 12 | 16 | 4 hours ||| 5 days | 1.5 mg/kg/day ||| GPIAS | 1 day | days 1 and 10 days ||| ||| ||| 1 and 10 days | two | 32 | 1 day ||| GPIAS | 36.4% | 14.1% | 16.4% | 18.8% | 10 days | 0.017 ||| two | hematoxylin ||| GPIAS ||| [SUMMARY] | null |
||||
Prevalence, risk factors, treatment and outcome of multidrug resistance Candida auris infections in Coronavirus disease (COVID-19) patients: A systematic review. | 35441748 | Candida auris is an emerging multidrug-resistant pathogen in intensive care settings (ICU). During the coronavirus disease 19 (COVID-19) pandemic, ICU admissions were overwhelmed, possibly contributing to the C. auris outbreak in COVID-19 patients. | BACKGROUND | MEDLINE, Scopus, Embase, Web of Science and LitCovid databases were systematically searched with appropriate keywords from 1 January 2020 to 31 December 2021. | METHODS | A total of 97 cases of C. auris were identified in COVID-19 patients. The pooled prevalence of C. auris infections (encompassing candidemia and non-candidemia cases) in COVID-19 patients was 14%. The major underlying diseases were diabetes mellitus (42.7%), hypertension (32.9%) and obesity (14.6%), followed by the iatrogenic risk factors such as a central venous catheter (76.8%%), intensive care unit (ICU) stay (75.6%) and broad-spectrum antibiotic usage (74.3%). There were no significant differences in underlying disease and iatrogenic risk factors among C. auris non-candidemia/colonisation and C. auris candidemia cases. The mortality rate of the total cohort is 44.4%, whereas, in C. auris candidemia patients, the mortality was 64.7%. | RESULTS | This study shows that the prevalence of C. auris infections remains unchanged in the COVID-19 pandemic. Hospital-acquired risk factors may contribute to the clinical illness. Proper infection control practices and hospital surveillance may stop future hospital outbreaks during the pandemic. | CONCLUSION | [
"Antifungal Agents",
"COVID-19",
"Candida",
"Candida auris",
"Candidemia",
"Drug Resistance, Multiple",
"Humans",
"Iatrogenic Disease",
"Microbial Sensitivity Tests",
"Pandemics",
"Prevalence",
"Risk Factors",
"Treatment Outcome"
] | 9115268 | INTRODUCTION | During the coronavirus disease 19 (COVID‐19) pandemic, the healthcare facility was overwhelmed with patients admitted to intensive care units (ICUs), and those patients were highly susceptible to bacterial and fungal co‐infections.
1
,
2
Candida auris is an emerging pathogen in ICU settings with high mortality and infection due to this pathogen has been reported across 44 countries.
3
,
4
Candida auris is a unique species, as this agent is multidrug‐resistant, and they can survive on inanimate objects in the hospital environment for more extended periods, leading to potential transmission among patients (hospital outbreaks), and difficulty in accurate laboratory identification makes them the pathogen of public interest globally.
5
,
6
,
7
,
8
Patients with underlying diseases such as diabetes mellitus, kidney disease, lung disease, trauma, ear diseases and hypertension, followed by iatrogenic risk factors such as prolonged ICU stay, central venous catheter, mechanical ventilation and prior antibiotic usage, were significantly associated with C. auris infections prior to the COVID‐19 pandemic.
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Like C. auris infections, diabetes mellitus, hypertension, malignancies, chronic kidney diseases, chronic liver disease and cardiovascular disease are associated with high mortality in severe COVID‐19 patients.
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Underlying disease and the invasive medical procedures during the hospital stay in COVID‐19 patients make them highly susceptible to C. auris infections/colonisation. The outbreak of C. auris infections has been reported during the COVID‐19 pandemic across different countries.
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Due to the overlapping features (underlying disease/risk factors) between the two disease groups, the disease epidemiology of C. auris infections in COVID‐19 patients will be interesting to study. In the present study, we systematically reviewed the C. auris infections/colonisation in COVID‐19 patients to determine the prevalence, underlying disease/risk factors, treatment and outcome. | null | null | RESULTS | The initial database search identified 291 articles; of those, 11 articles were included in the final analysis (Figure 1), and the data were extracted for qualitative and quantitative analysis. Quality assessment of the articles was done using the JBI tool; of the 11 articles assessed, 8 fulfilled the criteria as mentioned above,
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in 3 of the articles, the method of identification was unclear, and the rest of the criteria were fulfilled.
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A total of 97 cases of C. auris were identified in COVID‐19 patients across different countries (Figure 2). Majority of the cases were reported from the United States of America (n = 48, 49.5%), followed by Mexico (n = 12, 12.4%) and India (n = 10, 10.3%; Figure 2). The male to female ratio was 2.6:1. The mean age was 65.41 years (with a range of 1–101 years), and 99% (n = 96) of the patients with C. auris infections were adults (Table 1). For estimation of pooled prevalence of C. auris infections in the COVID‐19 patients, the data from the 5 studies were analysed (Figure 3).
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The prevalance of C. auris infections (including candidemia and non‐candidemia cases) among the COVID‐19 patients was 14%, with high heterogeneity among the included publications (I
2 = 99.88; Figure 3).
Candida auris cases in COVID‐19 patients across countries. References are given in square brackets
Risk factors, treatment and outcome of Candida auris cases in COVID‐19 patients
The values in the table are expressed in numbers (n) and percentages (%). * ‘p’ values <.05 were considered significant.
Abbreviations: ARDS, Acute Respiratory Distress Syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; TC, total cohort.
The data were extracted from the 11 studies which fulfilled inclusion criteria (TC).
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Age range, male/female ratio, details of clinical specimens, underlying disease, iatrogenic risk factors, antifungal therapy and clinical outcome of CANC and CAC cases were extracted from 10 studies.
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Mean age and mean days from first isolation of C. auris from admission for CANC and CAC cases were extracted from 9 studies.
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Renal diseases were chronic kidney disease (CKD) and acute kidney injury (AKI). [TC: CKD (n = 9), and AKI (n = 2); CANC cases: CKD (n = 2); CAC cases: CKD (n = 4) and AKI (n = 2)].
Heart diseases were ischemic heart disease (IHD) and coronary heart disease (CAD). [TC: IHD (n = 5), and CAD (n = 3); CANC cases: IHD (n = 2), and CAD (n = 1); CAC cases: IHD (n = 2) and CAD (n = 2)].
Liver and Biliary diseases were chronic liver disease (CLD) and biliary lithiasis (BL). [TC: CLD (n = 3), and BL (n = 1); CANC cases: BL (n = 1); CAC cases: CLD (n = 3)].
Thromboembolic diseases were pulmonary embolism (PE) and deep venous thrombosis (DVT). [TC: PE (n = 3), and DVT (n = 2); CANC cases: PE (n = 1), and DVT (n = 2); CAC cases: PE (n = 2)].
Miscellaneous respiratory diseases were asthma, chronic obstructive pulmonary disease (COPD), respiratory failure (RF) and pneumothorax (PT). [TC: asthma (n = 4), COPD (n = 3), RF (n = 1), and PT (n = 1); CANC cases: asthma (n = 1), COPD (n = 2), RF (n = 1), and PT (n = 1); CAC cases: asthma (n = 3), and COPD (n = 1)].
Other risk factors included wound infections (WI), hyperlipidaemia (HLD), hypothyroidism (HT), stroke, dementia, stem cell transplant (SCT), systemic lupus erythematosus (SLE). [TC: WI (n = 6), HLD (n = 3), HT (n = 2), stroke (n = 1), dementia (n = 1), SCT (n = 1), and SLE (n = 1); CANC cases: WI (n = 2), HLD (n = 1), HT (n = 1), stroke (n = 1), dementia (n = 1), and SLE (n = 1); CAC cases: HLD (n = 2), HT (n = 1), and SCT (n = 1).
In patients with antifungal therapy, many patients received combination of antifungals for treatment.
Forest plot of pooled prevalence of Candida auris infections in COVID‐19 patients. "Frequency" denotes total number of C. auris cases and "Total" denotes total number of COVID‐19 infected patients. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Clinical specimens, isolation and identification of Candida auris
Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.
As per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.
29
Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).
Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.
As per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.
29
Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).
Underlying disease and risk factors Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.
Pooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients
Abbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.
The data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).
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The denominators used in the analysis for the total cohort are n = 82.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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Denominators used in the analysis for comparison of CANC and CAC group is n = 62.
Refer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.
Similar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2
= 88.65) (Table 2).
Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.
Pooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients
Abbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.
The data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).
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The denominators used in the analysis for the total cohort are n = 82.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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Denominators used in the analysis for comparison of CANC and CAC group is n = 62.
Refer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.
Similar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2
= 88.65) (Table 2).
Antifungal therapy and outcome of Candida auris infections A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2
= 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).
Forest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. "Frequency" denotes total number of patients survived with C. auris infections and "Total" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Underlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases
The values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.
Abbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.
Underlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2
= 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).
Forest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. "Frequency" denotes total number of patients survived with C. auris infections and "Total" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Underlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases
The values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.
Abbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.
Underlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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Antifungal susceptibility of Candida auris isolates from COVID‐19 patients Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).
Antifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients
The data provided in the table were extracted from 5 studies.
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Abbreviations: MIC, minimum inhibitory concentration.
MIC50: MIC at which 50% of the tested isolates are inhibited.
MIC90: MIC at which 90% of the tested isolates are inhibited.
Furthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).
Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).
Antifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients
The data provided in the table were extracted from 5 studies.
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Abbreviations: MIC, minimum inhibitory concentration.
MIC50: MIC at which 50% of the tested isolates are inhibited.
MIC90: MIC at which 90% of the tested isolates are inhibited.
Furthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%). | CONCLUSION | The study highlights the role of hospital‐acquired C. auris infections in COVID‐19 patients. Despite the multiple risk factors possibly favouring the C. auris infections in COVID‐19 patients, the prevalence of the disease remains unchanged compared with the pre‐pandemic. However, one must be cautioned that COVID‐19 patients may be more vulnerable to C. auris infections because of the overlapping risk factors. Increased burden of colonised patients with C. auris in ICU settings may lead to person‐to‐person transmission. Hence, proper infection control practices and strict hospital surveillance for screening and isolating C. auris colonised patients in the COVID‐19 ICUs may prevent the potential outbreaks in hospital settings. | [
"INTRODUCTION",
"METHODOLOGY",
"Literature search and eligibility criteria",
"Study selection and data extraction",
"Risk of bias assessment",
"Statistical methods",
"Clinical specimens, isolation and identification of Candida auris\n",
"Underlying disease and risk factors",
"Antifungal therapy and outcome of Candida auris infections",
"Antifungal susceptibility of Candida auris isolates from COVID‐19 patients",
"AUTHOR CONTRIBUTIONS"
] | [
"During the coronavirus disease 19 (COVID‐19) pandemic, the healthcare facility was overwhelmed with patients admitted to intensive care units (ICUs), and those patients were highly susceptible to bacterial and fungal co‐infections.\n1\n, \n2\n\nCandida auris is an emerging pathogen in ICU settings with high mortality and infection due to this pathogen has been reported across 44 countries.\n3\n, \n4\n\nCandida auris is a unique species, as this agent is multidrug‐resistant, and they can survive on inanimate objects in the hospital environment for more extended periods, leading to potential transmission among patients (hospital outbreaks), and difficulty in accurate laboratory identification makes them the pathogen of public interest globally.\n5\n, \n6\n, \n7\n, \n8\n Patients with underlying diseases such as diabetes mellitus, kidney disease, lung disease, trauma, ear diseases and hypertension, followed by iatrogenic risk factors such as prolonged ICU stay, central venous catheter, mechanical ventilation and prior antibiotic usage, were significantly associated with C. auris infections prior to the COVID‐19 pandemic.\n3\n, \n4\n, \n9\n, \n10\n\n\nLike C. auris infections, diabetes mellitus, hypertension, malignancies, chronic kidney diseases, chronic liver disease and cardiovascular disease are associated with high mortality in severe COVID‐19 patients.\n11\n, \n12\n Underlying disease and the invasive medical procedures during the hospital stay in COVID‐19 patients make them highly susceptible to C. auris infections/colonisation. The outbreak of C. auris infections has been reported during the COVID‐19 pandemic across different countries.\n13\n, \n14\n, \n15\n, \n16\n Due to the overlapping features (underlying disease/risk factors) between the two disease groups, the disease epidemiology of C. auris infections in COVID‐19 patients will be interesting to study. In the present study, we systematically reviewed the C. auris infections/colonisation in COVID‐19 patients to determine the prevalence, underlying disease/risk factors, treatment and outcome.",
"Literature search and eligibility criteria The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).\nPRISMA flowchart describing the study selection process\nThe proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).\nPRISMA flowchart describing the study selection process\nStudy selection and data extraction After the literature search, the citations were uploaded to Rayyan QCRI software for screening.\n17\n Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).\nAfter the literature search, the citations were uploaded to Rayyan QCRI software for screening.\n17\n Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).\nRisk of bias assessment The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.\n18\n, \n19\n The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.\nThe risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.\n18\n, \n19\n The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.\nStatistical methods Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).\n20\n, \n21\n Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I\n2 statistics.\nComparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).\n20\n, \n21\n Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I\n2 statistics.",
"The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).\nPRISMA flowchart describing the study selection process",
"After the literature search, the citations were uploaded to Rayyan QCRI software for screening.\n17\n Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).",
"The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.\n18\n, \n19\n The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.",
"Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).\n20\n, \n21\n Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I\n2 statistics.",
"Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.\nAs per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.\n29\n Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).",
"Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.\nPooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients\nAbbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.\nThe data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).\n13\n, \n14\n, \n15\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n The denominators used in the analysis for the total cohort are n = 82.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n Denominators used in the analysis for comparison of CANC and CAC group is n = 62.\nRefer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.\nSimilar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2\n = 88.65) (Table 2).",
"A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2\n = 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).\nForest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. \"Frequency\" denotes total number of patients survived with C. auris infections and \"Total\" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval\nUnderlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases\nThe values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.\nAbbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.\nUnderlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n\n",
"Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).\nAntifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients\nThe data provided in the table were extracted from 5 studies.\n13\n, \n14\n, \n22\n, \n23\n, \n24\n\n\nAbbreviations: MIC, minimum inhibitory concentration.\nMIC50: MIC at which 50% of the tested isolates are inhibited.\nMIC90: MIC at which 90% of the tested isolates are inhibited.\nFurthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).",
"HPP conceptualised the idea of this study. HPP, KSV and KCP designed the study. HPP and KSV did primary data extraction for the study and wrote the manuscript. KCP and HPP performed the analysis. The final draft of the manuscript was corrected and proofread by all the authors."
] | [
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] | [
"INTRODUCTION",
"METHODOLOGY",
"Literature search and eligibility criteria",
"Study selection and data extraction",
"Risk of bias assessment",
"Statistical methods",
"RESULTS",
"Clinical specimens, isolation and identification of Candida auris\n",
"Underlying disease and risk factors",
"Antifungal therapy and outcome of Candida auris infections",
"Antifungal susceptibility of Candida auris isolates from COVID‐19 patients",
"DISCUSSION",
"CONCLUSION",
"CONFLICT OF INTEREST",
"AUTHOR CONTRIBUTIONS"
] | [
"During the coronavirus disease 19 (COVID‐19) pandemic, the healthcare facility was overwhelmed with patients admitted to intensive care units (ICUs), and those patients were highly susceptible to bacterial and fungal co‐infections.\n1\n, \n2\n\nCandida auris is an emerging pathogen in ICU settings with high mortality and infection due to this pathogen has been reported across 44 countries.\n3\n, \n4\n\nCandida auris is a unique species, as this agent is multidrug‐resistant, and they can survive on inanimate objects in the hospital environment for more extended periods, leading to potential transmission among patients (hospital outbreaks), and difficulty in accurate laboratory identification makes them the pathogen of public interest globally.\n5\n, \n6\n, \n7\n, \n8\n Patients with underlying diseases such as diabetes mellitus, kidney disease, lung disease, trauma, ear diseases and hypertension, followed by iatrogenic risk factors such as prolonged ICU stay, central venous catheter, mechanical ventilation and prior antibiotic usage, were significantly associated with C. auris infections prior to the COVID‐19 pandemic.\n3\n, \n4\n, \n9\n, \n10\n\n\nLike C. auris infections, diabetes mellitus, hypertension, malignancies, chronic kidney diseases, chronic liver disease and cardiovascular disease are associated with high mortality in severe COVID‐19 patients.\n11\n, \n12\n Underlying disease and the invasive medical procedures during the hospital stay in COVID‐19 patients make them highly susceptible to C. auris infections/colonisation. The outbreak of C. auris infections has been reported during the COVID‐19 pandemic across different countries.\n13\n, \n14\n, \n15\n, \n16\n Due to the overlapping features (underlying disease/risk factors) between the two disease groups, the disease epidemiology of C. auris infections in COVID‐19 patients will be interesting to study. In the present study, we systematically reviewed the C. auris infections/colonisation in COVID‐19 patients to determine the prevalence, underlying disease/risk factors, treatment and outcome.",
"Literature search and eligibility criteria The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).\nPRISMA flowchart describing the study selection process\nThe proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).\nPRISMA flowchart describing the study selection process\nStudy selection and data extraction After the literature search, the citations were uploaded to Rayyan QCRI software for screening.\n17\n Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).\nAfter the literature search, the citations were uploaded to Rayyan QCRI software for screening.\n17\n Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).\nRisk of bias assessment The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.\n18\n, \n19\n The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.\nThe risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.\n18\n, \n19\n The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.\nStatistical methods Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).\n20\n, \n21\n Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I\n2 statistics.\nComparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).\n20\n, \n21\n Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I\n2 statistics.",
"The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).\nPRISMA flowchart describing the study selection process",
"After the literature search, the citations were uploaded to Rayyan QCRI software for screening.\n17\n Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).",
"The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.\n18\n, \n19\n The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.",
"Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).\n20\n, \n21\n Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I\n2 statistics.",
"The initial database search identified 291 articles; of those, 11 articles were included in the final analysis (Figure 1), and the data were extracted for qualitative and quantitative analysis. Quality assessment of the articles was done using the JBI tool; of the 11 articles assessed, 8 fulfilled the criteria as mentioned above,\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n in 3 of the articles, the method of identification was unclear, and the rest of the criteria were fulfilled.\n15\n, \n27\n, \n28\n A total of 97 cases of C. auris were identified in COVID‐19 patients across different countries (Figure 2). Majority of the cases were reported from the United States of America (n = 48, 49.5%), followed by Mexico (n = 12, 12.4%) and India (n = 10, 10.3%; Figure 2). The male to female ratio was 2.6:1. The mean age was 65.41 years (with a range of 1–101 years), and 99% (n = 96) of the patients with C. auris infections were adults (Table 1). For estimation of pooled prevalence of C. auris infections in the COVID‐19 patients, the data from the 5 studies were analysed (Figure 3).\n15\n, \n16\n, \n23\n, \n25\n, \n26\n The prevalance of C. auris infections (including candidemia and non‐candidemia cases) among the COVID‐19 patients was 14%, with high heterogeneity among the included publications (I\n2 = 99.88; Figure 3).\n\nCandida auris cases in COVID‐19 patients across countries. References are given in square brackets\nRisk factors, treatment and outcome of Candida auris cases in COVID‐19 patients\nThe values in the table are expressed in numbers (n) and percentages (%). * ‘p’ values <.05 were considered significant.\nAbbreviations: ARDS, Acute Respiratory Distress Syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; TC, total cohort.\nThe data were extracted from the 11 studies which fulfilled inclusion criteria (TC).\n13\n, \n14\n, \n15\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n\n\nAge range, male/female ratio, details of clinical specimens, underlying disease, iatrogenic risk factors, antifungal therapy and clinical outcome of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n\n\nMean age and mean days from first isolation of C. auris from admission for CANC and CAC cases were extracted from 9 studies.\n13\n, \n14\n, \n15\n, \n16\n, \n23\n, \n24\n, \n25\n, \n27\n, \n28\n\n\nRenal diseases were chronic kidney disease (CKD) and acute kidney injury (AKI). [TC: CKD (n = 9), and AKI (n = 2); CANC cases: CKD (n = 2); CAC cases: CKD (n = 4) and AKI (n = 2)].\nHeart diseases were ischemic heart disease (IHD) and coronary heart disease (CAD). [TC: IHD (n = 5), and CAD (n = 3); CANC cases: IHD (n = 2), and CAD (n = 1); CAC cases: IHD (n = 2) and CAD (n = 2)].\nLiver and Biliary diseases were chronic liver disease (CLD) and biliary lithiasis (BL). [TC: CLD (n = 3), and BL (n = 1); CANC cases: BL (n = 1); CAC cases: CLD (n = 3)].\nThromboembolic diseases were pulmonary embolism (PE) and deep venous thrombosis (DVT). [TC: PE (n = 3), and DVT (n = 2); CANC cases: PE (n = 1), and DVT (n = 2); CAC cases: PE (n = 2)].\nMiscellaneous respiratory diseases were asthma, chronic obstructive pulmonary disease (COPD), respiratory failure (RF) and pneumothorax (PT). [TC: asthma (n = 4), COPD (n = 3), RF (n = 1), and PT (n = 1); CANC cases: asthma (n = 1), COPD (n = 2), RF (n = 1), and PT (n = 1); CAC cases: asthma (n = 3), and COPD (n = 1)].\nOther risk factors included wound infections (WI), hyperlipidaemia (HLD), hypothyroidism (HT), stroke, dementia, stem cell transplant (SCT), systemic lupus erythematosus (SLE). [TC: WI (n = 6), HLD (n = 3), HT (n = 2), stroke (n = 1), dementia (n = 1), SCT (n = 1), and SLE (n = 1); CANC cases: WI (n = 2), HLD (n = 1), HT (n = 1), stroke (n = 1), dementia (n = 1), and SLE (n = 1); CAC cases: HLD (n = 2), HT (n = 1), and SCT (n = 1).\nIn patients with antifungal therapy, many patients received combination of antifungals for treatment.\nForest plot of pooled prevalence of Candida auris infections in COVID‐19 patients. \"Frequency\" denotes total number of C. auris cases and \"Total\" denotes total number of COVID‐19 infected patients. References are given in square brackets. Abbreviations: C.I, Confidence Interval\nClinical specimens, isolation and identification of Candida auris\n Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.\nAs per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.\n29\n Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).\nOf those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.\nAs per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.\n29\n Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).\nUnderlying disease and risk factors Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.\nPooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients\nAbbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.\nThe data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).\n13\n, \n14\n, \n15\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n The denominators used in the analysis for the total cohort are n = 82.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n Denominators used in the analysis for comparison of CANC and CAC group is n = 62.\nRefer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.\nSimilar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2\n = 88.65) (Table 2).\nTable 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.\nPooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients\nAbbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.\nThe data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).\n13\n, \n14\n, \n15\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n The denominators used in the analysis for the total cohort are n = 82.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n Denominators used in the analysis for comparison of CANC and CAC group is n = 62.\nRefer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.\nSimilar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2\n = 88.65) (Table 2).\nAntifungal therapy and outcome of Candida auris infections A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2\n = 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).\nForest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. \"Frequency\" denotes total number of patients survived with C. auris infections and \"Total\" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval\nUnderlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases\nThe values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.\nAbbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.\nUnderlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n\n\nA total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2\n = 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).\nForest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. \"Frequency\" denotes total number of patients survived with C. auris infections and \"Total\" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval\nUnderlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases\nThe values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.\nAbbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.\nUnderlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n\n\nAntifungal susceptibility of Candida auris isolates from COVID‐19 patients Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).\nAntifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients\nThe data provided in the table were extracted from 5 studies.\n13\n, \n14\n, \n22\n, \n23\n, \n24\n\n\nAbbreviations: MIC, minimum inhibitory concentration.\nMIC50: MIC at which 50% of the tested isolates are inhibited.\nMIC90: MIC at which 90% of the tested isolates are inhibited.\nFurthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).\nTable 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).\nAntifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients\nThe data provided in the table were extracted from 5 studies.\n13\n, \n14\n, \n22\n, \n23\n, \n24\n\n\nAbbreviations: MIC, minimum inhibitory concentration.\nMIC50: MIC at which 50% of the tested isolates are inhibited.\nMIC90: MIC at which 90% of the tested isolates are inhibited.\nFurthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).",
"Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.\nAs per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.\n29\n Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).",
"Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.\nPooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients\nAbbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.\nThe data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).\n13\n, \n14\n, \n15\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n The denominators used in the analysis for the total cohort are n = 82.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n Denominators used in the analysis for comparison of CANC and CAC group is n = 62.\nRefer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.\nSimilar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2\n = 88.65) (Table 2).",
"A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2\n = 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).\nForest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. \"Frequency\" denotes total number of patients survived with C. auris infections and \"Total\" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval\nUnderlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases\nThe values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.\nAbbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.\nUnderlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.\nThe data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.\n13\n, \n14\n, \n16\n, \n22\n, \n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n\n",
"Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).\nAntifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients\nThe data provided in the table were extracted from 5 studies.\n13\n, \n14\n, \n22\n, \n23\n, \n24\n\n\nAbbreviations: MIC, minimum inhibitory concentration.\nMIC50: MIC at which 50% of the tested isolates are inhibited.\nMIC90: MIC at which 90% of the tested isolates are inhibited.\nFurthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).",
"The present systematic review analysed the published cases of C. auris in COVID‐19 patients, providing insight on prevalence, underlying disease/risk factors, treatment and disease outcome. The review was conducted per PRISMA guidelines, including only the articles that met the study criteria. This review has provided an update on the epidemiology of C. auris infections in COVID‐19 patients.\nIn the present study, majority of the C. auris‐infected patients were adults (99%), with one case in the paediatric population, similar to previous reports.\n4\n, \n6\n, \n10\n In this study, the pooled prevalence of C. auris infections in COVID‐19 patients was 14%. The true prevalence of C. auris infections in the global population is largely unknown. As per multiple studies, the prevalence of C. auris in candidemia patients (non‐COVID‐19 cases) ranges between 5‐30%.\n30\n Further, many C. auris colonisation patients have also been identified in countries like the USA, Europe, India, South Africa and henceforth.\n5\n, \n31\n, \n32\n, \n33\n\n\nAs per CDC recommendation on C. auris definition for colonisation (non‐invasive sites) and true infections (invasive sites),\n29\n in the present study, the cases were classified as C. auris non‐candidemia/colonised (CANC) and C. auris candidemia (CAC) cases, respectively. A total of 35 (56.5%) CAC cases and 27 (43.5%) CANC cases were identified. The underlying disease/iatrogenic risk factors in identified both groups were: hypertension (43.5%), diabetes mellitus (37%), obesity (19.4%), COVID‐19‐associated ARDS (14.5%) and renal diseases (13%); whereas the iatrogenic risk factors included broad‐spectrum antibiotic usage (98.4%), ICU stay (97%), steroid therapy (82.3%), the central venous catheter (75.8%), mechanical ventilation (74.2%) and steroid therapy (82.2%). The respective analysis of C. auris and COVID‐19 patients showed that both groups shared the underlying disease mentioned above.\n3\n, \n4\n, \n9\n, \n10\n, \n11\n, \n12\n Multiple studies have reported central venous catheter, previous antifungal exposure, mechanical ventilation, and prolonged ICU stay as significant risk factors for C. auris colonisation/infection.\n7\n, \n34\n, \n35\n Thus, the ongoing COVID‐19 pandemic would become a perfect battlefield for outbreaks of C. auris because of the similar underlying diseases and the risk factors, which increases the chance of C. auris infections in COVID‐19 patients. Further, a recent study from India confirmed these findings, that the treatment with interleukin‐6 antagonists such as tocilizumab, prolonged ICU stay, mechanical ventilation and raised ferritin levels were identified as significant risk factors of candidemia (majority of reported cases in the study were due to C. auris) in COVID‐19 patients.\n36\n\n\nFurthermore, during the COVID‐19 pandemic, the existing ICU facilities have seen overflowing patients, with a high burden of patients, there was a challenge in implementing the infection control practices. Of the included studies in this review, a few studies documented the source for the C. auris outbreak, and the infection prevention and control (IPCs) measures to contain the C. auris infection in COVID‐19 patients.\n13\n, \n14\n, \n16\n Allaw et al. reported C. auris outbreak in Lebanon in a tertiary care centre for 13 weeks. Following the first case of C. auris infection, IPC practices such as hand hygiene using antiseptics, disinfection of floors and surfaces of patient's room, screening for C. auris in environmental samples and skin colonisation in patients, 4% chlorhexidine bath for C. auris skin decolonisation was initiated. C. auris was not isolated from environmental samples; however, of the 26 patients screened for skin colonisation, one patient grew C. auris. Authors attributed the delay in reporting and identifying the first case of C. auris to the outbreak, which delayed the implementation of IPC measures.\n14\n Villanueva‐Lozano et al. reported C. auris outbreak in COVID‐19 patients in Mexico, and the authors isolated three environmental isolates from their bedrooms. The phylogenetic analysis of the clinical and environmental isolates clustered together, suggesting close similarities among the isolates.\n13\n These findings show that the environmental contamination of hospitals remains a possibility either by cross‐contamination of the equipment or by the healthcare providers. However, Chowdhary et al.\n23\n proposed that C. auris infection in COVID‐19 patients may not be transmitted by healthcare personnel because of personal protective equipment (PPE). Further, the authors cautioned that improper use of PPE may lead to contamination and disease transmission.\n23\n A study from Brazil documented the source of C. auris outbreak in the hospital settings; the environmental screening showed C. auris contamination from auxiliary digital thermometers (17%), bed rails (15%), and intravenous infusion pumps (11%) and tray tables (11%). The study reported that C. auris colonisation of digital thermometer was the significant risk factor associated with C. auris colonisation in patients, which correlated with the high isolation rate of C. auris from axillae of the patients.\n16\n Similarly, Eyre et al.\n7\n reported that skin surface axillary temperature reusable probes were significantly associated with C. auris colonisation/infection (86% in the patient group versus 34% in controls). Proper surveillance for C. auris colonisation and implementation of infection control practices may stop the spread of C. auris infections/colonisation in COVID‐19 ICU settings.\n7\n, \n37\n\n\n\nCandida auris multidrug‐resistant isolates have been reported from Asia, the USA, Europe and Africa.\n7\n, \n38\n, \n39\n, \n40\n, \n41\n, \n42\n In the present study, C. auris isolates were resistant to fluconazole (81%), followed by voriconazole (29.3%), amphotericin B (46.3%), caspofungin (12.8%), anidulafungin (5.1%), micafungin (3.7%) and 5‐flucytosine (43.8%). Similarly, antifungal susceptibility testing of C. auris isolates from multiple countries reported a large number of isolates are resistant to fluconazole (35%–100%), followed by amphotericin B resistance (8%–61%), echinocandins resistance (0.5%–3%) and voriconazole (14%–90%).\n7\n, \n38\n, \n39\n, \n40\n, \n41\n In the present study, multidrug resistance was noted in 53.6% of C. auris isolates, similar to the previously reported studies (6%–61%).\n40\n, \n41\n, \n42\n\n\nIn the present study, 44 (71%) patients from CANC (n = 11, 25%) and CAC (n = 33, 75%) group received antifungal therapy. Globally, C. auris isolates exhibit a higher susceptible pattern to echinocandins,\n7\n, \n38\n, \n39\n, \n40\n, \n41\n making them the drug of choice for C. auris infections. Similarly, echinocandins (75%) were the most common drug used in this study. The survival rate for CANC and CAC groups was 77.8% and 35.3%, respectively (p < .002). Multiple systematic reviews showed that mortality in C. auris cases was at 39% to 47.5%.\n4\n, \n43\n Similar to the present study's findings, Sayeed et al.\n10\n reported a survival rate of 54% in C. auris candidemia cases and 67% in colonised cases. Further, this study showed that iatrogenic risk factors such as prolonged ICU stay, mechanical ventilation, steroid therapy, broad‐spectrum antibiotics and central venous catheters were significantly associated with high mortality in CAC patients compared with the CANC group. These findings caution that healthcare personnel should be vigilant while treating severe COVID‐19 patients, as they are more vulnerable to C. auris infection. Despite the treatment with antifungals, a high mortality rate is seen in C. auris infections, making them a global threat.",
"The study highlights the role of hospital‐acquired C. auris infections in COVID‐19 patients. Despite the multiple risk factors possibly favouring the C. auris infections in COVID‐19 patients, the prevalence of the disease remains unchanged compared with the pre‐pandemic. However, one must be cautioned that COVID‐19 patients may be more vulnerable to C. auris infections because of the overlapping risk factors. Increased burden of colonised patients with C. auris in ICU settings may lead to person‐to‐person transmission. Hence, proper infection control practices and strict hospital surveillance for screening and isolating C. auris colonised patients in the COVID‐19 ICUs may prevent the potential outbreaks in hospital settings.",
"The authors declare no competing interest.",
"HPP conceptualised the idea of this study. HPP, KSV and KCP designed the study. HPP and KSV did primary data extraction for the study and wrote the manuscript. KCP and HPP performed the analysis. The final draft of the manuscript was corrected and proofread by all the authors."
] | [
null,
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"results",
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"discussion",
"conclusions",
"COI-statement",
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] | [
"\nCandida auris\n",
"candidemia",
"COVID‐19",
"mortality",
"prevalence",
"systematic review"
] | INTRODUCTION:
During the coronavirus disease 19 (COVID‐19) pandemic, the healthcare facility was overwhelmed with patients admitted to intensive care units (ICUs), and those patients were highly susceptible to bacterial and fungal co‐infections.
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Candida auris is an emerging pathogen in ICU settings with high mortality and infection due to this pathogen has been reported across 44 countries.
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Candida auris is a unique species, as this agent is multidrug‐resistant, and they can survive on inanimate objects in the hospital environment for more extended periods, leading to potential transmission among patients (hospital outbreaks), and difficulty in accurate laboratory identification makes them the pathogen of public interest globally.
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7
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Patients with underlying diseases such as diabetes mellitus, kidney disease, lung disease, trauma, ear diseases and hypertension, followed by iatrogenic risk factors such as prolonged ICU stay, central venous catheter, mechanical ventilation and prior antibiotic usage, were significantly associated with C. auris infections prior to the COVID‐19 pandemic.
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4
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9
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Like C. auris infections, diabetes mellitus, hypertension, malignancies, chronic kidney diseases, chronic liver disease and cardiovascular disease are associated with high mortality in severe COVID‐19 patients.
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Underlying disease and the invasive medical procedures during the hospital stay in COVID‐19 patients make them highly susceptible to C. auris infections/colonisation. The outbreak of C. auris infections has been reported during the COVID‐19 pandemic across different countries.
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Due to the overlapping features (underlying disease/risk factors) between the two disease groups, the disease epidemiology of C. auris infections in COVID‐19 patients will be interesting to study. In the present study, we systematically reviewed the C. auris infections/colonisation in COVID‐19 patients to determine the prevalence, underlying disease/risk factors, treatment and outcome.
METHODOLOGY:
Literature search and eligibility criteria The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).
PRISMA flowchart describing the study selection process
The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).
PRISMA flowchart describing the study selection process
Study selection and data extraction After the literature search, the citations were uploaded to Rayyan QCRI software for screening.
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Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).
After the literature search, the citations were uploaded to Rayyan QCRI software for screening.
17
Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).
Risk of bias assessment The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.
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The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.
The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.
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The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.
Statistical methods Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).
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Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I
2 statistics.
Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).
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Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I
2 statistics.
Literature search and eligibility criteria:
The proposal for the present systematic review was registered with PROSPERO (registration number: CRD42021252484). The systematic review was conducted as per PRISMA guidelines. The following databases (MEDLINE, Scopus, Embase, Web of Science, LitCovid, back‐reference of the manuscripts) were searched for articles published in the English language from 1 January 2020 to 31 December 2021. The study design is described in Figure 1. Studies encompassing details of C. auris infection in COVID‐19 patients, such as case reports, case series (≥2 cases), retrospective cohort studies and observational studies, were included in the review. Studies that failed to fulfil the inclusion criteria were excluded (infections other than C. auris, review articles, articles other than English and studies without details of the study population were excluded from the review).
PRISMA flowchart describing the study selection process
Study selection and data extraction:
After the literature search, the citations were uploaded to Rayyan QCRI software for screening.
17
Following duplicates removal, the title and abstract of the articles were screened for inclusion by two independent reviewers (KSV and HPP), and disparities were sorted by discussion and consensus (sought the opinion of third reviewer KCP); the qualified articles were screened for full text by two independent reviewers (KSV and HPP). Only the studies that qualified after full‐text screening were proceeded to the data extraction process by two independent reviewers (KSV and HPP) and verified by a third reviewer (KCP). The following data were extracted in Microsoft excel: study design, country of the study, number of C. auris cases reported, patients details such as age, sex, underlying diseases, iatrogenic risk factors, antifungal treatment and outcome, that is, mortality, details of clinical specimens, antifungal susceptibility results and drug‐resistance of the C. auris isolates. Further, for estimation of pooled prevalence of C. auris infections in COVID‐19 patients, only the studies that provided information on total number of C. auris cases and total number of COVID‐19 cases were included in the analysis (studies without denominators such as case reports and case series were excluded from the prevalence estimation analysis).
Risk of bias assessment:
The risk of bias assessment was done by two independent reviewers (KSV and HPP) using a modification of the Joanna Briggs Institute (JBI) tool for the case series.
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The risk of bias was assessed as low, high and unclear under the following domains: clear inclusion criteria, a valid identification method, clear reporting of the demographic information, clinical parameters, outcomes, the presenting site(s)/clinic(s) demographic information.
Statistical methods:
Comparison of underlying diseases, iatrogenic risk factors, antifungal therapy and the outcome of the disease between the C. auris non‐candidemia/colonisation and C. auris candidemia cases was performed using Fisher's exact test using MedCalc Statistical Software (MedCalc Statistical Software version 14.8.1 (MedCalc Software by, Ostend, Belgium; http://www.medcalc.org; 2014)). ‘p’ values <.05 was considered significant. Meta‐analysis was done using OpenMeta analyst software (version 10.10).
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Pooled prevalence was calculated using the binary random effect model (Restricted Maximum likelihood method). Heterogeneity among the studies was evaluated using I
2 statistics.
RESULTS:
The initial database search identified 291 articles; of those, 11 articles were included in the final analysis (Figure 1), and the data were extracted for qualitative and quantitative analysis. Quality assessment of the articles was done using the JBI tool; of the 11 articles assessed, 8 fulfilled the criteria as mentioned above,
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in 3 of the articles, the method of identification was unclear, and the rest of the criteria were fulfilled.
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A total of 97 cases of C. auris were identified in COVID‐19 patients across different countries (Figure 2). Majority of the cases were reported from the United States of America (n = 48, 49.5%), followed by Mexico (n = 12, 12.4%) and India (n = 10, 10.3%; Figure 2). The male to female ratio was 2.6:1. The mean age was 65.41 years (with a range of 1–101 years), and 99% (n = 96) of the patients with C. auris infections were adults (Table 1). For estimation of pooled prevalence of C. auris infections in the COVID‐19 patients, the data from the 5 studies were analysed (Figure 3).
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The prevalance of C. auris infections (including candidemia and non‐candidemia cases) among the COVID‐19 patients was 14%, with high heterogeneity among the included publications (I
2 = 99.88; Figure 3).
Candida auris cases in COVID‐19 patients across countries. References are given in square brackets
Risk factors, treatment and outcome of Candida auris cases in COVID‐19 patients
The values in the table are expressed in numbers (n) and percentages (%). * ‘p’ values <.05 were considered significant.
Abbreviations: ARDS, Acute Respiratory Distress Syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; TC, total cohort.
The data were extracted from the 11 studies which fulfilled inclusion criteria (TC).
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Age range, male/female ratio, details of clinical specimens, underlying disease, iatrogenic risk factors, antifungal therapy and clinical outcome of CANC and CAC cases were extracted from 10 studies.
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Mean age and mean days from first isolation of C. auris from admission for CANC and CAC cases were extracted from 9 studies.
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Renal diseases were chronic kidney disease (CKD) and acute kidney injury (AKI). [TC: CKD (n = 9), and AKI (n = 2); CANC cases: CKD (n = 2); CAC cases: CKD (n = 4) and AKI (n = 2)].
Heart diseases were ischemic heart disease (IHD) and coronary heart disease (CAD). [TC: IHD (n = 5), and CAD (n = 3); CANC cases: IHD (n = 2), and CAD (n = 1); CAC cases: IHD (n = 2) and CAD (n = 2)].
Liver and Biliary diseases were chronic liver disease (CLD) and biliary lithiasis (BL). [TC: CLD (n = 3), and BL (n = 1); CANC cases: BL (n = 1); CAC cases: CLD (n = 3)].
Thromboembolic diseases were pulmonary embolism (PE) and deep venous thrombosis (DVT). [TC: PE (n = 3), and DVT (n = 2); CANC cases: PE (n = 1), and DVT (n = 2); CAC cases: PE (n = 2)].
Miscellaneous respiratory diseases were asthma, chronic obstructive pulmonary disease (COPD), respiratory failure (RF) and pneumothorax (PT). [TC: asthma (n = 4), COPD (n = 3), RF (n = 1), and PT (n = 1); CANC cases: asthma (n = 1), COPD (n = 2), RF (n = 1), and PT (n = 1); CAC cases: asthma (n = 3), and COPD (n = 1)].
Other risk factors included wound infections (WI), hyperlipidaemia (HLD), hypothyroidism (HT), stroke, dementia, stem cell transplant (SCT), systemic lupus erythematosus (SLE). [TC: WI (n = 6), HLD (n = 3), HT (n = 2), stroke (n = 1), dementia (n = 1), SCT (n = 1), and SLE (n = 1); CANC cases: WI (n = 2), HLD (n = 1), HT (n = 1), stroke (n = 1), dementia (n = 1), and SLE (n = 1); CAC cases: HLD (n = 2), HT (n = 1), and SCT (n = 1).
In patients with antifungal therapy, many patients received combination of antifungals for treatment.
Forest plot of pooled prevalence of Candida auris infections in COVID‐19 patients. "Frequency" denotes total number of C. auris cases and "Total" denotes total number of COVID‐19 infected patients. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Clinical specimens, isolation and identification of Candida auris
Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.
As per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.
29
Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).
Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.
As per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.
29
Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).
Underlying disease and risk factors Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.
Pooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients
Abbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.
The data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).
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The denominators used in the analysis for the total cohort are n = 82.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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Denominators used in the analysis for comparison of CANC and CAC group is n = 62.
Refer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.
Similar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2
= 88.65) (Table 2).
Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.
Pooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients
Abbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.
The data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).
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The denominators used in the analysis for the total cohort are n = 82.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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Denominators used in the analysis for comparison of CANC and CAC group is n = 62.
Refer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.
Similar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2
= 88.65) (Table 2).
Antifungal therapy and outcome of Candida auris infections A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2
= 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).
Forest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. "Frequency" denotes total number of patients survived with C. auris infections and "Total" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Underlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases
The values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.
Abbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.
Underlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2
= 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).
Forest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. "Frequency" denotes total number of patients survived with C. auris infections and "Total" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Underlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases
The values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.
Abbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.
Underlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
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Antifungal susceptibility of Candida auris isolates from COVID‐19 patients Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).
Antifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients
The data provided in the table were extracted from 5 studies.
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Abbreviations: MIC, minimum inhibitory concentration.
MIC50: MIC at which 50% of the tested isolates are inhibited.
MIC90: MIC at which 90% of the tested isolates are inhibited.
Furthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).
Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).
Antifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients
The data provided in the table were extracted from 5 studies.
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Abbreviations: MIC, minimum inhibitory concentration.
MIC50: MIC at which 50% of the tested isolates are inhibited.
MIC90: MIC at which 90% of the tested isolates are inhibited.
Furthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).
Clinical specimens, isolation and identification of Candida auris
:
Of those 97 C. auris cases identified, 62 (64%) patients had details of clinical specimens from which C. auris was isolated. Majority of C. auris isolation was from blood (n = 35, 56.5%), followed by urine (n = 12, 19.4%), and respiratory specimens (n = 10, 16.1%) (Table 1). The identification of C. auris in seven studies was performed using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS); four studies additionally used DNA sequencing of the internal transcribed spacer (ITS) region for confirmation. Whereas one study used API 20C AUX system (bioMerieux) and phenotypic methods for identification, and in three studies, method of identification is not mentioned.
As per the Center for Disease Control (CDC) (https://ndc.services.cdc.gov/case‐definitions/candida‐auris‐2019/), the case definition of C. auris infections/colonisation was specimens collected from invasive infections (eg blood, cerebrospinal fluid) were designated as confirmed cases, whereas isolation of C. auris from non‐invasive sites (wound swabs, urine and the respiratory tract) may reflect colonisation and not true infection.
29
Based on the definition, in the present study, C. auris cases (n = 62) were grouped into the following: (a) C. auris candidemia (CAC) cases (n = 35, 56.5%), (b) C. auris non‐candidemia/colonised (CANC) cases (n = 27, 43.5%; Table 1). The mean days from admission to the first isolation of C. auris in CANC and CAC cases were 27.7 and 22.8 days, respectively (Table 1).
Underlying disease and risk factors:
Table 1 shows the underlying diseases associated with C. auris infections in COVID‐19 patients: diabetes mellitus (n = 35, 42.7%), hypertension (n = 27, 32.9%, obesity (n = 12, 14.6%), COVID‐19 associated acute respiratory distress syndrome (ARDS; n = 9, 11%) and malignancy (n = 8, 13.4%). Approximately 12% of the patients had no underlying disease. Further, CANC and CAC cases showed no significant differences with any underlying diseases analysed (Table 1). The pooled estimates of the underlying disease and iatrogenic risk factors of C. auris infections in COVID‐19 patients are depicted in Table 2.
Pooled estimates of underlying diseases and iatrogenic risk factors of Candida auris infections in COVID‐19 patients
Abbreviations: ARDS, acute respiratory distress syndrome; CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised; CI, confidence interval.
The data were extracted from the 11 studies which fulfilled inclusion criteria (Total cohort).
13
,
14
,
15
,
16
,
22
,
23
,
24
,
25
,
26
,
27
,
28
The denominators used in the analysis for the total cohort are n = 82.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
13
,
14
,
16
,
22
,
23
,
24
,
25
,
26
,
27
,
28
Denominators used in the analysis for comparison of CANC and CAC group is n = 62.
Refer to Table 1 for the different risk factors placed under the subheadings, such as renal, heart, liver, thromboembolic, miscellaneous respiratory disease and other risk factors.
Similar to underlying diseases, iatrogenic risk factors such as central venous catheter (n = 63, 76.8%), intensive care unit (ICU) stay (n = 62, 75.6%), broad‐spectrum antibiotic usage (n = 61, 74.3%), mechanical ventilation (n = 57, 69.5%), steroid therapy (n = 51, 62.2%) and urinary catheter (n = 47, 57.3%) showed no significance differences between the CANC and CAC groups. (Table 1). The pooled estimates of co‐infections in C. auris among the total cohort of COVID‐19 patients was 58% (with high heterogeneity I2
= 88.65) (Table 2).
Antifungal therapy and outcome of Candida auris infections:
A total of 44 patients received antifungal therapy (Table 1); echinocandins (n = 33, 75%) were commonly used, followed by amphotericin B (n = 13, 29.6%). No significant difference was observed between different antifungal medications and the survival (data not shown). The mortality rate of the total cohort (n = 81) was at 44.44% (Table 1), in comparison with CANC cases was at 22.2% (n = 6) and CAC case was at 64.7% (n = 22) (p = <.002). The pooled survival estimates of 61 patients in CANC and CAC groups were 37% and 16%, with low heterogeneity (I2
= 40.36 and 0%), respectively (Figure 4). The mortality rate in patients with underlying disease and iatrogenic risk factors such as diabetes mellitus, central venous catheter, ICU stay, broad‐spectrum antibiotic usage, mechanical ventilation, steroid therapy and urinary catheter were significantly associated with a higher mortality rate in the CAC group compared with the CANC group (Table 3).
Forest plot of pooled survival estimates of (A) Candida auris non‐candidemia/colonised (CANC) and (B) Candida auris candidemia (CAC) cases in COVID‐19 patients. "Frequency" denotes total number of patients survived with C. auris infections and "Total" denotes total number of C. auris cases reported in each study. References are given in square brackets. Abbreviations: C.I, Confidence Interval
Underlying disease and iatrogenic risk factors associated with mortality in Candida auris non‐candidemia/colonised (CANC) and Candida auris candidemia (CAC) cases
The values in the table are expressed in numbers (n). ‘n’ denotes the total number of patients. * ‘p’ values <.05 were considered significant.
Abbreviations: CAC, Candida auris candidemia; CANC, Candida auris non‐candidemia/colonised.
Underlying disease and mortality association was statistically analysed for diabetes mellitus and hypertension alone. The number of cases for in other underlying diseases were less (refer Table 1), hence no statistical analysis was performed.
The data for underlying diseases and iatrogenic risk factors of CANC and CAC cases were extracted from 10 studies.
13
,
14
,
16
,
22
,
23
,
24
,
25
,
26
,
27
,
28
Antifungal susceptibility of Candida auris isolates from COVID‐19 patients:
Table 4 shows the antifungal susceptibility testing of C. auris isolates (n = 41). Of those tested isolates, resistance was noted in 33 isolates (80.5%) to fluconazole (MIC ≥ 32 mg/L), followed by 19 (46.3%) to amphotericin B (MIC ≥ 2 mg/L), 5 (12.8%) to caspofungin (MIC ≥ 2 mg/L), 2 (5.1%) to anidulafungin (MIC ≥ 4 mg/L), 1 (3.7%) to micafungin (MIC ≥ 4 mg/L), and 7 (43.8%) to 5‐flucytosine (MIC ≥ 32 mg/L). Voriconazole non‐susceptibility (MIC ≥ 2 mg/L) was observed in 12 (29.3%) C. auris isolates (Table 4).
Antifungals minimum inhibitory concertation (MICs) for Candida auris isolates from COVID‐19 patients
The data provided in the table were extracted from 5 studies.
13
,
14
,
22
,
23
,
24
Abbreviations: MIC, minimum inhibitory concentration.
MIC50: MIC at which 50% of the tested isolates are inhibited.
MIC90: MIC at which 90% of the tested isolates are inhibited.
Furthermore, 8 (19.5%) isolates were resistant to fluconazole and voriconazole. Multidrug resistance (resistance to two different classes of antifungal drugs) was noted in 22 (53.6%) C. auris isolates; of those, 18 (81.8%) and 4 (18.2%) isolates were resistant to two and three classes of antifungal drugs, respectively. Amphotericin B plus azole resistance was noted in 10 (45.5%), followed by echinocandins and azole (n = 4, 18.2%), azole and 5‐flucytosine (n = 3, 13.6%), amphotericin B and echinocandins (n = 1, 4.6%), amphotericin B, azole and 5‐flucytosine (n = 3, 13.6%) and echinocandins, azole and 5‐flucytosine (n = 1, 4.6%).
DISCUSSION:
The present systematic review analysed the published cases of C. auris in COVID‐19 patients, providing insight on prevalence, underlying disease/risk factors, treatment and disease outcome. The review was conducted per PRISMA guidelines, including only the articles that met the study criteria. This review has provided an update on the epidemiology of C. auris infections in COVID‐19 patients.
In the present study, majority of the C. auris‐infected patients were adults (99%), with one case in the paediatric population, similar to previous reports.
4
,
6
,
10
In this study, the pooled prevalence of C. auris infections in COVID‐19 patients was 14%. The true prevalence of C. auris infections in the global population is largely unknown. As per multiple studies, the prevalence of C. auris in candidemia patients (non‐COVID‐19 cases) ranges between 5‐30%.
30
Further, many C. auris colonisation patients have also been identified in countries like the USA, Europe, India, South Africa and henceforth.
5
,
31
,
32
,
33
As per CDC recommendation on C. auris definition for colonisation (non‐invasive sites) and true infections (invasive sites),
29
in the present study, the cases were classified as C. auris non‐candidemia/colonised (CANC) and C. auris candidemia (CAC) cases, respectively. A total of 35 (56.5%) CAC cases and 27 (43.5%) CANC cases were identified. The underlying disease/iatrogenic risk factors in identified both groups were: hypertension (43.5%), diabetes mellitus (37%), obesity (19.4%), COVID‐19‐associated ARDS (14.5%) and renal diseases (13%); whereas the iatrogenic risk factors included broad‐spectrum antibiotic usage (98.4%), ICU stay (97%), steroid therapy (82.3%), the central venous catheter (75.8%), mechanical ventilation (74.2%) and steroid therapy (82.2%). The respective analysis of C. auris and COVID‐19 patients showed that both groups shared the underlying disease mentioned above.
3
,
4
,
9
,
10
,
11
,
12
Multiple studies have reported central venous catheter, previous antifungal exposure, mechanical ventilation, and prolonged ICU stay as significant risk factors for C. auris colonisation/infection.
7
,
34
,
35
Thus, the ongoing COVID‐19 pandemic would become a perfect battlefield for outbreaks of C. auris because of the similar underlying diseases and the risk factors, which increases the chance of C. auris infections in COVID‐19 patients. Further, a recent study from India confirmed these findings, that the treatment with interleukin‐6 antagonists such as tocilizumab, prolonged ICU stay, mechanical ventilation and raised ferritin levels were identified as significant risk factors of candidemia (majority of reported cases in the study were due to C. auris) in COVID‐19 patients.
36
Furthermore, during the COVID‐19 pandemic, the existing ICU facilities have seen overflowing patients, with a high burden of patients, there was a challenge in implementing the infection control practices. Of the included studies in this review, a few studies documented the source for the C. auris outbreak, and the infection prevention and control (IPCs) measures to contain the C. auris infection in COVID‐19 patients.
13
,
14
,
16
Allaw et al. reported C. auris outbreak in Lebanon in a tertiary care centre for 13 weeks. Following the first case of C. auris infection, IPC practices such as hand hygiene using antiseptics, disinfection of floors and surfaces of patient's room, screening for C. auris in environmental samples and skin colonisation in patients, 4% chlorhexidine bath for C. auris skin decolonisation was initiated. C. auris was not isolated from environmental samples; however, of the 26 patients screened for skin colonisation, one patient grew C. auris. Authors attributed the delay in reporting and identifying the first case of C. auris to the outbreak, which delayed the implementation of IPC measures.
14
Villanueva‐Lozano et al. reported C. auris outbreak in COVID‐19 patients in Mexico, and the authors isolated three environmental isolates from their bedrooms. The phylogenetic analysis of the clinical and environmental isolates clustered together, suggesting close similarities among the isolates.
13
These findings show that the environmental contamination of hospitals remains a possibility either by cross‐contamination of the equipment or by the healthcare providers. However, Chowdhary et al.
23
proposed that C. auris infection in COVID‐19 patients may not be transmitted by healthcare personnel because of personal protective equipment (PPE). Further, the authors cautioned that improper use of PPE may lead to contamination and disease transmission.
23
A study from Brazil documented the source of C. auris outbreak in the hospital settings; the environmental screening showed C. auris contamination from auxiliary digital thermometers (17%), bed rails (15%), and intravenous infusion pumps (11%) and tray tables (11%). The study reported that C. auris colonisation of digital thermometer was the significant risk factor associated with C. auris colonisation in patients, which correlated with the high isolation rate of C. auris from axillae of the patients.
16
Similarly, Eyre et al.
7
reported that skin surface axillary temperature reusable probes were significantly associated with C. auris colonisation/infection (86% in the patient group versus 34% in controls). Proper surveillance for C. auris colonisation and implementation of infection control practices may stop the spread of C. auris infections/colonisation in COVID‐19 ICU settings.
7
,
37
Candida auris multidrug‐resistant isolates have been reported from Asia, the USA, Europe and Africa.
7
,
38
,
39
,
40
,
41
,
42
In the present study, C. auris isolates were resistant to fluconazole (81%), followed by voriconazole (29.3%), amphotericin B (46.3%), caspofungin (12.8%), anidulafungin (5.1%), micafungin (3.7%) and 5‐flucytosine (43.8%). Similarly, antifungal susceptibility testing of C. auris isolates from multiple countries reported a large number of isolates are resistant to fluconazole (35%–100%), followed by amphotericin B resistance (8%–61%), echinocandins resistance (0.5%–3%) and voriconazole (14%–90%).
7
,
38
,
39
,
40
,
41
In the present study, multidrug resistance was noted in 53.6% of C. auris isolates, similar to the previously reported studies (6%–61%).
40
,
41
,
42
In the present study, 44 (71%) patients from CANC (n = 11, 25%) and CAC (n = 33, 75%) group received antifungal therapy. Globally, C. auris isolates exhibit a higher susceptible pattern to echinocandins,
7
,
38
,
39
,
40
,
41
making them the drug of choice for C. auris infections. Similarly, echinocandins (75%) were the most common drug used in this study. The survival rate for CANC and CAC groups was 77.8% and 35.3%, respectively (p < .002). Multiple systematic reviews showed that mortality in C. auris cases was at 39% to 47.5%.
4
,
43
Similar to the present study's findings, Sayeed et al.
10
reported a survival rate of 54% in C. auris candidemia cases and 67% in colonised cases. Further, this study showed that iatrogenic risk factors such as prolonged ICU stay, mechanical ventilation, steroid therapy, broad‐spectrum antibiotics and central venous catheters were significantly associated with high mortality in CAC patients compared with the CANC group. These findings caution that healthcare personnel should be vigilant while treating severe COVID‐19 patients, as they are more vulnerable to C. auris infection. Despite the treatment with antifungals, a high mortality rate is seen in C. auris infections, making them a global threat.
CONCLUSION:
The study highlights the role of hospital‐acquired C. auris infections in COVID‐19 patients. Despite the multiple risk factors possibly favouring the C. auris infections in COVID‐19 patients, the prevalence of the disease remains unchanged compared with the pre‐pandemic. However, one must be cautioned that COVID‐19 patients may be more vulnerable to C. auris infections because of the overlapping risk factors. Increased burden of colonised patients with C. auris in ICU settings may lead to person‐to‐person transmission. Hence, proper infection control practices and strict hospital surveillance for screening and isolating C. auris colonised patients in the COVID‐19 ICUs may prevent the potential outbreaks in hospital settings.
CONFLICT OF INTEREST:
The authors declare no competing interest.
AUTHOR CONTRIBUTIONS:
HPP conceptualised the idea of this study. HPP, KSV and KCP designed the study. HPP and KSV did primary data extraction for the study and wrote the manuscript. KCP and HPP performed the analysis. The final draft of the manuscript was corrected and proofread by all the authors.
| Background:
Candida auris is an emerging multidrug-resistant pathogen in intensive care settings (ICU). During the coronavirus disease 19 (COVID-19) pandemic, ICU admissions were overwhelmed, possibly contributing to the C. auris outbreak in COVID-19 patients.
Methods:
MEDLINE, Scopus, Embase, Web of Science and LitCovid databases were systematically searched with appropriate keywords from 1 January 2020 to 31 December 2021.
Results:
A total of 97 cases of C. auris were identified in COVID-19 patients. The pooled prevalence of C. auris infections (encompassing candidemia and non-candidemia cases) in COVID-19 patients was 14%. The major underlying diseases were diabetes mellitus (42.7%), hypertension (32.9%) and obesity (14.6%), followed by the iatrogenic risk factors such as a central venous catheter (76.8%%), intensive care unit (ICU) stay (75.6%) and broad-spectrum antibiotic usage (74.3%). There were no significant differences in underlying disease and iatrogenic risk factors among C. auris non-candidemia/colonisation and C. auris candidemia cases. The mortality rate of the total cohort is 44.4%, whereas, in C. auris candidemia patients, the mortality was 64.7%.
Conclusions:
This study shows that the prevalence of C. auris infections remains unchanged in the COVID-19 pandemic. Hospital-acquired risk factors may contribute to the clinical illness. Proper infection control practices and hospital surveillance may stop future hospital outbreaks during the pandemic. | INTRODUCTION:
During the coronavirus disease 19 (COVID‐19) pandemic, the healthcare facility was overwhelmed with patients admitted to intensive care units (ICUs), and those patients were highly susceptible to bacterial and fungal co‐infections.
1
,
2
Candida auris is an emerging pathogen in ICU settings with high mortality and infection due to this pathogen has been reported across 44 countries.
3
,
4
Candida auris is a unique species, as this agent is multidrug‐resistant, and they can survive on inanimate objects in the hospital environment for more extended periods, leading to potential transmission among patients (hospital outbreaks), and difficulty in accurate laboratory identification makes them the pathogen of public interest globally.
5
,
6
,
7
,
8
Patients with underlying diseases such as diabetes mellitus, kidney disease, lung disease, trauma, ear diseases and hypertension, followed by iatrogenic risk factors such as prolonged ICU stay, central venous catheter, mechanical ventilation and prior antibiotic usage, were significantly associated with C. auris infections prior to the COVID‐19 pandemic.
3
,
4
,
9
,
10
Like C. auris infections, diabetes mellitus, hypertension, malignancies, chronic kidney diseases, chronic liver disease and cardiovascular disease are associated with high mortality in severe COVID‐19 patients.
11
,
12
Underlying disease and the invasive medical procedures during the hospital stay in COVID‐19 patients make them highly susceptible to C. auris infections/colonisation. The outbreak of C. auris infections has been reported during the COVID‐19 pandemic across different countries.
13
,
14
,
15
,
16
Due to the overlapping features (underlying disease/risk factors) between the two disease groups, the disease epidemiology of C. auris infections in COVID‐19 patients will be interesting to study. In the present study, we systematically reviewed the C. auris infections/colonisation in COVID‐19 patients to determine the prevalence, underlying disease/risk factors, treatment and outcome.
CONCLUSION:
The study highlights the role of hospital‐acquired C. auris infections in COVID‐19 patients. Despite the multiple risk factors possibly favouring the C. auris infections in COVID‐19 patients, the prevalence of the disease remains unchanged compared with the pre‐pandemic. However, one must be cautioned that COVID‐19 patients may be more vulnerable to C. auris infections because of the overlapping risk factors. Increased burden of colonised patients with C. auris in ICU settings may lead to person‐to‐person transmission. Hence, proper infection control practices and strict hospital surveillance for screening and isolating C. auris colonised patients in the COVID‐19 ICUs may prevent the potential outbreaks in hospital settings. | Background:
Candida auris is an emerging multidrug-resistant pathogen in intensive care settings (ICU). During the coronavirus disease 19 (COVID-19) pandemic, ICU admissions were overwhelmed, possibly contributing to the C. auris outbreak in COVID-19 patients.
Methods:
MEDLINE, Scopus, Embase, Web of Science and LitCovid databases were systematically searched with appropriate keywords from 1 January 2020 to 31 December 2021.
Results:
A total of 97 cases of C. auris were identified in COVID-19 patients. The pooled prevalence of C. auris infections (encompassing candidemia and non-candidemia cases) in COVID-19 patients was 14%. The major underlying diseases were diabetes mellitus (42.7%), hypertension (32.9%) and obesity (14.6%), followed by the iatrogenic risk factors such as a central venous catheter (76.8%%), intensive care unit (ICU) stay (75.6%) and broad-spectrum antibiotic usage (74.3%). There were no significant differences in underlying disease and iatrogenic risk factors among C. auris non-candidemia/colonisation and C. auris candidemia cases. The mortality rate of the total cohort is 44.4%, whereas, in C. auris candidemia patients, the mortality was 64.7%.
Conclusions:
This study shows that the prevalence of C. auris infections remains unchanged in the COVID-19 pandemic. Hospital-acquired risk factors may contribute to the clinical illness. Proper infection control practices and hospital surveillance may stop future hospital outbreaks during the pandemic. | 10,754 | 288 | [
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Candida auris
| candidemia | COVID‐19 | mortality | prevalence | systematic review [SUMMARY] | null | [CONTENT]
Candida auris
| candidemia | COVID‐19 | mortality | prevalence | systematic review [SUMMARY] | [CONTENT]
Candida auris
| candidemia | COVID‐19 | mortality | prevalence | systematic review [SUMMARY] | [CONTENT]
Candida auris
| candidemia | COVID‐19 | mortality | prevalence | systematic review [SUMMARY] | [CONTENT]
Candida auris
| candidemia | COVID‐19 | mortality | prevalence | systematic review [SUMMARY] | [CONTENT] Antifungal Agents | COVID-19 | Candida | Candida auris | Candidemia | Drug Resistance, Multiple | Humans | Iatrogenic Disease | Microbial Sensitivity Tests | Pandemics | Prevalence | Risk Factors | Treatment Outcome [SUMMARY] | null | [CONTENT] Antifungal Agents | COVID-19 | Candida | Candida auris | Candidemia | Drug Resistance, Multiple | Humans | Iatrogenic Disease | Microbial Sensitivity Tests | Pandemics | Prevalence | Risk Factors | Treatment Outcome [SUMMARY] | [CONTENT] Antifungal Agents | COVID-19 | Candida | Candida auris | Candidemia | Drug Resistance, Multiple | Humans | Iatrogenic Disease | Microbial Sensitivity Tests | Pandemics | Prevalence | Risk Factors | Treatment Outcome [SUMMARY] | [CONTENT] Antifungal Agents | COVID-19 | Candida | Candida auris | Candidemia | Drug Resistance, Multiple | Humans | Iatrogenic Disease | Microbial Sensitivity Tests | Pandemics | Prevalence | Risk Factors | Treatment Outcome [SUMMARY] | [CONTENT] Antifungal Agents | COVID-19 | Candida | Candida auris | Candidemia | Drug Resistance, Multiple | Humans | Iatrogenic Disease | Microbial Sensitivity Tests | Pandemics | Prevalence | Risk Factors | Treatment Outcome [SUMMARY] | [CONTENT] candida auris isolates | susceptibility candida auris | mortality candida auris | auris infection covid | auris infections covid [SUMMARY] | null | [CONTENT] candida auris isolates | susceptibility candida auris | mortality candida auris | auris infection covid | auris infections covid [SUMMARY] | [CONTENT] candida auris isolates | susceptibility candida auris | mortality candida auris | auris infection covid | auris infections covid [SUMMARY] | [CONTENT] candida auris isolates | susceptibility candida auris | mortality candida auris | auris infection covid | auris infections covid [SUMMARY] | [CONTENT] candida auris isolates | susceptibility candida auris | mortality candida auris | auris infection covid | auris infections covid [SUMMARY] | [CONTENT] auris | patients | cases | 19 | covid 19 | covid | risk | studies | canc | cac [SUMMARY] | null | [CONTENT] auris | patients | cases | 19 | covid 19 | covid | risk | studies | canc | cac [SUMMARY] | [CONTENT] auris | patients | cases | 19 | covid 19 | covid | risk | studies | canc | cac [SUMMARY] | [CONTENT] auris | patients | cases | 19 | covid 19 | covid | risk | studies | canc | cac [SUMMARY] | [CONTENT] auris | patients | cases | 19 | covid 19 | covid | risk | studies | canc | cac [SUMMARY] | [CONTENT] disease | patients | auris | 19 | infections | covid 19 | covid | pathogen | auris infections | 19 pandemic [SUMMARY] | null | [CONTENT] auris | canc | cac | table | cases | mic | candida | candida auris | patients | cac cases [SUMMARY] | [CONTENT] hospital | patients | colonised patients | person | auris | covid | covid 19 | 19 | settings | auris infections [SUMMARY] | [CONTENT] auris | patients | cases | 19 | covid | covid 19 | studies | table | study | risk [SUMMARY] | [CONTENT] auris | patients | cases | 19 | covid | covid 19 | studies | table | study | risk [SUMMARY] | [CONTENT] ICU ||| 19 | COVID-19 | ICU | C. | COVID-19 [SUMMARY] | null | [CONTENT] 97 | C. | COVID-19 ||| C. auris infections | COVID-19 | 14% ||| 42.7% | 32.9% | 14.6% | 76.8%% | ICU | 75.6% | 74.3% ||| C. | C. ||| 44.4% | C. | 64.7% [SUMMARY] | [CONTENT] C. | COVID-19 ||| ||| [SUMMARY] | [CONTENT] ICU ||| 19 | COVID-19 | ICU | C. | COVID-19 ||| Embase | LitCovid | 1 January 2020 to 31 | December 2021 ||| ||| 97 | C. | COVID-19 ||| C. auris infections | COVID-19 | 14% ||| 42.7% | 32.9% | 14.6% | 76.8%% | ICU | 75.6% | 74.3% ||| C. | C. ||| 44.4% | C. | 64.7% ||| C. | COVID-19 ||| ||| [SUMMARY] | [CONTENT] ICU ||| 19 | COVID-19 | ICU | C. | COVID-19 ||| Embase | LitCovid | 1 January 2020 to 31 | December 2021 ||| ||| 97 | C. | COVID-19 ||| C. auris infections | COVID-19 | 14% ||| 42.7% | 32.9% | 14.6% | 76.8%% | ICU | 75.6% | 74.3% ||| C. | C. ||| 44.4% | C. | 64.7% ||| C. | COVID-19 ||| ||| [SUMMARY] |
|||||
COMPARATIVE ANALYSIS OF TISSULAR RESPONSE AFTER ABDOMINAL WALL REPAIR USING POLYPROPYLENE MESH AND BOVINE PERICARDIUM MESH. | 35019111 | The use of polypropylene meshes for surgical repair of the abdominal wall contributes to a reduction of the of recurrence rates of hernias or defects. However, its intra-abdominal use comes along with the formation of adhesions and several complications. The study and the search for alternative materials, including bovine pericardium, have been regarded as an option for the correction and treatment of resulting hernias with better adaptations and effectiveness. | BACKGROUND | Bovine pericardium mesh and polypropylene mesh were placed, both on the same animal. The first group had the mesh removed for analysis on day 20, and the second group on day 40. The variables congestion, granulation, giant cells, necrosis, acute inflammation, chronic inflammation and collagen were analyzed. | METHOD | All variables were found in greater numbers as a response to the polypropylene mesh, except for the collagen, which, on day 40, was greater in response to the bovine pericardium mesh. | RESULTS | The data in this study suggest that there is less inflammatory reaction in response to bovine pericardium mesh when compared to polypropylene mesh. | CONCLUSION | [
"Abdominal Wall",
"Animals",
"Cattle",
"Pericardium",
"Polypropylenes",
"Surgical Mesh",
"Tissue Adhesions"
] | 8735132 | INTRODUCTION | Surgical repair or reinforcement of the tissues that compose the wall of the abdominal cavity is one of the most frequent needs in human surgery
12
, due to both the scarcity of tissues and the high tension in the suture line, which results in the occurrence of high rates of incisional hernia
8
.
The use of meshes for surgical repair of hernia defects of the abdominal wall contributes to reducing the recurrence
11
,
17
. However, its intra-abdominal use comes along with the formation of adhesions and several complications
2
.
The first reported use of tissue for hernioplasty is dated 1958 by Usher et al
16
, who used a polypropylene mesh. This remains the most used material
18
because it is safe and offers several advantages in its use, including its availability, easy handling, low cost and good tolerance
9
.
Polypropylene is a synthetic material that produces little tissue reaction and good tensile strength, which strength lasts for several years after its use in living organisms. However, even with this possible choice, the use of meshes still has several side effects
4
, such as increased postoperative pain, abdominal adhesions and areas of fibrosis and increased risk of infection due to the placement of the prosthesis
18
.
Since the 1960s, biological membranes as implant material for the repair of organs and tissues have been used in Brazil. The advantages of using this type of material include the ease in obtaining, its low cost, simple preparation, viable sterilization, easy storage, little or no tissue reaction, and a long period of viability as an implant. Bovine pericardium is one of the most used biological membranes, being composed almost exclusively of collagen. Due to this characteristic, bovine pericardium easily adapts to the different situations to which it is submitted in the surgical practice
5
.
Although reports in the literature indicate that bovine pericardium has a greater tensile strength in relation to certain synthetic meshes, calcifications and immune rejection responses should be considered, taking into account the indications in the use of this material
13
. The bovine pericardium is the bovine heart surrounding membrane, being a biological tissue widely studied in the literature to produce products of medical use, many of which already in clinical use. The structure of this biological tissue basically comprises collagen and it is used in medicine after being physically and/or chemically treated for improving its mechanical and immunogenic properties and controlling its degradation or calcification. It is used for example in the production of cardiac and vascular prostheses, repair of ligaments, controlled drug delivery systems, hemostatic agents, grafts and others. Valve prostheses made with bovine pericardium have a good hydrodynamic performance and low thrombogenicity
3
.
Since the beginning of the last century, the use of prostheses has become constant in abdominal hernia surgery, especially for inguinal hernia. The study and search for alternative materials, such as bovine pericardium, have proved to be an option in the correction and treatment of hernias resulting in better adaptations and effectiveness
7
.
The objective of this study is to evaluate the inflammatory process and formation of collagen fibers in response to bovine pericardium compared to the inflammatory process and formation of collagen fibers in response to the synthetic polypropylene mesh. | METHODS | This study has a qualitative and quantitative experimental character, and therefore, the abdominal wall defect induction and subsequent correction with polypropylene mesh and bovine pericardium mesh was performed. It was approved under CEP number 1513/2016. The procedures with the animals complied with that recommended by the Ethics Committee on the Use of Animals (CEUA) of Evangelical Faculty of Paraná (FEPAR), Curitiba, PR, Brazil.
A total of 19 male guinea pigs, weighing between 300 and 400 g, were collected from the Paraná Institute of Technology (TECPAR) vivarium. The animals were kept during the experiment at IPEM’s vivarium, in 47x34x18 cm plastic boxes, with two animals each, lined with shavings, in 12-h light/dark cycle (light from 7am to 7pm) at a temperature of 22±2º C. The animals were treated daily with filtered water and appropriate ration fed freely.
The model used was abdominal wall defect correction using mesh, divided into two groups of nine guinea pigs in group 1 and 10 guinea pigs in group 2. In group 1 surgical correction using polypropylene synthetic mesh and bovine pericardium biological tissue with tissue removal and animal death after 20 days was done. In group 2 the same procedure was performed, but the animal death was after 40 days.
A mixture of xylazine hydrochloride 5 mg and ketamine hydrochloride 40 mg per body weight in kilograms intraperitoneally was used to anesthetize the animals, and they were positioned for surgery in the dorsal decubitus position. The surgical intervention began with trichotomy and disinfection of the abdominal region, followed by an incision to create a 1 cm defect in the abdominal wall through the epithelium to the peritoneal tissue, for subsequent correction with polypropylene mesh on the right side and bovine pericardium mesh on the left side (Figure 1).
The meshes were 0.5 x 0.5 cm and were accommodated in the abdominal cavity above the peritoneum.
The abdominal cavity was closed with separate stitches in the cutaneous plane, and the abdominal muscle layer was closed with continuous suture.
FIGURE 1Meshes inserted into the abdominal wall: A) Left side with polypropylene mesh; B) right side with bovine pericardium mesh; C) cut meshes; D) after abdominal closure.
Tramadol was used for postoperatively analgesia at a dose of 5 mg per kg every 12 h for four days and we also used amoxicillin at 20 mg per every 48 h for four days. The surgical wound was cleaned with iodine. For animal welfare concern, knowing that the environment influences the behavioral and physiological state of animals, we implemented environmental enrichment measures including: appropriate environment, feed, temperature, space and PVC pipe in the cages.
Death of the animals and tissue resection for histopathological analysis After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).
FIGURE 2Mesh analysis by transparency
The surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.
The material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis.
The inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition
6
.
On the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.
After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).
FIGURE 2Mesh analysis by transparency
The surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.
The material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis.
The inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition
6
.
On the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.
Histological evaluation Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al
18
, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.
The Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.
Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al
18
, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.
The Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.
Statistical analysis The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.
The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software. | RESULTS | The analysis was performed based on the data of nine animals of the group submitted to euthanasia after 20 days and 10 after 40 days. Both types of mesh (polypropylene and bovine pericardium) were used in each animal. For the comparative analysis between the types of screen (in the same animal) were included the cases that had evaluation of the variable in the two types of mesh. The “necrosis” variable was not analyzed, since all animals in the experiment had no characteristic of this characteristic.
In relation to congestion, there was presence of alteration in 66.7% of polypropylene tissues in group 1 and 60% in group 2 (p>0.05). In the group of tissues with bovine pericardium, there was congestion in 33.3% in group 1 and 10% in group 2 (p>0.05). Although without significant difference, the granulation variable was present in 100% of polypropylene mesh materials in group 1 and in 55.6% with bovine pericardium mesh. In group 2 this variable was present in 80% with polypropylene and only 20% in the pieces with bovine pericardium (Figure 3).
FIGURE 3Photomicrographs evidencing alterations in the abdominal wall (400x H&E): A) connective tissue with congested vessels (arrows); B) granulation tissue (arrow).
The presence of giant cells was evidenced in 66.7% of the analyzed pieces with polypropylene of group 1 and in 33.3% of the pieces with bovine pericardium. In group 2 this variable was present in 60% of the pieces with polypropylene and in no part with bovine pericardium (both with p>0.05, Figure 4).
FIGURE 4Photomicrograph demonstrating giant cell (400x H&E - arrow)
Regarding acute inflammation, no alteration was found in any part of the first group and did not present a statistically significant difference.
In relation to chronic inflammation, there were alterations in 66.7% of the pieces with polypropylene of group 1 and in 22.2% of the pieces with bovine pericardium of group 1, with p>0.05. However, in group 2, a significant difference (p=0.004) was found when compared to the presence of chronic inflammation in parts with polypropylene (90%) and bovine pericardium (0%, Figure 5).
FIGURE 5Photomicrographs evidencing changes of chronic inflammatory process (A - arrows) and minimal scar area (B - arrow) - 100x H&E
The collagen fibers were found in 66.7% of the pieces with polypropylene and in 33.3% of the pieces with bovine pericardium in group 1 (p>0.05). In group 2, the variable was found in 62.5% of the pieces with polypropylene and 87.5% of the pieces with bovine pericardium (p>0.05, Figure 6).
FIGURE 6Photomicrographs showing changes to Mallory’s trichrome: A) photomicrograph demonstrating on the arrow cuts of skin with presence of polypropylene and slight deposition of collagen around (Mallory 50x); B) photomicrograph demonstrating on the arrow area cicatricial with remnants of the polypropylene mesh and moderate degree of fibrosis around (Mallory 400x)
| CONCLUSION | The data of this study suggests that there is less inflammatory reaction and increased formation of collagen fibers in response to bovine pericardium mesh when compared to the polypropylene mesh. | [
"Death of the animals and tissue resection for histopathological analysis",
"Histological evaluation",
"Statistical analysis"
] | [
"After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).\n\nFIGURE 2Mesh analysis by transparency\n\nThe surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.\nThe material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis. \nThe inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition\n6\n.\nOn the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.",
"Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al\n18\n, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.\nThe Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.",
"The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software."
] | [
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"Death of the animals and tissue resection for histopathological analysis",
"Histological evaluation",
"Statistical analysis",
"RESULTS",
"DISCUSSION",
"CONCLUSION"
] | [
"Surgical repair or reinforcement of the tissues that compose the wall of the abdominal cavity is one of the most frequent needs in human surgery\n12\n, due to both the scarcity of tissues and the high tension in the suture line, which results in the occurrence of high rates of incisional hernia\n8\n.\nThe use of meshes for surgical repair of hernia defects of the abdominal wall contributes to reducing the recurrence\n11\n\n,\n\n17\n. However, its intra-abdominal use comes along with the formation of adhesions and several complications\n2\n.\nThe first reported use of tissue for hernioplasty is dated 1958 by Usher et al\n16\n, who used a polypropylene mesh. This remains the most used material\n18\n because it is safe and offers several advantages in its use, including its availability, easy handling, low cost and good tolerance\n9\n.\nPolypropylene is a synthetic material that produces little tissue reaction and good tensile strength, which strength lasts for several years after its use in living organisms. However, even with this possible choice, the use of meshes still has several side effects\n4\n, such as increased postoperative pain, abdominal adhesions and areas of fibrosis and increased risk of infection due to the placement of the prosthesis\n18\n.\nSince the 1960s, biological membranes as implant material for the repair of organs and tissues have been used in Brazil. The advantages of using this type of material include the ease in obtaining, its low cost, simple preparation, viable sterilization, easy storage, little or no tissue reaction, and a long period of viability as an implant. Bovine pericardium is one of the most used biological membranes, being composed almost exclusively of collagen. Due to this characteristic, bovine pericardium easily adapts to the different situations to which it is submitted in the surgical practice\n5\n. \nAlthough reports in the literature indicate that bovine pericardium has a greater tensile strength in relation to certain synthetic meshes, calcifications and immune rejection responses should be considered, taking into account the indications in the use of this material\n13\n. The bovine pericardium is the bovine heart surrounding membrane, being a biological tissue widely studied in the literature to produce products of medical use, many of which already in clinical use. The structure of this biological tissue basically comprises collagen and it is used in medicine after being physically and/or chemically treated for improving its mechanical and immunogenic properties and controlling its degradation or calcification. It is used for example in the production of cardiac and vascular prostheses, repair of ligaments, controlled drug delivery systems, hemostatic agents, grafts and others. Valve prostheses made with bovine pericardium have a good hydrodynamic performance and low thrombogenicity\n3\n.\nSince the beginning of the last century, the use of prostheses has become constant in abdominal hernia surgery, especially for inguinal hernia. The study and search for alternative materials, such as bovine pericardium, have proved to be an option in the correction and treatment of hernias resulting in better adaptations and effectiveness\n7\n.\nThe objective of this study is to evaluate the inflammatory process and formation of collagen fibers in response to bovine pericardium compared to the inflammatory process and formation of collagen fibers in response to the synthetic polypropylene mesh.",
"This study has a qualitative and quantitative experimental character, and therefore, the abdominal wall defect induction and subsequent correction with polypropylene mesh and bovine pericardium mesh was performed. It was approved under CEP number 1513/2016. The procedures with the animals complied with that recommended by the Ethics Committee on the Use of Animals (CEUA) of Evangelical Faculty of Paraná (FEPAR), Curitiba, PR, Brazil.\nA total of 19 male guinea pigs, weighing between 300 and 400 g, were collected from the Paraná Institute of Technology (TECPAR) vivarium. The animals were kept during the experiment at IPEM’s vivarium, in 47x34x18 cm plastic boxes, with two animals each, lined with shavings, in 12-h light/dark cycle (light from 7am to 7pm) at a temperature of 22±2º C. The animals were treated daily with filtered water and appropriate ration fed freely.\nThe model used was abdominal wall defect correction using mesh, divided into two groups of nine guinea pigs in group 1 and 10 guinea pigs in group 2. In group 1 surgical correction using polypropylene synthetic mesh and bovine pericardium biological tissue with tissue removal and animal death after 20 days was done. In group 2 the same procedure was performed, but the animal death was after 40 days.\nA mixture of xylazine hydrochloride 5 mg and ketamine hydrochloride 40 mg per body weight in kilograms intraperitoneally was used to anesthetize the animals, and they were positioned for surgery in the dorsal decubitus position. The surgical intervention began with trichotomy and disinfection of the abdominal region, followed by an incision to create a 1 cm defect in the abdominal wall through the epithelium to the peritoneal tissue, for subsequent correction with polypropylene mesh on the right side and bovine pericardium mesh on the left side (Figure 1).\nThe meshes were 0.5 x 0.5 cm and were accommodated in the abdominal cavity above the peritoneum.\nThe abdominal cavity was closed with separate stitches in the cutaneous plane, and the abdominal muscle layer was closed with continuous suture.\n\nFIGURE 1Meshes inserted into the abdominal wall: A) Left side with polypropylene mesh; B) right side with bovine pericardium mesh; C) cut meshes; D) after abdominal closure.\n\nTramadol was used for postoperatively analgesia at a dose of 5 mg per kg every 12 h for four days and we also used amoxicillin at 20 mg per every 48 h for four days. The surgical wound was cleaned with iodine. For animal welfare concern, knowing that the environment influences the behavioral and physiological state of animals, we implemented environmental enrichment measures including: appropriate environment, feed, temperature, space and PVC pipe in the cages.\n Death of the animals and tissue resection for histopathological analysis After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).\n\nFIGURE 2Mesh analysis by transparency\n\nThe surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.\nThe material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis. \nThe inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition\n6\n.\nOn the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.\nAfter the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).\n\nFIGURE 2Mesh analysis by transparency\n\nThe surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.\nThe material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis. \nThe inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition\n6\n.\nOn the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.\n Histological evaluation Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al\n18\n, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.\nThe Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.\nTwo staining colors were used for the histopathological analysis: H&E, according to Yasojima et al\n18\n, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.\nThe Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.\n Statistical analysis The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.\nThe results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.",
"After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).\n\nFIGURE 2Mesh analysis by transparency\n\nThe surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.\nThe material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis. \nThe inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition\n6\n.\nOn the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.",
"Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al\n18\n, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.\nThe Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.",
"The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.",
"The analysis was performed based on the data of nine animals of the group submitted to euthanasia after 20 days and 10 after 40 days. Both types of mesh (polypropylene and bovine pericardium) were used in each animal. For the comparative analysis between the types of screen (in the same animal) were included the cases that had evaluation of the variable in the two types of mesh. The “necrosis” variable was not analyzed, since all animals in the experiment had no characteristic of this characteristic.\nIn relation to congestion, there was presence of alteration in 66.7% of polypropylene tissues in group 1 and 60% in group 2 (p>0.05). In the group of tissues with bovine pericardium, there was congestion in 33.3% in group 1 and 10% in group 2 (p>0.05). Although without significant difference, the granulation variable was present in 100% of polypropylene mesh materials in group 1 and in 55.6% with bovine pericardium mesh. In group 2 this variable was present in 80% with polypropylene and only 20% in the pieces with bovine pericardium (Figure 3).\n\nFIGURE 3Photomicrographs evidencing alterations in the abdominal wall (400x H&E): A) connective tissue with congested vessels (arrows); B) granulation tissue (arrow).\n\nThe presence of giant cells was evidenced in 66.7% of the analyzed pieces with polypropylene of group 1 and in 33.3% of the pieces with bovine pericardium. In group 2 this variable was present in 60% of the pieces with polypropylene and in no part with bovine pericardium (both with p>0.05, Figure 4).\n\nFIGURE 4Photomicrograph demonstrating giant cell (400x H&E - arrow)\n\nRegarding acute inflammation, no alteration was found in any part of the first group and did not present a statistically significant difference.\nIn relation to chronic inflammation, there were alterations in 66.7% of the pieces with polypropylene of group 1 and in 22.2% of the pieces with bovine pericardium of group 1, with p>0.05. However, in group 2, a significant difference (p=0.004) was found when compared to the presence of chronic inflammation in parts with polypropylene (90%) and bovine pericardium (0%, Figure 5).\n\nFIGURE 5Photomicrographs evidencing changes of chronic inflammatory process (A - arrows) and minimal scar area (B - arrow) - 100x H&E \n\nThe collagen fibers were found in 66.7% of the pieces with polypropylene and in 33.3% of the pieces with bovine pericardium in group 1 (p>0.05). In group 2, the variable was found in 62.5% of the pieces with polypropylene and 87.5% of the pieces with bovine pericardium (p>0.05, Figure 6).\n\nFIGURE 6Photomicrographs showing changes to Mallory’s trichrome: A) photomicrograph demonstrating on the arrow cuts of skin with presence of polypropylene and slight deposition of collagen around (Mallory 50x); B) photomicrograph demonstrating on the arrow area cicatricial with remnants of the polypropylene mesh and moderate degree of fibrosis around (Mallory 400x) \n",
"The present study aims to analyze the healing of the abdominal wall tissues after the use of two meshes, so that we can compare the tissue response. The subject was chosen due to the high number of surgeries performed with meshes. A guinea pig was used for each two implants (bovine pericardium mesh and polypropylene mesh) to reduce the number of animals in the experiment. Caviaporcellus was chosen because its size and docile nature facilitate the manipulation of the abdominal tissues.\nWhen the tissue responses of the pericardium meshes are compared to each other on day 20 and day 40, they show a decrease in the prevalence of the variables granulation, congestion, giant cells and chronic inflammation, showing better results compared to polypropylene shares when compared to each other. This demonstrates that these factors can be reduced as the days pass when using the bovine pericardium biological mesh.\nIn the next step of the analyzes the variables for one mesh were compared to the other. When the results obtained by the death of the animals on day 20 are compared, we noticed that while 66.7% of the specimens with polypropylene mesh showed congestion, which figure decreased to 60 on day 40, for the pericardium mesh this value decreased from 33.3% to 10.0%. This decrease also occurred in relation to the variables giant cells and granulation, both resulting in p=0.031. This shows us the ability of the tissue surrounding the implant to adapt more quickly when in response to the biological tissue.\nThe variable acute inflammation is also improved when using the bovine pericardium patch. For the variable chronic inflammation, the polypropylene group presented the variable in 66.7% on day 20, while the pericardium group only had 22.2%. The analysis of the same variable at day 40 show otherwise that 90% of the polypropylene meshes caused chronic inflammation, while no bovine pericardium mesh presented this variable. This brings us a p=0.004, corroborating with studies\n15\n which characterize the synthetic screens as more inflammatory. It also shows the importance of a biological tissue in the tissue response, as already observed by Abouelnasr, K. S. et al\n1\n, and in studies related to the area of cardiac surgery.\nAnalyzing the variable collagen, while on day 20 the polypropylene mesh group presented the variable in 66.7%, only 33.3% was present in the pericardium group. However, on day 40, we noticed that while in the polypropylene group the variable was present in 62.5%, in the bovine pericardium group it was present in 87.5%. It is noted that there was a 54.2% increase in the presence of collagen in the bovine pericardium group.\nAnalyzing the results of the present study we note that although for all variables the polypropylene meshes resulted in increased tissue response and greater inflammation, the bovine pericardium meshes resulted in longer production of collagen for a longer term. The value of p=0.004 demonstrates the presence of much greater chronic inflation in response to polypropylene and corroborates with studies that show that synthetic meshes of this material have inflammation as a major side effect, generating an exacerbated response of the tissue, and may present adhesions and other complications\n14\n. On the other hand, when we analyze the variable collagen, we can assume that the increased collagen in the long term suggests that, although the bovine pericardium meshes do not cause intense local inflammation, they can form collagen that brings consistency and tensile strength to the abdominal wall after a defect repair surgery, as noted by Ricciardi, Bruno Filippi et al\n\n10\n, who showed the best tissue response and least adhesion in response to a collagen-enveloped mesh when compared to a collagen-free mesh.",
"The data of this study suggests that there is less inflammatory reaction and increased formation of collagen fibers in response to bovine pericardium mesh when compared to the polypropylene mesh."
] | [
"intro",
"methods",
null,
null,
null,
"results",
"discussion",
"conclusions"
] | [
"Bovine pericardium",
"Polypropylene",
"Inflammatory process",
"Pericárdio bovino",
"Polipropileno",
"Processo inflamatório"
] | INTRODUCTION:
Surgical repair or reinforcement of the tissues that compose the wall of the abdominal cavity is one of the most frequent needs in human surgery
12
, due to both the scarcity of tissues and the high tension in the suture line, which results in the occurrence of high rates of incisional hernia
8
.
The use of meshes for surgical repair of hernia defects of the abdominal wall contributes to reducing the recurrence
11
,
17
. However, its intra-abdominal use comes along with the formation of adhesions and several complications
2
.
The first reported use of tissue for hernioplasty is dated 1958 by Usher et al
16
, who used a polypropylene mesh. This remains the most used material
18
because it is safe and offers several advantages in its use, including its availability, easy handling, low cost and good tolerance
9
.
Polypropylene is a synthetic material that produces little tissue reaction and good tensile strength, which strength lasts for several years after its use in living organisms. However, even with this possible choice, the use of meshes still has several side effects
4
, such as increased postoperative pain, abdominal adhesions and areas of fibrosis and increased risk of infection due to the placement of the prosthesis
18
.
Since the 1960s, biological membranes as implant material for the repair of organs and tissues have been used in Brazil. The advantages of using this type of material include the ease in obtaining, its low cost, simple preparation, viable sterilization, easy storage, little or no tissue reaction, and a long period of viability as an implant. Bovine pericardium is one of the most used biological membranes, being composed almost exclusively of collagen. Due to this characteristic, bovine pericardium easily adapts to the different situations to which it is submitted in the surgical practice
5
.
Although reports in the literature indicate that bovine pericardium has a greater tensile strength in relation to certain synthetic meshes, calcifications and immune rejection responses should be considered, taking into account the indications in the use of this material
13
. The bovine pericardium is the bovine heart surrounding membrane, being a biological tissue widely studied in the literature to produce products of medical use, many of which already in clinical use. The structure of this biological tissue basically comprises collagen and it is used in medicine after being physically and/or chemically treated for improving its mechanical and immunogenic properties and controlling its degradation or calcification. It is used for example in the production of cardiac and vascular prostheses, repair of ligaments, controlled drug delivery systems, hemostatic agents, grafts and others. Valve prostheses made with bovine pericardium have a good hydrodynamic performance and low thrombogenicity
3
.
Since the beginning of the last century, the use of prostheses has become constant in abdominal hernia surgery, especially for inguinal hernia. The study and search for alternative materials, such as bovine pericardium, have proved to be an option in the correction and treatment of hernias resulting in better adaptations and effectiveness
7
.
The objective of this study is to evaluate the inflammatory process and formation of collagen fibers in response to bovine pericardium compared to the inflammatory process and formation of collagen fibers in response to the synthetic polypropylene mesh.
METHODS:
This study has a qualitative and quantitative experimental character, and therefore, the abdominal wall defect induction and subsequent correction with polypropylene mesh and bovine pericardium mesh was performed. It was approved under CEP number 1513/2016. The procedures with the animals complied with that recommended by the Ethics Committee on the Use of Animals (CEUA) of Evangelical Faculty of Paraná (FEPAR), Curitiba, PR, Brazil.
A total of 19 male guinea pigs, weighing between 300 and 400 g, were collected from the Paraná Institute of Technology (TECPAR) vivarium. The animals were kept during the experiment at IPEM’s vivarium, in 47x34x18 cm plastic boxes, with two animals each, lined with shavings, in 12-h light/dark cycle (light from 7am to 7pm) at a temperature of 22±2º C. The animals were treated daily with filtered water and appropriate ration fed freely.
The model used was abdominal wall defect correction using mesh, divided into two groups of nine guinea pigs in group 1 and 10 guinea pigs in group 2. In group 1 surgical correction using polypropylene synthetic mesh and bovine pericardium biological tissue with tissue removal and animal death after 20 days was done. In group 2 the same procedure was performed, but the animal death was after 40 days.
A mixture of xylazine hydrochloride 5 mg and ketamine hydrochloride 40 mg per body weight in kilograms intraperitoneally was used to anesthetize the animals, and they were positioned for surgery in the dorsal decubitus position. The surgical intervention began with trichotomy and disinfection of the abdominal region, followed by an incision to create a 1 cm defect in the abdominal wall through the epithelium to the peritoneal tissue, for subsequent correction with polypropylene mesh on the right side and bovine pericardium mesh on the left side (Figure 1).
The meshes were 0.5 x 0.5 cm and were accommodated in the abdominal cavity above the peritoneum.
The abdominal cavity was closed with separate stitches in the cutaneous plane, and the abdominal muscle layer was closed with continuous suture.
FIGURE 1Meshes inserted into the abdominal wall: A) Left side with polypropylene mesh; B) right side with bovine pericardium mesh; C) cut meshes; D) after abdominal closure.
Tramadol was used for postoperatively analgesia at a dose of 5 mg per kg every 12 h for four days and we also used amoxicillin at 20 mg per every 48 h for four days. The surgical wound was cleaned with iodine. For animal welfare concern, knowing that the environment influences the behavioral and physiological state of animals, we implemented environmental enrichment measures including: appropriate environment, feed, temperature, space and PVC pipe in the cages.
Death of the animals and tissue resection for histopathological analysis After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).
FIGURE 2Mesh analysis by transparency
The surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.
The material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis.
The inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition
6
.
On the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.
After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).
FIGURE 2Mesh analysis by transparency
The surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.
The material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis.
The inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition
6
.
On the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.
Histological evaluation Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al
18
, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.
The Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.
Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al
18
, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.
The Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.
Statistical analysis The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.
The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.
Death of the animals and tissue resection for histopathological analysis:
After the median abdominal incision, the meshes were found by transparency, so that the specimen was removed from the correct place (Figure 2).
FIGURE 2Mesh analysis by transparency
The surgical specimen was removed in a single block and immediately immersed in 10% buffered neutral formaldehyde, remaining in the fixer for 72 h at room temperature.
The material was cut in 6-micrometer sections and stained using hematoxylin-eosin (H&E) technique for the cellular and capillary elements, and the Mallory’s trichrome technique for fibrous collagen fibers. The analysis was made using an Olympus® optical microscope, model DX 50 to evaluate the inflammatory process of each material, i.e.: congestion, formation of granulation tissue, presence of acute inflammation, presence of chronic inflammation, presence of giant cells, and necrosis.
The inflammatory cells analyzed were histiocytes, neutrophils, lymphocytes, giant cells and calcified cells. The histiocyte considered in this histopathological study as a type of macrophage of endothelial reticulum origin, is typically immobile and inactive but, when stimulated, it can become active; the neutrophil, a polynuclear leukocyte with neutrophilic granulations; the lymphocyte, a type of leukocyte ranging between 10-12 microns in diameter, with round nucleus with condensed chromatin and scarce basophilic cytoplasm; the giant cell, as that formed by the union of several distinct cells. Regarding the presence of fibrosis, it was classified as absent, scarce and focal, diffuse and moderate or diffuse and intense deposition
6
.
On the last day of the experiment, day 20 for the first group and day 40 for the second, anesthesia was applied for the death of the animals. The anesthetic technique involved intraperitoneal administration using ketamine hydrochloride at the dose of 120 mg per kg of the animal and xylazine hydrochloride at a dose of 15 mg per kg of the animal.
Histological evaluation:
Two staining colors were used for the histopathological analysis: H&E, according to Yasojima et al
18
, and Mallory’s trichrome. A pathologist without previous knowledge of the division of the groups received a slide for each animal and evaluated the pattern of histological changes. In H&E staining the parameters of congestion, granulation, giant cells and necrosis were analyzed and classified as S=presence or N=absence. Acute and chronic inflammation are classified as 0=absence, 1=slight, 2=moderate, and 3=intense.
The Mallory’s trichrome quantified the arrangement of collagen fibers, being classified as 0=absence, 1=scarce and focal deposition, 2=moderate and diffuse deposition, and 3=intense and diffuse deposition.
Statistical analysis:
The results of H&E and collagen fiber variables were described by frequencies and percentages. Fisher’s exact test was used to compare the groups defined by the day of death of the animals (20 days or 40 days) in relation to the variables evaluated. The comparisons between the two types of meshes (polypropylene or bovine pericardium) were made using the binomial test. Values of p<0.05 indicated statistical significance. The data was analyzed using the IBM SPSS Statistics v.20 software.
RESULTS:
The analysis was performed based on the data of nine animals of the group submitted to euthanasia after 20 days and 10 after 40 days. Both types of mesh (polypropylene and bovine pericardium) were used in each animal. For the comparative analysis between the types of screen (in the same animal) were included the cases that had evaluation of the variable in the two types of mesh. The “necrosis” variable was not analyzed, since all animals in the experiment had no characteristic of this characteristic.
In relation to congestion, there was presence of alteration in 66.7% of polypropylene tissues in group 1 and 60% in group 2 (p>0.05). In the group of tissues with bovine pericardium, there was congestion in 33.3% in group 1 and 10% in group 2 (p>0.05). Although without significant difference, the granulation variable was present in 100% of polypropylene mesh materials in group 1 and in 55.6% with bovine pericardium mesh. In group 2 this variable was present in 80% with polypropylene and only 20% in the pieces with bovine pericardium (Figure 3).
FIGURE 3Photomicrographs evidencing alterations in the abdominal wall (400x H&E): A) connective tissue with congested vessels (arrows); B) granulation tissue (arrow).
The presence of giant cells was evidenced in 66.7% of the analyzed pieces with polypropylene of group 1 and in 33.3% of the pieces with bovine pericardium. In group 2 this variable was present in 60% of the pieces with polypropylene and in no part with bovine pericardium (both with p>0.05, Figure 4).
FIGURE 4Photomicrograph demonstrating giant cell (400x H&E - arrow)
Regarding acute inflammation, no alteration was found in any part of the first group and did not present a statistically significant difference.
In relation to chronic inflammation, there were alterations in 66.7% of the pieces with polypropylene of group 1 and in 22.2% of the pieces with bovine pericardium of group 1, with p>0.05. However, in group 2, a significant difference (p=0.004) was found when compared to the presence of chronic inflammation in parts with polypropylene (90%) and bovine pericardium (0%, Figure 5).
FIGURE 5Photomicrographs evidencing changes of chronic inflammatory process (A - arrows) and minimal scar area (B - arrow) - 100x H&E
The collagen fibers were found in 66.7% of the pieces with polypropylene and in 33.3% of the pieces with bovine pericardium in group 1 (p>0.05). In group 2, the variable was found in 62.5% of the pieces with polypropylene and 87.5% of the pieces with bovine pericardium (p>0.05, Figure 6).
FIGURE 6Photomicrographs showing changes to Mallory’s trichrome: A) photomicrograph demonstrating on the arrow cuts of skin with presence of polypropylene and slight deposition of collagen around (Mallory 50x); B) photomicrograph demonstrating on the arrow area cicatricial with remnants of the polypropylene mesh and moderate degree of fibrosis around (Mallory 400x)
DISCUSSION:
The present study aims to analyze the healing of the abdominal wall tissues after the use of two meshes, so that we can compare the tissue response. The subject was chosen due to the high number of surgeries performed with meshes. A guinea pig was used for each two implants (bovine pericardium mesh and polypropylene mesh) to reduce the number of animals in the experiment. Caviaporcellus was chosen because its size and docile nature facilitate the manipulation of the abdominal tissues.
When the tissue responses of the pericardium meshes are compared to each other on day 20 and day 40, they show a decrease in the prevalence of the variables granulation, congestion, giant cells and chronic inflammation, showing better results compared to polypropylene shares when compared to each other. This demonstrates that these factors can be reduced as the days pass when using the bovine pericardium biological mesh.
In the next step of the analyzes the variables for one mesh were compared to the other. When the results obtained by the death of the animals on day 20 are compared, we noticed that while 66.7% of the specimens with polypropylene mesh showed congestion, which figure decreased to 60 on day 40, for the pericardium mesh this value decreased from 33.3% to 10.0%. This decrease also occurred in relation to the variables giant cells and granulation, both resulting in p=0.031. This shows us the ability of the tissue surrounding the implant to adapt more quickly when in response to the biological tissue.
The variable acute inflammation is also improved when using the bovine pericardium patch. For the variable chronic inflammation, the polypropylene group presented the variable in 66.7% on day 20, while the pericardium group only had 22.2%. The analysis of the same variable at day 40 show otherwise that 90% of the polypropylene meshes caused chronic inflammation, while no bovine pericardium mesh presented this variable. This brings us a p=0.004, corroborating with studies
15
which characterize the synthetic screens as more inflammatory. It also shows the importance of a biological tissue in the tissue response, as already observed by Abouelnasr, K. S. et al
1
, and in studies related to the area of cardiac surgery.
Analyzing the variable collagen, while on day 20 the polypropylene mesh group presented the variable in 66.7%, only 33.3% was present in the pericardium group. However, on day 40, we noticed that while in the polypropylene group the variable was present in 62.5%, in the bovine pericardium group it was present in 87.5%. It is noted that there was a 54.2% increase in the presence of collagen in the bovine pericardium group.
Analyzing the results of the present study we note that although for all variables the polypropylene meshes resulted in increased tissue response and greater inflammation, the bovine pericardium meshes resulted in longer production of collagen for a longer term. The value of p=0.004 demonstrates the presence of much greater chronic inflation in response to polypropylene and corroborates with studies that show that synthetic meshes of this material have inflammation as a major side effect, generating an exacerbated response of the tissue, and may present adhesions and other complications
14
. On the other hand, when we analyze the variable collagen, we can assume that the increased collagen in the long term suggests that, although the bovine pericardium meshes do not cause intense local inflammation, they can form collagen that brings consistency and tensile strength to the abdominal wall after a defect repair surgery, as noted by Ricciardi, Bruno Filippi et al
10
, who showed the best tissue response and least adhesion in response to a collagen-enveloped mesh when compared to a collagen-free mesh.
CONCLUSION:
The data of this study suggests that there is less inflammatory reaction and increased formation of collagen fibers in response to bovine pericardium mesh when compared to the polypropylene mesh.
| Background:
The use of polypropylene meshes for surgical repair of the abdominal wall contributes to a reduction of the of recurrence rates of hernias or defects. However, its intra-abdominal use comes along with the formation of adhesions and several complications. The study and the search for alternative materials, including bovine pericardium, have been regarded as an option for the correction and treatment of resulting hernias with better adaptations and effectiveness.
Methods:
Bovine pericardium mesh and polypropylene mesh were placed, both on the same animal. The first group had the mesh removed for analysis on day 20, and the second group on day 40. The variables congestion, granulation, giant cells, necrosis, acute inflammation, chronic inflammation and collagen were analyzed.
Results:
All variables were found in greater numbers as a response to the polypropylene mesh, except for the collagen, which, on day 40, was greater in response to the bovine pericardium mesh.
Conclusions:
The data in this study suggest that there is less inflammatory reaction in response to bovine pericardium mesh when compared to polypropylene mesh. | INTRODUCTION:
Surgical repair or reinforcement of the tissues that compose the wall of the abdominal cavity is one of the most frequent needs in human surgery
12
, due to both the scarcity of tissues and the high tension in the suture line, which results in the occurrence of high rates of incisional hernia
8
.
The use of meshes for surgical repair of hernia defects of the abdominal wall contributes to reducing the recurrence
11
,
17
. However, its intra-abdominal use comes along with the formation of adhesions and several complications
2
.
The first reported use of tissue for hernioplasty is dated 1958 by Usher et al
16
, who used a polypropylene mesh. This remains the most used material
18
because it is safe and offers several advantages in its use, including its availability, easy handling, low cost and good tolerance
9
.
Polypropylene is a synthetic material that produces little tissue reaction and good tensile strength, which strength lasts for several years after its use in living organisms. However, even with this possible choice, the use of meshes still has several side effects
4
, such as increased postoperative pain, abdominal adhesions and areas of fibrosis and increased risk of infection due to the placement of the prosthesis
18
.
Since the 1960s, biological membranes as implant material for the repair of organs and tissues have been used in Brazil. The advantages of using this type of material include the ease in obtaining, its low cost, simple preparation, viable sterilization, easy storage, little or no tissue reaction, and a long period of viability as an implant. Bovine pericardium is one of the most used biological membranes, being composed almost exclusively of collagen. Due to this characteristic, bovine pericardium easily adapts to the different situations to which it is submitted in the surgical practice
5
.
Although reports in the literature indicate that bovine pericardium has a greater tensile strength in relation to certain synthetic meshes, calcifications and immune rejection responses should be considered, taking into account the indications in the use of this material
13
. The bovine pericardium is the bovine heart surrounding membrane, being a biological tissue widely studied in the literature to produce products of medical use, many of which already in clinical use. The structure of this biological tissue basically comprises collagen and it is used in medicine after being physically and/or chemically treated for improving its mechanical and immunogenic properties and controlling its degradation or calcification. It is used for example in the production of cardiac and vascular prostheses, repair of ligaments, controlled drug delivery systems, hemostatic agents, grafts and others. Valve prostheses made with bovine pericardium have a good hydrodynamic performance and low thrombogenicity
3
.
Since the beginning of the last century, the use of prostheses has become constant in abdominal hernia surgery, especially for inguinal hernia. The study and search for alternative materials, such as bovine pericardium, have proved to be an option in the correction and treatment of hernias resulting in better adaptations and effectiveness
7
.
The objective of this study is to evaluate the inflammatory process and formation of collagen fibers in response to bovine pericardium compared to the inflammatory process and formation of collagen fibers in response to the synthetic polypropylene mesh.
CONCLUSION:
The data of this study suggests that there is less inflammatory reaction and increased formation of collagen fibers in response to bovine pericardium mesh when compared to the polypropylene mesh. | Background:
The use of polypropylene meshes for surgical repair of the abdominal wall contributes to a reduction of the of recurrence rates of hernias or defects. However, its intra-abdominal use comes along with the formation of adhesions and several complications. The study and the search for alternative materials, including bovine pericardium, have been regarded as an option for the correction and treatment of resulting hernias with better adaptations and effectiveness.
Methods:
Bovine pericardium mesh and polypropylene mesh were placed, both on the same animal. The first group had the mesh removed for analysis on day 20, and the second group on day 40. The variables congestion, granulation, giant cells, necrosis, acute inflammation, chronic inflammation and collagen were analyzed.
Results:
All variables were found in greater numbers as a response to the polypropylene mesh, except for the collagen, which, on day 40, was greater in response to the bovine pericardium mesh.
Conclusions:
The data in this study suggest that there is less inflammatory reaction in response to bovine pericardium mesh when compared to polypropylene mesh. | 4,230 | 209 | [
350,
143,
88
] | 8 | [
"pericardium",
"bovine",
"bovine pericardium",
"polypropylene",
"group",
"mesh",
"collagen",
"tissue",
"cells",
"abdominal"
] | [
"use tissue hernioplasty",
"meshes polypropylene",
"abdominal incision meshes",
"compared polypropylene mesh",
"incisional hernia use"
] | [CONTENT] Bovine pericardium | Polypropylene | Inflammatory process | Pericárdio bovino | Polipropileno | Processo inflamatório [SUMMARY] | [CONTENT] Bovine pericardium | Polypropylene | Inflammatory process | Pericárdio bovino | Polipropileno | Processo inflamatório [SUMMARY] | [CONTENT] Bovine pericardium | Polypropylene | Inflammatory process | Pericárdio bovino | Polipropileno | Processo inflamatório [SUMMARY] | [CONTENT] Bovine pericardium | Polypropylene | Inflammatory process | Pericárdio bovino | Polipropileno | Processo inflamatório [SUMMARY] | [CONTENT] Bovine pericardium | Polypropylene | Inflammatory process | Pericárdio bovino | Polipropileno | Processo inflamatório [SUMMARY] | [CONTENT] Bovine pericardium | Polypropylene | Inflammatory process | Pericárdio bovino | Polipropileno | Processo inflamatório [SUMMARY] | [CONTENT] Abdominal Wall | Animals | Cattle | Pericardium | Polypropylenes | Surgical Mesh | Tissue Adhesions [SUMMARY] | [CONTENT] Abdominal Wall | Animals | Cattle | Pericardium | Polypropylenes | Surgical Mesh | Tissue Adhesions [SUMMARY] | [CONTENT] Abdominal Wall | Animals | Cattle | Pericardium | Polypropylenes | Surgical Mesh | Tissue Adhesions [SUMMARY] | [CONTENT] Abdominal Wall | Animals | Cattle | Pericardium | Polypropylenes | Surgical Mesh | Tissue Adhesions [SUMMARY] | [CONTENT] Abdominal Wall | Animals | Cattle | Pericardium | Polypropylenes | Surgical Mesh | Tissue Adhesions [SUMMARY] | [CONTENT] Abdominal Wall | Animals | Cattle | Pericardium | Polypropylenes | Surgical Mesh | Tissue Adhesions [SUMMARY] | [CONTENT] use tissue hernioplasty | meshes polypropylene | abdominal incision meshes | compared polypropylene mesh | incisional hernia use [SUMMARY] | [CONTENT] use tissue hernioplasty | meshes polypropylene | abdominal incision meshes | compared polypropylene mesh | incisional hernia use [SUMMARY] | [CONTENT] use tissue hernioplasty | meshes polypropylene | abdominal incision meshes | compared polypropylene mesh | incisional hernia use [SUMMARY] | [CONTENT] use tissue hernioplasty | meshes polypropylene | abdominal incision meshes | compared polypropylene mesh | incisional hernia use [SUMMARY] | [CONTENT] use tissue hernioplasty | meshes polypropylene | abdominal incision meshes | compared polypropylene mesh | incisional hernia use [SUMMARY] | [CONTENT] use tissue hernioplasty | meshes polypropylene | abdominal incision meshes | compared polypropylene mesh | incisional hernia use [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | polypropylene | group | mesh | collagen | tissue | cells | abdominal [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | polypropylene | group | mesh | collagen | tissue | cells | abdominal [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | polypropylene | group | mesh | collagen | tissue | cells | abdominal [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | polypropylene | group | mesh | collagen | tissue | cells | abdominal [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | polypropylene | group | mesh | collagen | tissue | cells | abdominal [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | polypropylene | group | mesh | collagen | tissue | cells | abdominal [SUMMARY] | [CONTENT] use | hernia | bovine | bovine pericardium | pericardium | repair | material | prostheses | good | low [SUMMARY] | [CONTENT] cells | animals | mg | abdominal | diffuse | classified | animal | presence | day | deposition [SUMMARY] | [CONTENT] group | pieces | polypropylene | figure | variable | pieces polypropylene | pieces bovine pericardium | arrow | pieces bovine | pericardium [SUMMARY] | [CONTENT] mesh | data study suggests | inflammatory reaction | reaction increased formation | reaction increased | mesh compared polypropylene mesh | mesh compared polypropylene | increased formation | inflammatory reaction increased formation | data study [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | mesh | polypropylene | group | day | collagen | variable | presence [SUMMARY] | [CONTENT] pericardium | bovine | bovine pericardium | mesh | polypropylene | group | day | collagen | variable | presence [SUMMARY] | [CONTENT] ||| ||| [SUMMARY] | [CONTENT] ||| first | day 20 | second | day 40 ||| [SUMMARY] | [CONTENT] day 40 [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] ||| ||| ||| ||| first | day 20 | second | day 40 ||| ||| ||| day 40 ||| [SUMMARY] | [CONTENT] ||| ||| ||| ||| first | day 20 | second | day 40 ||| ||| ||| day 40 ||| [SUMMARY] |
||||||
Determining dimensions of job satisfaction in healthcare using factor analysis. | 36303222 | Job satisfaction in health care has a great impact as it affects quality, productivity, effectiveness, and healthcare costs. In fact, it is an indicator of the well-being and quality of life of the organization's employees, as it has been variously linked with increased performance and negatively to absenteeism and turnover. Better knowledge of healthcare employees' job satisfaction and performance can directly contribute to the quality of the services provided to patients and is critical for the success of organizations. | BACKGROUND | The Cronbach's alpha coefficient, split-half reliability, exploratory factor and confirmatory factor analysis were employed to assess the reliability and validity of JSS. | METHODS | Six underlying dimensions were extracted (benefits and salary, management's attitude, supervision, communication, nature of work, and colleagues' support). Internal consistency reliability was satisfactory since Cronbach's alpha for the overall scale was 0.81 and for the various dimensions ranged from 0.61 to 0.81, respectively. Exploratory factor analysis showed a KMO value of 0.912. The confirmatory factor analysis indicated good fit: SRMR = 0.050, RMSEA = 0.055, IFI = 0.906 and CFI = 0.906. | RESULTS | Job satisfaction is a multidimensional construct that encompasses different facets of satisfaction. There is a lack of consensus as to which factors are more important and a researcher may find satisfaction with some factors while at the same time dissatisfaction with others. Our findings are significant for improving our understanding of the nature and assessment of job satisfaction in the Greek healthcare context, providing a more stable ground in a rapidly changing environment. A short JSS developed that could be much more widely used in the future. | CONCLUSION | [
"Humans",
"Job Satisfaction",
"Reproducibility of Results",
"Quality of Life",
"Surveys and Questionnaires",
"Factor Analysis, Statistical",
"Delivery of Health Care",
"Psychometrics"
] | 9610349 | Introduction | As employee knowledge and skills are intangible assets of any service organization,
employee satisfaction has become an issue of utmost importance. It has been defined as the positive emotional state resulting from the evaluation of one’s work or work experience [1]. Hoppock [2] was the first who brought forth the concept of job satisfaction in limelight and described it as “the employees’ subjective reflections or subjective feelings about their working conditions and working environment”. Since then, many researchers have recognized that satisfied employees are a key asset to an organization [3]. While the importance of job satisfaction is generally recognized, additional and ongoing investigations of satisfaction levels are necessary as external conditions and societal values are constantly changing. In this respect, job satisfaction has a significant role in the operation and performance of organizations.
An essential prerequisite for the development and long-term success of an organization is in fact the utilization of employee’s capabilities and the improvement of their working conditions [4]. The degree of job satisfaction is actually the overall level of satisfaction on a number of different dimensions of work and affects the behavior of employees that, in turn, impacts upon organizational functioning [5–7]. Swamy et al. [3] stated that satisfied employees are the key asset of an organization. Therefore, the issue of job satisfaction is very important especially for non-profit public organizations like hospitals, which are essential for a country’s provision of healthcare services and the population itself.
Employee satisfaction also affects patient satisfaction. As patients are the external customers and employees are the internal customers of the organization they form the current working environment and are willing to cooperate with the community to achieve organizational goals. Previous studies have documented associations between job fulfillment of health workforce and patient contentment with the type of health care services provided in health care facilities [8, 9]. Moreover, there seems to exist a positive correlation between the increase in job satisfaction and quality of care [10, 11]. Conversely, a low level of job satisfaction would create negative behaviors, including absenteeism, grievances, high level of stress, turnover, exhaustion, low morality, worse patient-provider ratios, longer wait times, psychological distress and increased medical errors [12–14].
Hospital managers have responsibilities to both patients and staff. It has been suggested that if you want to attain higher job productivity and efficiency, you should comprehend the domains of work which are decisive for job satisfaction amongst healthcare providers. In order to get employees contented with their job; the underlying factors that influence job satisfaction in that particular facility must be examined to guide proper managerial action [15, 16]. | null | null | Results | Study sample Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).
Table 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories
Characteristics
Doctors
Nurses
Other Health Professionals
Overall Sample
N = 803
%
N = 1,736
%
N = 739
%
N = 3,278
%
Gender
Male29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%
Age
< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%
Marital
Married38547.95%1,1767.40%49967.52%2,05462.66%
Status
Single39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%
Level of
Compulsory00.00%70.40%435.82%501.53%
Education
Secondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%
Employment
Permanent42552.93%1,5991.59%64086.60%2,65580.99%
Status
Temporary37847.07%1468.41%9913.40%62319.01%
Professional
< 5 years29036.11%22112.73%12416.78%63519.37%
Experience
6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%
Economic Situation
I cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%
Sociodemographic characteristics of the sample per professional category
Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).
Table 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories
Characteristics
Doctors
Nurses
Other Health Professionals
Overall Sample
N = 803
%
N = 1,736
%
N = 739
%
N = 3,278
%
Gender
Male29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%
Age
< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%
Marital
Married38547.95%1,1767.40%49967.52%2,05462.66%
Status
Single39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%
Level of
Compulsory00.00%70.40%435.82%501.53%
Education
Secondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%
Employment
Permanent42552.93%1,5991.59%64086.60%2,65580.99%
Status
Temporary37847.07%1468.41%9913.40%62319.01%
Professional
< 5 years29036.11%22112.73%12416.78%63519.37%
Experience
6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%
Economic Situation
I cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%
Sociodemographic characteristics of the sample per professional category | Conclusion | In total, this study applied quantitative methods to determine factors affecting job satisfaction. So, is important in terms of determining the specific factors that should be considered for job satisfaction, organizational engagement, managerial success, and high performance within hospitals. A short 20-item study for all healthcare staff can benefit hospitals to monitor employee satisfaction across all levels without overburdening employees and analysts with multiple or fielding several non-comparable types of research.
The findings suggest that effective communication and support from managers or supervisors to employees or among employees themselves will reduce stress and conflicts in the workplace. Additionally, it can be recommended that employee empowerment and training, collaboration in teamwork, and a systematic approach regarding innovative types of promotional opportunity, recognition, reward, and evaluation of hospital staff can lead to better results and benefits employees, quality of patient care, and healthcare organizations. Consequently, we believe that empowerment of management, achievement, promotion and evaluation should significantly improve job satisfaction respectively. This study showed that obtained factors are aligned with the findings of the prior studies in the literature [60, 61].
The results of this study should not be generalized extensively since the participants of the study come from a single geographical region of the country, only in hospitals in Athens, Greece. Nevertheless, the sample cannot be characterized as homogenous due to the fact that participants were working in different departments in the hospitals, so they deal with different tasks and procedures. Therefore, the findings and related conclusions may not be able to be generalized and compared with the rest regions of the country. | [
"Measurement of job satisfaction",
"Objectives",
"Research instrument translation and adaptation",
"Research design and procedure",
"Statistical analysis",
"Study sample",
"Normality analysis",
"Descriptive statistics results",
"Exploratory factor analysis",
"Confirmatory factor analysis"
] | [
"Due to its importance, a wide range of instruments have been designed to quantify and conceptualize job satisfaction during the past decades. They were developed to capture the entirety of various aspects of job satisfaction be it personal, social, environmental, organizational, and the nature of the job itself. A valuable and widely used measure of job satisfaction is the Job Satisfaction Survey (JSS) that was originally developed by Spector [17]. JSS provides sufficient reliability and validity and is available for researchers free of charge for use for non-commercial purposes. The instrument contains 36 items expressed on a Likert scale measuring nine dimensions of job satisfaction, as mentioned below:\n\nPay includes salaries and wages. Unfair distribution can negatively affect employees’ emotions and therefore their behavior in the organization [18].Promotion is an important aspect of a employee’s career. It refers to progression to a higher position with more challenges, authority and responsibilities [19]. Only a meritocratic promotion system with evaluation conditions known in advance can lead to satisfaction.Fringe Benefits, can be financial or non-financial compensations. Financial compensations consist of direct (e.g. bonuses) and indirect compensation (e.g. retirement plans). Non-financial compensations consist of the job itself (e.g. autonomy), job environment (e.g. working conditions) and workplace flexibility (e.g. part-time work) [20].Contingent Rewards, are referred to as promises and exchanges of rewards and recognition for good work. Is a valuable tool for motivating employees because they want to be paid well for the job they perform both for their self-esteem and as useful means of a living [21].Supervision, is defined as the perception of employees regarding the support received from supervisors in an organization besides coworkers. Usually, employees are satisfied when they are supported to achieve their goals [22].Operating Procedures, are described as steps of finishing tasks that have to follow a certain standard based on regulations, provincial laws, policies, procedures and standards. Inadequacy of equipment and resources, lighting, ventilation, and cleanliness can result in a stressful work environment that leads to job dissatisfaction among employees [23].Co-workers, are referred as people working in an organization (besides supervisors). Employees with the same values, attitudes and philosophies can improve satisfaction in an organization [24]. Support from colleagues can enhance job satisfaction and decrease job stress and burnout.Nature of Work, is defined as the variability of the given work. It refers to the daily and non-daily tasks carried out as part of the job scope and includes job challenges, feedback, autonomy, and skill variety [25]. Further, this can increase the motivational level of employees which will ultimately raise their internal happiness of employees, and the internal happiness will cause satisfaction.Communication, is referred as informing the current employees. Communication between supervisors or the managerial level with employees consistently enables managers to know whether their staff is satisfied and happy with its employment or not [26]. There is a positive association between communication and job satisfaction. Effective communication at the workplace is essential in ensuring organizational objectives, social support.\n\nPay includes salaries and wages. Unfair distribution can negatively affect employees’ emotions and therefore their behavior in the organization [18].\nPromotion is an important aspect of a employee’s career. It refers to progression to a higher position with more challenges, authority and responsibilities [19]. Only a meritocratic promotion system with evaluation conditions known in advance can lead to satisfaction.\nFringe Benefits, can be financial or non-financial compensations. Financial compensations consist of direct (e.g. bonuses) and indirect compensation (e.g. retirement plans). Non-financial compensations consist of the job itself (e.g. autonomy), job environment (e.g. working conditions) and workplace flexibility (e.g. part-time work) [20].\nContingent Rewards, are referred to as promises and exchanges of rewards and recognition for good work. Is a valuable tool for motivating employees because they want to be paid well for the job they perform both for their self-esteem and as useful means of a living [21].\nSupervision, is defined as the perception of employees regarding the support received from supervisors in an organization besides coworkers. Usually, employees are satisfied when they are supported to achieve their goals [22].\nOperating Procedures, are described as steps of finishing tasks that have to follow a certain standard based on regulations, provincial laws, policies, procedures and standards. Inadequacy of equipment and resources, lighting, ventilation, and cleanliness can result in a stressful work environment that leads to job dissatisfaction among employees [23].\nCo-workers, are referred as people working in an organization (besides supervisors). Employees with the same values, attitudes and philosophies can improve satisfaction in an organization [24]. Support from colleagues can enhance job satisfaction and decrease job stress and burnout.\nNature of Work, is defined as the variability of the given work. It refers to the daily and non-daily tasks carried out as part of the job scope and includes job challenges, feedback, autonomy, and skill variety [25]. Further, this can increase the motivational level of employees which will ultimately raise their internal happiness of employees, and the internal happiness will cause satisfaction.\nCommunication, is referred as informing the current employees. Communication between supervisors or the managerial level with employees consistently enables managers to know whether their staff is satisfied and happy with its employment or not [26]. There is a positive association between communication and job satisfaction. Effective communication at the workplace is essential in ensuring organizational objectives, social support.\nEvery dimension incorporates four items. Several previous studies have shown that JSS has high internal consistency and validity [27, 28].",
"This research aimed to explore (a) the underlying factorial structure of the JSS when applied to Greek hospital employees, (b) its psychometric properties. Undoubtedly, job satisfaction is a complex concept, so there is always a need to research this phenomenon and related factors to explore the development of optimal human resources strategies in the context of healthcare institutions. Moreover, there is a compelling need for developing constructs in the field of management rather than adapting the constructs that have been developed already.",
"The JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.\nThe reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].\n\nTable 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\n\nJob Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha\n* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\nPrior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].\nAdditionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).\n\nTable 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77\n\nSplit-Half reliability analysis\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.\nThe items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.",
"The survey was carried out in the region of Attiki with its capital Athens, with around 3.75 million inhabitants or approximately 35% of the total Greek population. The 1st Regional Health Authority of Attica has the responsibility for 27 public hospitals. Our survey was conducted between July 2019 and December 2020 in thirteen of those who provided healthcare services to 438,745 patients. The main criteria for the selection of these hospitals were (Table 3): (a) the categories of hospitals; for this reason, the survey was introduced into four different categories (general, pediatric, maternity, oncology), (b) a large number of different clinics, (c) hospitals with a large number of beds but without ignoring the role of smaller hospitals, (d) the large number of patients treated in these hospitals, (e) the large number of health care employees who work in these hospitals, and (f) the necessary approval of the research by hospital committees.\nThe researchers distributed the printed questionnaire along with a consent form to the participants in person at their workplaces. They were adults (over 18 years), health care professionals belonging to medical, nursing, administrative, and technical departments serving public healthcare. The main aim of selecting employees from various fields is to get the opinions of a diverse group of people so that the results can be generalized on s vast group of the overall population. They had worked for more than six months in the respective hospital facilities at the time of the research and consented to the study. The study excluded interns, volunteers, and those declining to consent to the study. The participants had one week to complete the questionnaire. All employees had the right to refuse or discontinue their participation in the survey at any time. The researcher guaranteed the anonymity and confidentiality of all data collected. We remained considerate of the names, safety, and well-being of participants, and also the organizations remained anonymous by using codes, such as H01, H02, and so on (Table 3). Finally, of the 4,000 questionnaires distributed, 3,278 (81.95%) were returned.\n\nTable 3The population of research per hospital and category. Distributed Questionnaires and Response Rate\nPeriod of research\n\nProfessional Category\n\n2019\n\n2020\n\nDoctors\n\nNurses\n\nOther Health Professionals\n\nOverall Sample\n\nNumber\n\nNumber\n\nDays of\n\nHospital\n\n3Q\n\n4Q\n\n1Q\n\n2Q\n\n3Q\n\n4Q\n\n( n )\n\n( % )\n\n( n )\n\n( % )\n\n( n )\n\n( % )\n\n( n )\n\n( % )\n\nοf Beds*\n\nοf Patients*\n\nHospitalization*\nH 01XXXXXX10813.45%1327.60%435.82%2838.64%H 0216820.92%29016.71%15821.38%61618.80%H 03XXXXXX293.61%995.70%7410.01%2026.17%H 049011.21%21012.10%648.66%36411.11%H 05XXXXXX597.35%895.13%628.39%2106.41%H 06516.35%1227.03%699.34%2427.39%H 07XXXXXX617.60%18110.43%395.28%2818.58%H 088710.83%905.18%324.33%2096.38%H 09XXXXXX364.48%1196.85%577.71%2126.47%H 10455.60%1116.39%506.77%2066.29%H 11XXXXXX293.61%1367.83%364.87%2016.14%H 12323.99%1327.60%385.14%2026.17%H 13XXXXXX81.00%251.44%172.30%501.53%[1] Hospitals of research(n = 13)80324.50%1,73652.96%73922.54%3,278100%6,511438,7451,443,660[2] Hospitals in 1st Regional Health Authority of Attica(n = 27)6,277**31.71%8,174**41.29%5,345**27.00%19,796**100%8,860567,8171,912,488[3] Hospitals in the National Health System(n = 127)36,4412,160,5967,343,348[4] Percentages of Hospitals of research / Hospitals of Attica= [1] / [2]12.79%21.24%13.83%16.56%73.49%77.27%[5] Percentages of Hospitals of research / NHS= [1] / [3]17.87%20.31%19.66%[6] Distributed Questionnaires of research1,00025.00%2,00050.00%1,00025.00%4,000100%[7] Response Rate= [1] / [6]80.30%86.80%73.90%81.95%Notes: * Data of year 2020 ** Data of year 2016X = research conduct\n\nThe population of research per hospital and category. Distributed Questionnaires and Response Rate\nNotes: * Data of year 2020 ** Data of year 2016\nX = research conduct\n Statistical analysis The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).\nExploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].\nAfter using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].\nThe data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).\nExploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].\nAfter using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].",
"The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).\nExploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].\nAfter using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].",
"Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).\n\nTable 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories\nCharacteristics\n\nDoctors\n\nNurses\n\nOther Health Professionals\n\nOverall Sample\n\nN = 803\n\n%\n\nN = 1,736\n\n%\n\nN = 739\n\n%\n\nN = 3,278\n\n%\n\nGender\nMale29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%\nAge\n< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%\nMarital\nMarried38547.95%1,1767.40%49967.52%2,05462.66%\nStatus\nSingle39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%\nLevel of\nCompulsory00.00%70.40%435.82%501.53%\nEducation\nSecondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%\nEmployment\nPermanent42552.93%1,5991.59%64086.60%2,65580.99%\nStatus\nTemporary37847.07%1468.41%9913.40%62319.01%\nProfessional\n< 5 years29036.11%22112.73%12416.78%63519.37%\nExperience\n6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%\nEconomic Situation\nI cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%\n\nSociodemographic characteristics of the sample per professional category",
"The Kolmogorov-Smirnov and Shapiro-Wilk normality tests were performed and showed that the data was not normally distributed (p < 0.05).",
"Descriptive statistics for the items of the questionnaire are shown in Table 5. The results indicate that the minimum value of the items is 1 while the maximum is 6.\n\nTable 5Surveys questions and descriptive statistics (N = 3,278)Itemno.Survey QuestionsMin.Max.MeanMedianPercentilesStd.DeviationVarianceStatisticSum\nStatistic\n\nStd. Error\n\n25\n\n50\n\n75\nJS 1I feel I am being paid a fair amount for the work I do162.460.0212.002.002.003.001.1931.4248049JS 2There is really too little chance for promotion on my job162.720.0223.002.003.003.001.2601.5878905JS 3My supervisor is quite competent in doing his/her job164.770.0185.004.005.005.001.0501.10315,649JS 4I am not satisfied with the benefits I receive162.930.0213.002.003.004.001.2021.4449598JS 5When I do a good job, I receive the recognition for it that I should receive163.260.0223.002.003.004.001.2841.64910,678JS 6Many of our rules and procedures make doing a good job difficult162.710.0193.002.003.003.001.0651.1358870JS 7I like the people I work with165.100.0135.005.005.006.000.7330.53816,717JS 8I sometimes feel my job is meaningless163.900.0234.003.004.005.001.3441.80612,790JS 9Communications seem good within this organization164.000.0194.004.004.005.001.0971.20413,114JS 10Raises are too few and far between161.730.0191.001.001.002.001.0701.1445657JS 11Those who do well on the job stand a fair chance of being promoted162.430.0212.001.002.003.001.2301.5127956JS 12My supervisor is unfair to me164.710.0205.004.005.005.001.1331.28415,448JS 13The benefits we receive are as good as most other organizations offer162.280.0202.001.002.003.001.1541.3327488JS 14I do not feel that the work I do is appreciated162.890.0223.002.003.004.001.2371.5309483JS 15My efforts to do a good job are seldom blocked by red tape163.450.0234.003.004.004.001.2921.67011,308JS 16I find I have to work harder at my job because of the incompetence of people I work with163.800.0224.003.004.005.001.2761.62912,458JS 17I like doing the things I do at work164.870.0155.005.005.005.000.8770.76915,979JS 18The goals of this organization are not clear to me163.150.0213.002.003.004.001.1901.41510,311JS 19I feel unappreciated by the organization when I think about what they pay me162.330.0202.002.002.003.001.1641.3567636JS 20People get ahead as fast here as they do in other places162.300.0202.001.002.003.001.1481.3187547JS 21My supervisor shows too little interest in the feelings of subordinates164.290.0224.004.004.005.001.2691.61014,049JS 22The benefit package we have is equitable162.790.0213.002.003.004.001.1751.3809137JS 23There are few rewards for those who work here162.720.0213.002.003.003.001.1921.4228909JS 24I have too much to do at work162.140.0162.002.002.003.000.8970.8057001JS 25I enjoy my coworkers164.810.0165.004.005.005.000.8980.80715,760JS 26I often feel that I do not know what is going on with the organization163.820.0224.003.004.005.001.2581.58212,532JS 27I feel a sense of pride in doing my job164.820.0185.004.005.005.001.0131.02715,802JS 28I feel satisfied with my chances for salary increases161.980.0192.001.002.002.001.1071.2266486JS 29There are benefits we do not have which we should have162.670.0213.002.003.003.001.1891.4148736JS 30I like my supervisor164.860.0165.004.005.005.000.9360.87615,941JS 31I have too much paperwork162.970.0233.002.003.004.001.2931.6719742JS 32I don’t feel my efforts are rewarded the way they should be162.790.0203.002.003.004.001.1671.3629132JS 33I am satisfied with my chances for promotion162.350.0212.001.002.003.001.1761.3837715JS 34There is too much bickering and fighting at work163.310.0203.003.003.004.001.1661.36110,855JS 35My job is enjoyable163.750.0214.003.004.004.001.1811.39512,293JS 36Work assignments are not fully explained164.170.0214.004.004.005.001.1871.41013,673\n\nSurveys questions and descriptive statistics (N = 3,278)\nThe highest mean values were found for Item–7 and Item–17 while the lowest ones for Item–10 and Item–28. The average variability of the items around mean values was relatively small.",
"According to the analysis result, the KMO (Kaiser-Meyer-Olkin) statistic of 0.912 confirmed that the sample used was quite sufficient. We can therefore be confident that the factor analysis fits into our data set. Next, Barlett’s test of sphericity (χ2 = 31831.572, df = 528, p = 0.000) demonstrated that the correlation matrix is not an identity matrix, therefore providing justification for the use of factor analysis [37, 44]. In PCA the eigenvalue provides the fraction of the variation accounted for by the corresponding component (eigenvector). We adopted a combined criteria method as suggested by Lings and Greenley [45], and Larose [46] to identify items and factors for inclusion in the final factorial solution. More specifically, to evaluate the factor structures, we used four criteria. First, items factor loadings should be at least equal to or greater than 0.5. Second, a scale should have more than two items or if it has only two they should be strongly correlated. Third, if an item loads more than one dimension and their difference is lower than 0.02, it will be deleted. Moreover, the difference in loadings, equal to or greater than 0.2, implies the item’s inclusion in the dimension with the highest factor load. Finally, in order to maintain an item, it would also have to conceptually match the factor [47–49].\nBased on an eigenvalue greater than one, as one eigenvalue represents a significant amount of variation, factors considered in subsequent analyses. Hence, another eigenvalue-based approach was used to examine Cattell’s “scree” plot, by looking for a spot in the plot where it abruptly levels out. By employing both methods, a six-factor model was identified (see Table 6) [50]. The final factorial structure explains 56.23% of the total variance of the dataset. According to the results obtained, the first factor had 23.78% of the total variance, the second factor 11.52%, the third factor 6.64%, the fourth factor 6.30%, the fifth factor 4.17%, and the sixth factor 3.81%. The total variance explanatory rates of the factors after rotation were as follows: 14.13%, 10.53%, 10.49%, 8.19%. 6.92% and 5.97%.\n\nTable 6Eigenvalues and the explained total variance of the extracted factorsInitial EigenvaluesExtraction Sums of Squared LoadingsRotation Sums of Squared LoadingsComponentTotal% of VarianceCumulative %Total% of VarianceCumulative %Total% of VarianceCumulative %16.1823.7823.786.1823.7823.783.6714.1314.1323.0011.5235.313.0011.5235.312.7410.5324.6631.736.6441.941.736.6441.942.7310.4935.1441.646.3048.241.646.3048.242.138.1943.3451.094.1752.411.094.1752.411.806.9250.2661.003.8156.231.003.8156.231.555.9756.23Extraction Method: Principal Component Analysis\n\nEigenvalues and the explained total variance of the extracted factors\nExtraction Method: Principal Component Analysis\nVarimax rotation was used for the rotation of the original solution as our sampling has a heterogeneous population [51, 52]. Twenty variables were included within six factors. The resulting six factors were: Factor 1 which indicates employees’ benefits and salary includes items: 11, 20, 28, 33. Factor 2 represents the management’s attitude and includes items: 14, 19, 24, 29. Factor 3 supervision and includes items: 3, 12, 21, 30. Factor 4 represents employees’ communication, includes items: 18, 26 and 36. Factor 5 mainly indicates the nature of work and includes items: 17, 27,35 and finally Factor 6 consists colleagues support and includes items: 7 and 25 (Table 7).\n\nTable 7Standardized loadings of items for each factorFactor 1Factor 2Factor 3Factor 4Factor 5Factor 6\nItem\n\nno.\n\nQuestions\n\nDimensions\n\nBenefits and Salary\n\nManagement’s attitude\n\nSupervision\n\nCommunication\n\nNature of work\n\nColleagues Support\nJS 33I am satisfied with my chances for promotionPromotion0.83JS 28I feel satisfied with my chances for salary increasesPay0.77JS 20People get ahead as fast here as they do in other placesPromotion0.76JS 11Those who do well on the job stand a fair chance of being promotedPromotion0.72JS 24I have too much to do at workOperating procedures0.83JS 29There are benefits we do not have which we should haveFringe Benefits0.70JS 19I feel unappreciated by the organization when I think about what they pay mePay0.56JS 14I do not feel that the work I do is appreciatedContingent rewards0.54JS 3My supervisor is quite competent in doing his/her jobSupervision0.83JS 30I like my supervisorSupervision0.81JS 21My supervisor shows too little interest in the feelings of subordinatesSupervision0.77JS 12My supervisor is unfair to meSupervision0.74JS 26I often feel that I do not know what is going on with the organizationCommunication0.78JS 18The goals of this organization are not clear to meCommunication0.68JS 36Work assignments are not fully explainedCommunication0.67JS 17I like doing the things I do at workNature of work0.81JS 27I feel a sense of pride in doing my jobNature of work0.75JS 35My job is enjoyableNature of work0.63JS 7I like the people I work withCoworkers0.82JS 25I enjoy my coworkersCoworkers0.81Notes: (1) The weights of extracted factors from exploratory factor analysis with varimax rotation (weights less than 0.4 are not displayed). (2) Extraction Method: Principal Component Analysis. (3) Factor loading > 0.5\n\nStandardized loadings of items for each factor\n\nItem\n\n\nno.\n\nNotes: (1) The weights of extracted factors from exploratory factor analysis with varimax rotation (weights less than 0.4 are not displayed). (2) Extraction Method: Principal Component Analysis. (3) Factor loading > 0.5\nThe reliability coefficient Cronbach’s alpha of new construction of scales after application of factor analysis for the overall scale was 0.81 and we concluded that the questionnaire has very good reliability. The results showed that obtained reliability figures (Alphas) range from 0.60 to 0.81 for the various job satisfaction dimensions. These findings provide support for the internal consistency of the sub-scales, so we can state that the scale of the survey questions used in the analysis was acceptable (Table 8).\n\nTable 8Reliability analysis of scalesFactor IDFactor NameCronbach’s Alpha (CA)Overall ItemsItemsAverage Item ScoreFactor 1Benefits and Salary0.74411, 20, 28, 332.27Factor 2Management’s attitude0.67414, 19, 24, 292.51Factor 3Supervision0.8143, 12, 21, 304.66Factor 4Communication0.60318, 26, 363.71Factor 5Nature of work0.61317, 27, 354.48Factor 6Colleagues Support0.6627, 254.96\nOverall Satisfaction\n\n0.81\n\n20\n3.76\n\nReliability analysis of scales",
"Confirmatory Factor Analysis (CFA) is a statistical technique used to evaluate the measurement models that represent hypotheses about relations between indicators and factors. The CFA assessed the fit of the six-factor structure and the model fitted the data well as defined from the SRMR, RMSEA, CFI and IFI values that were equal to 0.050 (≤ 0.800), 0.055 (≤ 0.800), 0.906 (≥ 0.900) and 0.906 (≥ 0.900) respectively. It was suggested that the fitting optimization index was acceptable and the structure of the model was designed reasonably (Fig. 1).\n\nFig. 1Result of confirmatory factor analysis (CFA)\n\nResult of confirmatory factor analysis (CFA)"
] | [
null,
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null,
null,
null,
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null,
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] | [
"Introduction",
"Measurement of job satisfaction",
"Objectives",
"Materials and methods",
"Research instrument translation and adaptation",
"Research design and procedure",
"Statistical analysis",
"Results",
"Study sample",
"Normality analysis",
"Descriptive statistics results",
"Exploratory factor analysis",
"Confirmatory factor analysis",
"Discussion",
"Conclusion"
] | [
"As employee knowledge and skills are intangible assets of any service organization,\nemployee satisfaction has become an issue of utmost importance. It has been defined as the positive emotional state resulting from the evaluation of one’s work or work experience [1]. Hoppock [2] was the first who brought forth the concept of job satisfaction in limelight and described it as “the employees’ subjective reflections or subjective feelings about their working conditions and working environment”. Since then, many researchers have recognized that satisfied employees are a key asset to an organization [3]. While the importance of job satisfaction is generally recognized, additional and ongoing investigations of satisfaction levels are necessary as external conditions and societal values are constantly changing. In this respect, job satisfaction has a significant role in the operation and performance of organizations.\nAn essential prerequisite for the development and long-term success of an organization is in fact the utilization of employee’s capabilities and the improvement of their working conditions [4]. The degree of job satisfaction is actually the overall level of satisfaction on a number of different dimensions of work and affects the behavior of employees that, in turn, impacts upon organizational functioning [5–7]. Swamy et al. [3] stated that satisfied employees are the key asset of an organization. Therefore, the issue of job satisfaction is very important especially for non-profit public organizations like hospitals, which are essential for a country’s provision of healthcare services and the population itself.\nEmployee satisfaction also affects patient satisfaction. As patients are the external customers and employees are the internal customers of the organization they form the current working environment and are willing to cooperate with the community to achieve organizational goals. Previous studies have documented associations between job fulfillment of health workforce and patient contentment with the type of health care services provided in health care facilities [8, 9]. Moreover, there seems to exist a positive correlation between the increase in job satisfaction and quality of care [10, 11]. Conversely, a low level of job satisfaction would create negative behaviors, including absenteeism, grievances, high level of stress, turnover, exhaustion, low morality, worse patient-provider ratios, longer wait times, psychological distress and increased medical errors [12–14].\nHospital managers have responsibilities to both patients and staff. It has been suggested that if you want to attain higher job productivity and efficiency, you should comprehend the domains of work which are decisive for job satisfaction amongst healthcare providers. In order to get employees contented with their job; the underlying factors that influence job satisfaction in that particular facility must be examined to guide proper managerial action [15, 16].",
"Due to its importance, a wide range of instruments have been designed to quantify and conceptualize job satisfaction during the past decades. They were developed to capture the entirety of various aspects of job satisfaction be it personal, social, environmental, organizational, and the nature of the job itself. A valuable and widely used measure of job satisfaction is the Job Satisfaction Survey (JSS) that was originally developed by Spector [17]. JSS provides sufficient reliability and validity and is available for researchers free of charge for use for non-commercial purposes. The instrument contains 36 items expressed on a Likert scale measuring nine dimensions of job satisfaction, as mentioned below:\n\nPay includes salaries and wages. Unfair distribution can negatively affect employees’ emotions and therefore their behavior in the organization [18].Promotion is an important aspect of a employee’s career. It refers to progression to a higher position with more challenges, authority and responsibilities [19]. Only a meritocratic promotion system with evaluation conditions known in advance can lead to satisfaction.Fringe Benefits, can be financial or non-financial compensations. Financial compensations consist of direct (e.g. bonuses) and indirect compensation (e.g. retirement plans). Non-financial compensations consist of the job itself (e.g. autonomy), job environment (e.g. working conditions) and workplace flexibility (e.g. part-time work) [20].Contingent Rewards, are referred to as promises and exchanges of rewards and recognition for good work. Is a valuable tool for motivating employees because they want to be paid well for the job they perform both for their self-esteem and as useful means of a living [21].Supervision, is defined as the perception of employees regarding the support received from supervisors in an organization besides coworkers. Usually, employees are satisfied when they are supported to achieve their goals [22].Operating Procedures, are described as steps of finishing tasks that have to follow a certain standard based on regulations, provincial laws, policies, procedures and standards. Inadequacy of equipment and resources, lighting, ventilation, and cleanliness can result in a stressful work environment that leads to job dissatisfaction among employees [23].Co-workers, are referred as people working in an organization (besides supervisors). Employees with the same values, attitudes and philosophies can improve satisfaction in an organization [24]. Support from colleagues can enhance job satisfaction and decrease job stress and burnout.Nature of Work, is defined as the variability of the given work. It refers to the daily and non-daily tasks carried out as part of the job scope and includes job challenges, feedback, autonomy, and skill variety [25]. Further, this can increase the motivational level of employees which will ultimately raise their internal happiness of employees, and the internal happiness will cause satisfaction.Communication, is referred as informing the current employees. Communication between supervisors or the managerial level with employees consistently enables managers to know whether their staff is satisfied and happy with its employment or not [26]. There is a positive association between communication and job satisfaction. Effective communication at the workplace is essential in ensuring organizational objectives, social support.\n\nPay includes salaries and wages. Unfair distribution can negatively affect employees’ emotions and therefore their behavior in the organization [18].\nPromotion is an important aspect of a employee’s career. It refers to progression to a higher position with more challenges, authority and responsibilities [19]. Only a meritocratic promotion system with evaluation conditions known in advance can lead to satisfaction.\nFringe Benefits, can be financial or non-financial compensations. Financial compensations consist of direct (e.g. bonuses) and indirect compensation (e.g. retirement plans). Non-financial compensations consist of the job itself (e.g. autonomy), job environment (e.g. working conditions) and workplace flexibility (e.g. part-time work) [20].\nContingent Rewards, are referred to as promises and exchanges of rewards and recognition for good work. Is a valuable tool for motivating employees because they want to be paid well for the job they perform both for their self-esteem and as useful means of a living [21].\nSupervision, is defined as the perception of employees regarding the support received from supervisors in an organization besides coworkers. Usually, employees are satisfied when they are supported to achieve their goals [22].\nOperating Procedures, are described as steps of finishing tasks that have to follow a certain standard based on regulations, provincial laws, policies, procedures and standards. Inadequacy of equipment and resources, lighting, ventilation, and cleanliness can result in a stressful work environment that leads to job dissatisfaction among employees [23].\nCo-workers, are referred as people working in an organization (besides supervisors). Employees with the same values, attitudes and philosophies can improve satisfaction in an organization [24]. Support from colleagues can enhance job satisfaction and decrease job stress and burnout.\nNature of Work, is defined as the variability of the given work. It refers to the daily and non-daily tasks carried out as part of the job scope and includes job challenges, feedback, autonomy, and skill variety [25]. Further, this can increase the motivational level of employees which will ultimately raise their internal happiness of employees, and the internal happiness will cause satisfaction.\nCommunication, is referred as informing the current employees. Communication between supervisors or the managerial level with employees consistently enables managers to know whether their staff is satisfied and happy with its employment or not [26]. There is a positive association between communication and job satisfaction. Effective communication at the workplace is essential in ensuring organizational objectives, social support.\nEvery dimension incorporates four items. Several previous studies have shown that JSS has high internal consistency and validity [27, 28].",
"This research aimed to explore (a) the underlying factorial structure of the JSS when applied to Greek hospital employees, (b) its psychometric properties. Undoubtedly, job satisfaction is a complex concept, so there is always a need to research this phenomenon and related factors to explore the development of optimal human resources strategies in the context of healthcare institutions. Moreover, there is a compelling need for developing constructs in the field of management rather than adapting the constructs that have been developed already.",
" Research instrument translation and adaptation The JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.\nThe reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].\n\nTable 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\n\nJob Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha\n* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\nPrior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].\nAdditionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).\n\nTable 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77\n\nSplit-Half reliability analysis\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.\nThe items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.\nThe JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.\nThe reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].\n\nTable 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\n\nJob Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha\n* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\nPrior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].\nAdditionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).\n\nTable 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77\n\nSplit-Half reliability analysis\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.\nThe items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.",
"The JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.\nThe reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].\n\nTable 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\n\nJob Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha\n* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)\nPrior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].\nAdditionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).\n\nTable 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77\n\nSplit-Half reliability analysis\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.\n\nThe items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.\nThe items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.",
"The survey was carried out in the region of Attiki with its capital Athens, with around 3.75 million inhabitants or approximately 35% of the total Greek population. The 1st Regional Health Authority of Attica has the responsibility for 27 public hospitals. Our survey was conducted between July 2019 and December 2020 in thirteen of those who provided healthcare services to 438,745 patients. The main criteria for the selection of these hospitals were (Table 3): (a) the categories of hospitals; for this reason, the survey was introduced into four different categories (general, pediatric, maternity, oncology), (b) a large number of different clinics, (c) hospitals with a large number of beds but without ignoring the role of smaller hospitals, (d) the large number of patients treated in these hospitals, (e) the large number of health care employees who work in these hospitals, and (f) the necessary approval of the research by hospital committees.\nThe researchers distributed the printed questionnaire along with a consent form to the participants in person at their workplaces. They were adults (over 18 years), health care professionals belonging to medical, nursing, administrative, and technical departments serving public healthcare. The main aim of selecting employees from various fields is to get the opinions of a diverse group of people so that the results can be generalized on s vast group of the overall population. They had worked for more than six months in the respective hospital facilities at the time of the research and consented to the study. The study excluded interns, volunteers, and those declining to consent to the study. The participants had one week to complete the questionnaire. All employees had the right to refuse or discontinue their participation in the survey at any time. The researcher guaranteed the anonymity and confidentiality of all data collected. We remained considerate of the names, safety, and well-being of participants, and also the organizations remained anonymous by using codes, such as H01, H02, and so on (Table 3). Finally, of the 4,000 questionnaires distributed, 3,278 (81.95%) were returned.\n\nTable 3The population of research per hospital and category. Distributed Questionnaires and Response Rate\nPeriod of research\n\nProfessional Category\n\n2019\n\n2020\n\nDoctors\n\nNurses\n\nOther Health Professionals\n\nOverall Sample\n\nNumber\n\nNumber\n\nDays of\n\nHospital\n\n3Q\n\n4Q\n\n1Q\n\n2Q\n\n3Q\n\n4Q\n\n( n )\n\n( % )\n\n( n )\n\n( % )\n\n( n )\n\n( % )\n\n( n )\n\n( % )\n\nοf Beds*\n\nοf Patients*\n\nHospitalization*\nH 01XXXXXX10813.45%1327.60%435.82%2838.64%H 0216820.92%29016.71%15821.38%61618.80%H 03XXXXXX293.61%995.70%7410.01%2026.17%H 049011.21%21012.10%648.66%36411.11%H 05XXXXXX597.35%895.13%628.39%2106.41%H 06516.35%1227.03%699.34%2427.39%H 07XXXXXX617.60%18110.43%395.28%2818.58%H 088710.83%905.18%324.33%2096.38%H 09XXXXXX364.48%1196.85%577.71%2126.47%H 10455.60%1116.39%506.77%2066.29%H 11XXXXXX293.61%1367.83%364.87%2016.14%H 12323.99%1327.60%385.14%2026.17%H 13XXXXXX81.00%251.44%172.30%501.53%[1] Hospitals of research(n = 13)80324.50%1,73652.96%73922.54%3,278100%6,511438,7451,443,660[2] Hospitals in 1st Regional Health Authority of Attica(n = 27)6,277**31.71%8,174**41.29%5,345**27.00%19,796**100%8,860567,8171,912,488[3] Hospitals in the National Health System(n = 127)36,4412,160,5967,343,348[4] Percentages of Hospitals of research / Hospitals of Attica= [1] / [2]12.79%21.24%13.83%16.56%73.49%77.27%[5] Percentages of Hospitals of research / NHS= [1] / [3]17.87%20.31%19.66%[6] Distributed Questionnaires of research1,00025.00%2,00050.00%1,00025.00%4,000100%[7] Response Rate= [1] / [6]80.30%86.80%73.90%81.95%Notes: * Data of year 2020 ** Data of year 2016X = research conduct\n\nThe population of research per hospital and category. Distributed Questionnaires and Response Rate\nNotes: * Data of year 2020 ** Data of year 2016\nX = research conduct\n Statistical analysis The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).\nExploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].\nAfter using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].\nThe data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).\nExploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].\nAfter using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].",
"The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).\nExploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].\nAfter using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].",
" Study sample Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).\n\nTable 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories\nCharacteristics\n\nDoctors\n\nNurses\n\nOther Health Professionals\n\nOverall Sample\n\nN = 803\n\n%\n\nN = 1,736\n\n%\n\nN = 739\n\n%\n\nN = 3,278\n\n%\n\nGender\nMale29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%\nAge\n< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%\nMarital\nMarried38547.95%1,1767.40%49967.52%2,05462.66%\nStatus\nSingle39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%\nLevel of\nCompulsory00.00%70.40%435.82%501.53%\nEducation\nSecondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%\nEmployment\nPermanent42552.93%1,5991.59%64086.60%2,65580.99%\nStatus\nTemporary37847.07%1468.41%9913.40%62319.01%\nProfessional\n< 5 years29036.11%22112.73%12416.78%63519.37%\nExperience\n6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%\nEconomic Situation\nI cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%\n\nSociodemographic characteristics of the sample per professional category\nAmong the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).\n\nTable 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories\nCharacteristics\n\nDoctors\n\nNurses\n\nOther Health Professionals\n\nOverall Sample\n\nN = 803\n\n%\n\nN = 1,736\n\n%\n\nN = 739\n\n%\n\nN = 3,278\n\n%\n\nGender\nMale29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%\nAge\n< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%\nMarital\nMarried38547.95%1,1767.40%49967.52%2,05462.66%\nStatus\nSingle39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%\nLevel of\nCompulsory00.00%70.40%435.82%501.53%\nEducation\nSecondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%\nEmployment\nPermanent42552.93%1,5991.59%64086.60%2,65580.99%\nStatus\nTemporary37847.07%1468.41%9913.40%62319.01%\nProfessional\n< 5 years29036.11%22112.73%12416.78%63519.37%\nExperience\n6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%\nEconomic Situation\nI cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%\n\nSociodemographic characteristics of the sample per professional category",
"Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).\n\nTable 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories\nCharacteristics\n\nDoctors\n\nNurses\n\nOther Health Professionals\n\nOverall Sample\n\nN = 803\n\n%\n\nN = 1,736\n\n%\n\nN = 739\n\n%\n\nN = 3,278\n\n%\n\nGender\nMale29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%\nAge\n< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%\nMarital\nMarried38547.95%1,1767.40%49967.52%2,05462.66%\nStatus\nSingle39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%\nLevel of\nCompulsory00.00%70.40%435.82%501.53%\nEducation\nSecondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%\nEmployment\nPermanent42552.93%1,5991.59%64086.60%2,65580.99%\nStatus\nTemporary37847.07%1468.41%9913.40%62319.01%\nProfessional\n< 5 years29036.11%22112.73%12416.78%63519.37%\nExperience\n6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%\nEconomic Situation\nI cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%\n\nSociodemographic characteristics of the sample per professional category",
"The Kolmogorov-Smirnov and Shapiro-Wilk normality tests were performed and showed that the data was not normally distributed (p < 0.05).",
"Descriptive statistics for the items of the questionnaire are shown in Table 5. The results indicate that the minimum value of the items is 1 while the maximum is 6.\n\nTable 5Surveys questions and descriptive statistics (N = 3,278)Itemno.Survey QuestionsMin.Max.MeanMedianPercentilesStd.DeviationVarianceStatisticSum\nStatistic\n\nStd. Error\n\n25\n\n50\n\n75\nJS 1I feel I am being paid a fair amount for the work I do162.460.0212.002.002.003.001.1931.4248049JS 2There is really too little chance for promotion on my job162.720.0223.002.003.003.001.2601.5878905JS 3My supervisor is quite competent in doing his/her job164.770.0185.004.005.005.001.0501.10315,649JS 4I am not satisfied with the benefits I receive162.930.0213.002.003.004.001.2021.4449598JS 5When I do a good job, I receive the recognition for it that I should receive163.260.0223.002.003.004.001.2841.64910,678JS 6Many of our rules and procedures make doing a good job difficult162.710.0193.002.003.003.001.0651.1358870JS 7I like the people I work with165.100.0135.005.005.006.000.7330.53816,717JS 8I sometimes feel my job is meaningless163.900.0234.003.004.005.001.3441.80612,790JS 9Communications seem good within this organization164.000.0194.004.004.005.001.0971.20413,114JS 10Raises are too few and far between161.730.0191.001.001.002.001.0701.1445657JS 11Those who do well on the job stand a fair chance of being promoted162.430.0212.001.002.003.001.2301.5127956JS 12My supervisor is unfair to me164.710.0205.004.005.005.001.1331.28415,448JS 13The benefits we receive are as good as most other organizations offer162.280.0202.001.002.003.001.1541.3327488JS 14I do not feel that the work I do is appreciated162.890.0223.002.003.004.001.2371.5309483JS 15My efforts to do a good job are seldom blocked by red tape163.450.0234.003.004.004.001.2921.67011,308JS 16I find I have to work harder at my job because of the incompetence of people I work with163.800.0224.003.004.005.001.2761.62912,458JS 17I like doing the things I do at work164.870.0155.005.005.005.000.8770.76915,979JS 18The goals of this organization are not clear to me163.150.0213.002.003.004.001.1901.41510,311JS 19I feel unappreciated by the organization when I think about what they pay me162.330.0202.002.002.003.001.1641.3567636JS 20People get ahead as fast here as they do in other places162.300.0202.001.002.003.001.1481.3187547JS 21My supervisor shows too little interest in the feelings of subordinates164.290.0224.004.004.005.001.2691.61014,049JS 22The benefit package we have is equitable162.790.0213.002.003.004.001.1751.3809137JS 23There are few rewards for those who work here162.720.0213.002.003.003.001.1921.4228909JS 24I have too much to do at work162.140.0162.002.002.003.000.8970.8057001JS 25I enjoy my coworkers164.810.0165.004.005.005.000.8980.80715,760JS 26I often feel that I do not know what is going on with the organization163.820.0224.003.004.005.001.2581.58212,532JS 27I feel a sense of pride in doing my job164.820.0185.004.005.005.001.0131.02715,802JS 28I feel satisfied with my chances for salary increases161.980.0192.001.002.002.001.1071.2266486JS 29There are benefits we do not have which we should have162.670.0213.002.003.003.001.1891.4148736JS 30I like my supervisor164.860.0165.004.005.005.000.9360.87615,941JS 31I have too much paperwork162.970.0233.002.003.004.001.2931.6719742JS 32I don’t feel my efforts are rewarded the way they should be162.790.0203.002.003.004.001.1671.3629132JS 33I am satisfied with my chances for promotion162.350.0212.001.002.003.001.1761.3837715JS 34There is too much bickering and fighting at work163.310.0203.003.003.004.001.1661.36110,855JS 35My job is enjoyable163.750.0214.003.004.004.001.1811.39512,293JS 36Work assignments are not fully explained164.170.0214.004.004.005.001.1871.41013,673\n\nSurveys questions and descriptive statistics (N = 3,278)\nThe highest mean values were found for Item–7 and Item–17 while the lowest ones for Item–10 and Item–28. The average variability of the items around mean values was relatively small.",
"According to the analysis result, the KMO (Kaiser-Meyer-Olkin) statistic of 0.912 confirmed that the sample used was quite sufficient. We can therefore be confident that the factor analysis fits into our data set. Next, Barlett’s test of sphericity (χ2 = 31831.572, df = 528, p = 0.000) demonstrated that the correlation matrix is not an identity matrix, therefore providing justification for the use of factor analysis [37, 44]. In PCA the eigenvalue provides the fraction of the variation accounted for by the corresponding component (eigenvector). We adopted a combined criteria method as suggested by Lings and Greenley [45], and Larose [46] to identify items and factors for inclusion in the final factorial solution. More specifically, to evaluate the factor structures, we used four criteria. First, items factor loadings should be at least equal to or greater than 0.5. Second, a scale should have more than two items or if it has only two they should be strongly correlated. Third, if an item loads more than one dimension and their difference is lower than 0.02, it will be deleted. Moreover, the difference in loadings, equal to or greater than 0.2, implies the item’s inclusion in the dimension with the highest factor load. Finally, in order to maintain an item, it would also have to conceptually match the factor [47–49].\nBased on an eigenvalue greater than one, as one eigenvalue represents a significant amount of variation, factors considered in subsequent analyses. Hence, another eigenvalue-based approach was used to examine Cattell’s “scree” plot, by looking for a spot in the plot where it abruptly levels out. By employing both methods, a six-factor model was identified (see Table 6) [50]. The final factorial structure explains 56.23% of the total variance of the dataset. According to the results obtained, the first factor had 23.78% of the total variance, the second factor 11.52%, the third factor 6.64%, the fourth factor 6.30%, the fifth factor 4.17%, and the sixth factor 3.81%. The total variance explanatory rates of the factors after rotation were as follows: 14.13%, 10.53%, 10.49%, 8.19%. 6.92% and 5.97%.\n\nTable 6Eigenvalues and the explained total variance of the extracted factorsInitial EigenvaluesExtraction Sums of Squared LoadingsRotation Sums of Squared LoadingsComponentTotal% of VarianceCumulative %Total% of VarianceCumulative %Total% of VarianceCumulative %16.1823.7823.786.1823.7823.783.6714.1314.1323.0011.5235.313.0011.5235.312.7410.5324.6631.736.6441.941.736.6441.942.7310.4935.1441.646.3048.241.646.3048.242.138.1943.3451.094.1752.411.094.1752.411.806.9250.2661.003.8156.231.003.8156.231.555.9756.23Extraction Method: Principal Component Analysis\n\nEigenvalues and the explained total variance of the extracted factors\nExtraction Method: Principal Component Analysis\nVarimax rotation was used for the rotation of the original solution as our sampling has a heterogeneous population [51, 52]. Twenty variables were included within six factors. The resulting six factors were: Factor 1 which indicates employees’ benefits and salary includes items: 11, 20, 28, 33. Factor 2 represents the management’s attitude and includes items: 14, 19, 24, 29. Factor 3 supervision and includes items: 3, 12, 21, 30. Factor 4 represents employees’ communication, includes items: 18, 26 and 36. Factor 5 mainly indicates the nature of work and includes items: 17, 27,35 and finally Factor 6 consists colleagues support and includes items: 7 and 25 (Table 7).\n\nTable 7Standardized loadings of items for each factorFactor 1Factor 2Factor 3Factor 4Factor 5Factor 6\nItem\n\nno.\n\nQuestions\n\nDimensions\n\nBenefits and Salary\n\nManagement’s attitude\n\nSupervision\n\nCommunication\n\nNature of work\n\nColleagues Support\nJS 33I am satisfied with my chances for promotionPromotion0.83JS 28I feel satisfied with my chances for salary increasesPay0.77JS 20People get ahead as fast here as they do in other placesPromotion0.76JS 11Those who do well on the job stand a fair chance of being promotedPromotion0.72JS 24I have too much to do at workOperating procedures0.83JS 29There are benefits we do not have which we should haveFringe Benefits0.70JS 19I feel unappreciated by the organization when I think about what they pay mePay0.56JS 14I do not feel that the work I do is appreciatedContingent rewards0.54JS 3My supervisor is quite competent in doing his/her jobSupervision0.83JS 30I like my supervisorSupervision0.81JS 21My supervisor shows too little interest in the feelings of subordinatesSupervision0.77JS 12My supervisor is unfair to meSupervision0.74JS 26I often feel that I do not know what is going on with the organizationCommunication0.78JS 18The goals of this organization are not clear to meCommunication0.68JS 36Work assignments are not fully explainedCommunication0.67JS 17I like doing the things I do at workNature of work0.81JS 27I feel a sense of pride in doing my jobNature of work0.75JS 35My job is enjoyableNature of work0.63JS 7I like the people I work withCoworkers0.82JS 25I enjoy my coworkersCoworkers0.81Notes: (1) The weights of extracted factors from exploratory factor analysis with varimax rotation (weights less than 0.4 are not displayed). (2) Extraction Method: Principal Component Analysis. (3) Factor loading > 0.5\n\nStandardized loadings of items for each factor\n\nItem\n\n\nno.\n\nNotes: (1) The weights of extracted factors from exploratory factor analysis with varimax rotation (weights less than 0.4 are not displayed). (2) Extraction Method: Principal Component Analysis. (3) Factor loading > 0.5\nThe reliability coefficient Cronbach’s alpha of new construction of scales after application of factor analysis for the overall scale was 0.81 and we concluded that the questionnaire has very good reliability. The results showed that obtained reliability figures (Alphas) range from 0.60 to 0.81 for the various job satisfaction dimensions. These findings provide support for the internal consistency of the sub-scales, so we can state that the scale of the survey questions used in the analysis was acceptable (Table 8).\n\nTable 8Reliability analysis of scalesFactor IDFactor NameCronbach’s Alpha (CA)Overall ItemsItemsAverage Item ScoreFactor 1Benefits and Salary0.74411, 20, 28, 332.27Factor 2Management’s attitude0.67414, 19, 24, 292.51Factor 3Supervision0.8143, 12, 21, 304.66Factor 4Communication0.60318, 26, 363.71Factor 5Nature of work0.61317, 27, 354.48Factor 6Colleagues Support0.6627, 254.96\nOverall Satisfaction\n\n0.81\n\n20\n3.76\n\nReliability analysis of scales",
"Confirmatory Factor Analysis (CFA) is a statistical technique used to evaluate the measurement models that represent hypotheses about relations between indicators and factors. The CFA assessed the fit of the six-factor structure and the model fitted the data well as defined from the SRMR, RMSEA, CFI and IFI values that were equal to 0.050 (≤ 0.800), 0.055 (≤ 0.800), 0.906 (≥ 0.900) and 0.906 (≥ 0.900) respectively. It was suggested that the fitting optimization index was acceptable and the structure of the model was designed reasonably (Fig. 1).\n\nFig. 1Result of confirmatory factor analysis (CFA)\n\nResult of confirmatory factor analysis (CFA)",
"To sum up the discussion, the basic purpose of this study was to validate Spector’s JSS instrument and develop a valid, short and reliable instrument that can measure employee job satisfaction for public hospitals in Athens, Greece. There were 3,278 responses received from the employees of thirteen different hospitals. Factor analysis was conducted due to anticipated dimensionality of factors that are involved in measuring job satisfaction. The values of Cronbach’s Alpha coefficients were computed in order to assess the internal consistency reliability.\nOverall, the job satisfaction scale developed in this research illustrates valid and reliable measures for assessing hospital employees’ satisfaction levels with their work. Yet in reality, job satisfaction is a complex multidimensional concept. The study is based on the premise that an organization’s intellectual capital is its most important asset. For this purpose, our survey used a personalized “bottom-up” approach, which studied the properties of employees, their behavior at the workplace, motivators, dissatisfiers, and other properties of the job environment. Satisfied human resource is the most valuable asset for high productivity, commitment, efficiency, and quality of care in a healthcare organization [53]. Aiming we get answers to basic questions: “How do employees feel in their workplace? What makes them behave in the workplace the way they do? What would motivate them to perform well and according to the hospital’s goals?“ The employees are motivated (or not) to perform as they do because of a combination of internal and external factors, which should be investigated, measured and improved as much as possible.\nThe statistical analyses identified six predominant components to quantify job satisfaction, namely Benefits and Salary (F1), Management’s attitude (F2), Supervision (F3), Communication (F4), Nature of work (F5), Colleagues Support (F6). Meanwhile, among the affecting factors of job satisfaction, monetary benefits have the most influence, relationships with superiors and colleagues, training and enhancement of employee skills, the perceived fairness of the promotion system, the quality of the working conditions, and a sense of belonging are vital to the development of job satisfaction.\nAn important strength of this study is that a short JSS questionnaire was developed for healthcare organizations that can be used much more widely in a rapidly changing environment. This newly developed questionnaire will prove very useful in providing continuous feedback to top management as well as health policy makers regarding the level of job satisfaction. Such feedback provided by the existing health workforce will immediately alert them to any adverse working conditions that present themselves as factors leading to job dissatisfaction.\nIn Greece, the results of this study are important in terms of determining factors that should be considered for success within organizations. This research is valuable because it has both a practical and humanitarian application, as it gives a better understanding of employee satisfaction which in turn will lead to improved organizational behavior and employee attitudes that directly affect the improvement of health quality. Gaining employee’s commitment to their organization’s goals is believed to unlock their potential and achieve heightened levels of performance. Opposite results can lead employees to dissatisfaction or tend to lose interest in their work, higher levels of burnout and stress, absenteeism, intention to quit, and consequently suboptimal healthcare delivery and poor clinical outcomes [54]. Managers of health services organizations in cooperation with the Ministry of Health (MoH) must elicit cooperation and performance of the employees to ensure the quality of care and the morbidities and mortalities may be improved undoubtedly. Most researchers agree that employees with high job satisfaction levels have improved mental and physical health, job involvement, and improved quality of life. Eliciting such commitment from employees is not easy to obtain especially under uncertain or difficult working conditions [55–58].\nMore than ever, due to the globalization evolution of the Covid-19 pandemic, health systems need satisfied employees who can cope with very difficult conditions, refine health care services, and up surging the level of patient satisfaction. The study of job satisfaction is gaining more and more importance with the passage of time because of its nature and impact on society. The need to understand employee satisfaction resurfaces as everyone understands that they serve the ultimate human good, health [59].",
"In total, this study applied quantitative methods to determine factors affecting job satisfaction. So, is important in terms of determining the specific factors that should be considered for job satisfaction, organizational engagement, managerial success, and high performance within hospitals. A short 20-item study for all healthcare staff can benefit hospitals to monitor employee satisfaction across all levels without overburdening employees and analysts with multiple or fielding several non-comparable types of research.\nThe findings suggest that effective communication and support from managers or supervisors to employees or among employees themselves will reduce stress and conflicts in the workplace. Additionally, it can be recommended that employee empowerment and training, collaboration in teamwork, and a systematic approach regarding innovative types of promotional opportunity, recognition, reward, and evaluation of hospital staff can lead to better results and benefits employees, quality of patient care, and healthcare organizations. Consequently, we believe that empowerment of management, achievement, promotion and evaluation should significantly improve job satisfaction respectively. This study showed that obtained factors are aligned with the findings of the prior studies in the literature [60, 61].\nThe results of this study should not be generalized extensively since the participants of the study come from a single geographical region of the country, only in hospitals in Athens, Greece. Nevertheless, the sample cannot be characterized as homogenous due to the fact that participants were working in different departments in the hospitals, so they deal with different tasks and procedures. Therefore, the findings and related conclusions may not be able to be generalized and compared with the rest regions of the country."
] | [
"introduction",
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"materials|methods",
null,
null,
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"results",
null,
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"discussion",
"conclusion"
] | [
"Healthcare",
"Job satisfaction",
"Exploratory factor analysis",
"Principal component analysis",
"Confirmatory factor analysis",
"Greece"
] | Introduction:
As employee knowledge and skills are intangible assets of any service organization,
employee satisfaction has become an issue of utmost importance. It has been defined as the positive emotional state resulting from the evaluation of one’s work or work experience [1]. Hoppock [2] was the first who brought forth the concept of job satisfaction in limelight and described it as “the employees’ subjective reflections or subjective feelings about their working conditions and working environment”. Since then, many researchers have recognized that satisfied employees are a key asset to an organization [3]. While the importance of job satisfaction is generally recognized, additional and ongoing investigations of satisfaction levels are necessary as external conditions and societal values are constantly changing. In this respect, job satisfaction has a significant role in the operation and performance of organizations.
An essential prerequisite for the development and long-term success of an organization is in fact the utilization of employee’s capabilities and the improvement of their working conditions [4]. The degree of job satisfaction is actually the overall level of satisfaction on a number of different dimensions of work and affects the behavior of employees that, in turn, impacts upon organizational functioning [5–7]. Swamy et al. [3] stated that satisfied employees are the key asset of an organization. Therefore, the issue of job satisfaction is very important especially for non-profit public organizations like hospitals, which are essential for a country’s provision of healthcare services and the population itself.
Employee satisfaction also affects patient satisfaction. As patients are the external customers and employees are the internal customers of the organization they form the current working environment and are willing to cooperate with the community to achieve organizational goals. Previous studies have documented associations between job fulfillment of health workforce and patient contentment with the type of health care services provided in health care facilities [8, 9]. Moreover, there seems to exist a positive correlation between the increase in job satisfaction and quality of care [10, 11]. Conversely, a low level of job satisfaction would create negative behaviors, including absenteeism, grievances, high level of stress, turnover, exhaustion, low morality, worse patient-provider ratios, longer wait times, psychological distress and increased medical errors [12–14].
Hospital managers have responsibilities to both patients and staff. It has been suggested that if you want to attain higher job productivity and efficiency, you should comprehend the domains of work which are decisive for job satisfaction amongst healthcare providers. In order to get employees contented with their job; the underlying factors that influence job satisfaction in that particular facility must be examined to guide proper managerial action [15, 16].
Measurement of job satisfaction:
Due to its importance, a wide range of instruments have been designed to quantify and conceptualize job satisfaction during the past decades. They were developed to capture the entirety of various aspects of job satisfaction be it personal, social, environmental, organizational, and the nature of the job itself. A valuable and widely used measure of job satisfaction is the Job Satisfaction Survey (JSS) that was originally developed by Spector [17]. JSS provides sufficient reliability and validity and is available for researchers free of charge for use for non-commercial purposes. The instrument contains 36 items expressed on a Likert scale measuring nine dimensions of job satisfaction, as mentioned below:
Pay includes salaries and wages. Unfair distribution can negatively affect employees’ emotions and therefore their behavior in the organization [18].Promotion is an important aspect of a employee’s career. It refers to progression to a higher position with more challenges, authority and responsibilities [19]. Only a meritocratic promotion system with evaluation conditions known in advance can lead to satisfaction.Fringe Benefits, can be financial or non-financial compensations. Financial compensations consist of direct (e.g. bonuses) and indirect compensation (e.g. retirement plans). Non-financial compensations consist of the job itself (e.g. autonomy), job environment (e.g. working conditions) and workplace flexibility (e.g. part-time work) [20].Contingent Rewards, are referred to as promises and exchanges of rewards and recognition for good work. Is a valuable tool for motivating employees because they want to be paid well for the job they perform both for their self-esteem and as useful means of a living [21].Supervision, is defined as the perception of employees regarding the support received from supervisors in an organization besides coworkers. Usually, employees are satisfied when they are supported to achieve their goals [22].Operating Procedures, are described as steps of finishing tasks that have to follow a certain standard based on regulations, provincial laws, policies, procedures and standards. Inadequacy of equipment and resources, lighting, ventilation, and cleanliness can result in a stressful work environment that leads to job dissatisfaction among employees [23].Co-workers, are referred as people working in an organization (besides supervisors). Employees with the same values, attitudes and philosophies can improve satisfaction in an organization [24]. Support from colleagues can enhance job satisfaction and decrease job stress and burnout.Nature of Work, is defined as the variability of the given work. It refers to the daily and non-daily tasks carried out as part of the job scope and includes job challenges, feedback, autonomy, and skill variety [25]. Further, this can increase the motivational level of employees which will ultimately raise their internal happiness of employees, and the internal happiness will cause satisfaction.Communication, is referred as informing the current employees. Communication between supervisors or the managerial level with employees consistently enables managers to know whether their staff is satisfied and happy with its employment or not [26]. There is a positive association between communication and job satisfaction. Effective communication at the workplace is essential in ensuring organizational objectives, social support.
Pay includes salaries and wages. Unfair distribution can negatively affect employees’ emotions and therefore their behavior in the organization [18].
Promotion is an important aspect of a employee’s career. It refers to progression to a higher position with more challenges, authority and responsibilities [19]. Only a meritocratic promotion system with evaluation conditions known in advance can lead to satisfaction.
Fringe Benefits, can be financial or non-financial compensations. Financial compensations consist of direct (e.g. bonuses) and indirect compensation (e.g. retirement plans). Non-financial compensations consist of the job itself (e.g. autonomy), job environment (e.g. working conditions) and workplace flexibility (e.g. part-time work) [20].
Contingent Rewards, are referred to as promises and exchanges of rewards and recognition for good work. Is a valuable tool for motivating employees because they want to be paid well for the job they perform both for their self-esteem and as useful means of a living [21].
Supervision, is defined as the perception of employees regarding the support received from supervisors in an organization besides coworkers. Usually, employees are satisfied when they are supported to achieve their goals [22].
Operating Procedures, are described as steps of finishing tasks that have to follow a certain standard based on regulations, provincial laws, policies, procedures and standards. Inadequacy of equipment and resources, lighting, ventilation, and cleanliness can result in a stressful work environment that leads to job dissatisfaction among employees [23].
Co-workers, are referred as people working in an organization (besides supervisors). Employees with the same values, attitudes and philosophies can improve satisfaction in an organization [24]. Support from colleagues can enhance job satisfaction and decrease job stress and burnout.
Nature of Work, is defined as the variability of the given work. It refers to the daily and non-daily tasks carried out as part of the job scope and includes job challenges, feedback, autonomy, and skill variety [25]. Further, this can increase the motivational level of employees which will ultimately raise their internal happiness of employees, and the internal happiness will cause satisfaction.
Communication, is referred as informing the current employees. Communication between supervisors or the managerial level with employees consistently enables managers to know whether their staff is satisfied and happy with its employment or not [26]. There is a positive association between communication and job satisfaction. Effective communication at the workplace is essential in ensuring organizational objectives, social support.
Every dimension incorporates four items. Several previous studies have shown that JSS has high internal consistency and validity [27, 28].
Objectives:
This research aimed to explore (a) the underlying factorial structure of the JSS when applied to Greek hospital employees, (b) its psychometric properties. Undoubtedly, job satisfaction is a complex concept, so there is always a need to research this phenomenon and related factors to explore the development of optimal human resources strategies in the context of healthcare institutions. Moreover, there is a compelling need for developing constructs in the field of management rather than adapting the constructs that have been developed already.
Materials and methods:
Research instrument translation and adaptation The JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.
The reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].
Table 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)
Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha
* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)
Prior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].
Additionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).
Table 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77
Split-Half reliability analysis
The items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.
The items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.
The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.
The JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.
The reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].
Table 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)
Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha
* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)
Prior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].
Additionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).
Table 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77
Split-Half reliability analysis
The items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.
The items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.
The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.
Research instrument translation and adaptation:
The JSS has been translated in several languages and found to be valid and reliable among different categories of employees. Spector’s original JSS tool was translated into the Greek language and adapted by Tsounis and Sarafis [27] to be administered to employees of the Greek Therapy Centre for Dependent Individuals. In this context, the JSS was translated into Greek using the forward-backward translation process. Firstly, the original English of the JSS was translated into the Greek language by two experienced translators. The assessment of forwarding translation drafts was performed by two other researchers who worked independently and asked to review each translated item and choose the most adequate in terms of clarity, common language, and cultural diversity. Secondly, a retranslation of the agreed Greek text to the English language was held by a researcher who had not previously seen the original version. Thirdly, the backward translation was compared with the original version of the survey, and judgments about potential inaccuracies were made by two other researchers. Finally, the resulting differences were checked by another scientist who made the necessary adjustments.
The reliability and validity of the tool has been documented worldwide in a variety of settings. Reliability coefficients of prior and current research are presented in Table 1. The measures whose Cronbach’s Alpha exceeds 0.6 are considered to be the reliable ones and indicates an acceptable level of reliability [29–31]. Schmitt [32] has suggested that there is no general level (such as 0.7) where alpha becomes acceptable. In reality, a key feature of the alpha coefficient is that it is highly dependent on the number of items involved. Thus, if we wish to reduce the items in our survey (e.g. EFA), because of this, a small number of well-correlated items may have a fairly low alpha coefficient. Conversely, since there are more items, the value of alpha can be quite high despite the low correlation between many of these items. Addionnaly, Ursally [33] showed that important differences in the values of Cronbach Alpha are possible due to indirect influences from external factors - respondents’ age, gender, level of study, religiousness, rural/urban living, and survey type of the research subject for the participants to the survey [34, 35].
Table 1Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s AlphaDimensionsDescriptions of DimensionsItemsSpector, 1985Greek Sample Tsounis & Sarafis, 2018Present study*PayPay and remuneration1, 10, 19, 280.750.620.66PromotionPromotion opportunities2, 11, 20, 330.730.670.65SupervisionImmediate supervisor3, 12, 21, 300.820.870.81Fringe BenefitsMonetary and nonmonetary fringe benefits4, 13, 22, 290.730.730.68Contingent rewardsAppreciation, recognition and rewards for good work5, 14, 23, 320.760.710.74Operating proceduresOperating policies and procedures6, 15, 24, 310.620.480.41Co-workersPeople you work with7, 16, 25, 340.600.670.62Nature of workJob tasks themselves8, 17, 27, 350.780.740.62CommunicationCommunication within the organization9, 18, 26, 360.710.710.64Overall SatisfactionAll items0.910.870.89* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)
Job Satisfaction Survey Dimensions, Descriptions and Cronbach’s Alpha
* To calculate Cronbach’s Alpha coefficients, we took into consideration the creator’s suggestion to reverse 19 of the statements (2,4,6,8,10,12,14,16,18,19,21,23, 24,26,29,31,32,34,36)
Prior reliability analysis of the translated and adapted Greek version of the instrument seems to have some issues. First of all, one facet of job satisfaction had Cronbach’s alpha below 0.6 (i.e., 0.48 for “Operating procedures”). Second, the JSS was applied and evaluated on 239 employees of various specialties in drug addiction treatment of one only medical center with common structure. This implies that the sample size might be rather small for factor analysis and that its findings might not even be generalizable [31, 36].
Additionally for this research, Split-half reliability analysis (Table 2) was assessed by dividing the instrument into two halves; Part 1: consisted of the first 18 items, and Part 2: consisted of the remaining 18 items of the scale. The findings showed that JSS had good split-half reliability as assessed through the Guttman Split-Half Coefficient (0.77).
Table 2Split-Half reliability analysisCronbach’s AlphaPart 1Value0.81 N of Items18aPart 2Value0.83 N of Items18bTotal N of Items36Correlation Between Forms0.63Spearman-Brown CoefficientEqual Length0.77Unequal Length0.77Guttman Split-Half Coefficient0.77
Split-Half reliability analysis
The items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.
The items are: Q1, Q2, Q3, Q5, Q7, Q9, Q11, Q13, Q15, Q17, Q20, Q22, Q25, Q27, Q28, Q30, Q33, Q35.
The items are: Q4, Q6, Q8, Q10, Q12, Q14, Q16, Q18, Q19, Q21, Q23, Q24, Q26, Q29, Q31, Q32, Q34, Q36.
Research design and procedure:
The survey was carried out in the region of Attiki with its capital Athens, with around 3.75 million inhabitants or approximately 35% of the total Greek population. The 1st Regional Health Authority of Attica has the responsibility for 27 public hospitals. Our survey was conducted between July 2019 and December 2020 in thirteen of those who provided healthcare services to 438,745 patients. The main criteria for the selection of these hospitals were (Table 3): (a) the categories of hospitals; for this reason, the survey was introduced into four different categories (general, pediatric, maternity, oncology), (b) a large number of different clinics, (c) hospitals with a large number of beds but without ignoring the role of smaller hospitals, (d) the large number of patients treated in these hospitals, (e) the large number of health care employees who work in these hospitals, and (f) the necessary approval of the research by hospital committees.
The researchers distributed the printed questionnaire along with a consent form to the participants in person at their workplaces. They were adults (over 18 years), health care professionals belonging to medical, nursing, administrative, and technical departments serving public healthcare. The main aim of selecting employees from various fields is to get the opinions of a diverse group of people so that the results can be generalized on s vast group of the overall population. They had worked for more than six months in the respective hospital facilities at the time of the research and consented to the study. The study excluded interns, volunteers, and those declining to consent to the study. The participants had one week to complete the questionnaire. All employees had the right to refuse or discontinue their participation in the survey at any time. The researcher guaranteed the anonymity and confidentiality of all data collected. We remained considerate of the names, safety, and well-being of participants, and also the organizations remained anonymous by using codes, such as H01, H02, and so on (Table 3). Finally, of the 4,000 questionnaires distributed, 3,278 (81.95%) were returned.
Table 3The population of research per hospital and category. Distributed Questionnaires and Response Rate
Period of research
Professional Category
2019
2020
Doctors
Nurses
Other Health Professionals
Overall Sample
Number
Number
Days of
Hospital
3Q
4Q
1Q
2Q
3Q
4Q
( n )
( % )
( n )
( % )
( n )
( % )
( n )
( % )
οf Beds*
οf Patients*
Hospitalization*
H 01XXXXXX10813.45%1327.60%435.82%2838.64%H 0216820.92%29016.71%15821.38%61618.80%H 03XXXXXX293.61%995.70%7410.01%2026.17%H 049011.21%21012.10%648.66%36411.11%H 05XXXXXX597.35%895.13%628.39%2106.41%H 06516.35%1227.03%699.34%2427.39%H 07XXXXXX617.60%18110.43%395.28%2818.58%H 088710.83%905.18%324.33%2096.38%H 09XXXXXX364.48%1196.85%577.71%2126.47%H 10455.60%1116.39%506.77%2066.29%H 11XXXXXX293.61%1367.83%364.87%2016.14%H 12323.99%1327.60%385.14%2026.17%H 13XXXXXX81.00%251.44%172.30%501.53%[1] Hospitals of research(n = 13)80324.50%1,73652.96%73922.54%3,278100%6,511438,7451,443,660[2] Hospitals in 1st Regional Health Authority of Attica(n = 27)6,277**31.71%8,174**41.29%5,345**27.00%19,796**100%8,860567,8171,912,488[3] Hospitals in the National Health System(n = 127)36,4412,160,5967,343,348[4] Percentages of Hospitals of research / Hospitals of Attica= [1] / [2]12.79%21.24%13.83%16.56%73.49%77.27%[5] Percentages of Hospitals of research / NHS= [1] / [3]17.87%20.31%19.66%[6] Distributed Questionnaires of research1,00025.00%2,00050.00%1,00025.00%4,000100%[7] Response Rate= [1] / [6]80.30%86.80%73.90%81.95%Notes: * Data of year 2020 ** Data of year 2016X = research conduct
The population of research per hospital and category. Distributed Questionnaires and Response Rate
Notes: * Data of year 2020 ** Data of year 2016
X = research conduct
Statistical analysis The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).
Exploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].
After using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].
The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).
Exploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].
After using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].
Statistical analysis:
The data collected were analyzed using SPSS software (version 24.0). The mean (M) and standard deviation (SD) of each JSS item were determined. The reliability coefficient was examined. As a rule of thumb, values of Cronbach’s α ≥ 0.6 are thought to be acceptable [31]. Validity was evaluated using convergent and discriminant validity, as well as factor analysis consisting of exploratory factor analysis (EFA) and Confirmatory Factor Analysis (CFA).
Exploratory factor analysis (EFA) was conducted by utilizing principle component analysis (PCA) with the varimax rotation method, which had applied an Eigenvalue of > 1 for this purpose. For EFA we used the Kaiser-Meyer-Olkin (KMO) statistic was employed to assess whether the sample data are suitable for factor analysis. According to Kaiser [37], a value above 0.5 is considered acceptable; between 0.5 and 0.7 is moderate; between 0.7 and 0.8 is good; between 0.8 and 0.9 is very good; and 0.9 and above is superb. Also, Bartlett’s Test was applied to verify if the data was appropriate for factor analysis and indicated that correlations between items were sufficiently large for PCA. Retained and excluded factors were also explored visually on a screen plot along with the parallel analysis. Many studies reported that factor loadings should be greater than 0.5 for better results [38–40]. Principal Component Analysis was chosen as the suitable extraction method for obtaining the initial factor solution and reducing the number of factors. PCA is a robust method that is psychometric and less complex conceptually than other methods and is also preferred because it resembles many aspects of discriminate analysis. Varimax rotation of the factors was also applied to produce the factor structure. The advantage of Varimax rotation is that maximizes distribution within the factors, thus introducing a small number of variable loads and more easily interpretable factor clusters into each factor load. Cross-loaded statements also were deleted [38–41].
After using EFA to identify the factor structure present in a set of variables, the model fit was then assessed by using Confirmatory Factor Analysis (CFA). A CFA with a maximum likelihood method (ml) in AMOS (version 24.0) was also performed. The fit of the CFA model was assessed using the incremental and absolute indexes, namely: the comparative fit index (CFI), incremental fit index (IFI), the standard mean root square residual (SRMR) and the root mean square error of approximation (RMSEA). The following cut-off values were assumed: CFI, and IFI ≥ 0.900, SRMR and RMSEA ≤ 0.800 [42, 43].
Results:
Study sample Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).
Table 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories
Characteristics
Doctors
Nurses
Other Health Professionals
Overall Sample
N = 803
%
N = 1,736
%
N = 739
%
N = 3,278
%
Gender
Male29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%
Age
< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%
Marital
Married38547.95%1,1767.40%49967.52%2,05462.66%
Status
Single39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%
Level of
Compulsory00.00%70.40%435.82%501.53%
Education
Secondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%
Employment
Permanent42552.93%1,5991.59%64086.60%2,65580.99%
Status
Temporary37847.07%1468.41%9913.40%62319.01%
Professional
< 5 years29036.11%22112.73%12416.78%63519.37%
Experience
6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%
Economic Situation
I cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%
Sociodemographic characteristics of the sample per professional category
Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).
Table 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories
Characteristics
Doctors
Nurses
Other Health Professionals
Overall Sample
N = 803
%
N = 1,736
%
N = 739
%
N = 3,278
%
Gender
Male29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%
Age
< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%
Marital
Married38547.95%1,1767.40%49967.52%2,05462.66%
Status
Single39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%
Level of
Compulsory00.00%70.40%435.82%501.53%
Education
Secondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%
Employment
Permanent42552.93%1,5991.59%64086.60%2,65580.99%
Status
Temporary37847.07%1468.41%9913.40%62319.01%
Professional
< 5 years29036.11%22112.73%12416.78%63519.37%
Experience
6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%
Economic Situation
I cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%
Sociodemographic characteristics of the sample per professional category
Study sample:
Among the sample participants 612 (18.67%) were male and 2,666 (81.33%) were female. Regarding their age, 1.49% was under 25 years old, 15.86% were 26–35, 33.25% were between 36 and 45, 38.16% between 46 and 55. The remaining 11.23% were older than 56 years. As far as the educational level is concerned, the majority was university graduates (59.55%), 19.37% had post-graduate studies, only 1.53% had compulsory education and the remaining 19.55% had secondary education. Concerning employment status, the majority worked as permanent staff (80.99%). As regards length of service, 19.37% had less than 5 years, 11.90% of study participants had worked from 6 to 10 years, 17.63% from 11 to 15 years, 22.45% from 16 to 20 years, while 28.65% had worked for more than 20 years. With respect to income, the majority of employees stated that they managed without having much money left aside (see Table 4).
Table 4Sociodemographic characteristics of the sample per professional categoryProfessional Categories
Characteristics
Doctors
Nurses
Other Health Professionals
Overall Sample
N = 803
%
N = 1,736
%
N = 739
%
N = 3,278
%
Gender
Male29436.61%1508.64%16822.73%61218.67%Female50963.39%1,58691.36%57177.27%2,66681.33%
Age
< 25 years50.62%321.84%121.62%491.49%26–35 years23629.39%24314.00%415.55%52015.86%36–45 years27334.00%61235.25%20527.74%1,0933.25%46–55 years21026.15%72341.65%31843.03%1,25138.16%56 > years799.84%1267.26%16322.06%36811.23%
Marital
Married38547.95%1,1767.40%49967.52%2,05462.66%
Status
Single39348.94%43124.83%15220.57%97629.77%Divorced242.99%1247.14%628.39%2106.41%Widowed10.12%110.63%263.52%381.16%
Level of
Compulsory00.00%70.40%435.82%501.53%
Education
Secondary00.00%31318.03%32844.38%64119.55%Bachelor55969.61%1,09963.31%29439.78%1,95259.55%Master’s / PhD24430.39%31718.26%7410.01%63519.37%
Employment
Permanent42552.93%1,5991.59%64086.60%2,65580.99%
Status
Temporary37847.07%1468.41%9913.40%62319.01%
Professional
< 5 years29036.11%22112.73%12416.78%63519.37%
Experience
6–10 years15819.68%1589.10%7410.01%39011.90%11–15 years11414.20%37621.66%8811.91%57817.63%16–20 years13516.81%45726.32%14419.49%73622.45%20 > years10613.20%52430.18%30941.81%93928.65%
Economic Situation
I cannot cope with my financial obligations20.25%704.03%557.44%1273.87%I manage financially with great difficulties10813.45%71641.24%36349.12%1,18736.21%I manage financially but I do not have much left aside57070.98%87150.17%27437.08%1,71552.32%I am financially comfortable10513.08%311.79%253.38%1614.91%I do not know / I do not answer182.24%482.76%222.98%882.68%
Sociodemographic characteristics of the sample per professional category
Normality analysis:
The Kolmogorov-Smirnov and Shapiro-Wilk normality tests were performed and showed that the data was not normally distributed (p < 0.05).
Descriptive statistics results:
Descriptive statistics for the items of the questionnaire are shown in Table 5. The results indicate that the minimum value of the items is 1 while the maximum is 6.
Table 5Surveys questions and descriptive statistics (N = 3,278)Itemno.Survey QuestionsMin.Max.MeanMedianPercentilesStd.DeviationVarianceStatisticSum
Statistic
Std. Error
25
50
75
JS 1I feel I am being paid a fair amount for the work I do162.460.0212.002.002.003.001.1931.4248049JS 2There is really too little chance for promotion on my job162.720.0223.002.003.003.001.2601.5878905JS 3My supervisor is quite competent in doing his/her job164.770.0185.004.005.005.001.0501.10315,649JS 4I am not satisfied with the benefits I receive162.930.0213.002.003.004.001.2021.4449598JS 5When I do a good job, I receive the recognition for it that I should receive163.260.0223.002.003.004.001.2841.64910,678JS 6Many of our rules and procedures make doing a good job difficult162.710.0193.002.003.003.001.0651.1358870JS 7I like the people I work with165.100.0135.005.005.006.000.7330.53816,717JS 8I sometimes feel my job is meaningless163.900.0234.003.004.005.001.3441.80612,790JS 9Communications seem good within this organization164.000.0194.004.004.005.001.0971.20413,114JS 10Raises are too few and far between161.730.0191.001.001.002.001.0701.1445657JS 11Those who do well on the job stand a fair chance of being promoted162.430.0212.001.002.003.001.2301.5127956JS 12My supervisor is unfair to me164.710.0205.004.005.005.001.1331.28415,448JS 13The benefits we receive are as good as most other organizations offer162.280.0202.001.002.003.001.1541.3327488JS 14I do not feel that the work I do is appreciated162.890.0223.002.003.004.001.2371.5309483JS 15My efforts to do a good job are seldom blocked by red tape163.450.0234.003.004.004.001.2921.67011,308JS 16I find I have to work harder at my job because of the incompetence of people I work with163.800.0224.003.004.005.001.2761.62912,458JS 17I like doing the things I do at work164.870.0155.005.005.005.000.8770.76915,979JS 18The goals of this organization are not clear to me163.150.0213.002.003.004.001.1901.41510,311JS 19I feel unappreciated by the organization when I think about what they pay me162.330.0202.002.002.003.001.1641.3567636JS 20People get ahead as fast here as they do in other places162.300.0202.001.002.003.001.1481.3187547JS 21My supervisor shows too little interest in the feelings of subordinates164.290.0224.004.004.005.001.2691.61014,049JS 22The benefit package we have is equitable162.790.0213.002.003.004.001.1751.3809137JS 23There are few rewards for those who work here162.720.0213.002.003.003.001.1921.4228909JS 24I have too much to do at work162.140.0162.002.002.003.000.8970.8057001JS 25I enjoy my coworkers164.810.0165.004.005.005.000.8980.80715,760JS 26I often feel that I do not know what is going on with the organization163.820.0224.003.004.005.001.2581.58212,532JS 27I feel a sense of pride in doing my job164.820.0185.004.005.005.001.0131.02715,802JS 28I feel satisfied with my chances for salary increases161.980.0192.001.002.002.001.1071.2266486JS 29There are benefits we do not have which we should have162.670.0213.002.003.003.001.1891.4148736JS 30I like my supervisor164.860.0165.004.005.005.000.9360.87615,941JS 31I have too much paperwork162.970.0233.002.003.004.001.2931.6719742JS 32I don’t feel my efforts are rewarded the way they should be162.790.0203.002.003.004.001.1671.3629132JS 33I am satisfied with my chances for promotion162.350.0212.001.002.003.001.1761.3837715JS 34There is too much bickering and fighting at work163.310.0203.003.003.004.001.1661.36110,855JS 35My job is enjoyable163.750.0214.003.004.004.001.1811.39512,293JS 36Work assignments are not fully explained164.170.0214.004.004.005.001.1871.41013,673
Surveys questions and descriptive statistics (N = 3,278)
The highest mean values were found for Item–7 and Item–17 while the lowest ones for Item–10 and Item–28. The average variability of the items around mean values was relatively small.
Exploratory factor analysis:
According to the analysis result, the KMO (Kaiser-Meyer-Olkin) statistic of 0.912 confirmed that the sample used was quite sufficient. We can therefore be confident that the factor analysis fits into our data set. Next, Barlett’s test of sphericity (χ2 = 31831.572, df = 528, p = 0.000) demonstrated that the correlation matrix is not an identity matrix, therefore providing justification for the use of factor analysis [37, 44]. In PCA the eigenvalue provides the fraction of the variation accounted for by the corresponding component (eigenvector). We adopted a combined criteria method as suggested by Lings and Greenley [45], and Larose [46] to identify items and factors for inclusion in the final factorial solution. More specifically, to evaluate the factor structures, we used four criteria. First, items factor loadings should be at least equal to or greater than 0.5. Second, a scale should have more than two items or if it has only two they should be strongly correlated. Third, if an item loads more than one dimension and their difference is lower than 0.02, it will be deleted. Moreover, the difference in loadings, equal to or greater than 0.2, implies the item’s inclusion in the dimension with the highest factor load. Finally, in order to maintain an item, it would also have to conceptually match the factor [47–49].
Based on an eigenvalue greater than one, as one eigenvalue represents a significant amount of variation, factors considered in subsequent analyses. Hence, another eigenvalue-based approach was used to examine Cattell’s “scree” plot, by looking for a spot in the plot where it abruptly levels out. By employing both methods, a six-factor model was identified (see Table 6) [50]. The final factorial structure explains 56.23% of the total variance of the dataset. According to the results obtained, the first factor had 23.78% of the total variance, the second factor 11.52%, the third factor 6.64%, the fourth factor 6.30%, the fifth factor 4.17%, and the sixth factor 3.81%. The total variance explanatory rates of the factors after rotation were as follows: 14.13%, 10.53%, 10.49%, 8.19%. 6.92% and 5.97%.
Table 6Eigenvalues and the explained total variance of the extracted factorsInitial EigenvaluesExtraction Sums of Squared LoadingsRotation Sums of Squared LoadingsComponentTotal% of VarianceCumulative %Total% of VarianceCumulative %Total% of VarianceCumulative %16.1823.7823.786.1823.7823.783.6714.1314.1323.0011.5235.313.0011.5235.312.7410.5324.6631.736.6441.941.736.6441.942.7310.4935.1441.646.3048.241.646.3048.242.138.1943.3451.094.1752.411.094.1752.411.806.9250.2661.003.8156.231.003.8156.231.555.9756.23Extraction Method: Principal Component Analysis
Eigenvalues and the explained total variance of the extracted factors
Extraction Method: Principal Component Analysis
Varimax rotation was used for the rotation of the original solution as our sampling has a heterogeneous population [51, 52]. Twenty variables were included within six factors. The resulting six factors were: Factor 1 which indicates employees’ benefits and salary includes items: 11, 20, 28, 33. Factor 2 represents the management’s attitude and includes items: 14, 19, 24, 29. Factor 3 supervision and includes items: 3, 12, 21, 30. Factor 4 represents employees’ communication, includes items: 18, 26 and 36. Factor 5 mainly indicates the nature of work and includes items: 17, 27,35 and finally Factor 6 consists colleagues support and includes items: 7 and 25 (Table 7).
Table 7Standardized loadings of items for each factorFactor 1Factor 2Factor 3Factor 4Factor 5Factor 6
Item
no.
Questions
Dimensions
Benefits and Salary
Management’s attitude
Supervision
Communication
Nature of work
Colleagues Support
JS 33I am satisfied with my chances for promotionPromotion0.83JS 28I feel satisfied with my chances for salary increasesPay0.77JS 20People get ahead as fast here as they do in other placesPromotion0.76JS 11Those who do well on the job stand a fair chance of being promotedPromotion0.72JS 24I have too much to do at workOperating procedures0.83JS 29There are benefits we do not have which we should haveFringe Benefits0.70JS 19I feel unappreciated by the organization when I think about what they pay mePay0.56JS 14I do not feel that the work I do is appreciatedContingent rewards0.54JS 3My supervisor is quite competent in doing his/her jobSupervision0.83JS 30I like my supervisorSupervision0.81JS 21My supervisor shows too little interest in the feelings of subordinatesSupervision0.77JS 12My supervisor is unfair to meSupervision0.74JS 26I often feel that I do not know what is going on with the organizationCommunication0.78JS 18The goals of this organization are not clear to meCommunication0.68JS 36Work assignments are not fully explainedCommunication0.67JS 17I like doing the things I do at workNature of work0.81JS 27I feel a sense of pride in doing my jobNature of work0.75JS 35My job is enjoyableNature of work0.63JS 7I like the people I work withCoworkers0.82JS 25I enjoy my coworkersCoworkers0.81Notes: (1) The weights of extracted factors from exploratory factor analysis with varimax rotation (weights less than 0.4 are not displayed). (2) Extraction Method: Principal Component Analysis. (3) Factor loading > 0.5
Standardized loadings of items for each factor
Item
no.
Notes: (1) The weights of extracted factors from exploratory factor analysis with varimax rotation (weights less than 0.4 are not displayed). (2) Extraction Method: Principal Component Analysis. (3) Factor loading > 0.5
The reliability coefficient Cronbach’s alpha of new construction of scales after application of factor analysis for the overall scale was 0.81 and we concluded that the questionnaire has very good reliability. The results showed that obtained reliability figures (Alphas) range from 0.60 to 0.81 for the various job satisfaction dimensions. These findings provide support for the internal consistency of the sub-scales, so we can state that the scale of the survey questions used in the analysis was acceptable (Table 8).
Table 8Reliability analysis of scalesFactor IDFactor NameCronbach’s Alpha (CA)Overall ItemsItemsAverage Item ScoreFactor 1Benefits and Salary0.74411, 20, 28, 332.27Factor 2Management’s attitude0.67414, 19, 24, 292.51Factor 3Supervision0.8143, 12, 21, 304.66Factor 4Communication0.60318, 26, 363.71Factor 5Nature of work0.61317, 27, 354.48Factor 6Colleagues Support0.6627, 254.96
Overall Satisfaction
0.81
20
3.76
Reliability analysis of scales
Confirmatory factor analysis:
Confirmatory Factor Analysis (CFA) is a statistical technique used to evaluate the measurement models that represent hypotheses about relations between indicators and factors. The CFA assessed the fit of the six-factor structure and the model fitted the data well as defined from the SRMR, RMSEA, CFI and IFI values that were equal to 0.050 (≤ 0.800), 0.055 (≤ 0.800), 0.906 (≥ 0.900) and 0.906 (≥ 0.900) respectively. It was suggested that the fitting optimization index was acceptable and the structure of the model was designed reasonably (Fig. 1).
Fig. 1Result of confirmatory factor analysis (CFA)
Result of confirmatory factor analysis (CFA)
Discussion:
To sum up the discussion, the basic purpose of this study was to validate Spector’s JSS instrument and develop a valid, short and reliable instrument that can measure employee job satisfaction for public hospitals in Athens, Greece. There were 3,278 responses received from the employees of thirteen different hospitals. Factor analysis was conducted due to anticipated dimensionality of factors that are involved in measuring job satisfaction. The values of Cronbach’s Alpha coefficients were computed in order to assess the internal consistency reliability.
Overall, the job satisfaction scale developed in this research illustrates valid and reliable measures for assessing hospital employees’ satisfaction levels with their work. Yet in reality, job satisfaction is a complex multidimensional concept. The study is based on the premise that an organization’s intellectual capital is its most important asset. For this purpose, our survey used a personalized “bottom-up” approach, which studied the properties of employees, their behavior at the workplace, motivators, dissatisfiers, and other properties of the job environment. Satisfied human resource is the most valuable asset for high productivity, commitment, efficiency, and quality of care in a healthcare organization [53]. Aiming we get answers to basic questions: “How do employees feel in their workplace? What makes them behave in the workplace the way they do? What would motivate them to perform well and according to the hospital’s goals?“ The employees are motivated (or not) to perform as they do because of a combination of internal and external factors, which should be investigated, measured and improved as much as possible.
The statistical analyses identified six predominant components to quantify job satisfaction, namely Benefits and Salary (F1), Management’s attitude (F2), Supervision (F3), Communication (F4), Nature of work (F5), Colleagues Support (F6). Meanwhile, among the affecting factors of job satisfaction, monetary benefits have the most influence, relationships with superiors and colleagues, training and enhancement of employee skills, the perceived fairness of the promotion system, the quality of the working conditions, and a sense of belonging are vital to the development of job satisfaction.
An important strength of this study is that a short JSS questionnaire was developed for healthcare organizations that can be used much more widely in a rapidly changing environment. This newly developed questionnaire will prove very useful in providing continuous feedback to top management as well as health policy makers regarding the level of job satisfaction. Such feedback provided by the existing health workforce will immediately alert them to any adverse working conditions that present themselves as factors leading to job dissatisfaction.
In Greece, the results of this study are important in terms of determining factors that should be considered for success within organizations. This research is valuable because it has both a practical and humanitarian application, as it gives a better understanding of employee satisfaction which in turn will lead to improved organizational behavior and employee attitudes that directly affect the improvement of health quality. Gaining employee’s commitment to their organization’s goals is believed to unlock their potential and achieve heightened levels of performance. Opposite results can lead employees to dissatisfaction or tend to lose interest in their work, higher levels of burnout and stress, absenteeism, intention to quit, and consequently suboptimal healthcare delivery and poor clinical outcomes [54]. Managers of health services organizations in cooperation with the Ministry of Health (MoH) must elicit cooperation and performance of the employees to ensure the quality of care and the morbidities and mortalities may be improved undoubtedly. Most researchers agree that employees with high job satisfaction levels have improved mental and physical health, job involvement, and improved quality of life. Eliciting such commitment from employees is not easy to obtain especially under uncertain or difficult working conditions [55–58].
More than ever, due to the globalization evolution of the Covid-19 pandemic, health systems need satisfied employees who can cope with very difficult conditions, refine health care services, and up surging the level of patient satisfaction. The study of job satisfaction is gaining more and more importance with the passage of time because of its nature and impact on society. The need to understand employee satisfaction resurfaces as everyone understands that they serve the ultimate human good, health [59].
Conclusion:
In total, this study applied quantitative methods to determine factors affecting job satisfaction. So, is important in terms of determining the specific factors that should be considered for job satisfaction, organizational engagement, managerial success, and high performance within hospitals. A short 20-item study for all healthcare staff can benefit hospitals to monitor employee satisfaction across all levels without overburdening employees and analysts with multiple or fielding several non-comparable types of research.
The findings suggest that effective communication and support from managers or supervisors to employees or among employees themselves will reduce stress and conflicts in the workplace. Additionally, it can be recommended that employee empowerment and training, collaboration in teamwork, and a systematic approach regarding innovative types of promotional opportunity, recognition, reward, and evaluation of hospital staff can lead to better results and benefits employees, quality of patient care, and healthcare organizations. Consequently, we believe that empowerment of management, achievement, promotion and evaluation should significantly improve job satisfaction respectively. This study showed that obtained factors are aligned with the findings of the prior studies in the literature [60, 61].
The results of this study should not be generalized extensively since the participants of the study come from a single geographical region of the country, only in hospitals in Athens, Greece. Nevertheless, the sample cannot be characterized as homogenous due to the fact that participants were working in different departments in the hospitals, so they deal with different tasks and procedures. Therefore, the findings and related conclusions may not be able to be generalized and compared with the rest regions of the country.
| Background:
Job satisfaction in health care has a great impact as it affects quality, productivity, effectiveness, and healthcare costs. In fact, it is an indicator of the well-being and quality of life of the organization's employees, as it has been variously linked with increased performance and negatively to absenteeism and turnover. Better knowledge of healthcare employees' job satisfaction and performance can directly contribute to the quality of the services provided to patients and is critical for the success of organizations.
Methods:
The Cronbach's alpha coefficient, split-half reliability, exploratory factor and confirmatory factor analysis were employed to assess the reliability and validity of JSS.
Results:
Six underlying dimensions were extracted (benefits and salary, management's attitude, supervision, communication, nature of work, and colleagues' support). Internal consistency reliability was satisfactory since Cronbach's alpha for the overall scale was 0.81 and for the various dimensions ranged from 0.61 to 0.81, respectively. Exploratory factor analysis showed a KMO value of 0.912. The confirmatory factor analysis indicated good fit: SRMR = 0.050, RMSEA = 0.055, IFI = 0.906 and CFI = 0.906.
Conclusions:
Job satisfaction is a multidimensional construct that encompasses different facets of satisfaction. There is a lack of consensus as to which factors are more important and a researcher may find satisfaction with some factors while at the same time dissatisfaction with others. Our findings are significant for improving our understanding of the nature and assessment of job satisfaction in the Greek healthcare context, providing a more stable ground in a rapidly changing environment. A short JSS developed that could be much more widely used in the future. | Introduction:
As employee knowledge and skills are intangible assets of any service organization,
employee satisfaction has become an issue of utmost importance. It has been defined as the positive emotional state resulting from the evaluation of one’s work or work experience [1]. Hoppock [2] was the first who brought forth the concept of job satisfaction in limelight and described it as “the employees’ subjective reflections or subjective feelings about their working conditions and working environment”. Since then, many researchers have recognized that satisfied employees are a key asset to an organization [3]. While the importance of job satisfaction is generally recognized, additional and ongoing investigations of satisfaction levels are necessary as external conditions and societal values are constantly changing. In this respect, job satisfaction has a significant role in the operation and performance of organizations.
An essential prerequisite for the development and long-term success of an organization is in fact the utilization of employee’s capabilities and the improvement of their working conditions [4]. The degree of job satisfaction is actually the overall level of satisfaction on a number of different dimensions of work and affects the behavior of employees that, in turn, impacts upon organizational functioning [5–7]. Swamy et al. [3] stated that satisfied employees are the key asset of an organization. Therefore, the issue of job satisfaction is very important especially for non-profit public organizations like hospitals, which are essential for a country’s provision of healthcare services and the population itself.
Employee satisfaction also affects patient satisfaction. As patients are the external customers and employees are the internal customers of the organization they form the current working environment and are willing to cooperate with the community to achieve organizational goals. Previous studies have documented associations between job fulfillment of health workforce and patient contentment with the type of health care services provided in health care facilities [8, 9]. Moreover, there seems to exist a positive correlation between the increase in job satisfaction and quality of care [10, 11]. Conversely, a low level of job satisfaction would create negative behaviors, including absenteeism, grievances, high level of stress, turnover, exhaustion, low morality, worse patient-provider ratios, longer wait times, psychological distress and increased medical errors [12–14].
Hospital managers have responsibilities to both patients and staff. It has been suggested that if you want to attain higher job productivity and efficiency, you should comprehend the domains of work which are decisive for job satisfaction amongst healthcare providers. In order to get employees contented with their job; the underlying factors that influence job satisfaction in that particular facility must be examined to guide proper managerial action [15, 16].
Conclusion:
In total, this study applied quantitative methods to determine factors affecting job satisfaction. So, is important in terms of determining the specific factors that should be considered for job satisfaction, organizational engagement, managerial success, and high performance within hospitals. A short 20-item study for all healthcare staff can benefit hospitals to monitor employee satisfaction across all levels without overburdening employees and analysts with multiple or fielding several non-comparable types of research.
The findings suggest that effective communication and support from managers or supervisors to employees or among employees themselves will reduce stress and conflicts in the workplace. Additionally, it can be recommended that employee empowerment and training, collaboration in teamwork, and a systematic approach regarding innovative types of promotional opportunity, recognition, reward, and evaluation of hospital staff can lead to better results and benefits employees, quality of patient care, and healthcare organizations. Consequently, we believe that empowerment of management, achievement, promotion and evaluation should significantly improve job satisfaction respectively. This study showed that obtained factors are aligned with the findings of the prior studies in the literature [60, 61].
The results of this study should not be generalized extensively since the participants of the study come from a single geographical region of the country, only in hospitals in Athens, Greece. Nevertheless, the sample cannot be characterized as homogenous due to the fact that participants were working in different departments in the hospitals, so they deal with different tasks and procedures. Therefore, the findings and related conclusions may not be able to be generalized and compared with the rest regions of the country. | Background:
Job satisfaction in health care has a great impact as it affects quality, productivity, effectiveness, and healthcare costs. In fact, it is an indicator of the well-being and quality of life of the organization's employees, as it has been variously linked with increased performance and negatively to absenteeism and turnover. Better knowledge of healthcare employees' job satisfaction and performance can directly contribute to the quality of the services provided to patients and is critical for the success of organizations.
Methods:
The Cronbach's alpha coefficient, split-half reliability, exploratory factor and confirmatory factor analysis were employed to assess the reliability and validity of JSS.
Results:
Six underlying dimensions were extracted (benefits and salary, management's attitude, supervision, communication, nature of work, and colleagues' support). Internal consistency reliability was satisfactory since Cronbach's alpha for the overall scale was 0.81 and for the various dimensions ranged from 0.61 to 0.81, respectively. Exploratory factor analysis showed a KMO value of 0.912. The confirmatory factor analysis indicated good fit: SRMR = 0.050, RMSEA = 0.055, IFI = 0.906 and CFI = 0.906.
Conclusions:
Job satisfaction is a multidimensional construct that encompasses different facets of satisfaction. There is a lack of consensus as to which factors are more important and a researcher may find satisfaction with some factors while at the same time dissatisfaction with others. Our findings are significant for improving our understanding of the nature and assessment of job satisfaction in the Greek healthcare context, providing a more stable ground in a rapidly changing environment. A short JSS developed that could be much more widely used in the future. | 10,929 | 324 | [
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|||||
Analysis of nickel distribution by synchrotron radiation X-ray fluorescence in nickel-induced early- and late-phase allergic contact dermatitis in Hartley guinea pigs. | 31373908 | Nickel-induced allergic contact dermatitis (Ni-ACD) is a global health problem. More detailed knowledge on the skin uptake of haptens is required. This study aimed to investigate the penetration process and distribution of nickel in skin tissues with late phase and early phase of Ni-ACD to understand the mechanisms of metal allergy. | BACKGROUND | Forty Hartley guinea pigs were divided into four groups according to the NiSO4 sensitizing concentration and the NiSO4 challenged concentration: the 5% NiSO4-group, 5% to 10% (sensitization-challenge; late phase group); 10% NiSO4-group, 10% to 10% (sensitization-challenge; early-phase group); and the positive and negative controls. Pathological biopsies were performed on each group. The depth profile of nickel element concentration in the skin of guinea pigs was detected by synchrotron radiation micro X-ray fluorescence spectroscopy (SR-μ-XRF) and micro X-ray absorption near-edge spectroscopy (μ-XANES). | METHODS | In each section, the nickel element concentration in both the 5% NiSO4-group and 10% NiSO4-group was significantly higher than that in the negative control group. In the upper 300-μm section of skin for the early phase group, the nickel element concentration was significantly higher than that in the lower section of skin. In deeper sections (>200 μm) of skin, the concentration of nickel in the early phase group was approximately equal to that in the late phase group. The curve of the late phase group was flat, which means that the nickel element concentration was distributed uniformly by SR-μ-XRF. According to the XANES data for the 10% NiSO4 metal salt solution, structural changes occurred in the skin model sample, indicating that nickel was not present in the Ni aqueous ionic state but in the nickel-binding protein. | RESULTS | This study showed that the distribution of the nickel element concentration in ACD skin tissue was different between the early phase and late phase groups. The nickel element was not present in the Ni aqueous ionic state but bound with certain proteins to form a complex in the stratum corneum in ACD model tissue. | CONCLUSIONS | [
"Animals",
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] | 6708687 | Introduction | Contact allergies represent a common form of dermatitis worldwide, especially in developed countries such as the United States and Europe.[1] Nickel is a common contact allergen on patch testing in western countries, and it affects 10% of women and 1% to 2% of men.[2] The differences between sexes were mainly explained by an increased frequency of jewelry exposure in women. Despite efforts to minimize prevalence, for example, by nickel restriction within the Europe,[3] up to 21% of the market accessories studied were found to release nickel.[4] In coins made of alloys, nickel was detected, and the concentration of nickel was beyond the safe exposure threshold.[5] Thus, the stresses caused by exposure to nickel are still high.
Within the past few decades, the interest in nickel among scientists has increased as a result of the progressive industrial and commercial significance of nickel as well as the improvement of analytical methods for nickel by electrothermal atomic absorption spectrometry. Currently, measurements in many countries, including China, indicate that the concentrations of nickel in the environment (air, water, soil, and food) do not exceed legislative limits and should not be dangerous for the general population.[6] However, people should keep in mind that, at present, nickel, although not released extensively into the environment, may represent a hazard to human health.
Nickel elements are potent allergens or haptens that can trigger skin inflammation, and nickel can penetrate through the stratum corneum to the epidermis. Therefore, efforts have been undertaken to investigate the penetration and distribution of haptens through the skin. Synchrotron radiation X-ray fluorescence spectroscopy (SR-XRF) and micro-focused particle-induced X-ray emission were used to analyze nickel allergy patch application in normal mouse skin.[7] Time of flight secondary ion mass spectrometry was used for investigation of the penetration and distribution of nickel in normal human skin.[8] Although these nickel-exposed animals developed metal allergy, the precise mechanism underlying this allergy remains unknown.
As reported, metal allergy is an inflammatory disease categorized as a delayed-type hypersensitivity reaction, in which skin inflammation is mediated by hapten-specific T cells.[9,10] Therefore, there is a need to identify the clinical relevance and the elemental- and chemical-specific distribution of nickel within tissues. This study investigates the internal distribution of nickel in skin tissues with nickel-induced allergic contact dermatitis (Ni-ACD) using microprobe-SR-XRF to identify the spatial distribution of nickel and its colocalization with endogenous elements. X-ray absorption near-edge structure (XANES) spectroscopy can also be used to determine the oxidation state of nickel bound to the biotic ligands. This study used correlative microprobe SR-XRF and XANES to investigate the penetration process and distribution of nickel in skin tissues with late phase and early phase of Ni-ACD to understand the mechanisms of metal allergy. | Methods | Ethical approval Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.
Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.
Animals Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.
Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.
Chemicals Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.
Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.
Treatments The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.
Control guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.
The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.
Control guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.
Skin reaction (erythema and swelling) evaluation The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.
The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.
Skin specimen preparation The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).
The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).
SR-μ-XRF spectroscopy The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]
The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]
SR-μ-XANES spectroscopy The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.
The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.
Statistical analysis Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.
Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant. | Results | Skin reactions (erythema and swelling) Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.
Evaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.
Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.
Evaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.
Pathohistological images of skin tissues The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.
Pathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.
The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.
Pathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.
Distribution of nickel element by SR-XRF from skin cross-sections SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.
The main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.
The relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.
SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.
The main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.
The relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.
Chemical species of nickel element in the skin model by SR-XANES XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]
X-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.
XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]
X-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy. | null | null | [
"Ethical approval",
"Animals",
"Chemicals",
"Treatments",
"Skin reaction (erythema and swelling) evaluation",
"Skin specimen preparation",
"SR-μ-XRF spectroscopy",
"SR-μ-XANES spectroscopy",
"Statistical analysis",
"Skin reactions (erythema and swelling)",
"Pathohistological images of skin tissues",
"Distribution of nickel element by SR-XRF from skin cross-sections",
"Chemical species of nickel element in the skin model by SR-XANES",
"Acknowledgements",
"Funding",
"Conflicts of interest"
] | [
"Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.",
"Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.",
"Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.",
"The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.\nControl guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.",
"The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.",
"The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).",
"The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]",
"The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.",
"Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.",
"Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.\nEvaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.",
"The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.\nPathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.",
"SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.\nThe main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.\nThe relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.",
"XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]\nX-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.",
"The authors thank Bo Chen at pathology department of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (CAMS) for giving advices in this study.",
"This research was supported by the grants from National Natural Science Foundation of China (No. 81373175), and CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-1003).",
"None."
] | [
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] | [
"Introduction",
"Methods",
"Ethical approval",
"Animals",
"Chemicals",
"Treatments",
"Skin reaction (erythema and swelling) evaluation",
"Skin specimen preparation",
"SR-μ-XRF spectroscopy",
"SR-μ-XANES spectroscopy",
"Statistical analysis",
"Results",
"Skin reactions (erythema and swelling)",
"Pathohistological images of skin tissues",
"Distribution of nickel element by SR-XRF from skin cross-sections",
"Chemical species of nickel element in the skin model by SR-XANES",
"Discussion",
"Acknowledgements",
"Funding",
"Conflicts of interest"
] | [
"Contact allergies represent a common form of dermatitis worldwide, especially in developed countries such as the United States and Europe.[1] Nickel is a common contact allergen on patch testing in western countries, and it affects 10% of women and 1% to 2% of men.[2] The differences between sexes were mainly explained by an increased frequency of jewelry exposure in women. Despite efforts to minimize prevalence, for example, by nickel restriction within the Europe,[3] up to 21% of the market accessories studied were found to release nickel.[4] In coins made of alloys, nickel was detected, and the concentration of nickel was beyond the safe exposure threshold.[5] Thus, the stresses caused by exposure to nickel are still high.\nWithin the past few decades, the interest in nickel among scientists has increased as a result of the progressive industrial and commercial significance of nickel as well as the improvement of analytical methods for nickel by electrothermal atomic absorption spectrometry. Currently, measurements in many countries, including China, indicate that the concentrations of nickel in the environment (air, water, soil, and food) do not exceed legislative limits and should not be dangerous for the general population.[6] However, people should keep in mind that, at present, nickel, although not released extensively into the environment, may represent a hazard to human health.\nNickel elements are potent allergens or haptens that can trigger skin inflammation, and nickel can penetrate through the stratum corneum to the epidermis. Therefore, efforts have been undertaken to investigate the penetration and distribution of haptens through the skin. Synchrotron radiation X-ray fluorescence spectroscopy (SR-XRF) and micro-focused particle-induced X-ray emission were used to analyze nickel allergy patch application in normal mouse skin.[7] Time of flight secondary ion mass spectrometry was used for investigation of the penetration and distribution of nickel in normal human skin.[8] Although these nickel-exposed animals developed metal allergy, the precise mechanism underlying this allergy remains unknown.\nAs reported, metal allergy is an inflammatory disease categorized as a delayed-type hypersensitivity reaction, in which skin inflammation is mediated by hapten-specific T cells.[9,10] Therefore, there is a need to identify the clinical relevance and the elemental- and chemical-specific distribution of nickel within tissues. This study investigates the internal distribution of nickel in skin tissues with nickel-induced allergic contact dermatitis (Ni-ACD) using microprobe-SR-XRF to identify the spatial distribution of nickel and its colocalization with endogenous elements. X-ray absorption near-edge structure (XANES) spectroscopy can also be used to determine the oxidation state of nickel bound to the biotic ligands. This study used correlative microprobe SR-XRF and XANES to investigate the penetration process and distribution of nickel in skin tissues with late phase and early phase of Ni-ACD to understand the mechanisms of metal allergy.",
" Ethical approval Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.\nForty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.\n Animals Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.\nHalf male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.\n Chemicals Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.\nNickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.\n Treatments The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.\nControl guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.\nThe animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.\nControl guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.\n Skin reaction (erythema and swelling) evaluation The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.\nThe evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.\n Skin specimen preparation The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).\nThe patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).\n SR-μ-XRF spectroscopy The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]\nThe micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]\n SR-μ-XANES spectroscopy The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.\nThe XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.\n Statistical analysis Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.\nStatistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.",
"Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.",
"Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.",
"Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.",
"The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.\nControl guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.",
"The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.",
"The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).",
"The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]",
"The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.",
"Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.",
" Skin reactions (erythema and swelling) Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.\nEvaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.\nFigure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.\nEvaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.\n Pathohistological images of skin tissues The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.\nPathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.\nThe H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.\nPathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.\n Distribution of nickel element by SR-XRF from skin cross-sections SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.\nThe main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.\nThe relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.\nSR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.\nThe main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.\nThe relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.\n Chemical species of nickel element in the skin model by SR-XANES XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]\nX-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.\nXANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]\nX-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.",
"Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.\nEvaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.",
"The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.\nPathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.",
"SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.\nThe main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.\nThe relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.",
"XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]\nX-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.",
"In the present study, Ni-induced ACD models were produced by modified protocols with SP formulations for guinea pigs based on traditional skin sensitization methods. We used SR-XRF to investigate the distribution of the nickel element in the skin tissues of two different Ni-ACD models (late phase and early phase), and the chemical species of the nickel element was also revealed via SR-XANES. In addition, H&E stained imaging of the target tissue sections provided histopathological information. Hence, the feasibility of these models and the distribution of metal permeation behavior in the skin tissues was confirmed.\nNi-ACD is a type IV allergy reaction.[14] For a hapten to be able to initiate this reaction, it has to penetrate through the stratum corneum to the epidermis.[15] Therefore, efforts have been undertaken to investigate the penetration and distribution of haptens through the skin.[11,14,16–18] For normal human and mouse skin, imaging mass spectrometry and SR-XRF were used to analyze the penetration and distribution of nickel.[7,8] However, these methods do not give detailed information on nickel element penetration and distribution in the skin in the pathogenesis of contact dermatitis. Therefore, there is a need to investigate clinically relevant haptens and their distribution in the epidermis of contact dermatitis with non-destructive inspection.\nThe evaluation of erythema on skin reactions is used to judge the concentration of nickel indirectly.[19] According to our study, we found that symptoms were dependent on the time of nickel penetration. An in vitro study evaluating nickel element permeation through the human stratum corneum also reported that the permeation rate of aqueous NiSO4 solution was the highest after approximately 24 h, regardless of the nickel salt species, which is in agreement with our results.[8]\nThe two groups, including both early phase and late phase ACD models, displayed different symptoms during the experiment. For each depth of skin, the nickel element concentration in the early phase group was higher than that in the late phase group. In our study, it was observed that the skin nickel element concentration from 100 to 200 μm depth in the early phase group was higher than that under 300 μm depth. Similar results were also found by Sugiyama et al.[7] However, the lord-balanced permeated nickel was diffused in the cross-section of the late phase group. The concentration of nickel caused this phenomenon. Permeation of high concentrations of nickel into the skin was reported to increase blood flow.[20,21] Thus, the removal of nickel-binding proteins by blood flow may keep nickel concentration well-balanced under deeper tissues. Corresponding to the evaluation of erythema in skin reactions, the states of swelling and skin lesions contributed to the depth of tissues, which might enlarge scales of the stratum corneum and epidermis in a severe lesion condition.\nComparing the XANES data for the 10% NiSO4 solution treatment, structural changes occurred in the skin model sample. We demonstrated that nickel was not present in the Ni2+ aqueous ionic state but bound to nickel-binding proteins in vivo. Some reports found and proved this phenomenon of nickel metal-binding amino acids, peptides, or proteins in vitro.[22–24] Our study demonstrated nickel binding events for nickel contact allergy in vivo by the measurement of XANES. Some relative studies have reported that Ni2+ triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4) in vitro, and they demonstrated that Ni2+-induced ACD symptoms were species-specific, as the mouse TLR family could not generate this response; mouse TLR senses microbial pathogens and endogenous ligands.[13,25] A recent study has reported that nickel (II) ions bind particularly at histidine residues, which are in the region required for the recognition of nickel signals.[26] However, it is not clear which proteins of hapten Ni2+ were combined to trigger Ni-induced ACD. Future research is required to explore this further.\nIn conclusion, this study showed that the distribution of the nickel element in ACD skin tissue was different between the early phase and late phase groups. The nickel element was not present in the Ni2+ aqueous ionic state but bound with certain proteins to form a complex in the stratum corneum nickel in ACD model tissue. SR-XRF and XANES technologies were non-invasive, and depth-resolved structure analysis was used for the conventional histopathological specimens. Therefore, these technologies may be applicable for the screening of metal-affected lesions and the evaluation of the effects of permeated metal elements on skin tissue lesions.",
"The authors thank Bo Chen at pathology department of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (CAMS) for giving advices in this study.",
"This research was supported by the grants from National Natural Science Foundation of China (No. 81373175), and CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-1003).",
"None."
] | [
"intro",
"methods",
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null,
null,
null,
null,
null,
null,
null,
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"results",
null,
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null,
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"discussion",
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] | [
"Synchrotron radiation micro X-ray fluorescence spectroscopy",
"Micro X-ray absorption near-edge spectroscopy",
"Dermatitis, Allergic contact",
"Nickel-induced allergic contact dermatitis",
"Dermatology"
] | Introduction:
Contact allergies represent a common form of dermatitis worldwide, especially in developed countries such as the United States and Europe.[1] Nickel is a common contact allergen on patch testing in western countries, and it affects 10% of women and 1% to 2% of men.[2] The differences between sexes were mainly explained by an increased frequency of jewelry exposure in women. Despite efforts to minimize prevalence, for example, by nickel restriction within the Europe,[3] up to 21% of the market accessories studied were found to release nickel.[4] In coins made of alloys, nickel was detected, and the concentration of nickel was beyond the safe exposure threshold.[5] Thus, the stresses caused by exposure to nickel are still high.
Within the past few decades, the interest in nickel among scientists has increased as a result of the progressive industrial and commercial significance of nickel as well as the improvement of analytical methods for nickel by electrothermal atomic absorption spectrometry. Currently, measurements in many countries, including China, indicate that the concentrations of nickel in the environment (air, water, soil, and food) do not exceed legislative limits and should not be dangerous for the general population.[6] However, people should keep in mind that, at present, nickel, although not released extensively into the environment, may represent a hazard to human health.
Nickel elements are potent allergens or haptens that can trigger skin inflammation, and nickel can penetrate through the stratum corneum to the epidermis. Therefore, efforts have been undertaken to investigate the penetration and distribution of haptens through the skin. Synchrotron radiation X-ray fluorescence spectroscopy (SR-XRF) and micro-focused particle-induced X-ray emission were used to analyze nickel allergy patch application in normal mouse skin.[7] Time of flight secondary ion mass spectrometry was used for investigation of the penetration and distribution of nickel in normal human skin.[8] Although these nickel-exposed animals developed metal allergy, the precise mechanism underlying this allergy remains unknown.
As reported, metal allergy is an inflammatory disease categorized as a delayed-type hypersensitivity reaction, in which skin inflammation is mediated by hapten-specific T cells.[9,10] Therefore, there is a need to identify the clinical relevance and the elemental- and chemical-specific distribution of nickel within tissues. This study investigates the internal distribution of nickel in skin tissues with nickel-induced allergic contact dermatitis (Ni-ACD) using microprobe-SR-XRF to identify the spatial distribution of nickel and its colocalization with endogenous elements. X-ray absorption near-edge structure (XANES) spectroscopy can also be used to determine the oxidation state of nickel bound to the biotic ligands. This study used correlative microprobe SR-XRF and XANES to investigate the penetration process and distribution of nickel in skin tissues with late phase and early phase of Ni-ACD to understand the mechanisms of metal allergy.
Methods:
Ethical approval Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.
Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.
Animals Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.
Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.
Chemicals Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.
Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.
Treatments The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.
Control guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.
The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.
Control guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.
Skin reaction (erythema and swelling) evaluation The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.
The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.
Skin specimen preparation The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).
The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).
SR-μ-XRF spectroscopy The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]
The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]
SR-μ-XANES spectroscopy The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.
The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.
Statistical analysis Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.
Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.
Ethical approval:
Forty Hartley guinea pigs were used in the study after approval by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Peking Union Medical College (No. ILAS-GLP-2013-042). All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.
Animals:
Half male and half female Hartley guinea pigs (wide type genetic), weighing 240 to 270 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.: a joint venture of Charles River Laboratories in China. The animals were housed in individual cages, maintained under standardized conditions. The animals were given free access to filtered tap water and food and were maintained in specific pathogen-free conditions in our animal facility with a 12-h light/dark cycle.
Chemicals:
Nickel sulfate (5% and 10%) (NiSO4·6H2O analytical grade, Xilong Chemical Co. Ltd, Beijing, China) was prepared in synthetic perspiration (SP) formulations (0.01 g/mL sodium chloride, 0.005 g/mL ammonium chloride, 0.005 g/mL urea, 0.0025 g/mL acetic acid, and 0.005 g/mL lactic acid was adjusted to pH 5.5 by the addition of ammonia). 2,4-Dinitrochlorobenzene (DNCB) was prepared with 1% acetone (Beijing Chemical Works, Beijing, China). Normal saline (NS) (Beijing Chemical Works) was prepared before the study.
Treatments:
The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Guinea pigs were allowed to acclimate for 1 week before use. Aliquots of the nickel sulfate solution (0.1 mL) were administered percutaneously to a clipped and shaved skin area (3× 3 cm) located on the left flank of the animals. The animals were treated three times on days 0, 1, and 2 for the sensitizing phase; the treated areas were patched for 6 h. The animals were challenged by occlusive patch tests on the right flank site using the previously described nickel sulfate solution on the shaved skin area on day 7. The treated areas were under occlusion for 24 h. After the challenge, we waited for 24 h and then recorded delayed hypersensitive response. In this study, we observed the skin change of 24 h (defined as early phase) and 72 h (defined as late phase), then we collected these skin biopsies after patch removement.
Control guinea pigs were treated with 1% to 1% (sensitization-challenge) DNCB in acetone and NS as the positive and negative control (NC) groups, respectively, and were challenged under identical conditions on day 7.
Skin reaction (erythema and swelling) evaluation:
The evaluation time should be the immediate time of the removement of the patch and at least 24 h later to observe the delayed hypersensitive response. Skin reaction (erythema and swelling) was recorded by visual assessment after challenge. The intensity of the reaction was scored according to the following criteria[11]: 0, no visible change; 1, slight or discrete erythema and barely visible swelling; 2, moderate and confluent erythema and visible border swelling; 3, intense erythema and swelling; and 4, purplish-red eschar and over 1 mm of swelling. Response score averages were calculated for each group. Swelling and erythema were also documented by photography.
Skin specimen preparation:
The patch was removed, and the flank skin was gently wiped after the guinea pigs were sacrificed. The skin that contacted the patch test was excised and gently wiped in each group. The specimen was cut into sections (5× 5 mm, 1 mm thick) and fixed in 10% neutral buffered formalin for use. The sectioned specimens in the 5% NiSO4-group, 10% NiSO4-group and NC group were placed on Kapton film (12.5 μm; Du Pont-Toray, Tokyo, Japan) and subjected to elemental distribution analyses. After that, specimen slices in all groups were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) (Beijing Chemical Works) staining for histopathological analysis. Stained tissues were observed using a light microscope (original magnification ×40).
SR-μ-XRF spectroscopy:
The micro X-ray fluorescence (μ-XRF) spectroscopy experiment was performed at 4W1B beamline, at the Beijing Synchrotron Radiation Facility (BSRF), which runs 2.5 GeV electron with current from 150 to 250 mA. The incident X-ray energy was monochromatized by W/B4C double-multilayer-monochromator at 15 keV and was focused down to 50 μm in diameter by the polycarpellary lens. Two-dimensional mapping was acquired by step-mode: the sample was held on a precision motor-driven stage, scanning 100 μm stepwise. The Si (Li) solid-state detector was used to detect X-ray fluorescence emission lines with a live time of 100 s. Here, XRF scans of the first point (0 μm) were determined to be at the position of the maximum change point of the profile. Data reduction and processing were performed using the PyMca package, which is freely distributed for non-commercial applications, and the code can be downloaded from http://www.esrf.fr/computing/bliss/downloads.[12]
SR-μ-XANES spectroscopy:
The XANES experiments at a 1W2B wiggler beamline of BSRF were performed, where the storage ring was run with electrons at an energy level of 2.5 GeV and 150 to 250 mA ring current. The micro-XANES spectra (irradiated spot 50× 50 μm) at the nickel K-edge of the skin sample were collected in fluorescence mode using a Vortex-EM silicon drift detector. The XANES spectrum of the reference nickel ion solution (10% NiSO4) was also collected in fluorescence mode for comparison. The photon energy was calibrated against the K-edge of nickel foil at 8333 ± 0.3 eV. The spectra were processed using Bruce Ravel's program ATHENA based on Matt Newville's IFEFFIT library.
Statistical analysis:
Statistics analysis was performed with SPSS 18.0.0 software (IBM Corporation, Armonk, New York, USA) and Prism 7.0 software (GraphPad, La Jolla, CA, USA). The animals were randomly assigned to either the test or the control group by the random number table. The random allocation sequence was generated and was concealed from the main investigator. Kolmogorov-Smirnov test was used to test the normality of samples. To analyze the evaluation of erythema, we used the non-parametric Mann-Whitney U test, data were presented as median (Q1, Q3). To compare the fluorescence intensity of nickel, the Student's t test was used for statistical analysis. Data were presented as mean ± standard deviation. Levene test was used to test equality of variances. If the variances were heterogeneity, we used the Welch approximate t test to analyze data. A P < 0.05 was considered statistically significant.
Results:
Skin reactions (erythema and swelling) Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.
Evaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.
Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.
Evaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.
Pathohistological images of skin tissues The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.
Pathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.
The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.
Pathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.
Distribution of nickel element by SR-XRF from skin cross-sections SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.
The main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.
The relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.
SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.
The main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.
The relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.
Chemical species of nickel element in the skin model by SR-XANES XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]
X-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.
XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]
X-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.
Skin reactions (erythema and swelling):
Figure 1 shows the evaluation of erythema surrounding skin reactions in all study groups during the experimental time periods (at 6, 12, 24, 48, and 72 h after the challenge). The average erythema scores peaked at 24 h in 5% to 10% group (sensitization-challenge NiSO4 solution) and 10% to 10% group (sensitization-challenge NiSO4 solution). At 12 h, the score in the 10% to 10% group was significantly higher than that in the 5% to 10% group (3 [2, 3] vs. 1 [1, 1], Z = –3.953, P < 0.0001). The skin reaction in two groups simultaneously declined gradually after 24 h. These symptoms disappeared after 72 h in two groups.
Evaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.
Pathohistological images of skin tissues:
The H&E stained histopathological images from the skin cross-sections treated with patches for 24 h are shown in Figure 2. Gross changes observed by the naked eye revealed that the allergic responses on the surface of the skin tissues of guinea pigs were erythema and scabbing, which are likely due to the itching and redness found in patients with nickel ACD, with different degrees of change observed. In the late phase group, we observed late-phase histological features such as epidermal thickening, hyperkeratosis, and/or para-asteatosis and acanthosis in the epidermis, as well as angiotelectasis and inflammatory cell infiltration in the epidermis and dermis. Histopathological findings included hyperkeratosis with parakeratosis, epidermal hyperplasia, dermal telangiectasia, and hyperemia [Figure 2]. In the early phase group, with 24 h challenge, there was substantially more inflammatory cell infiltration in the dermis than that in the late phase group. The skin lesions showed intra-cellular edema, liquefaction, and denaturation of the basal layer, as well as dermis vasodilatation and dermal cell infiltration.
Pathological alterations in skin of model after challenge with NiSO4 at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.
Distribution of nickel element by SR-XRF from skin cross-sections:
SR-XRF of the main elements from the skin cross-sections [Figure 3] showed clear peaks for not only nickel but also the metal elements Fe and Zn. The significantly higher signal intensity of nickel suggested the feasibility of the Ni-induced ACD model. The depth-resolved XRF was acquired to determine the distribution of nickel in the ACD model tissue. The photon energy corresponds to absorption above the Ni K edge excitation threshold and should be independent of any change in white line intensity due to oxidation state variation. The fluorescence intensity was proportional to the concentration of nickel [Figure 4]. In the early phase group, the content of nickel increased from 0 to 200 μm (epidermis in Figure 2), and it decreased gradually with deeper position. In contrast, the content of nickel element in the late phase group was slightly reduced and showed a stable fluctuation over the full depth. Obviously, within the deep position of 200 μm, the nickel content in the early phase group was much higher than that in the late phase group (0.00092 ± 1.961 × 10–5vs. 0.00053 ± 9.487 × 10–6, t = 17.91, degree of freedom [df] = 13.00, P < 0.0001). In deeper positions of 500 μm, the nickel element concentration in the two groups was approximately similar (0.00040 ± 9.803 × 10–6vs. 0.00038 ± 1.992 × 10–5, t = 0.90, df = 13.12, P = 0.380) [Figure 4]. These results suggested that the permeated nickel content was concentrated in the epidermis first. Then, the content penetrated to the dermis beyond the basal layer, especially in the early phase group.
The main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.
The relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.
Chemical species of nickel element in the skin model by SR-XANES:
XANES studies were performed to investigate the nickel binding properties in ACD model tissues [Figure 5]. The comparison of the XANES data for nickel in the 10% NiSO4 solution and the ACD model tissue showed that a structural change occurred in the nickel site. Nickel had vacancies in the 3d manifold, and peaks associated with 1s–3d transitions were observed in the pre-edge XANES for nickel in the 10% NiSO4 solution [Figure 5], suggesting that nickel was in the aqueous ionic state. In the case of ACD model tissues, small shoulders associated with peaks involving 1s–4pz transitions were observed. The peaks depended on the coordination number and geometry of the metal site. By comparing the peaks with samples of known coordination numbers and geometries, nickel in ACD model tissue was not present in the Ni2+ aqueous ionic state but in nickel-binding protein.[13]
X-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO4 solution (NiSO4 solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.
Discussion:
In the present study, Ni-induced ACD models were produced by modified protocols with SP formulations for guinea pigs based on traditional skin sensitization methods. We used SR-XRF to investigate the distribution of the nickel element in the skin tissues of two different Ni-ACD models (late phase and early phase), and the chemical species of the nickel element was also revealed via SR-XANES. In addition, H&E stained imaging of the target tissue sections provided histopathological information. Hence, the feasibility of these models and the distribution of metal permeation behavior in the skin tissues was confirmed.
Ni-ACD is a type IV allergy reaction.[14] For a hapten to be able to initiate this reaction, it has to penetrate through the stratum corneum to the epidermis.[15] Therefore, efforts have been undertaken to investigate the penetration and distribution of haptens through the skin.[11,14,16–18] For normal human and mouse skin, imaging mass spectrometry and SR-XRF were used to analyze the penetration and distribution of nickel.[7,8] However, these methods do not give detailed information on nickel element penetration and distribution in the skin in the pathogenesis of contact dermatitis. Therefore, there is a need to investigate clinically relevant haptens and their distribution in the epidermis of contact dermatitis with non-destructive inspection.
The evaluation of erythema on skin reactions is used to judge the concentration of nickel indirectly.[19] According to our study, we found that symptoms were dependent on the time of nickel penetration. An in vitro study evaluating nickel element permeation through the human stratum corneum also reported that the permeation rate of aqueous NiSO4 solution was the highest after approximately 24 h, regardless of the nickel salt species, which is in agreement with our results.[8]
The two groups, including both early phase and late phase ACD models, displayed different symptoms during the experiment. For each depth of skin, the nickel element concentration in the early phase group was higher than that in the late phase group. In our study, it was observed that the skin nickel element concentration from 100 to 200 μm depth in the early phase group was higher than that under 300 μm depth. Similar results were also found by Sugiyama et al.[7] However, the lord-balanced permeated nickel was diffused in the cross-section of the late phase group. The concentration of nickel caused this phenomenon. Permeation of high concentrations of nickel into the skin was reported to increase blood flow.[20,21] Thus, the removal of nickel-binding proteins by blood flow may keep nickel concentration well-balanced under deeper tissues. Corresponding to the evaluation of erythema in skin reactions, the states of swelling and skin lesions contributed to the depth of tissues, which might enlarge scales of the stratum corneum and epidermis in a severe lesion condition.
Comparing the XANES data for the 10% NiSO4 solution treatment, structural changes occurred in the skin model sample. We demonstrated that nickel was not present in the Ni2+ aqueous ionic state but bound to nickel-binding proteins in vivo. Some reports found and proved this phenomenon of nickel metal-binding amino acids, peptides, or proteins in vitro.[22–24] Our study demonstrated nickel binding events for nickel contact allergy in vivo by the measurement of XANES. Some relative studies have reported that Ni2+ triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4) in vitro, and they demonstrated that Ni2+-induced ACD symptoms were species-specific, as the mouse TLR family could not generate this response; mouse TLR senses microbial pathogens and endogenous ligands.[13,25] A recent study has reported that nickel (II) ions bind particularly at histidine residues, which are in the region required for the recognition of nickel signals.[26] However, it is not clear which proteins of hapten Ni2+ were combined to trigger Ni-induced ACD. Future research is required to explore this further.
In conclusion, this study showed that the distribution of the nickel element in ACD skin tissue was different between the early phase and late phase groups. The nickel element was not present in the Ni2+ aqueous ionic state but bound with certain proteins to form a complex in the stratum corneum nickel in ACD model tissue. SR-XRF and XANES technologies were non-invasive, and depth-resolved structure analysis was used for the conventional histopathological specimens. Therefore, these technologies may be applicable for the screening of metal-affected lesions and the evaluation of the effects of permeated metal elements on skin tissue lesions.
Acknowledgements:
The authors thank Bo Chen at pathology department of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (CAMS) for giving advices in this study.
Funding:
This research was supported by the grants from National Natural Science Foundation of China (No. 81373175), and CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-1003).
Conflicts of interest:
None.
| Background:
Nickel-induced allergic contact dermatitis (Ni-ACD) is a global health problem. More detailed knowledge on the skin uptake of haptens is required. This study aimed to investigate the penetration process and distribution of nickel in skin tissues with late phase and early phase of Ni-ACD to understand the mechanisms of metal allergy.
Methods:
Forty Hartley guinea pigs were divided into four groups according to the NiSO4 sensitizing concentration and the NiSO4 challenged concentration: the 5% NiSO4-group, 5% to 10% (sensitization-challenge; late phase group); 10% NiSO4-group, 10% to 10% (sensitization-challenge; early-phase group); and the positive and negative controls. Pathological biopsies were performed on each group. The depth profile of nickel element concentration in the skin of guinea pigs was detected by synchrotron radiation micro X-ray fluorescence spectroscopy (SR-μ-XRF) and micro X-ray absorption near-edge spectroscopy (μ-XANES).
Results:
In each section, the nickel element concentration in both the 5% NiSO4-group and 10% NiSO4-group was significantly higher than that in the negative control group. In the upper 300-μm section of skin for the early phase group, the nickel element concentration was significantly higher than that in the lower section of skin. In deeper sections (>200 μm) of skin, the concentration of nickel in the early phase group was approximately equal to that in the late phase group. The curve of the late phase group was flat, which means that the nickel element concentration was distributed uniformly by SR-μ-XRF. According to the XANES data for the 10% NiSO4 metal salt solution, structural changes occurred in the skin model sample, indicating that nickel was not present in the Ni aqueous ionic state but in the nickel-binding protein.
Conclusions:
This study showed that the distribution of the nickel element concentration in ACD skin tissue was different between the early phase and late phase groups. The nickel element was not present in the Ni aqueous ionic state but bound with certain proteins to form a complex in the stratum corneum in ACD model tissue. | null | null | 9,780 | 426 | [
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] | null | null | [CONTENT] Synchrotron radiation micro X-ray fluorescence spectroscopy | Micro X-ray absorption near-edge spectroscopy | Dermatitis, Allergic contact | Nickel-induced allergic contact dermatitis | Dermatology [SUMMARY] | [CONTENT] Synchrotron radiation micro X-ray fluorescence spectroscopy | Micro X-ray absorption near-edge spectroscopy | Dermatitis, Allergic contact | Nickel-induced allergic contact dermatitis | Dermatology [SUMMARY] | [CONTENT] Synchrotron radiation micro X-ray fluorescence spectroscopy | Micro X-ray absorption near-edge spectroscopy | Dermatitis, Allergic contact | Nickel-induced allergic contact dermatitis | Dermatology [SUMMARY] | null | [CONTENT] Synchrotron radiation micro X-ray fluorescence spectroscopy | Micro X-ray absorption near-edge spectroscopy | Dermatitis, Allergic contact | Nickel-induced allergic contact dermatitis | Dermatology [SUMMARY] | null | [CONTENT] Animals | Dermatitis, Allergic Contact | Female | Guinea Pigs | Male | Nickel | Random Allocation | Skin | Spectrometry, X-Ray Emission [SUMMARY] | [CONTENT] Animals | Dermatitis, Allergic Contact | Female | Guinea Pigs | Male | Nickel | Random Allocation | Skin | Spectrometry, X-Ray Emission [SUMMARY] | [CONTENT] Animals | Dermatitis, Allergic Contact | Female | Guinea Pigs | Male | Nickel | Random Allocation | Skin | Spectrometry, X-Ray Emission [SUMMARY] | null | [CONTENT] Animals | Dermatitis, Allergic Contact | Female | Guinea Pigs | Male | Nickel | Random Allocation | Skin | Spectrometry, X-Ray Emission [SUMMARY] | null | [CONTENT] concentrations nickel environment | nickel tissues study | prevalence example nickel | skin nickel exposed | analyze nickel allergy [SUMMARY] | [CONTENT] concentrations nickel environment | nickel tissues study | prevalence example nickel | skin nickel exposed | analyze nickel allergy [SUMMARY] | [CONTENT] concentrations nickel environment | nickel tissues study | prevalence example nickel | skin nickel exposed | analyze nickel allergy [SUMMARY] | null | [CONTENT] concentrations nickel environment | nickel tissues study | prevalence example nickel | skin nickel exposed | analyze nickel allergy [SUMMARY] | null | [CONTENT] nickel | skin | group | phase | 10 | model | acd | xrf | erythema | 24 [SUMMARY] | [CONTENT] nickel | skin | group | phase | 10 | model | acd | xrf | erythema | 24 [SUMMARY] | [CONTENT] nickel | skin | group | phase | 10 | model | acd | xrf | erythema | 24 [SUMMARY] | null | [CONTENT] nickel | skin | group | phase | 10 | model | acd | xrf | erythema | 24 [SUMMARY] | null | [CONTENT] nickel | allergy | distribution | distribution nickel | skin | metal allergy | countries | exposure | penetration | metal [SUMMARY] | [CONTENT] test | ml | swelling | beijing | animals | skin | erythema | group | patch | nickel [SUMMARY] | [CONTENT] nickel | model | skin | group | acd | phase group | acd model | phase | 10 | figure [SUMMARY] | null | [CONTENT] nickel | skin | group | phase | 10 | acd | model | test | erythema | 24 [SUMMARY] | null | [CONTENT] ||| ||| [SUMMARY] | [CONTENT] Forty | four | NiSO4 | NiSO4 | 5% | 5% to 10% | 10% | 10% to 10% ||| ||| [SUMMARY] | [CONTENT] 5% | 10% ||| 300-μm ||| 200 μm ||| SR-μ-XRF ||| XANES | 10% | Ni [SUMMARY] | null | [CONTENT] ||| ||| ||| Forty | four | NiSO4 | NiSO4 | 5% | 5% to 10% | 10% | 10% to 10% ||| ||| ||| ||| 5% | 10% ||| 300-μm ||| 200 μm ||| SR-μ-XRF ||| XANES | 10% | Ni ||| ACD ||| Ni | ACD [SUMMARY] | null |
||||
LncRNA LIFR-AS1 overexpression suppressed the progression of serous ovarian carcinoma. | 35778954 | Serous ovarian carcinoma (SOC) is a common malignant tumor in female reproductive system. Long noncoding RNA (lncRNA) LIFR-AS1 is a tumor suppressor gene in colorectal cancer, but its effect and underlying mechanism in SOC are still unclear. Therefore, this study focuses on unveiling the regulatory mechanism of LIFR-AS1 in SOC. | BACKGROUND | The relationship between LIFR-AS1 expression and prognosis of SOC patients was analyzed by TCGA database and Starbase, and then, the LIFR-AS1 expression in SOC tissues and cells was detected by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Besides, the relationship between LIFR-AS1 and clinical characteristics was analyzed. Also, the effects of LIFR-AS1 on the biological behaviors of SOC cells were measured by Cell Counting Kit-8, colony formation, and wound-healing and Transwell assays, respectively. Western blot and qRT-PCR were employed to determine the protein expressions of genes related to proliferation (PCNA), apoptosis (cleaved caspase-3), epithelial-mesenchymal transition (E-cadherin, N-cadherin, and Snail). | METHODS | LIFR-AS1 was lowly expressed in SOC, which was correlated with the poor prognosis of SOC patients. Low expression of LIFR-AS1 in SOC was associated with the tumor size, clinical stage, lymph node metastasis, and distant metastasis. LIFR-AS1 overexpression promoted the expressions of cleaved caspase-3 and E-cadherin while suppressing the malignant behaviors (proliferation, migration, and invasion) of SOC cells, the expressions of PCNA, N-cadherin, and Snail. Besides, silencing LIFR-AS1 exerted the effects opposite to overexpressed LIFR-AS1. | RESULTS | LIFR-AS1 overexpression inhibits biological behaviors of SOC cells, which may be a new therapeutic method. | CONCLUSION | [
"Cadherins",
"Carcinoma, Ovarian Epithelial",
"Caspase 3",
"Cell Line, Tumor",
"Cell Movement",
"Cell Proliferation",
"Female",
"Gene Expression Regulation, Neoplastic",
"Humans",
"Leukemia Inhibitory Factor Receptor alpha Subunit",
"Ovarian Neoplasms",
"Proliferating Cell Nuclear Antigen",
"RNA, Long Noncoding"
] | 9396205 | INTRODUCTION | Ovarian cancer (OC) is one of the most common malignancies of the female reproductive system, and the WHO estimates that there are approximately 225, 500 newly diagnosed cases of OC and approximately 140, 200 newly emerged deaths worldwide each year.
1
Although the diagnosis and treatment of malignant tumors have gradually improved in recent years, the mortality rate of OC still ranks first in gynecological malignancies.
2
The most common pathological type of OC is epithelial ovarian cancer, 75% of which are serous ovarian carcinomas (SOCs).
3
SOC has been classified into various stages, including ovarian serous cystadenoma, borderline ovarian serous, low‐grade SOC, and high‐grade SOC, with different expressions of specific tumor markers at various stages.
3
SOC lacks a reliable early diagnostic indicator and typical early symptoms.
4
As such, about 75% of patients, at the time of diagnosis, are at clinically advanced stage III or IV.
5
Due to the susceptibility to recurrence and the postoperative resistance to chemotherapy drugs, the prognosis of patients is very poor and 5‐year survival rate is low.
6
Therefore, exploring the molecular mechanisms associated with the biological behavior of SOC is essential for the early diagnosis and prognosis evaluation of SOC.
Long noncoding RNAs (lncRNAs) are RNAs that cannot encode proteins due to the lack of a meaningful open reading frame.
7
Several recent studies have discovered that lncRNAs have powerful gene regulatory function and participate in a variety of pathophysiological processes,
8
,
9
playing vital roles in cancer progression,
10
as well as the progression of SOC. For instance, lncRNA MAGI2‐AS3 leads to the tumor suppression of high‐grade SOC,
11
and lncRNA CTD‐2020 K17.1 promotes metastasis and proliferation of SOC cells.
12
In addition, lncRNA NEAT1 facilitates the malignant phenotype of SOC by mediating miR‐506.
13
There is also evidence that the mechanism of action of some lncRNAs is dependent on their genomic location (sense, antisense, bidirection, intron, and intergene), particularly the positional relationship with neighboring genes,
14
,
15
in which antisense lncRNAs are noncoding RNAs encoded by genes located on the opposite strand of protein‐coding genes and often completely or partially complementary to protein‐coding genes.
16
A large body of evidence has indicated that antisense lncRNAs are pervasive, abundantly present within cells, and have specific cellular localizations, heralding that such molecules may have important biological implications.
17
,
18
However, less attention has been paid to this part, and the specific role of antisense lncRNAs in SOC has not been clearly elucidated. Combined with relevant literature and bioinformatics analysis, our study singled out LIFR‐AS1 to unveil its effects on SOC.
LIFR‐AS1 is a newly discovered lncRNA and has a strong association with tumor progression, which is transcribed in an antisense fashion from the leukemia inhibitory factor receptor (LIFR) gene.
19
LIFR could regulate cell proliferation, differentiation, and phenotype in various cancers, such as colorectal cancer and breast cancer.
20
,
21
,
22
Few literatures have indicated that LIFR‐AS1 exerts suppressive effects on the initiation and progression of assorted cancers, such as breast cancer,
23
lung cancer,
24
and glioma.
25
Nevertheless, the effect and underlying mechanism of LIFR‐AS1 in SOC are still dim, which is the direction of this current study. | null | null | RESULTS | Low expression of LIFR‐AS1 in SOC was associated with the poor prognosis of SOC patients As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).
Expression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).
Expression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
LIFR‐AS1 overexpression inhibited viability and proliferation of SOC cells while silencing LIFR‐AS1 had the opposite effect Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).
Effects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **
p < 0.01; ***
p < 0.001 vs. HOSE; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).
Effects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **
p < 0.01; ***
p < 0.001 vs. HOSE; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
LIFR‐AS1 overexpression inhibited the migration and invasion of SOC cells while silencing LIFR‐AS1 had the opposite effect As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).
Effects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation
As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).
Effects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation
LIFR‐AS1 regulated the expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in SOC cells It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).
Effects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation
It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).
Effects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation | null | null | [
"INTRODUCTION",
"Ethics statement and specimen collection",
"Bioinformatics assay",
"Cell culture",
"In situ hybridization (ISH)",
"Transfection",
"QRT‐PCR",
"Cell counting kit‐8 (CCK‐8) assay",
"Colony formation assay",
"Wound‐healing assay",
"Transwell assay",
"Western blot",
"Statistical analysis",
"Low expression of LIFR‐AS1 in SOC was associated with the poor prognosis of SOC patients",
"\nLIFR‐AS1 overexpression inhibited viability and proliferation of SOC cells while silencing LIFR‐AS1 had the opposite effect",
"\nLIFR‐AS1 overexpression inhibited the migration and invasion of SOC cells while silencing LIFR‐AS1 had the opposite effect",
"\nLIFR‐AS1 regulated the expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in SOC cells"
] | [
"Ovarian cancer (OC) is one of the most common malignancies of the female reproductive system, and the WHO estimates that there are approximately 225, 500 newly diagnosed cases of OC and approximately 140, 200 newly emerged deaths worldwide each year.\n1\n Although the diagnosis and treatment of malignant tumors have gradually improved in recent years, the mortality rate of OC still ranks first in gynecological malignancies.\n2\n The most common pathological type of OC is epithelial ovarian cancer, 75% of which are serous ovarian carcinomas (SOCs).\n3\n SOC has been classified into various stages, including ovarian serous cystadenoma, borderline ovarian serous, low‐grade SOC, and high‐grade SOC, with different expressions of specific tumor markers at various stages.\n3\n SOC lacks a reliable early diagnostic indicator and typical early symptoms.\n4\n As such, about 75% of patients, at the time of diagnosis, are at clinically advanced stage III or IV.\n5\n Due to the susceptibility to recurrence and the postoperative resistance to chemotherapy drugs, the prognosis of patients is very poor and 5‐year survival rate is low.\n6\n Therefore, exploring the molecular mechanisms associated with the biological behavior of SOC is essential for the early diagnosis and prognosis evaluation of SOC.\nLong noncoding RNAs (lncRNAs) are RNAs that cannot encode proteins due to the lack of a meaningful open reading frame.\n7\n Several recent studies have discovered that lncRNAs have powerful gene regulatory function and participate in a variety of pathophysiological processes,\n8\n, \n9\n playing vital roles in cancer progression,\n10\n as well as the progression of SOC. For instance, lncRNA MAGI2‐AS3 leads to the tumor suppression of high‐grade SOC,\n11\n and lncRNA CTD‐2020 K17.1 promotes metastasis and proliferation of SOC cells.\n12\n In addition, lncRNA NEAT1 facilitates the malignant phenotype of SOC by mediating miR‐506.\n13\n There is also evidence that the mechanism of action of some lncRNAs is dependent on their genomic location (sense, antisense, bidirection, intron, and intergene), particularly the positional relationship with neighboring genes,\n14\n, \n15\n in which antisense lncRNAs are noncoding RNAs encoded by genes located on the opposite strand of protein‐coding genes and often completely or partially complementary to protein‐coding genes.\n16\n A large body of evidence has indicated that antisense lncRNAs are pervasive, abundantly present within cells, and have specific cellular localizations, heralding that such molecules may have important biological implications.\n17\n, \n18\n However, less attention has been paid to this part, and the specific role of antisense lncRNAs in SOC has not been clearly elucidated. Combined with relevant literature and bioinformatics analysis, our study singled out LIFR‐AS1 to unveil its effects on SOC.\nLIFR‐AS1 is a newly discovered lncRNA and has a strong association with tumor progression, which is transcribed in an antisense fashion from the leukemia inhibitory factor receptor (LIFR) gene.\n19\n LIFR could regulate cell proliferation, differentiation, and phenotype in various cancers, such as colorectal cancer and breast cancer.\n20\n, \n21\n, \n22\n Few literatures have indicated that LIFR‐AS1 exerts suppressive effects on the initiation and progression of assorted cancers, such as breast cancer,\n23\n lung cancer,\n24\n and glioma.\n25\n Nevertheless, the effect and underlying mechanism of LIFR‐AS1 in SOC are still dim, which is the direction of this current study.",
"The diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.\n26\n The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).\nClinical characteristics of SOC patients",
"Data on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).",
"Human Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.",
"The tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).",
"The overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).",
"Cell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.\n27\n\n\nPrimers for quantitative real‐time polymerase chain reaction",
"A2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).",
"For the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).",
"A2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).",
"The 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).",
"RIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).",
"Based on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.",
"As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).\nExpression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation",
"Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).\nEffects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **\np < 0.01; ***\np < 0.001 vs. HOSE; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation",
"As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).\nEffects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation",
"It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).\nEffects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation"
] | [
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] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Ethics statement and specimen collection",
"Bioinformatics assay",
"Cell culture",
"In situ hybridization (ISH)",
"Transfection",
"QRT‐PCR",
"Cell counting kit‐8 (CCK‐8) assay",
"Colony formation assay",
"Wound‐healing assay",
"Transwell assay",
"Western blot",
"Statistical analysis",
"RESULTS",
"Low expression of LIFR‐AS1 in SOC was associated with the poor prognosis of SOC patients",
"\nLIFR‐AS1 overexpression inhibited viability and proliferation of SOC cells while silencing LIFR‐AS1 had the opposite effect",
"\nLIFR‐AS1 overexpression inhibited the migration and invasion of SOC cells while silencing LIFR‐AS1 had the opposite effect",
"\nLIFR‐AS1 regulated the expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in SOC cells",
"DISCUSSION",
"CONFLICT OF INTEREST"
] | [
"Ovarian cancer (OC) is one of the most common malignancies of the female reproductive system, and the WHO estimates that there are approximately 225, 500 newly diagnosed cases of OC and approximately 140, 200 newly emerged deaths worldwide each year.\n1\n Although the diagnosis and treatment of malignant tumors have gradually improved in recent years, the mortality rate of OC still ranks first in gynecological malignancies.\n2\n The most common pathological type of OC is epithelial ovarian cancer, 75% of which are serous ovarian carcinomas (SOCs).\n3\n SOC has been classified into various stages, including ovarian serous cystadenoma, borderline ovarian serous, low‐grade SOC, and high‐grade SOC, with different expressions of specific tumor markers at various stages.\n3\n SOC lacks a reliable early diagnostic indicator and typical early symptoms.\n4\n As such, about 75% of patients, at the time of diagnosis, are at clinically advanced stage III or IV.\n5\n Due to the susceptibility to recurrence and the postoperative resistance to chemotherapy drugs, the prognosis of patients is very poor and 5‐year survival rate is low.\n6\n Therefore, exploring the molecular mechanisms associated with the biological behavior of SOC is essential for the early diagnosis and prognosis evaluation of SOC.\nLong noncoding RNAs (lncRNAs) are RNAs that cannot encode proteins due to the lack of a meaningful open reading frame.\n7\n Several recent studies have discovered that lncRNAs have powerful gene regulatory function and participate in a variety of pathophysiological processes,\n8\n, \n9\n playing vital roles in cancer progression,\n10\n as well as the progression of SOC. For instance, lncRNA MAGI2‐AS3 leads to the tumor suppression of high‐grade SOC,\n11\n and lncRNA CTD‐2020 K17.1 promotes metastasis and proliferation of SOC cells.\n12\n In addition, lncRNA NEAT1 facilitates the malignant phenotype of SOC by mediating miR‐506.\n13\n There is also evidence that the mechanism of action of some lncRNAs is dependent on their genomic location (sense, antisense, bidirection, intron, and intergene), particularly the positional relationship with neighboring genes,\n14\n, \n15\n in which antisense lncRNAs are noncoding RNAs encoded by genes located on the opposite strand of protein‐coding genes and often completely or partially complementary to protein‐coding genes.\n16\n A large body of evidence has indicated that antisense lncRNAs are pervasive, abundantly present within cells, and have specific cellular localizations, heralding that such molecules may have important biological implications.\n17\n, \n18\n However, less attention has been paid to this part, and the specific role of antisense lncRNAs in SOC has not been clearly elucidated. Combined with relevant literature and bioinformatics analysis, our study singled out LIFR‐AS1 to unveil its effects on SOC.\nLIFR‐AS1 is a newly discovered lncRNA and has a strong association with tumor progression, which is transcribed in an antisense fashion from the leukemia inhibitory factor receptor (LIFR) gene.\n19\n LIFR could regulate cell proliferation, differentiation, and phenotype in various cancers, such as colorectal cancer and breast cancer.\n20\n, \n21\n, \n22\n Few literatures have indicated that LIFR‐AS1 exerts suppressive effects on the initiation and progression of assorted cancers, such as breast cancer,\n23\n lung cancer,\n24\n and glioma.\n25\n Nevertheless, the effect and underlying mechanism of LIFR‐AS1 in SOC are still dim, which is the direction of this current study.",
"Ethics statement and specimen collection The diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.\n26\n The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).\nClinical characteristics of SOC patients\nThe diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.\n26\n The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).\nClinical characteristics of SOC patients\nBioinformatics assay Data on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).\nData on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).\nCell culture Human Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.\nHuman Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.\nIn situ hybridization (ISH) The tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).\nThe tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).\nTransfection The overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).\nThe overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).\nQRT‐PCR Cell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.\n27\n\n\nPrimers for quantitative real‐time polymerase chain reaction\nCell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.\n27\n\n\nPrimers for quantitative real‐time polymerase chain reaction\nCell counting kit‐8 (CCK‐8) assay A2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).\nA2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).\nColony formation assay For the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).\nFor the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).\nWound‐healing assay A2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).\nA2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).\nTranswell assay The 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).\nThe 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).\nWestern blot RIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).\nRIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).\nStatistical analysis Based on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.\nBased on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.",
"The diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.\n26\n The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).\nClinical characteristics of SOC patients",
"Data on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).",
"Human Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.",
"The tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).",
"The overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).",
"Cell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.\n27\n\n\nPrimers for quantitative real‐time polymerase chain reaction",
"A2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).",
"For the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).",
"A2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).",
"The 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).",
"RIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).",
"Based on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.",
"Low expression of LIFR‐AS1 in SOC was associated with the poor prognosis of SOC patients As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).\nExpression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation\nAs depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).\nExpression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation\n\nLIFR‐AS1 overexpression inhibited viability and proliferation of SOC cells while silencing LIFR‐AS1 had the opposite effect Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).\nEffects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **\np < 0.01; ***\np < 0.001 vs. HOSE; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation\nSubsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).\nEffects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **\np < 0.01; ***\np < 0.001 vs. HOSE; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation\n\nLIFR‐AS1 overexpression inhibited the migration and invasion of SOC cells while silencing LIFR‐AS1 had the opposite effect As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).\nEffects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation\nAs the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).\nEffects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation\n\nLIFR‐AS1 regulated the expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in SOC cells It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).\nEffects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation\nIt can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).\nEffects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation",
"As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).\nExpression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation",
"Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).\nEffects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **\np < 0.01; ***\np < 0.001 vs. HOSE; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation",
"As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).\nEffects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation",
"It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).\nEffects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^\np < 0.05; ^^\np < 0.01; ^^^\np < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation",
"As an important regulator, lncRNA has become a hotspot in researching SOC. Although lncRNAs could not encode protein, they can exert some effects on cellular behaviors such as cell proliferation, apoptosis, senescence, and migration by regulating the expressions of relevant functional genes.\n15\n The study of Ni et al. showed that down‐regulated LINC00515 is correlated with the tumor growth of SOC.\n28\n Guo et al. proved that lncRNA SOCAR aggravates the development of SOC via the Wnt/β‐catenin pathway.\n29\n Additionally, Gokulnath et al. indicated that lncRNA HAND2‐AS1 has the function to suppress the tumor growth of SOC.\n30\n LIFR‐AS1, which is an antisense lncRNA for the LIFR coding gene, has been noted that its aberrant expression is closely related to the pathogenesis and prognosis of patients with some tumors. The study by Okamura et al. found that LIFR‐AS1 can inhibit the proliferation and migration of hepatocellular carcinoma (HCC) cells and is an important indicator of poor prognosis in HCC.\n31\n Another study revealed that LIFR‐AS1 up‐regulation is positively correlated with more advanced and aggressive gastric cancer features.\n32\n Based on previous studies, this study explored the expression of LIFR‐AS1 in SOC cells and its role in SOC progression. The results of this study signified that LIFR‐AS1 was lowly expressed in SOC tissues and cells (A2780, OV‐56, SK‐OV‐3, and OVCAR3). LIFR‐AS1 overexpression could inhibit the cell viability, proliferation, migration, and invasion in A2780 and SK‐OV‐3 cells.\nThe indefinite proliferation and hampered apoptosis of the cells are the main characteristics of the tumor cells, such as SOC cells.\n33\n, \n34\n PCNA is a nuclear protein widely expressed in S phase of cell cycle and is only synthesized and expressed in proliferative cells. The positive expression of PCNA indicates that the cell is in proliferative state.\n35\n, \n36\n As a tumor marker protein, PCNA can reflect the synthesis and metabolism of RNA and DNA in tumor cells, its expression level can be measured to assess the proliferative activity of tissues and various cancer cells, contributing to better determining progression, aggressiveness, and prognosis of the lesions.\n36\n It has proved that down‐regulation of the expression of PCNA repressed G1/S cell cycle transition of human OC cells.\n37\n Additionally, lncRNA DLX6‐AS1 inhibited the proliferation of OC cells via reducing the expression of PCNA.\n38\n Caspase‐3 is the most important final executor of apoptosis in caspase family and has a strong ability to induce apoptosis.\n39\n Studies evidenced that lncRNA NNT‐AS1 knockdown or lncRNA PCAT‐1 overexpression weakens the activity of caspase‐3 to impede the apoptosis of human OC cells.\n40\n, \n41\n Similar to these studies, our study got the knowledge that LIFR‐AS1 overexpression decreased the expression of PCNA and increased the cleaved caspase‐3 expression of SOC cells, and silencing of LIFR‐AS1 reversely regulated these expressions, indicating that LIFR‐AS1 overexpression hindered proliferation and induced apoptosis of SOC cells by regulating the levels of related molecules.\nInvasion and metastasis are important contributors to the vast majority of tumor‐associated metastasis and recurrence. Epithelial–mesenchymal transition (EMT) is an important mechanism of invasion and metastasis.\n42\n It refers to the pathophysiological process of epithelial cells with polar and tight adhesion into non‐polar and highly mobile stromal cells.\n42\n EMT has been observed in a variety of human malignant tumors,\n43\n, \n44\n including SOC.\n45\n In this study, we detected the expressions of three EMT‐related proteins, including E‐cadherin, N‐cadherin, and Snail. E‐cadherin participates in the adhesion and connection between homotypic cells and maintains cell polarity, which plays an important role in maintaining the integrity of epithelial cell morphology and tissue structure. The overexpression of E‐cadherin protein may inhibit tumor occurrence and metastasis, while the effect of N‐cadherin is opposite to that of E‐cadherin, and its expression can promote tumor invasion and metastasis.\n46\n Besides, Snail, as one of EMT‐related transcription factors, could regulate the expression level of E‐cadherin, and is also a key factor in EMT.\n47\n Massive amounts of evidence proved that lncRNAs enhance the migratory and invasive abilities of OC or SOC cells via regulating the EMT‐regulated genes. For instance, lncRNA OIP5‐AS1 up‐regulates Snail expression to promote OC cell invasion and migration,\n48\n lncRNA EBIC promotes metastasis of OC cells through up‐regulating the E‐cadherin expression,\n49\n and lncRNA HAL suppresses the metastasis of SOC cells by regulating the expressions of E‐cadherin and N‐cadherin to inhibit EMT signaling pathway.\n50\n Consistent with the results described in the previous literature, our study discovered that LIFR‐AS1 overexpression facilitated an anti‐metastatic phenotype in SOC by regulating EMT‐related genes. Concretely, LIFR‐AS1 overexpression increased the expression of E‐cadherin, but decreased N‐cadherin and Snail expressions. Moreover, silencing LIFR‐AS1 regulated these expressions in a reverse way.\nTo sum up, our study demonstrates that LIFR‐AS1 expression is down‐regulated in SOC, and LIFR‐AS1 overexpression inhibits SOC cell viability, proliferation, invasion, and migration by regulating the expressions of PCNA, cleaved caspase‐3, and EMT‐related genes. Our findings may provide the potential of LIFR‐AS1, as a therapeutic target for SOC.",
"The authors declare no conflicts of interest."
] | [
null,
"materials-and-methods",
null,
null,
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null,
null,
null,
null,
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null,
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null,
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"results",
null,
null,
null,
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"discussion",
"COI-statement"
] | [
"epithelial–mesenchymal transition",
"LIFR‐AS1",
"proliferation",
"serous ovarian carcinoma"
] | INTRODUCTION:
Ovarian cancer (OC) is one of the most common malignancies of the female reproductive system, and the WHO estimates that there are approximately 225, 500 newly diagnosed cases of OC and approximately 140, 200 newly emerged deaths worldwide each year.
1
Although the diagnosis and treatment of malignant tumors have gradually improved in recent years, the mortality rate of OC still ranks first in gynecological malignancies.
2
The most common pathological type of OC is epithelial ovarian cancer, 75% of which are serous ovarian carcinomas (SOCs).
3
SOC has been classified into various stages, including ovarian serous cystadenoma, borderline ovarian serous, low‐grade SOC, and high‐grade SOC, with different expressions of specific tumor markers at various stages.
3
SOC lacks a reliable early diagnostic indicator and typical early symptoms.
4
As such, about 75% of patients, at the time of diagnosis, are at clinically advanced stage III or IV.
5
Due to the susceptibility to recurrence and the postoperative resistance to chemotherapy drugs, the prognosis of patients is very poor and 5‐year survival rate is low.
6
Therefore, exploring the molecular mechanisms associated with the biological behavior of SOC is essential for the early diagnosis and prognosis evaluation of SOC.
Long noncoding RNAs (lncRNAs) are RNAs that cannot encode proteins due to the lack of a meaningful open reading frame.
7
Several recent studies have discovered that lncRNAs have powerful gene regulatory function and participate in a variety of pathophysiological processes,
8
,
9
playing vital roles in cancer progression,
10
as well as the progression of SOC. For instance, lncRNA MAGI2‐AS3 leads to the tumor suppression of high‐grade SOC,
11
and lncRNA CTD‐2020 K17.1 promotes metastasis and proliferation of SOC cells.
12
In addition, lncRNA NEAT1 facilitates the malignant phenotype of SOC by mediating miR‐506.
13
There is also evidence that the mechanism of action of some lncRNAs is dependent on their genomic location (sense, antisense, bidirection, intron, and intergene), particularly the positional relationship with neighboring genes,
14
,
15
in which antisense lncRNAs are noncoding RNAs encoded by genes located on the opposite strand of protein‐coding genes and often completely or partially complementary to protein‐coding genes.
16
A large body of evidence has indicated that antisense lncRNAs are pervasive, abundantly present within cells, and have specific cellular localizations, heralding that such molecules may have important biological implications.
17
,
18
However, less attention has been paid to this part, and the specific role of antisense lncRNAs in SOC has not been clearly elucidated. Combined with relevant literature and bioinformatics analysis, our study singled out LIFR‐AS1 to unveil its effects on SOC.
LIFR‐AS1 is a newly discovered lncRNA and has a strong association with tumor progression, which is transcribed in an antisense fashion from the leukemia inhibitory factor receptor (LIFR) gene.
19
LIFR could regulate cell proliferation, differentiation, and phenotype in various cancers, such as colorectal cancer and breast cancer.
20
,
21
,
22
Few literatures have indicated that LIFR‐AS1 exerts suppressive effects on the initiation and progression of assorted cancers, such as breast cancer,
23
lung cancer,
24
and glioma.
25
Nevertheless, the effect and underlying mechanism of LIFR‐AS1 in SOC are still dim, which is the direction of this current study.
MATERIALS AND METHODS:
Ethics statement and specimen collection The diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.
26
The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).
Clinical characteristics of SOC patients
The diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.
26
The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).
Clinical characteristics of SOC patients
Bioinformatics assay Data on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).
Data on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).
Cell culture Human Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.
Human Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.
In situ hybridization (ISH) The tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).
The tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).
Transfection The overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).
The overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).
QRT‐PCR Cell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.
27
Primers for quantitative real‐time polymerase chain reaction
Cell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.
27
Primers for quantitative real‐time polymerase chain reaction
Cell counting kit‐8 (CCK‐8) assay A2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).
A2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).
Colony formation assay For the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).
For the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).
Wound‐healing assay A2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).
A2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).
Transwell assay The 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).
The 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).
Western blot RIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).
RIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).
Statistical analysis Based on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.
Based on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.
Ethics statement and specimen collection:
The diagnostic and staging criteria of SOC were based on the study of Steffen Hauptmann et al.
26
The SOC (n = 87) and adjacent healthy fallopian tube tissues (n = 38, more than 2 cm away from tumor tissues) were obtained from SOC patients in the Second Affiliated Hospital of Jiaxing University. The clinical characteristics of SOC patients are depicted in Table 1. Tissue samples were cut from tumor and adjacent tissues about 0.5 cm in diameter, and taken back immediately after the operation. Following liquid–nitrogen cryogenic treatment, tissue samples were stored in a−80°C refrigerator. This research was conducted on the premise that patients agreed to provide their tissue for clinical research, and the clinical trial program was reviewed and approved by the Ethics Committee of The Second Affiliated Hospital of Jiaxing University (JXEY‐ZFYJ045).
Clinical characteristics of SOC patients
Bioinformatics assay:
Data on LIFR‐AS1 expression in SOC tissues were retrieved from TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and overall survival (150 months) of SOC patients was analyzed by Starbase (http://starbase.sysu.edu.cn/).
Cell culture:
Human Ovarian Surface Epithelial (HOSE) cells (7310, Yuhengfeng biotech,) were grown in Ovarian Epithelial Cell Medium (OEpiCM, 7311, Yuhengfeng biotech). A2780 cells (CBP60283, Cobioer,) were cultivated in RPMI‐1640 medium (R0883, Sigma‐Aldrich,) with 10% fetal bovine serum (FBS, 12007C, Sigma‐Aldrich). OV‐56 cells (96020759, ECACC,) were cultured in the Dulbecco's Modified Eagle Medium (DMEM, 56499C, Sigma‐Aldrich,) and HAMS F12 (51651C, Sigma‐Aldrich,) (1:1) supplemented with 2 mM glutamine, 5% FBS, 0.5 μg/ml hydrocortisone, and 10 μg/ml insulin. OVCAR3 cells (HTB‐161, ATCC,) were incubated in RPMI‐1640 medium containing 20% FBS and 0.01 mg/mL bovine insulin. SK‐OV‐3 cells (HTB‐77, ATCC) were maintained in McCoy's 5a Medium Modified (M9309, Sigma‐Aldrich) added with 10% FBS. The above cells were cultured in 37°C with 5% CO2.
In situ hybridization (ISH):
The tissue sections were put into the mixture of 0.8% Pepsin/Hydrochloric Acid solution (EHJ‐CAS0164999, JiaHui Biotech,) and digested in a water bath (TSGP28, Thermo Scientific,) at 37°C for 10 min (min). Then tissues were washed with tris‐buffered saline (TBS, 28358, Thermo Scientific,) for 3 times (5 min (min) for each time), dehydrated by gradient ethanol, and dried at room temperature. The DNA probe of LIFR‐AS1 (5’‐GCGCGCGGGTGCTCCAAG‐3′) was dripped into the section, covered with cover glass, denatured at 98°C for 10 min, annealed in ice bath, and hybridized in 37°C water bath for 1 h. Next, tissues were washed with TBS for additional 3 times (5 min each time). After addition of digoxigenin (DIG) antibody (1:1000, 11,093,274, Roche,) dropwise, the tissues were incubated with Alkaline Phosphatase (IVGN2208, Invitrogen,) at room temperature for 30 min. Following the washing with phosphate‐buffered saline (PBS, 10010049, Gibco,) twice for 5 min, the tissues were supplemented with DAB (8801–4965‐72, Invitrogen,) to develop for 5 min in the dark. Subsequently, the tissues were routinely dehydrated and transparently sealed. Finally, the coloration of LIFR‐AS1 in tissues was observed under a microscope (×200, Eclipse 80i, Nikon,).
Transfection:
The overexpression plasmid of LIFR‐AS1 was constructed using pCMV6‐Entry vector (PS100001, Origene), and short hairpin RNA targeting LIFR‐AS1 (shLIFR‐AS1, C02003, 5’‐TGGGACTTTGCGAATTACCTAAA‐3′) were purchased from GenePharma. LIFR‐AS1 overexpression plasmid, shLIFR‐AS1, and empty vector (negative control) were transfected into the A2780 and SK‐OV‐3 cells under the help of Lipofectamine 3000 Reagent (L3000001, Thermo Fisher,). Briefly, cells were seeded onto 6‐well plates at a density of 3 × 105 cells/well until the cells reached 70%–90% confluence. Then, the serum‐free medium was utilized to dilute transfection reagent and LIFR‐AS1 overexpression plasmid, empty vector, or shLIFR‐AS1 to form the reagent/sample mixture, followed by 48 h culture of SOC cells and mixture. The success of transfection was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR).
QRT‐PCR:
Cell/Tissue Total RNA Isolation Kit (RK02009, Biomarker,) was utilized to isolate the total RNAs, and cDNA synthesis was then operated using RT Master Mix (HY‐K0510A, MedChemExpress,). QRT‐PCR was utilized for detecting the mRNA expression levels of LIFR‐AS1, proliferating cell nuclear antigen (PCNA), E‐cadherin, N‐cadherin, and Snail using SYBR Green Fast qPCR Mix (RK02001, Biomarker,) in a D10 PCR gene amplification instrument (XuSensmart,). PCR conditions were as follows: 40 cycles of denaturation at 95°C for 20 s(s), annealing at 58°C for 20 s, and extension at 72°C for 20 s. The sequences of the primers are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was chosen as the internal control to normalize the gene expressions using the 2−ΔΔCT method.
27
Primers for quantitative real‐time polymerase chain reaction
Cell counting kit‐8 (CCK‐8) assay:
A2780 and SK‐OV‐3 cells inoculated on 96‐well plates (5 × 103 cells/well) were cultured for 24, 48, and 72 h(h) after various treatments, followed by the incubation with CCK‐8 reagent (96,992, 10 μl, Sigma‐Aldrich,) for 4 h. The optical density (OD) value was recorded at a wavelength of 450 nm with the RNE‐90002 microplate reader (Reagen,).
Colony formation assay:
For the determination of colony formation, 1 × 103 SOC cells that suspended in culture media with 10% FBS were placed in 6‐well plates, which were subsequently subjected to the incubation at 37 °C with 5% CO2 for 14 days. Thereafter, the fixation (15 min) and staining (20 min) of A2780 and SK‐OV‐3 cells were performed with 4% paraformaldehyde (M009, Gefanbio,) and 0.5% crystal violet solution (C0121, Beyotime,). Finally, the condition of cell colony formation was observed under a DMi8 microscope (×100, Leica).
Wound‐healing assay:
A2780 and SK‐OV‐3 cells were seeded in a 6‐well plate at a density of 5 × 104 cells/well until the cells reached 80% confluence. Then, wounds were created every 0.5 cm with a pipette tip. After being rinsed with PBS, the images of wound closure were obtained at 0 and 24 h with the microscope (×100).
Transwell assay:
The 24‐well Transwell chamber (8 μm pores, Corning,) covered with Matrigel (354,234, Corning) was applied in the invasion assay. In brief, SOC cells maintained in serum‐free medium (100 μl, 5 × 105 cells/mL) were put into the upper chamber, while those cultured in 600 μl medium with 10% FBS were put into the lower chamber. Following the incubation for 24 h, cells on the lower surface of membrane were fixed with 4% paraformaldehyde (BL‐G002, Sbjbio,), followed by being stained with 0.5% crystal violet solution (G1062, Solarbio,) and observed under the microscope (×250).
Western blot:
RIPA lysis buffer (C500007, Sangon,) was applied to extract the total proteins from SOC cells. Following that, the measurement of protein concentration was performed using the BCA kit (E112‐01, Vazyme,). Thereafter, 50 μg of total proteins and 5 μl of prestained protein marker (MP102‐01, Vazyme,) were loaded into the SDS‐PAGE gel (P0688, Beyotime,) and then shifted onto the PVDF membranes (FFP32, Beyotime,). Later, the membranes were blocked with 5% non‐fat milk and cultured with primary antibodies at 4°C overnight, followed by being washed with Tris‐buffered Saline with Tween‐20 (TBST; BI‐WB025, Sbjbio,) for 30 min. Subsequently, the membranes were incubated with secondary antibodies goat anti‐rabbit IgG (1:1000, ab6702, Abcam,) and goat anti‐mouse (1:2000, ab150113, Abcam,) at room temperature for 2 h. Next, the membranes were immersed in ECL luminescence reagent (R30199‐100 ml, Pierce,) to observe its completion after being developed and photographed in the dark with a GEL‐PRO‐ANALYZER software (Bethesda,). GAPDH was selected as the internal reference. The primary antibodies involved in this assay mainly comprised those against PCNA (1:1000; Mouse; ab29, Abcam, 29 kDa), cleaved caspase‐3 (1:500; Rabbit; ab2302, Abcam, 17 kDa), E‐cadherin (1:1000; Mouse; ab76055, Abcam, 97 kDa), N‐cadherin (1:1000; Rabbit; ab18203, Abcam, 130 kDa), Snail (1:1000; Rabbit; ab216347, Abcam, 29 kDa), and GAPDH (1:10000; Rabbit; ab181602, Abcam, 36 kDa).
Statistical analysis:
Based on the statistical analysis that conducted by Graphpad prism 8.0, measurement data were expressed as mean ± standard deviation (SD). The comparison on expression difference in LIFR‐AS1 in adjacent and SOC tissues was conducted by paired sample t test, and comparisons among multiple groups were performed using one‐way analysis of variance (ANOVA) and followed by Bonferroni post hoc test. The enumeration data in Table 1 were compared by chi‐square test. The statistical significance was indicated by p < 0.05.
RESULTS:
Low expression of LIFR‐AS1 in SOC was associated with the poor prognosis of SOC patients As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).
Expression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).
Expression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
LIFR‐AS1 overexpression inhibited viability and proliferation of SOC cells while silencing LIFR‐AS1 had the opposite effect Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).
Effects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **
p < 0.01; ***
p < 0.001 vs. HOSE; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).
Effects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **
p < 0.01; ***
p < 0.001 vs. HOSE; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
LIFR‐AS1 overexpression inhibited the migration and invasion of SOC cells while silencing LIFR‐AS1 had the opposite effect As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).
Effects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation
As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).
Effects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation
LIFR‐AS1 regulated the expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in SOC cells It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).
Effects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation
It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).
Effects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation
Low expression of LIFR‐AS1 in SOC was associated with the poor prognosis of SOC patients:
As depicted in Figure 1A, TCGA‐OV database indicated that LIFR‐AS1 expression was remarkably lowered in tumor samples as compared to that in healthy samples, and Starbase revealed that the low expression of LIFR‐AS1 was associated with poor survival of SOC (Figure 1A, p = 0.018). In addition, the result of qRT‐PCR further confirmed that LIFR‐AS1 expression was lower in SOC tissues than that in adjacent tissues (Figure 1B, p < 0.001). Moreover, compared with SOC tissues, the adjacent tissues were obviously stained brown (Figure 1C), demonstrating that LIFR‐AS1 expression was largely decreased in SOC tissues. Besides, low expression of LIFR‐AS1 in SOC was associated with higher levels of tumor size, clinical stage, lymph node metastasis, and distant metastasis (p < 0.05, Table 1).
Expression of LIFR‐AS1 in SOC. (A) The analysis of LIFR‐AS1 expression was performed using TCGA database (https://www.cancer.gov/about‐nci/organization/ccg/research/structural‐genomics/tcga), and Starbase (http://starbase.sysu.edu.cn/) analyzed the relationship between LIFR‐AS1 high (n = 187) or low expression (n = 187) and the overall survival of SOC patients (p = 0.018). (B) LIFR‐AS1 expression in adjacent (n = 38) and SOC (n = 87) tissues was detected by qRT‐PCR. GAPDH was served as the internal reference (p < 0.001). (C) ISH was used to detect the expression of LIFR‐AS1 in SOC and adjacent normal tissues (magnification ×200). The data were presented as the mean ± SD of three independent experiments. Abbreviation: SOC, serous ovarian carcinoma; TCGA, the Cancer Genome Atlas; qRT‐PCR, quantitative real‐time PCR; ISH, in situ hybridization; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
LIFR‐AS1 overexpression inhibited viability and proliferation of SOC cells while silencing LIFR‐AS1 had the opposite effect:
Subsequently, we detected the LIFR‐AS1 expression in normal (HOSE) and SOC cells (A2780, OV‐56, SK‐OV‐3, OVCAR3). In SOC cell lines, LIFR‐AS1 expression was lower in SOC cells than that in HOSE cells, and among these SOC cells, LIFR‐AS1 in OV‐56 cells exhibited the highest expression (p < 0.01) and SK‐OV‐3 cells presented the lowest expression (p < 0.001, Figure 2A). Because SK‐OV‐3 and A2780 cells showed the most significant difference, we selected these two kinds of cells in the following experiments. Thereafter, the transfection of LIFR‐AS1 overexpression plasmid or sh‐LIFR‐AS1 into A2780 and SK‐OV‐3 cells was successfully conducted that overexpression or silencing vector observably up‐regulated or down‐regulated the expression of LIFR‐AS1 (p < 0.05, Figure 2B). It is worth noting that LIFR‐AS1 overexpression contributed to the decrease in cell viability and proliferation, while LIFR‐AS1 knockdown exerted the opposite effects (p < 0.05, Figure 2C–E).
Effects of LIFR‐AS1 on SOC cell viability and proliferation. (A) The expression of LIFR‐AS1 in SOC cells was quantified by qRT‐PCR. GAPDH was served as the internal reference. (B) QRT‐PCR was utilized to measure the LIFR‐AS1 expression in control, empty vector, sh‐LIFR‐AS1, and LIFR‐AS1 groups. GAPDH was served as the internal control. (C) The viability of A2780 and SK‐OV‐3 cells was accessed by CCK‐8 assay. (D,E) A2780 and SK‐OV‐3 cell proliferation was analyzed using colony formation assay. The data from three independent experiments were presented as the mean ± SD; **
p < 0.01; ***
p < 0.001 vs. HOSE; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation
LIFR‐AS1 overexpression inhibited the migration and invasion of SOC cells while silencing LIFR‐AS1 had the opposite effect:
As the data suggested, the migration rates of A2780 and SK‐OV‐3 cells were reduced by overexpressed LIFR‐AS1 and elevated by sh‐LIFR‐AS1 (p < 0.001, Figure 3A,B). Additionally, LIFR‐AS1 overexpression weakened the invasive ability of A2780 and SK‐OV‐3 cells (p < 0.01, Figure 3C,D), but LIFR‐AS1 silencing exerted the opposite effect and enhanced the ability of cell invasion (p < 0.001, Figure 3C,D).
Effects of LIFR‐AS1 on migration and invasion of SOC cells. (A,B) The migration rates of A2780 and SK‐OV‐3 cells were detected by wound healing assay (magnification ×100). (C,D) Transwell assay was used to determine the invasion of A2780 and SK‐OV‐3 cells (magnification ×250). All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: SOC, serous ovarian carcinoma; SD, standard deviation
LIFR‐AS1 regulated the expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in SOC cells:
It can be observed in Figure 4 that LIFR‐AS1 overexpression decreased the mRNA expressions of PCNA, N‐cadherin, and Snail in A2780 and SK‐OV‐3 cells (p < 0.01, Figure 4A and D), while increasing E‐cadherin expression (p < 0.001, Figure 4A and D). Conversely, sh‐LIFR‐AS1 up‐regulated the mRNA expressions of PCNA, N‐cadherin, and Snail (p < 0.01, Figure 4A and D), but down‐regulated the expression of E‐cadherin in A2780 and SK‐OV‐3 cells (p < 0.05, Figure 4A and D). Simultaneously, the cleaved caspase‐3 and E‐cadherin protein expressions were increased (p < 0.05, Figure 4B,C and 4E,F), while PCNA, N‐cadherin, and Snail expressions were decreased in LIFR‐AS1 group when compared to empty vector group (p < 0.05, Figure 4B,C and 4E,F). Besides, LIFR‐AS1 knockdown reversely regulated the protein expressions in A2780 and SK‐OV‐3 cells, which brought about the elevation of PCNA, N‐cadherin, and Snail expressions (p < 0.05, Figure 4B,C and 4E,F), and the reduction in cleaved caspase‐3 and E‐cadherin expressions (p < 0.05, Figure 4B,C and 4E,F).
Effects of LIFR‐AS1 on the expressions of genes related to proliferation and epithelial‐mesenchymal transition in SOC cells. (A) The mRNA expressions of PCNA, E‐cadherin, N‐cadherin, and Snail in A2780 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (B,C) The protein expressions of PCNA, cleaved caspase‐3, E‐cadherin, N‐cadherin, and Snail in A2780 cells were detected by Western blot. GAPDH was employed as the internal reference. (D) The mRNA expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were measured by qRT‐PCR. GAPDH was served as the internal reference. (E,F) The protein expressions of genes related to proliferation and epithelial‐mesenchymal transition in SK‐OV‐3 cells were determined by Western blot. GAPDH was served as the internal reference. All experiments were repeated three times to average. The data from three independent experiments were presented as the mean ± SD; ^
p < 0.05; ^^
p < 0.01; ^^^
p < 0.001 vs. Empty vector. Abbreviation: qRT‐PCR, quantitative real‐time PCR; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; SD, standard deviation
DISCUSSION:
As an important regulator, lncRNA has become a hotspot in researching SOC. Although lncRNAs could not encode protein, they can exert some effects on cellular behaviors such as cell proliferation, apoptosis, senescence, and migration by regulating the expressions of relevant functional genes.
15
The study of Ni et al. showed that down‐regulated LINC00515 is correlated with the tumor growth of SOC.
28
Guo et al. proved that lncRNA SOCAR aggravates the development of SOC via the Wnt/β‐catenin pathway.
29
Additionally, Gokulnath et al. indicated that lncRNA HAND2‐AS1 has the function to suppress the tumor growth of SOC.
30
LIFR‐AS1, which is an antisense lncRNA for the LIFR coding gene, has been noted that its aberrant expression is closely related to the pathogenesis and prognosis of patients with some tumors. The study by Okamura et al. found that LIFR‐AS1 can inhibit the proliferation and migration of hepatocellular carcinoma (HCC) cells and is an important indicator of poor prognosis in HCC.
31
Another study revealed that LIFR‐AS1 up‐regulation is positively correlated with more advanced and aggressive gastric cancer features.
32
Based on previous studies, this study explored the expression of LIFR‐AS1 in SOC cells and its role in SOC progression. The results of this study signified that LIFR‐AS1 was lowly expressed in SOC tissues and cells (A2780, OV‐56, SK‐OV‐3, and OVCAR3). LIFR‐AS1 overexpression could inhibit the cell viability, proliferation, migration, and invasion in A2780 and SK‐OV‐3 cells.
The indefinite proliferation and hampered apoptosis of the cells are the main characteristics of the tumor cells, such as SOC cells.
33
,
34
PCNA is a nuclear protein widely expressed in S phase of cell cycle and is only synthesized and expressed in proliferative cells. The positive expression of PCNA indicates that the cell is in proliferative state.
35
,
36
As a tumor marker protein, PCNA can reflect the synthesis and metabolism of RNA and DNA in tumor cells, its expression level can be measured to assess the proliferative activity of tissues and various cancer cells, contributing to better determining progression, aggressiveness, and prognosis of the lesions.
36
It has proved that down‐regulation of the expression of PCNA repressed G1/S cell cycle transition of human OC cells.
37
Additionally, lncRNA DLX6‐AS1 inhibited the proliferation of OC cells via reducing the expression of PCNA.
38
Caspase‐3 is the most important final executor of apoptosis in caspase family and has a strong ability to induce apoptosis.
39
Studies evidenced that lncRNA NNT‐AS1 knockdown or lncRNA PCAT‐1 overexpression weakens the activity of caspase‐3 to impede the apoptosis of human OC cells.
40
,
41
Similar to these studies, our study got the knowledge that LIFR‐AS1 overexpression decreased the expression of PCNA and increased the cleaved caspase‐3 expression of SOC cells, and silencing of LIFR‐AS1 reversely regulated these expressions, indicating that LIFR‐AS1 overexpression hindered proliferation and induced apoptosis of SOC cells by regulating the levels of related molecules.
Invasion and metastasis are important contributors to the vast majority of tumor‐associated metastasis and recurrence. Epithelial–mesenchymal transition (EMT) is an important mechanism of invasion and metastasis.
42
It refers to the pathophysiological process of epithelial cells with polar and tight adhesion into non‐polar and highly mobile stromal cells.
42
EMT has been observed in a variety of human malignant tumors,
43
,
44
including SOC.
45
In this study, we detected the expressions of three EMT‐related proteins, including E‐cadherin, N‐cadherin, and Snail. E‐cadherin participates in the adhesion and connection between homotypic cells and maintains cell polarity, which plays an important role in maintaining the integrity of epithelial cell morphology and tissue structure. The overexpression of E‐cadherin protein may inhibit tumor occurrence and metastasis, while the effect of N‐cadherin is opposite to that of E‐cadherin, and its expression can promote tumor invasion and metastasis.
46
Besides, Snail, as one of EMT‐related transcription factors, could regulate the expression level of E‐cadherin, and is also a key factor in EMT.
47
Massive amounts of evidence proved that lncRNAs enhance the migratory and invasive abilities of OC or SOC cells via regulating the EMT‐regulated genes. For instance, lncRNA OIP5‐AS1 up‐regulates Snail expression to promote OC cell invasion and migration,
48
lncRNA EBIC promotes metastasis of OC cells through up‐regulating the E‐cadherin expression,
49
and lncRNA HAL suppresses the metastasis of SOC cells by regulating the expressions of E‐cadherin and N‐cadherin to inhibit EMT signaling pathway.
50
Consistent with the results described in the previous literature, our study discovered that LIFR‐AS1 overexpression facilitated an anti‐metastatic phenotype in SOC by regulating EMT‐related genes. Concretely, LIFR‐AS1 overexpression increased the expression of E‐cadherin, but decreased N‐cadherin and Snail expressions. Moreover, silencing LIFR‐AS1 regulated these expressions in a reverse way.
To sum up, our study demonstrates that LIFR‐AS1 expression is down‐regulated in SOC, and LIFR‐AS1 overexpression inhibits SOC cell viability, proliferation, invasion, and migration by regulating the expressions of PCNA, cleaved caspase‐3, and EMT‐related genes. Our findings may provide the potential of LIFR‐AS1, as a therapeutic target for SOC.
CONFLICT OF INTEREST:
The authors declare no conflicts of interest.
| Background:
Serous ovarian carcinoma (SOC) is a common malignant tumor in female reproductive system. Long noncoding RNA (lncRNA) LIFR-AS1 is a tumor suppressor gene in colorectal cancer, but its effect and underlying mechanism in SOC are still unclear. Therefore, this study focuses on unveiling the regulatory mechanism of LIFR-AS1 in SOC.
Methods:
The relationship between LIFR-AS1 expression and prognosis of SOC patients was analyzed by TCGA database and Starbase, and then, the LIFR-AS1 expression in SOC tissues and cells was detected by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Besides, the relationship between LIFR-AS1 and clinical characteristics was analyzed. Also, the effects of LIFR-AS1 on the biological behaviors of SOC cells were measured by Cell Counting Kit-8, colony formation, and wound-healing and Transwell assays, respectively. Western blot and qRT-PCR were employed to determine the protein expressions of genes related to proliferation (PCNA), apoptosis (cleaved caspase-3), epithelial-mesenchymal transition (E-cadherin, N-cadherin, and Snail).
Results:
LIFR-AS1 was lowly expressed in SOC, which was correlated with the poor prognosis of SOC patients. Low expression of LIFR-AS1 in SOC was associated with the tumor size, clinical stage, lymph node metastasis, and distant metastasis. LIFR-AS1 overexpression promoted the expressions of cleaved caspase-3 and E-cadherin while suppressing the malignant behaviors (proliferation, migration, and invasion) of SOC cells, the expressions of PCNA, N-cadherin, and Snail. Besides, silencing LIFR-AS1 exerted the effects opposite to overexpressed LIFR-AS1.
Conclusions:
LIFR-AS1 overexpression inhibits biological behaviors of SOC cells, which may be a new therapeutic method. | null | null | 11,651 | 350 | [
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"ovarian serous cystadenoma",
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] | null | null | null | [CONTENT] epithelial–mesenchymal transition | LIFR‐AS1 | proliferation | serous ovarian carcinoma [SUMMARY] | null | [CONTENT] epithelial–mesenchymal transition | LIFR‐AS1 | proliferation | serous ovarian carcinoma [SUMMARY] | null | [CONTENT] epithelial–mesenchymal transition | LIFR‐AS1 | proliferation | serous ovarian carcinoma [SUMMARY] | null | [CONTENT] Cadherins | Carcinoma, Ovarian Epithelial | Caspase 3 | Cell Line, Tumor | Cell Movement | Cell Proliferation | Female | Gene Expression Regulation, Neoplastic | Humans | Leukemia Inhibitory Factor Receptor alpha Subunit | Ovarian Neoplasms | Proliferating Cell Nuclear Antigen | RNA, Long Noncoding [SUMMARY] | null | [CONTENT] Cadherins | Carcinoma, Ovarian Epithelial | Caspase 3 | Cell Line, Tumor | Cell Movement | Cell Proliferation | Female | Gene Expression Regulation, Neoplastic | Humans | Leukemia Inhibitory Factor Receptor alpha Subunit | Ovarian Neoplasms | Proliferating Cell Nuclear Antigen | RNA, Long Noncoding [SUMMARY] | null | [CONTENT] Cadherins | Carcinoma, Ovarian Epithelial | Caspase 3 | Cell Line, Tumor | Cell Movement | Cell Proliferation | Female | Gene Expression Regulation, Neoplastic | Humans | Leukemia Inhibitory Factor Receptor alpha Subunit | Ovarian Neoplasms | Proliferating Cell Nuclear Antigen | RNA, Long Noncoding [SUMMARY] | null | [CONTENT] ovarian serous cystadenoma | epithelial ovarian cancer | ovarian cancer oc | serous ovarian carcinoma | ovarian carcinomas socs [SUMMARY] | null | [CONTENT] ovarian serous cystadenoma | epithelial ovarian cancer | ovarian cancer oc | serous ovarian carcinoma | ovarian carcinomas socs [SUMMARY] | null | [CONTENT] ovarian serous cystadenoma | epithelial ovarian cancer | ovarian cancer oc | serous ovarian carcinoma | ovarian carcinomas socs [SUMMARY] | null | [CONTENT] as1 | cells | lifr | lifr as1 | soc | expression | ov | cadherin | sk ov | sk [SUMMARY] | null | [CONTENT] as1 | cells | lifr | lifr as1 | soc | expression | ov | cadherin | sk ov | sk [SUMMARY] | null | [CONTENT] as1 | cells | lifr | lifr as1 | soc | expression | ov | cadherin | sk ov | sk [SUMMARY] | null | [CONTENT] lncrnas | soc | cancer | antisense | progression | lncrna | oc | ovarian | diagnosis | antisense lncrnas [SUMMARY] | null | [CONTENT] lifr | lifr as1 | as1 | figure | cells | expression | soc | cadherin | ov | expressions [SUMMARY] | null | [CONTENT] cells | as1 | lifr | lifr as1 | soc | expression | ov | tissues | figure | cadherin [SUMMARY] | null | [CONTENT] SOC ||| SOC ||| SOC [SUMMARY] | null | [CONTENT] SOC | SOC ||| LIFR-AS1 | SOC ||| SOC | PCNA | Snail ||| LIFR-AS1 [SUMMARY] | null | [CONTENT] SOC ||| SOC ||| SOC ||| SOC | TCGA | LIFR | SOC | PCR | ISH ||| ||| SOC | Transwell ||| Snail ||| SOC | SOC ||| LIFR-AS1 | SOC ||| SOC | PCNA | Snail ||| LIFR-AS1 ||| SOC [SUMMARY] | null |
|||
Antimicrobial activity of customary medicinal plants of the Yaegl Aboriginal community of northern New South Wales, Australia: a preliminary study. | 26122212 | This study is a collaboration between Macquarie University researchers and the Yaegl Aboriginal Community of northern NSW, Australia to investigate the antimicrobial potential of plants used in the topical treatment of wounds, sores and skin infections. Based on previously documented medicinal applications, aqueous and aqueous ethanolic extracts of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasiliensis, Lophostemon suaveolens and Syncarpia glomulifera and the aqueous extracts of Smilax australis and Smilax glyciphylla were tested against common wound pathogens, including antibiotic resistant bacterial strains. | BACKGROUND | Plant material was prepared as aqueous extractions modelled on customary preparations and using 80% aqueous ethanol. Extracts were assayed against a selection of clinically relevant Gram positive (Streptococcus pyogenes and sensitive and resistant strains of Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium) bacteria and a fungus (Candida albicans) using disc diffusion and MTT microdilution methods. Viability of treated microorganisms was determined by subculturing from microdilution assays. | METHODS | The extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels of antimicrobial activity (MIC 31-1,000 µg/mL) against both antibiotic sensitive and resistant Staphylococcus aureus as well as the fungus Candida albicans (clinical isolate). | RESULTS | Aqueous and 80% aqueous ethanolic extracts of Lophostemon suaveolens, Corymbia intermedia and Syncarpia glomulifera exhibited promising levels of antimicrobial activity against a range of both antibiotic sensitive and resistant strains of microorganisms. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments. | CONCLUSION | [
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"New South Wales",
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] | 4485878 | Background | Australian Aboriginal people have over 40,000 years of knowledge of flora and fauna as sources of food, healing agents and other resources. Studies of customary (traditional and contemporary) medicinal plant preparations, especially in recent years, have revealed interesting medicinal properties and valuable biologically active compounds [1]. Australian Aboriginal medicinal flora have had limited biological screening studies aligned with their medicinal uses. Furthermore, the knowledge of their medicinal uses is quickly disappearing [2], particularly in some regions of the country, such as the southern states of Australia. Assessment of the bioactive potential of these plants with long historical use may support their wider application and also provide a source for safe, accessible alternative medicines and leads for the discovery of new drug-like molecules [3].
The ongoing crisis of antimicrobial resistance [4] calls for the investigation of novel sources of antimicrobials. Working with Indigenous communities who have been using natural remedies over the centuries can provide novel sources of antimicrobial therapies and/or lead compounds for the development of new medicines [3].
Discussions with Elders of the Yaegl Aboriginal community of New South Wales, Australia on their customary (traditional and contemporary medicines) identified plants used in the topical treatment of conditions of a microbial aetiology [5]. These included Alocasia brisbanensis for burns and boils, cuts, sores and open wounds; Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for antiseptic purposes; Canavalia rosea for boils and sores, Corymbia intermedia for the treatment of wounds; Hibbertia scandens for sores and rashes; and Ipomoea brasiliensis as a poultice for boils.
There has been little investigation into these plants identified by the Yaegl community for their antimicrobial potential. This study looks into the antimicrobial activity of these plants against eight pathogenic organisms commonly implicated in wounds, sores and skin infections. | Methods | Ethics protocol This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.
This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics. | Results | Aqueous or 80% aqueous ethanolic extracts from nine plants used by the Yaegl community were initially tested against the Gram positive and Gram negative antibiotic sensitive bacteria, S. aureus (ATCC 29213), E. coli (β-lactamase negative, ATCC 25922) and P. aeruginosa (ATCC 27853) using the disc diffusion and MTT assays. The plant extracts showed activity only against S. aureus (in both disc diffusion and MTT assays) and P. aeruginosa (in disc diffusion assay only). L. suaveolens, C. intermedia and S. glomulifera extracts had the highest levels of antimicrobial activity (Table 1).
Antimicrobial activity for mixtures or crude plant extracts of between 0.1 and 1 mg/mL have been suggested as being appropriate for further investigation [15]. L. suaveolens, C. intermedia and S. glomulifera crude plant extracts were therefore further investigated as they had MIC values ≤500 µg/mL (Table 1). These extracts were tested against a broader spectrum of microorganisms viz. Candida albicans, Streptococcus pyogenes, Salmonella typhimurium and resistant strains of S. aureus—multidrug resistant (MDRSA) and community acquired methicillin resistant (CMRSA). Table 2 (and Additional file 1) presents the activities of these extracts. Results for S. typhimurium are not included as no activity was observed with any of the three plants. The 80% aqueous ethanolic extract of S. glomulifera was the most active of the extracts, based on low MIC values (31 µg/mL) against the tested bacteria as well as C. albicans. The 80% aqueous ethanolic extracts of both L. suaveolens and S. glomulifera were microbicidal in their activity against the susceptible microorganisms.Table 2Broad spectrum antimicrobial screening of extracts of selected plantsMicrobeA:CACMRSAMDRSASPPlantExtB
Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)
Corymbia intermedia
(R.T. Baker) K.D. Hill & L.A.S. Johnson (NSW 792670)H2O6.5 (6–8)500 (250–500)3 (2.5–3)500 (250–500)3 (2–4)500nanaEtOH2 (0–6.5)500 (250–500)2.5 (2–5.5)375 (250–500)2.5 (1.5–4)250 (250–500)na250 (125–>1,000)
Lophostemon suaveolens
(Sol. ex Gaertn.) Peter G. Wilson & J.T. Waterh (MQ 73008908)H2O6 (5.5–6)1,000 (500–1,000)2.5 (2–3)1,000 (500–1,000)2.5 (2–3)1,000 (250–1,000)nanaEtOH6.5 (6–7)125 (31–125)3.5 (3–4.5)63 (63–125)3.5 (3–5)125 (63–250)na125 (16–>1,000)
Syncarpia glomulifera
subsp. glomulifera (Sm.) Nied. (MQ 73009066)H2O6.5 (5.5–7.5)500 (250–1,000)3 (3–4)375 (250–500)2.5 (2–3)250 (31–500)nanaEtOH5.5 (5–6)31 (16–1,000)5 (2.5–5)31 (16–1,000)3.5 (2–5)31 (16–>1,000)3 (2–4.5)31 (31–>1,000)Antibiotic control17a
2.8b
3c
1.5c
2c
1.6c
8d
0.02d
Antibiotic controls: afluconazole, bmiconazole ,cvancomycin, dpenicillin.
na no activity detected.
ACA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).
BExtraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.
CValues given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).
DMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates.
Broad spectrum antimicrobial screening of extracts of selected plants
Antibiotic controls: afluconazole, bmiconazole ,cvancomycin, dpenicillin.
na no activity detected.
ACA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).
BExtraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.
CValues given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).
DMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates. | Conclusion | 80% aqueous ethanolic extracts of Lophostemon suaveolens and Syncarpia glomulifera showed microbicidal activity at concentrations of <200 µg/mL against C. albicans and both sensitive and resistant strains of S. aureus. Aqueous extracts of L. suaveolens and S. glomulifera and aqueous and ethanolic extracts of Corymbia intermedia exhibited microbicidal activity against a range of both antibiotic sensitive and resistant strains of microorganisms at levels below 1,000 µg/mL. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments.
| [
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"Aqueous ethanolic extraction of samples for antimicrobial screening",
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] | [
"Australian Aboriginal people have over 40,000 years of knowledge of flora and fauna as sources of food, healing agents and other resources. Studies of customary (traditional and contemporary) medicinal plant preparations, especially in recent years, have revealed interesting medicinal properties and valuable biologically active compounds [1]. Australian Aboriginal medicinal flora have had limited biological screening studies aligned with their medicinal uses. Furthermore, the knowledge of their medicinal uses is quickly disappearing [2], particularly in some regions of the country, such as the southern states of Australia. Assessment of the bioactive potential of these plants with long historical use may support their wider application and also provide a source for safe, accessible alternative medicines and leads for the discovery of new drug-like molecules [3].\nThe ongoing crisis of antimicrobial resistance [4] calls for the investigation of novel sources of antimicrobials. Working with Indigenous communities who have been using natural remedies over the centuries can provide novel sources of antimicrobial therapies and/or lead compounds for the development of new medicines [3].\nDiscussions with Elders of the Yaegl Aboriginal community of New South Wales, Australia on their customary (traditional and contemporary medicines) identified plants used in the topical treatment of conditions of a microbial aetiology [5]. These included Alocasia brisbanensis for burns and boils, cuts, sores and open wounds; Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for antiseptic purposes; Canavalia rosea for boils and sores, Corymbia intermedia for the treatment of wounds; Hibbertia scandens for sores and rashes; and Ipomoea brasiliensis as a poultice for boils.\nThere has been little investigation into these plants identified by the Yaegl community for their antimicrobial potential. This study looks into the antimicrobial activity of these plants against eight pathogenic organisms commonly implicated in wounds, sores and skin infections.",
"This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.",
"Plants were collected in March 2011 after being located and identified with the assistance of the knowledge holders of the Yaegl community. Plant identification was confirmed by and voucher specimens deposited at the Herbarium of the Royal Botanic Gardens of NSW or the Macquarie University Herbarium [registered with the Index Herbariorum, New York Botanic Gardens (http://sciweb.nybg.org/science2/IndexHerbariorum.asp)], with the prefix MQU.\nFor all plants the material used was leaves, except for I. brasiliensis where the stem and leaves were used. Plant material was weighed and washed twice with water to remove surface contaminants and extraneous material. Approximately 50 g of fresh plant material for each extraction method was collected (except for S. australis and S. glyciphylla, where 10 g was collected for the aqueous extraction only, due to inadequate availability of the plant material in the area).",
"Unless otherwise indicated, all chemicals were sourced from Sigma Aldrich, St Louis, USA. All media listed below were sourced from Bacto Laboratories Pty Ltd, Australia and prepared as per the manufacturer’s instructions.",
" Aqueous extraction of samples for antimicrobial screening Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.\nAqueous extractions were prepared as either a decoction or an infusion as described below.\nDecoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.\nHot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.\nCold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.\nAll crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.\nPlant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.\nAqueous extractions were prepared as either a decoction or an infusion as described below.\nDecoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.\nHot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.\nCold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.\nAll crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.\n Aqueous ethanolic extraction of samples for antimicrobial screening Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.\nFresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.",
"Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.\nAqueous extractions were prepared as either a decoction or an infusion as described below.\nDecoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.\nHot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.\nCold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.\nAll crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.",
"Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.",
"All extracts were initially screened against one Gram positive (Staphylococcus aureus ATCC 29213) and two Gram negative (Escherichia coliβ-lactamase negative ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacterial strains, representing common pathogens associated with wounds, sores and skin infections [10, 11]. Selected extracts were also assayed against a broader range of microorganisms including: Streptococcus pyogenes—Group A, clinical isolate; Salmonella typhimurium—Group B, clinical isolate; resistant S. aureus strains (ATCC BAA 1026 community acquired methicillin resistant (CMRSA); wild multidrug resistant MRSA clinical isolate) and a yeast (Candida albicans—clinical isolate).\nAll microbial strains were kindly provided by Dr John Merlino (Department of Microbiology, Concord Hospital, Sydney) and the work was approved by the Macquarie University Biosafety Committee (Approval Reference 08/06/LAB, JOP250512BHA, TAN180512BHA). Müller Hinton II (MH) broth was used as the liquid medium for all bacteria; MH agar was used for the growth of E. coli, S. aureus, S. typhimurium and P. aeruginosa strains; horse blood agar (Edwards Instruments, Australia), was used for S. pyogenes. Potato dextrose agar (PDA, Oxoid, Basingstoke, UK) and Difco Sabouraud dextrose broth (SAB) were used for C. albicans. Gentamycin was used as the antibiotic control for the sensitive strains of S. aureus, E. coli, S. typhimurium and P. aeruginosa, vancomycin for the resistant strains of S. aureus and penicillin G for S. pyogenes (all antibiotics were supplied by Amresco, Ohio, USA). Fluconazole (MP Biomedicals, Ohio, USA) or miconazole (MP Biomedicals, LLC, France) was used as the antifungal control for the assays involving C. albicans.",
"Dried plant material was dissolved in DMSO at 50 mg/mL. Antibiotic controls were prepared in broth at a concentration of 0.1 mg/mL. Sterile paper discs (∅ 6 mm, Whatman, Maidstone, UK) were impregnated with 2 × 10 µL of plant extract (1 mg/disc) or antibiotic control (2 µg/disc) and air-dried. 20 µL DMSO impregnated discs were included as a negative control. Discs were placed on appropriate agar plates (horse blood agar for S. pyogenes, potato dextrose agar for C. albicans or MH agar for other microorganisms), inoculated with broth cultures at A600 = 0.08 (bacteria: 107–108 cfu/mL, C. albicans: 105 cfu/mL) and incubated at 37°C for 18–24 h (30°C for 48 h for C. albicans). The antimicrobial activity was measured as the zone of inhibition from the edge of the disc. Each sample and microbial combination was tested as at least four replicates.",
"The assay was performed as outlined by Steenkamp et al., with minor modifications [12]. Plant extracts were prepared at 10 mg/mL in MH broth/20% DMSO (or SAB/20% DMSO for C. albicans) and serially diluted to give 20 μL final plant sample concentrations of 2–1,000 µg/mL (0.02–10 µg/mL of antibiotic control) in 96-well clear bottom microtitre plates. Test samples were inoculated with 175 µL of microbial culture (A600 = 0.08 diluted 1/100 in MH broth); a sterile broth control was included. The plates were incubated at 37°C for 18 h (30°C for C. albicans), 5 µL of a sterile methanolic solution (5 mg/mL) of MTT was added to each well and incubated at 37°C for 60 min. The MIC was the lowest concentration of test material in which no growth (as measured by no blue colour) was observed [13].",
"Plant extracts with antimicrobial activity (Table 1), as determined by the MTT microdilution assay, were assessed for their microbicidal/static nature by subculturing onto fresh agar plates after the assay [14]. Subcultures not producing colonies were deemed to have been affected by the plant extract by a microbicidal mechanism. The minimum microbicidal concentration was defined as the concentration of the plant extract in the last well showing no further microbial growth on subculture.Table 1Initial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditionsPlanta (voucher number)Extractb\n\nPseudomonas aeruginosa (ATCC 27853)\nStaphylococcus aureus (ATCC 29213)Disc diffusionc\nMTTd\nDisc diffusionc\nMTTd\nAverage (range)MIC median (range)Average (range)MIC median (range)Apple gum Lophostemon suaveolens (Sol. ex Gaertn.) Peter G. Wilson & J.T. Waterh (MQ 73008908)H2O (I)nana2 (1–3)188 (125–250)EtOHnana3 (2.5–3.5)93 (63–125) Syncarpia glomulifera subsp. glomulifera (Sm.) Nied. (MQ 73009066)H2O (I)5.5 (0–10)na6.5 (5–10)300 (250–500)EtOHnana3 (3–6)63 (31–63)Beach morning glory Ipomoea brasiliensis (L.) Sweet (MQ 73007958)A\nH2O (D)nanananaEtOHnanananaBloodwood Corymbia intermedia (R.T. Baker) K.D. Hill & L.A.S. Johnson (NSW 792670)H2O (I)1 (1)na2 (1–3.5)250 (250–500)EtOHnana2.5 (2–4)299 (174–500)Coastal jackbean Canavalia rosea (Sw.) DC. (MQ 73008909)H2O (D)nanananaEtOHnanananaCunjevoi Alocasia brisbanensis Domin (MQ 73008737)H2O (D)nanananaEtOHnanananaSarsaparilla Smilax australis R.Br. (wide leaf) (NSW 792674)H2O (I)nananana Smilax glyciphylla Sm. (narrow leaf) (NSW 792380)H2O (I)nanananaYellowvine Hibbertia scandens (Willd.) Gilg (MQ 73008905)H2O (D)nanananaEtOHnananana Antibiotic control (Gentamycin)8 (8)1.258 (8)0.5\nna no activity detected.\naLeaves of plants used in all cases except where indicated.\nbExtraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.\ncValues given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.\ndMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.\nALeaves and stems.\nInitial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditions\n\nna no activity detected.\n\naLeaves of plants used in all cases except where indicated.\n\nbExtraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.\n\ncValues given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.\n\ndMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.\n\nALeaves and stems."
] | [
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] | [
"Background",
"Methods",
"Ethics protocol",
"Plant collection and identification",
"Chemicals",
"Extraction of plant material",
"Aqueous extraction of samples for antimicrobial screening",
"Aqueous ethanolic extraction of samples for antimicrobial screening",
"Microbial strains",
"Disc diffusion assay",
"MTT microdilution assay",
"Microbial viability determination",
"Results",
"Discussion",
"Conclusion"
] | [
"Australian Aboriginal people have over 40,000 years of knowledge of flora and fauna as sources of food, healing agents and other resources. Studies of customary (traditional and contemporary) medicinal plant preparations, especially in recent years, have revealed interesting medicinal properties and valuable biologically active compounds [1]. Australian Aboriginal medicinal flora have had limited biological screening studies aligned with their medicinal uses. Furthermore, the knowledge of their medicinal uses is quickly disappearing [2], particularly in some regions of the country, such as the southern states of Australia. Assessment of the bioactive potential of these plants with long historical use may support their wider application and also provide a source for safe, accessible alternative medicines and leads for the discovery of new drug-like molecules [3].\nThe ongoing crisis of antimicrobial resistance [4] calls for the investigation of novel sources of antimicrobials. Working with Indigenous communities who have been using natural remedies over the centuries can provide novel sources of antimicrobial therapies and/or lead compounds for the development of new medicines [3].\nDiscussions with Elders of the Yaegl Aboriginal community of New South Wales, Australia on their customary (traditional and contemporary medicines) identified plants used in the topical treatment of conditions of a microbial aetiology [5]. These included Alocasia brisbanensis for burns and boils, cuts, sores and open wounds; Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for antiseptic purposes; Canavalia rosea for boils and sores, Corymbia intermedia for the treatment of wounds; Hibbertia scandens for sores and rashes; and Ipomoea brasiliensis as a poultice for boils.\nThere has been little investigation into these plants identified by the Yaegl community for their antimicrobial potential. This study looks into the antimicrobial activity of these plants against eight pathogenic organisms commonly implicated in wounds, sores and skin infections.",
" Ethics protocol This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.\nThis research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.",
"This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.",
"Plants were collected in March 2011 after being located and identified with the assistance of the knowledge holders of the Yaegl community. Plant identification was confirmed by and voucher specimens deposited at the Herbarium of the Royal Botanic Gardens of NSW or the Macquarie University Herbarium [registered with the Index Herbariorum, New York Botanic Gardens (http://sciweb.nybg.org/science2/IndexHerbariorum.asp)], with the prefix MQU.\nFor all plants the material used was leaves, except for I. brasiliensis where the stem and leaves were used. Plant material was weighed and washed twice with water to remove surface contaminants and extraneous material. Approximately 50 g of fresh plant material for each extraction method was collected (except for S. australis and S. glyciphylla, where 10 g was collected for the aqueous extraction only, due to inadequate availability of the plant material in the area).",
"Unless otherwise indicated, all chemicals were sourced from Sigma Aldrich, St Louis, USA. All media listed below were sourced from Bacto Laboratories Pty Ltd, Australia and prepared as per the manufacturer’s instructions.",
" Aqueous extraction of samples for antimicrobial screening Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.\nAqueous extractions were prepared as either a decoction or an infusion as described below.\nDecoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.\nHot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.\nCold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.\nAll crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.\nPlant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.\nAqueous extractions were prepared as either a decoction or an infusion as described below.\nDecoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.\nHot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.\nCold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.\nAll crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.\n Aqueous ethanolic extraction of samples for antimicrobial screening Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.\nFresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.",
"Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.\nAqueous extractions were prepared as either a decoction or an infusion as described below.\nDecoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.\nHot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.\nCold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.\nAll crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.",
"Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.",
"All extracts were initially screened against one Gram positive (Staphylococcus aureus ATCC 29213) and two Gram negative (Escherichia coliβ-lactamase negative ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacterial strains, representing common pathogens associated with wounds, sores and skin infections [10, 11]. Selected extracts were also assayed against a broader range of microorganisms including: Streptococcus pyogenes—Group A, clinical isolate; Salmonella typhimurium—Group B, clinical isolate; resistant S. aureus strains (ATCC BAA 1026 community acquired methicillin resistant (CMRSA); wild multidrug resistant MRSA clinical isolate) and a yeast (Candida albicans—clinical isolate).\nAll microbial strains were kindly provided by Dr John Merlino (Department of Microbiology, Concord Hospital, Sydney) and the work was approved by the Macquarie University Biosafety Committee (Approval Reference 08/06/LAB, JOP250512BHA, TAN180512BHA). Müller Hinton II (MH) broth was used as the liquid medium for all bacteria; MH agar was used for the growth of E. coli, S. aureus, S. typhimurium and P. aeruginosa strains; horse blood agar (Edwards Instruments, Australia), was used for S. pyogenes. Potato dextrose agar (PDA, Oxoid, Basingstoke, UK) and Difco Sabouraud dextrose broth (SAB) were used for C. albicans. Gentamycin was used as the antibiotic control for the sensitive strains of S. aureus, E. coli, S. typhimurium and P. aeruginosa, vancomycin for the resistant strains of S. aureus and penicillin G for S. pyogenes (all antibiotics were supplied by Amresco, Ohio, USA). Fluconazole (MP Biomedicals, Ohio, USA) or miconazole (MP Biomedicals, LLC, France) was used as the antifungal control for the assays involving C. albicans.",
"Dried plant material was dissolved in DMSO at 50 mg/mL. Antibiotic controls were prepared in broth at a concentration of 0.1 mg/mL. Sterile paper discs (∅ 6 mm, Whatman, Maidstone, UK) were impregnated with 2 × 10 µL of plant extract (1 mg/disc) or antibiotic control (2 µg/disc) and air-dried. 20 µL DMSO impregnated discs were included as a negative control. Discs were placed on appropriate agar plates (horse blood agar for S. pyogenes, potato dextrose agar for C. albicans or MH agar for other microorganisms), inoculated with broth cultures at A600 = 0.08 (bacteria: 107–108 cfu/mL, C. albicans: 105 cfu/mL) and incubated at 37°C for 18–24 h (30°C for 48 h for C. albicans). The antimicrobial activity was measured as the zone of inhibition from the edge of the disc. Each sample and microbial combination was tested as at least four replicates.",
"The assay was performed as outlined by Steenkamp et al., with minor modifications [12]. Plant extracts were prepared at 10 mg/mL in MH broth/20% DMSO (or SAB/20% DMSO for C. albicans) and serially diluted to give 20 μL final plant sample concentrations of 2–1,000 µg/mL (0.02–10 µg/mL of antibiotic control) in 96-well clear bottom microtitre plates. Test samples were inoculated with 175 µL of microbial culture (A600 = 0.08 diluted 1/100 in MH broth); a sterile broth control was included. The plates were incubated at 37°C for 18 h (30°C for C. albicans), 5 µL of a sterile methanolic solution (5 mg/mL) of MTT was added to each well and incubated at 37°C for 60 min. The MIC was the lowest concentration of test material in which no growth (as measured by no blue colour) was observed [13].",
"Plant extracts with antimicrobial activity (Table 1), as determined by the MTT microdilution assay, were assessed for their microbicidal/static nature by subculturing onto fresh agar plates after the assay [14]. Subcultures not producing colonies were deemed to have been affected by the plant extract by a microbicidal mechanism. The minimum microbicidal concentration was defined as the concentration of the plant extract in the last well showing no further microbial growth on subculture.Table 1Initial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditionsPlanta (voucher number)Extractb\n\nPseudomonas aeruginosa (ATCC 27853)\nStaphylococcus aureus (ATCC 29213)Disc diffusionc\nMTTd\nDisc diffusionc\nMTTd\nAverage (range)MIC median (range)Average (range)MIC median (range)Apple gum Lophostemon suaveolens (Sol. ex Gaertn.) Peter G. Wilson & J.T. Waterh (MQ 73008908)H2O (I)nana2 (1–3)188 (125–250)EtOHnana3 (2.5–3.5)93 (63–125) Syncarpia glomulifera subsp. glomulifera (Sm.) Nied. (MQ 73009066)H2O (I)5.5 (0–10)na6.5 (5–10)300 (250–500)EtOHnana3 (3–6)63 (31–63)Beach morning glory Ipomoea brasiliensis (L.) Sweet (MQ 73007958)A\nH2O (D)nanananaEtOHnanananaBloodwood Corymbia intermedia (R.T. Baker) K.D. Hill & L.A.S. Johnson (NSW 792670)H2O (I)1 (1)na2 (1–3.5)250 (250–500)EtOHnana2.5 (2–4)299 (174–500)Coastal jackbean Canavalia rosea (Sw.) DC. (MQ 73008909)H2O (D)nanananaEtOHnanananaCunjevoi Alocasia brisbanensis Domin (MQ 73008737)H2O (D)nanananaEtOHnanananaSarsaparilla Smilax australis R.Br. (wide leaf) (NSW 792674)H2O (I)nananana Smilax glyciphylla Sm. (narrow leaf) (NSW 792380)H2O (I)nanananaYellowvine Hibbertia scandens (Willd.) Gilg (MQ 73008905)H2O (D)nanananaEtOHnananana Antibiotic control (Gentamycin)8 (8)1.258 (8)0.5\nna no activity detected.\naLeaves of plants used in all cases except where indicated.\nbExtraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.\ncValues given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.\ndMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.\nALeaves and stems.\nInitial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditions\n\nna no activity detected.\n\naLeaves of plants used in all cases except where indicated.\n\nbExtraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.\n\ncValues given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.\n\ndMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.\n\nALeaves and stems.",
"Aqueous or 80% aqueous ethanolic extracts from nine plants used by the Yaegl community were initially tested against the Gram positive and Gram negative antibiotic sensitive bacteria, S. aureus (ATCC 29213), E. coli (β-lactamase negative, ATCC 25922) and P. aeruginosa (ATCC 27853) using the disc diffusion and MTT assays. The plant extracts showed activity only against S. aureus (in both disc diffusion and MTT assays) and P. aeruginosa (in disc diffusion assay only). L. suaveolens, C. intermedia and S. glomulifera extracts had the highest levels of antimicrobial activity (Table 1).\nAntimicrobial activity for mixtures or crude plant extracts of between 0.1 and 1 mg/mL have been suggested as being appropriate for further investigation [15]. L. suaveolens, C. intermedia and S. glomulifera crude plant extracts were therefore further investigated as they had MIC values ≤500 µg/mL (Table 1). These extracts were tested against a broader spectrum of microorganisms viz. Candida albicans, Streptococcus pyogenes, Salmonella typhimurium and resistant strains of S. aureus—multidrug resistant (MDRSA) and community acquired methicillin resistant (CMRSA). Table 2 (and Additional file 1) presents the activities of these extracts. Results for S. typhimurium are not included as no activity was observed with any of the three plants. The 80% aqueous ethanolic extract of S. glomulifera was the most active of the extracts, based on low MIC values (31 µg/mL) against the tested bacteria as well as C. albicans. The 80% aqueous ethanolic extracts of both L. suaveolens and S. glomulifera were microbicidal in their activity against the susceptible microorganisms.Table 2Broad spectrum antimicrobial screening of extracts of selected plantsMicrobeA:CACMRSAMDRSASPPlantExtB\nDisc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)\nCorymbia intermedia\n(R.T. Baker) K.D. Hill & L.A.S. Johnson (NSW 792670)H2O6.5 (6–8)500 (250–500)3 (2.5–3)500 (250–500)3 (2–4)500nanaEtOH2 (0–6.5)500 (250–500)2.5 (2–5.5)375 (250–500)2.5 (1.5–4)250 (250–500)na250 (125–>1,000)\nLophostemon suaveolens\n(Sol. ex Gaertn.) Peter G. Wilson & J.T. Waterh (MQ 73008908)H2O6 (5.5–6)1,000 (500–1,000)2.5 (2–3)1,000 (500–1,000)2.5 (2–3)1,000 (250–1,000)nanaEtOH6.5 (6–7)125 (31–125)3.5 (3–4.5)63 (63–125)3.5 (3–5)125 (63–250)na125 (16–>1,000)\nSyncarpia glomulifera\nsubsp. glomulifera (Sm.) Nied. (MQ 73009066)H2O6.5 (5.5–7.5)500 (250–1,000)3 (3–4)375 (250–500)2.5 (2–3)250 (31–500)nanaEtOH5.5 (5–6)31 (16–1,000)5 (2.5–5)31 (16–1,000)3.5 (2–5)31 (16–>1,000)3 (2–4.5)31 (31–>1,000)Antibiotic control17a\n2.8b\n3c\n1.5c\n2c\n1.6c\n8d\n0.02d\nAntibiotic controls: afluconazole, bmiconazole ,cvancomycin, dpenicillin.\nna no activity detected.\nACA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).\nBExtraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.\nCValues given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).\nDMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates.\nBroad spectrum antimicrobial screening of extracts of selected plants\nAntibiotic controls: afluconazole, bmiconazole ,cvancomycin, dpenicillin.\n\nna no activity detected.\n\nACA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).\n\nBExtraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.\n\nCValues given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).\n\nDMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates.",
"Ethnobotanical studies conducted by us with Yaegl Aboriginal Elders of northern New South Wales, Australia, described the use of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasiliensis, Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for the treatment of wounds, sores and skin infections [5]. None of these plants have been investigated for their antimicrobial activity, with the exception of C. rosea [16]. The success of finding antimicrobial activity and bioactive compounds of medicinal value from traditional/customary medicines [3], prompted the investigation of these plants.\nThe plants were extracted with water to mimic the common customary practice of the community and/or with 80% aqueous ethanol, as a standard natural products research method [9]. The plant parts extracted were the same as those used by the Yaegl community, except for bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), where customarily the sap or latex was used in the treatment of wounds. However, as sap or latex were not readily accessible, we tested the leaves at the request of the Yaegl Elders.\nThe microorganisms selected for the assays are commonly implicated in wounds, sores and skin infections [11]. The two assay methods were chosen as they complement each other in that the disc diffusion assay is simple but not always appropriate for non-polar and large molecules [17] and the more sensitive, versatile and quantitative MTT microdilution assay can have interference with the colour and redox activity of plant extracts.\nThe extracts of C. intermedia, L. suaveolens and S. glomulifera had reasonable levels of antimicrobial activity (MIC 0.1–1 mg/mL; [15]) against antibiotic sensitive and resistant Staphylococcus aureus as well as the fungus Candida albicans (clinical isolate). Although antibacterial activity of the ethanolic extract of C. rosea leaves has been previously reported against S. aureus, albeit at a low level [16], no antibacterial activity was seen in this study, possibly due to the different bacterial strains and assay conditions used.\nThe 80% aqueous ethanolic extracts of C. intermedia, L. suaveolens and S. glomulifera were substantially more active than the aqueous extracts. This indicates that the antimicrobial components in these plants were more easily extractable in 80% aqueous ethanol. S. glomulifera was the most active of the extracts (Table 2). The 80% aqueous ethanolic extracts of both L. suaveolens and S. glomulifera were microbicidal in their activity against the susceptible microorganisms.\nSap from bloodwood (C. intermedia) has been used by the Yaegl community in the treatment of wounds [5]. No chemical or biological studies have been reported for C. intermedia. Milky sap and ash from the bark of the apple gum (Lophostemon suaveolens or Syncarpia glomulifera) have been used as antiseptics by the Yaegl community [5]. These two plants are sometimes interchangeably used due to their similar morphology.\nNo antimicrobial activity studies have been reported for L. suaveolens or the leaves of S. glomulifera. However, both plants have been found to contain the following compounds that have been independently reported for antimicrobial activity [18–22]. Leaves of L. suaveolens have been found to contain α-pinene, β-caryophyllene, aromadendrene, globulol and spathulenol [23], which have been independently reported for antimicrobial activity. The leaves of S. glomulifera also contain α-pinene, aromadendrene and globulol [24]. Eucalyptin, which has been reported to be antibacterial, has been found in the leaf wax of S. glomulifera [25]. Additionally, S. glomulifera bark has been reported to be antibacterial and to contain the antibacterial compounds betulinic acid, oleanolic acid-3-acetate and ursolic acid-3-acetate [26].\nThis is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. These results warrant further investigation of the active ingredients of these medicinal plants.",
"80% aqueous ethanolic extracts of Lophostemon suaveolens and Syncarpia glomulifera showed microbicidal activity at concentrations of <200 µg/mL against C. albicans and both sensitive and resistant strains of S. aureus. Aqueous extracts of L. suaveolens and S. glomulifera and aqueous and ethanolic extracts of Corymbia intermedia exhibited microbicidal activity against a range of both antibiotic sensitive and resistant strains of microorganisms at levels below 1,000 µg/mL. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments.\n"
] | [
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] | [
"Antimicrobials",
"Multidrug resistant",
"Wound healing",
"Traditional medicine",
"Ethnobotany"
] | Background:
Australian Aboriginal people have over 40,000 years of knowledge of flora and fauna as sources of food, healing agents and other resources. Studies of customary (traditional and contemporary) medicinal plant preparations, especially in recent years, have revealed interesting medicinal properties and valuable biologically active compounds [1]. Australian Aboriginal medicinal flora have had limited biological screening studies aligned with their medicinal uses. Furthermore, the knowledge of their medicinal uses is quickly disappearing [2], particularly in some regions of the country, such as the southern states of Australia. Assessment of the bioactive potential of these plants with long historical use may support their wider application and also provide a source for safe, accessible alternative medicines and leads for the discovery of new drug-like molecules [3].
The ongoing crisis of antimicrobial resistance [4] calls for the investigation of novel sources of antimicrobials. Working with Indigenous communities who have been using natural remedies over the centuries can provide novel sources of antimicrobial therapies and/or lead compounds for the development of new medicines [3].
Discussions with Elders of the Yaegl Aboriginal community of New South Wales, Australia on their customary (traditional and contemporary medicines) identified plants used in the topical treatment of conditions of a microbial aetiology [5]. These included Alocasia brisbanensis for burns and boils, cuts, sores and open wounds; Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for antiseptic purposes; Canavalia rosea for boils and sores, Corymbia intermedia for the treatment of wounds; Hibbertia scandens for sores and rashes; and Ipomoea brasiliensis as a poultice for boils.
There has been little investigation into these plants identified by the Yaegl community for their antimicrobial potential. This study looks into the antimicrobial activity of these plants against eight pathogenic organisms commonly implicated in wounds, sores and skin infections.
Methods:
Ethics protocol This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.
This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.
Ethics protocol:
This research was approved by the Human Ethics Committee at Macquarie University, (HE27 JUL2007-R05356 and 5201200763). It was conducted under the framework of best ethical practice, working in partnerships with Indigenous people [6, 7] and was governed by a cooperative research agreement with the Yaegl community [8]. The choice of plants and the biological testing to be undertaken were determined in consultation with the Yaegl community through the Yaegl Local Aboriginal Land Council as the appropriate authorising body. The proposed methods and workflow were discussed before work commenced, and the results of the screening were presented back to the Yaegl community in the form of regular reports and presentations. The regular contact with the community during all phases of this research project ensured that the progress of the project remained in accordance with the requirements of the community and academics.
Plant collection and identification:
Plants were collected in March 2011 after being located and identified with the assistance of the knowledge holders of the Yaegl community. Plant identification was confirmed by and voucher specimens deposited at the Herbarium of the Royal Botanic Gardens of NSW or the Macquarie University Herbarium [registered with the Index Herbariorum, New York Botanic Gardens (http://sciweb.nybg.org/science2/IndexHerbariorum.asp)], with the prefix MQU.
For all plants the material used was leaves, except for I. brasiliensis where the stem and leaves were used. Plant material was weighed and washed twice with water to remove surface contaminants and extraneous material. Approximately 50 g of fresh plant material for each extraction method was collected (except for S. australis and S. glyciphylla, where 10 g was collected for the aqueous extraction only, due to inadequate availability of the plant material in the area).
Chemicals:
Unless otherwise indicated, all chemicals were sourced from Sigma Aldrich, St Louis, USA. All media listed below were sourced from Bacto Laboratories Pty Ltd, Australia and prepared as per the manufacturer’s instructions.
Extraction of plant material:
Aqueous extraction of samples for antimicrobial screening Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.
Aqueous extractions were prepared as either a decoction or an infusion as described below.
Decoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.
Hot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.
Cold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.
All crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.
Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.
Aqueous extractions were prepared as either a decoction or an infusion as described below.
Decoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.
Hot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.
Cold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.
All crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.
Aqueous ethanolic extraction of samples for antimicrobial screening Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.
Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.
Aqueous extraction of samples for antimicrobial screening:
Plant material was, where possible, prepared in a way consistent with the preparation methods described by the Yaegl Elders [5] within 48 h of collection. In the case of pink bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), sap or latex while customarily used. Due to the risk of considerable damage to the tree, sap or latex was not collected and the Elders requested the leaves be collected and tested as an accessible and sustainable option. For C. rosea and H. scandens, the specifics of the preparation method could not be recalled by the knowledge holders.
Aqueous extractions were prepared as either a decoction or an infusion as described below.
Decoction (D): Plant material was pounded and boiled in 10× (w/v) tap water for 10 min, before being left to steep and cool (covered, for approximately 60 min). The decoction was filtered and the filtrate collected.
Hot infusion (I): Freshly boiled water was poured over roughly chopped or torn plant material (10× w/v). The infusion was covered and allowed to steep for 2 h. The infusion was filtered and the filtrate collected.
Cold infusion (CI): Plant material was roughly chopped or torn and submerged in 10× (w/v) water at room temperature. The infusion was shaken gently for 24 h, the mixture was vacuum filtered and the solid was shaken with fresh water (10× w/v) for an additional 24 h and filtered. The filtrates were pooled.
All crude aqueous extracts were dried by rotary evaporation (Büchi, Germany) at reduced pressure at approximately 50°C, followed by freeze drying (Labconco freeze dryer, USA). Extracts were stored at −20°C.
Aqueous ethanolic extraction of samples for antimicrobial screening:
Fresh plant material was ground to a coarse powder using a coffee grinder (IKA® A11 Basic, Selangor, Malaysia) and shaken in 10× w/v of 80% v/v aqueous ethanol at room temperature [9] for approximately 48 h. The mixture was filtered and the solid shaken with fresh 80% v/v aqueous ethanol (10× w/v) for an additional 48 h, and the filtrates pooled. The ethanolic portion was removed by rotary evaporation under reduced pressure at a low heat (<40°C), leaving the water portion, which was freeze dried and stored at −20°C.
Microbial strains:
All extracts were initially screened against one Gram positive (Staphylococcus aureus ATCC 29213) and two Gram negative (Escherichia coliβ-lactamase negative ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacterial strains, representing common pathogens associated with wounds, sores and skin infections [10, 11]. Selected extracts were also assayed against a broader range of microorganisms including: Streptococcus pyogenes—Group A, clinical isolate; Salmonella typhimurium—Group B, clinical isolate; resistant S. aureus strains (ATCC BAA 1026 community acquired methicillin resistant (CMRSA); wild multidrug resistant MRSA clinical isolate) and a yeast (Candida albicans—clinical isolate).
All microbial strains were kindly provided by Dr John Merlino (Department of Microbiology, Concord Hospital, Sydney) and the work was approved by the Macquarie University Biosafety Committee (Approval Reference 08/06/LAB, JOP250512BHA, TAN180512BHA). Müller Hinton II (MH) broth was used as the liquid medium for all bacteria; MH agar was used for the growth of E. coli, S. aureus, S. typhimurium and P. aeruginosa strains; horse blood agar (Edwards Instruments, Australia), was used for S. pyogenes. Potato dextrose agar (PDA, Oxoid, Basingstoke, UK) and Difco Sabouraud dextrose broth (SAB) were used for C. albicans. Gentamycin was used as the antibiotic control for the sensitive strains of S. aureus, E. coli, S. typhimurium and P. aeruginosa, vancomycin for the resistant strains of S. aureus and penicillin G for S. pyogenes (all antibiotics were supplied by Amresco, Ohio, USA). Fluconazole (MP Biomedicals, Ohio, USA) or miconazole (MP Biomedicals, LLC, France) was used as the antifungal control for the assays involving C. albicans.
Disc diffusion assay:
Dried plant material was dissolved in DMSO at 50 mg/mL. Antibiotic controls were prepared in broth at a concentration of 0.1 mg/mL. Sterile paper discs (∅ 6 mm, Whatman, Maidstone, UK) were impregnated with 2 × 10 µL of plant extract (1 mg/disc) or antibiotic control (2 µg/disc) and air-dried. 20 µL DMSO impregnated discs were included as a negative control. Discs were placed on appropriate agar plates (horse blood agar for S. pyogenes, potato dextrose agar for C. albicans or MH agar for other microorganisms), inoculated with broth cultures at A600 = 0.08 (bacteria: 107–108 cfu/mL, C. albicans: 105 cfu/mL) and incubated at 37°C for 18–24 h (30°C for 48 h for C. albicans). The antimicrobial activity was measured as the zone of inhibition from the edge of the disc. Each sample and microbial combination was tested as at least four replicates.
MTT microdilution assay:
The assay was performed as outlined by Steenkamp et al., with minor modifications [12]. Plant extracts were prepared at 10 mg/mL in MH broth/20% DMSO (or SAB/20% DMSO for C. albicans) and serially diluted to give 20 μL final plant sample concentrations of 2–1,000 µg/mL (0.02–10 µg/mL of antibiotic control) in 96-well clear bottom microtitre plates. Test samples were inoculated with 175 µL of microbial culture (A600 = 0.08 diluted 1/100 in MH broth); a sterile broth control was included. The plates were incubated at 37°C for 18 h (30°C for C. albicans), 5 µL of a sterile methanolic solution (5 mg/mL) of MTT was added to each well and incubated at 37°C for 60 min. The MIC was the lowest concentration of test material in which no growth (as measured by no blue colour) was observed [13].
Microbial viability determination:
Plant extracts with antimicrobial activity (Table 1), as determined by the MTT microdilution assay, were assessed for their microbicidal/static nature by subculturing onto fresh agar plates after the assay [14]. Subcultures not producing colonies were deemed to have been affected by the plant extract by a microbicidal mechanism. The minimum microbicidal concentration was defined as the concentration of the plant extract in the last well showing no further microbial growth on subculture.Table 1Initial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditionsPlanta (voucher number)Extractb
Pseudomonas aeruginosa (ATCC 27853)
Staphylococcus aureus (ATCC 29213)Disc diffusionc
MTTd
Disc diffusionc
MTTd
Average (range)MIC median (range)Average (range)MIC median (range)Apple gum Lophostemon suaveolens (Sol. ex Gaertn.) Peter G. Wilson & J.T. Waterh (MQ 73008908)H2O (I)nana2 (1–3)188 (125–250)EtOHnana3 (2.5–3.5)93 (63–125) Syncarpia glomulifera subsp. glomulifera (Sm.) Nied. (MQ 73009066)H2O (I)5.5 (0–10)na6.5 (5–10)300 (250–500)EtOHnana3 (3–6)63 (31–63)Beach morning glory Ipomoea brasiliensis (L.) Sweet (MQ 73007958)A
H2O (D)nanananaEtOHnanananaBloodwood Corymbia intermedia (R.T. Baker) K.D. Hill & L.A.S. Johnson (NSW 792670)H2O (I)1 (1)na2 (1–3.5)250 (250–500)EtOHnana2.5 (2–4)299 (174–500)Coastal jackbean Canavalia rosea (Sw.) DC. (MQ 73008909)H2O (D)nanananaEtOHnanananaCunjevoi Alocasia brisbanensis Domin (MQ 73008737)H2O (D)nanananaEtOHnanananaSarsaparilla Smilax australis R.Br. (wide leaf) (NSW 792674)H2O (I)nananana Smilax glyciphylla Sm. (narrow leaf) (NSW 792380)H2O (I)nanananaYellowvine Hibbertia scandens (Willd.) Gilg (MQ 73008905)H2O (D)nanananaEtOHnananana Antibiotic control (Gentamycin)8 (8)1.258 (8)0.5
na no activity detected.
aLeaves of plants used in all cases except where indicated.
bExtraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.
cValues given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.
dMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.
ALeaves and stems.
Initial screening for antimicrobial activity of extracts of plants used customarily to treat skin conditions
na no activity detected.
aLeaves of plants used in all cases except where indicated.
bExtraction method used: H2O—based on traditional preparation (D, decoction; I, infusion; CI, cold infusion), EtOH—80% aqueous ethanol.
cValues given are inhibition in mm from the edge of the disc; average (range) of 4 replicates.
dMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of four replicates.
ALeaves and stems.
Results:
Aqueous or 80% aqueous ethanolic extracts from nine plants used by the Yaegl community were initially tested against the Gram positive and Gram negative antibiotic sensitive bacteria, S. aureus (ATCC 29213), E. coli (β-lactamase negative, ATCC 25922) and P. aeruginosa (ATCC 27853) using the disc diffusion and MTT assays. The plant extracts showed activity only against S. aureus (in both disc diffusion and MTT assays) and P. aeruginosa (in disc diffusion assay only). L. suaveolens, C. intermedia and S. glomulifera extracts had the highest levels of antimicrobial activity (Table 1).
Antimicrobial activity for mixtures or crude plant extracts of between 0.1 and 1 mg/mL have been suggested as being appropriate for further investigation [15]. L. suaveolens, C. intermedia and S. glomulifera crude plant extracts were therefore further investigated as they had MIC values ≤500 µg/mL (Table 1). These extracts were tested against a broader spectrum of microorganisms viz. Candida albicans, Streptococcus pyogenes, Salmonella typhimurium and resistant strains of S. aureus—multidrug resistant (MDRSA) and community acquired methicillin resistant (CMRSA). Table 2 (and Additional file 1) presents the activities of these extracts. Results for S. typhimurium are not included as no activity was observed with any of the three plants. The 80% aqueous ethanolic extract of S. glomulifera was the most active of the extracts, based on low MIC values (31 µg/mL) against the tested bacteria as well as C. albicans. The 80% aqueous ethanolic extracts of both L. suaveolens and S. glomulifera were microbicidal in their activity against the susceptible microorganisms.Table 2Broad spectrum antimicrobial screening of extracts of selected plantsMicrobeA:CACMRSAMDRSASPPlantExtB
Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)Disc diffusionC Average (range)MTT MICD Median (range)
Corymbia intermedia
(R.T. Baker) K.D. Hill & L.A.S. Johnson (NSW 792670)H2O6.5 (6–8)500 (250–500)3 (2.5–3)500 (250–500)3 (2–4)500nanaEtOH2 (0–6.5)500 (250–500)2.5 (2–5.5)375 (250–500)2.5 (1.5–4)250 (250–500)na250 (125–>1,000)
Lophostemon suaveolens
(Sol. ex Gaertn.) Peter G. Wilson & J.T. Waterh (MQ 73008908)H2O6 (5.5–6)1,000 (500–1,000)2.5 (2–3)1,000 (500–1,000)2.5 (2–3)1,000 (250–1,000)nanaEtOH6.5 (6–7)125 (31–125)3.5 (3–4.5)63 (63–125)3.5 (3–5)125 (63–250)na125 (16–>1,000)
Syncarpia glomulifera
subsp. glomulifera (Sm.) Nied. (MQ 73009066)H2O6.5 (5.5–7.5)500 (250–1,000)3 (3–4)375 (250–500)2.5 (2–3)250 (31–500)nanaEtOH5.5 (5–6)31 (16–1,000)5 (2.5–5)31 (16–1,000)3.5 (2–5)31 (16–>1,000)3 (2–4.5)31 (31–>1,000)Antibiotic control17a
2.8b
3c
1.5c
2c
1.6c
8d
0.02d
Antibiotic controls: afluconazole, bmiconazole ,cvancomycin, dpenicillin.
na no activity detected.
ACA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).
BExtraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.
CValues given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).
DMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates.
Broad spectrum antimicrobial screening of extracts of selected plants
Antibiotic controls: afluconazole, bmiconazole ,cvancomycin, dpenicillin.
na no activity detected.
ACA: Candida albicans (clinical isolate); CMRSA: S. aureus (ATCC BAA 1026); MDRSA: S. aureus (multidrug resistant clinical isolate); SP: Streptococcus pyogenes (Group A—clinical isolate).
BExtraction method used: H2O, traditional prep; EtOH, 80% aqueous ethanol.
CValues given are inhibition in mm from the edge of the disc; average of four replicates (plant extracts tested at 1 mg/disc and antibiotic controls tested at 2 µg/disc).
DMIC noted as µg/mL, based on the last dilution of extract not to exhibit yellow–blue colour change in MTT assay; values given are median (range) of 4 replicates.
Discussion:
Ethnobotanical studies conducted by us with Yaegl Aboriginal Elders of northern New South Wales, Australia, described the use of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasiliensis, Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for the treatment of wounds, sores and skin infections [5]. None of these plants have been investigated for their antimicrobial activity, with the exception of C. rosea [16]. The success of finding antimicrobial activity and bioactive compounds of medicinal value from traditional/customary medicines [3], prompted the investigation of these plants.
The plants were extracted with water to mimic the common customary practice of the community and/or with 80% aqueous ethanol, as a standard natural products research method [9]. The plant parts extracted were the same as those used by the Yaegl community, except for bloodwood (C. intermedia) and apple gum (L. suaveolens or S. glomulifera), where customarily the sap or latex was used in the treatment of wounds. However, as sap or latex were not readily accessible, we tested the leaves at the request of the Yaegl Elders.
The microorganisms selected for the assays are commonly implicated in wounds, sores and skin infections [11]. The two assay methods were chosen as they complement each other in that the disc diffusion assay is simple but not always appropriate for non-polar and large molecules [17] and the more sensitive, versatile and quantitative MTT microdilution assay can have interference with the colour and redox activity of plant extracts.
The extracts of C. intermedia, L. suaveolens and S. glomulifera had reasonable levels of antimicrobial activity (MIC 0.1–1 mg/mL; [15]) against antibiotic sensitive and resistant Staphylococcus aureus as well as the fungus Candida albicans (clinical isolate). Although antibacterial activity of the ethanolic extract of C. rosea leaves has been previously reported against S. aureus, albeit at a low level [16], no antibacterial activity was seen in this study, possibly due to the different bacterial strains and assay conditions used.
The 80% aqueous ethanolic extracts of C. intermedia, L. suaveolens and S. glomulifera were substantially more active than the aqueous extracts. This indicates that the antimicrobial components in these plants were more easily extractable in 80% aqueous ethanol. S. glomulifera was the most active of the extracts (Table 2). The 80% aqueous ethanolic extracts of both L. suaveolens and S. glomulifera were microbicidal in their activity against the susceptible microorganisms.
Sap from bloodwood (C. intermedia) has been used by the Yaegl community in the treatment of wounds [5]. No chemical or biological studies have been reported for C. intermedia. Milky sap and ash from the bark of the apple gum (Lophostemon suaveolens or Syncarpia glomulifera) have been used as antiseptics by the Yaegl community [5]. These two plants are sometimes interchangeably used due to their similar morphology.
No antimicrobial activity studies have been reported for L. suaveolens or the leaves of S. glomulifera. However, both plants have been found to contain the following compounds that have been independently reported for antimicrobial activity [18–22]. Leaves of L. suaveolens have been found to contain α-pinene, β-caryophyllene, aromadendrene, globulol and spathulenol [23], which have been independently reported for antimicrobial activity. The leaves of S. glomulifera also contain α-pinene, aromadendrene and globulol [24]. Eucalyptin, which has been reported to be antibacterial, has been found in the leaf wax of S. glomulifera [25]. Additionally, S. glomulifera bark has been reported to be antibacterial and to contain the antibacterial compounds betulinic acid, oleanolic acid-3-acetate and ursolic acid-3-acetate [26].
This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. These results warrant further investigation of the active ingredients of these medicinal plants.
Conclusion:
80% aqueous ethanolic extracts of Lophostemon suaveolens and Syncarpia glomulifera showed microbicidal activity at concentrations of <200 µg/mL against C. albicans and both sensitive and resistant strains of S. aureus. Aqueous extracts of L. suaveolens and S. glomulifera and aqueous and ethanolic extracts of Corymbia intermedia exhibited microbicidal activity against a range of both antibiotic sensitive and resistant strains of microorganisms at levels below 1,000 µg/mL. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments.
| Background:
This study is a collaboration between Macquarie University researchers and the Yaegl Aboriginal Community of northern NSW, Australia to investigate the antimicrobial potential of plants used in the topical treatment of wounds, sores and skin infections. Based on previously documented medicinal applications, aqueous and aqueous ethanolic extracts of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasiliensis, Lophostemon suaveolens and Syncarpia glomulifera and the aqueous extracts of Smilax australis and Smilax glyciphylla were tested against common wound pathogens, including antibiotic resistant bacterial strains.
Methods:
Plant material was prepared as aqueous extractions modelled on customary preparations and using 80% aqueous ethanol. Extracts were assayed against a selection of clinically relevant Gram positive (Streptococcus pyogenes and sensitive and resistant strains of Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium) bacteria and a fungus (Candida albicans) using disc diffusion and MTT microdilution methods. Viability of treated microorganisms was determined by subculturing from microdilution assays.
Results:
The extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels of antimicrobial activity (MIC 31-1,000 µg/mL) against both antibiotic sensitive and resistant Staphylococcus aureus as well as the fungus Candida albicans (clinical isolate).
Conclusions:
Aqueous and 80% aqueous ethanolic extracts of Lophostemon suaveolens, Corymbia intermedia and Syncarpia glomulifera exhibited promising levels of antimicrobial activity against a range of both antibiotic sensitive and resistant strains of microorganisms. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments. | Background:
Australian Aboriginal people have over 40,000 years of knowledge of flora and fauna as sources of food, healing agents and other resources. Studies of customary (traditional and contemporary) medicinal plant preparations, especially in recent years, have revealed interesting medicinal properties and valuable biologically active compounds [1]. Australian Aboriginal medicinal flora have had limited biological screening studies aligned with their medicinal uses. Furthermore, the knowledge of their medicinal uses is quickly disappearing [2], particularly in some regions of the country, such as the southern states of Australia. Assessment of the bioactive potential of these plants with long historical use may support their wider application and also provide a source for safe, accessible alternative medicines and leads for the discovery of new drug-like molecules [3].
The ongoing crisis of antimicrobial resistance [4] calls for the investigation of novel sources of antimicrobials. Working with Indigenous communities who have been using natural remedies over the centuries can provide novel sources of antimicrobial therapies and/or lead compounds for the development of new medicines [3].
Discussions with Elders of the Yaegl Aboriginal community of New South Wales, Australia on their customary (traditional and contemporary medicines) identified plants used in the topical treatment of conditions of a microbial aetiology [5]. These included Alocasia brisbanensis for burns and boils, cuts, sores and open wounds; Lophostemon suaveolens, Smilax australis, Smilax glyciphylla and Syncarpia glomulifera for antiseptic purposes; Canavalia rosea for boils and sores, Corymbia intermedia for the treatment of wounds; Hibbertia scandens for sores and rashes; and Ipomoea brasiliensis as a poultice for boils.
There has been little investigation into these plants identified by the Yaegl community for their antimicrobial potential. This study looks into the antimicrobial activity of these plants against eight pathogenic organisms commonly implicated in wounds, sores and skin infections.
Conclusion:
80% aqueous ethanolic extracts of Lophostemon suaveolens and Syncarpia glomulifera showed microbicidal activity at concentrations of <200 µg/mL against C. albicans and both sensitive and resistant strains of S. aureus. Aqueous extracts of L. suaveolens and S. glomulifera and aqueous and ethanolic extracts of Corymbia intermedia exhibited microbicidal activity against a range of both antibiotic sensitive and resistant strains of microorganisms at levels below 1,000 µg/mL. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments.
| Background:
This study is a collaboration between Macquarie University researchers and the Yaegl Aboriginal Community of northern NSW, Australia to investigate the antimicrobial potential of plants used in the topical treatment of wounds, sores and skin infections. Based on previously documented medicinal applications, aqueous and aqueous ethanolic extracts of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasiliensis, Lophostemon suaveolens and Syncarpia glomulifera and the aqueous extracts of Smilax australis and Smilax glyciphylla were tested against common wound pathogens, including antibiotic resistant bacterial strains.
Methods:
Plant material was prepared as aqueous extractions modelled on customary preparations and using 80% aqueous ethanol. Extracts were assayed against a selection of clinically relevant Gram positive (Streptococcus pyogenes and sensitive and resistant strains of Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium) bacteria and a fungus (Candida albicans) using disc diffusion and MTT microdilution methods. Viability of treated microorganisms was determined by subculturing from microdilution assays.
Results:
The extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels of antimicrobial activity (MIC 31-1,000 µg/mL) against both antibiotic sensitive and resistant Staphylococcus aureus as well as the fungus Candida albicans (clinical isolate).
Conclusions:
Aqueous and 80% aqueous ethanolic extracts of Lophostemon suaveolens, Corymbia intermedia and Syncarpia glomulifera exhibited promising levels of antimicrobial activity against a range of both antibiotic sensitive and resistant strains of microorganisms. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments. | 5,472 | 312 | [
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"traditional contemporary medicinal",
"medicines identified plants",
"medicinal flora",
"compounds australian aboriginal",
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] | [CONTENT] Antimicrobials | Multidrug resistant | Wound healing | Traditional medicine | Ethnobotany [SUMMARY] | [CONTENT] Antimicrobials | Multidrug resistant | Wound healing | Traditional medicine | Ethnobotany [SUMMARY] | [CONTENT] Antimicrobials | Multidrug resistant | Wound healing | Traditional medicine | Ethnobotany [SUMMARY] | [CONTENT] Antimicrobials | Multidrug resistant | Wound healing | Traditional medicine | Ethnobotany [SUMMARY] | [CONTENT] Antimicrobials | Multidrug resistant | Wound healing | Traditional medicine | Ethnobotany [SUMMARY] | [CONTENT] Antimicrobials | Multidrug resistant | Wound healing | Traditional medicine | Ethnobotany [SUMMARY] | [CONTENT] Anti-Bacterial Agents | Antifungal Agents | Drug Resistance, Multiple, Bacterial | Drug Resistance, Multiple, Fungal | Ethnobotany | Humans | Medicine, Traditional | Native Hawaiian or Other Pacific Islander | New South Wales | Phytotherapy | Plant Extracts | Plants, Medicinal | Skin Diseases [SUMMARY] | [CONTENT] Anti-Bacterial Agents | Antifungal Agents | Drug Resistance, Multiple, Bacterial | Drug Resistance, Multiple, Fungal | Ethnobotany | Humans | Medicine, Traditional | Native Hawaiian or Other Pacific Islander | New South Wales | Phytotherapy | Plant Extracts | Plants, Medicinal | Skin Diseases [SUMMARY] | [CONTENT] Anti-Bacterial Agents | Antifungal Agents | Drug Resistance, Multiple, Bacterial | Drug Resistance, Multiple, Fungal | Ethnobotany | Humans | Medicine, Traditional | Native Hawaiian or Other Pacific Islander | New South Wales | Phytotherapy | Plant Extracts | Plants, Medicinal | Skin Diseases [SUMMARY] | [CONTENT] Anti-Bacterial Agents | Antifungal Agents | Drug Resistance, Multiple, Bacterial | Drug Resistance, Multiple, Fungal | Ethnobotany | Humans | Medicine, Traditional | Native Hawaiian or Other Pacific Islander | New South Wales | Phytotherapy | Plant Extracts | Plants, Medicinal | Skin Diseases [SUMMARY] | [CONTENT] Anti-Bacterial Agents | Antifungal Agents | Drug Resistance, Multiple, Bacterial | Drug Resistance, Multiple, Fungal | Ethnobotany | Humans | Medicine, Traditional | Native Hawaiian or Other Pacific Islander | New South Wales | Phytotherapy | Plant Extracts | Plants, Medicinal | Skin Diseases [SUMMARY] | [CONTENT] Anti-Bacterial Agents | Antifungal Agents | Drug Resistance, Multiple, Bacterial | Drug Resistance, Multiple, Fungal | Ethnobotany | Humans | Medicine, Traditional | Native Hawaiian or Other Pacific Islander | New South Wales | Phytotherapy | Plant Extracts | Plants, Medicinal | Skin Diseases [SUMMARY] | [CONTENT] traditional contemporary medicinal | medicines identified plants | medicinal flora | compounds australian aboriginal | australian aboriginal medicinal [SUMMARY] | [CONTENT] traditional contemporary medicinal | medicines identified plants | medicinal flora | compounds australian aboriginal | australian aboriginal medicinal [SUMMARY] | [CONTENT] traditional contemporary medicinal | medicines identified plants | medicinal flora | compounds australian aboriginal | australian aboriginal medicinal [SUMMARY] | [CONTENT] traditional contemporary medicinal | medicines identified plants | medicinal flora | compounds australian aboriginal | australian aboriginal medicinal [SUMMARY] | [CONTENT] traditional contemporary medicinal | medicines identified plants | medicinal flora | compounds australian aboriginal | australian aboriginal medicinal [SUMMARY] | [CONTENT] traditional contemporary medicinal | medicines identified plants | medicinal flora | compounds australian aboriginal | australian aboriginal medicinal [SUMMARY] | [CONTENT] plant | extracts | aqueous | 10 | glomulifera | activity | antimicrobial | community | yaegl | plants [SUMMARY] | [CONTENT] plant | extracts | aqueous | 10 | glomulifera | activity | antimicrobial | community | yaegl | plants [SUMMARY] | [CONTENT] plant | extracts | aqueous | 10 | glomulifera | activity | antimicrobial | community | yaegl | plants [SUMMARY] | [CONTENT] plant | extracts | aqueous | 10 | glomulifera | activity | antimicrobial | community | yaegl | plants [SUMMARY] | [CONTENT] plant | extracts | aqueous | 10 | glomulifera | activity | antimicrobial | community | yaegl | plants [SUMMARY] | [CONTENT] plant | extracts | aqueous | 10 | glomulifera | activity | antimicrobial | community | yaegl | plants [SUMMARY] | [CONTENT] medicinal | sores | boils | medicines | sources | wounds | plants | antimicrobial | aboriginal | new [SUMMARY] | [CONTENT] community | research | yaegl | yaegl community | project | regular | ethics | presented yaegl community | project ensured progress | project ensured [SUMMARY] | [CONTENT] 500 | 250 | disc | 000 | range | 31 | extracts | 500 250 | mtt | average [SUMMARY] | [CONTENT] sensitive resistant strains | sensitive resistant | glomulifera | suaveolens | resistant strains | ethanolic extracts | aqueous ethanolic extracts | microbicidal activity | extracts | aqueous [SUMMARY] | [CONTENT] community | plant | aqueous | material | extracts | 10 | yaegl | infusion | glomulifera | plant material [SUMMARY] | [CONTENT] community | plant | aqueous | material | extracts | 10 | yaegl | infusion | glomulifera | plant material [SUMMARY] | [CONTENT] Macquarie University | the Yaegl Aboriginal Community | Australia ||| Alocasia | Canavalia | Corymbia | Hibbertia | Ipomoea | Lophostemon | Syncarpia | Smilax | Smilax [SUMMARY] | [CONTENT] 80% ||| Gram | Staphylococcus | Gram negative | Escherichia | Salmonella | MTT ||| [SUMMARY] | [CONTENT] Corymbia | Lophostemon | Syncarpia | MIC 31-1,000 | Staphylococcus aureus [SUMMARY] | [CONTENT] 80% | Lophostemon | Corymbia | Syncarpia ||| first | C. | L. ||| [SUMMARY] | [CONTENT] ||| Macquarie University | the Yaegl Aboriginal Community | Australia ||| Alocasia | Canavalia | Corymbia | Hibbertia | Ipomoea | Lophostemon | Syncarpia | Smilax | Smilax ||| 80% ||| Gram | Staphylococcus | Gram negative | Escherichia | Salmonella | MTT ||| ||| Corymbia | Lophostemon | Syncarpia | MIC 31-1,000 | Staphylococcus aureus ||| 80% | Lophostemon | Corymbia | Syncarpia ||| first | C. | L. ||| [SUMMARY] | [CONTENT] ||| Macquarie University | the Yaegl Aboriginal Community | Australia ||| Alocasia | Canavalia | Corymbia | Hibbertia | Ipomoea | Lophostemon | Syncarpia | Smilax | Smilax ||| 80% ||| Gram | Staphylococcus | Gram negative | Escherichia | Salmonella | MTT ||| ||| Corymbia | Lophostemon | Syncarpia | MIC 31-1,000 | Staphylococcus aureus ||| 80% | Lophostemon | Corymbia | Syncarpia ||| first | C. | L. ||| [SUMMARY] |
||||||
Serotypes of group B streptococci in western Sweden and comparison with serotypes in two previous studies starting from 1988. | 26553333 | Group B Streptococci (GBS) are the most common neonatal pathogens and infect immunocompromised and elderly individuals. The species has 10 different serotypes. Serotypes have been studied in the south-west area of Sweden in 1988-1997 and 1998-2001. The aim of this study was to study serotypes in the same area from 2004 to 2009. | BACKGROUND | Invasive GBS isolates were collected prospectively from 2004 to 2009 in two counties in western Sweden with a population of 1.8 million, and were serotyped by latex agglutination. Clinical data were obtained from hospital records. During the study period 410 invasive GBS isolates from 398 patients were collected (multiple episodes ≥ 1 month apart). Clinical data were not available for two patients who are excluded. Four isolates were from stillborn neonates, 88 were from live born neonates and infants, and 318 from adults. | METHODS | Serotype III was the most common serotype (48%) in neonates and infants followed by serotypes Ia (18%) and V (16%). In adults serotype V (39%) dominated followed by serotypes III (20%) and Ib (14%). There was a significant increase of serotype V in comparison with the first study (1988-1997) but there were no significant changes in the serotype distribution between the present study and the second study (1998-2001). There were a few cases of serotype VI-IX, both in children and adults, not seen in the previous studies. Serotype V was more common among patients with arthritis than with any other manifestation. | RESULTS | Changes in GBS serotypes occur over time in the same region, which must be considered when GBS vaccines are formulated. | CONCLUSIONS | [
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"Male",
"Middle Aged",
"Prospective Studies",
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"Young Adult"
] | 4640215 | Background | Group B Streptococci (GBS) remain a leading cause of invasive neonatal infections and an important cause of infections in pregnant women, immune compromised adults and the elderly [1–3]. GBS possess ten distinct capsular polysaccharide (CPS) types: Ia, Ib and II-IX [4]. The principal disease-associated serotypes are currently Ia, Ib, II, III and V [2, 5–7] according to studies from North America and Norway.
Most centers in Sweden have been using a risk-based prophylaxis strategy which identifies candidates for intra partum antimicrobial prophylaxis (IAP) according to the presence of any of the following risk factors: a previous GBS-infected baby, positive for GBS in urine, delivering at <37 weeks’ gestation, having an intra-partum temperature ≥38.0°, or rupture of membranes for ≥18 h. There is no plan to begin with universal prenatal GBS screening followed by IAP of all GBS carriers as is recommended by the US guidelines from 2002 [8, 9]. Both strategies have dramatically reduced the incidence and mortality among early onset (EO, <7 days after birth) GBS infections, but they have not reduced the incidence of late onset (LO, 7–27 days after birth) or very late onset (VLO, 28 days–4 months after birth) disease [1, 10–13].
There are no efficacy studies of GBS vaccines, but most likely protection would be achieved by anticapsular serum antibodies as it is for other bacteria with polysaccharide capsules. The first vaccines against GBS consisted of pure polysaccharides and had disappointingly low immunogenicity. Polysaccharide-protein conjugates are more immunogenic and have been given in phase 1 to 2 clinical trials (including pregnant women with 1 to 5 serotypes. Proteins used have been tetanus toxoid or genetically inactivated diphtheria toxoid [14]. To make the best possible GBS vaccines they must be directed against the polysaccharides of the most common GBS serotypes.
It is important to follow the distribution of serotypes since it may differ over time and between populations [3].
The main aim of this study was to survey the current serotype distribution of invasive GBS infections in a Swedish population and to compare it with previous studies in the same area to detect any changes over time. | null | null | Results | Neonates and infants A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.
Serotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
A total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.
A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.
Serotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
A total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.
Adults There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).
Serotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa
Early onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410
aIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera
1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
Serotype distribution among invasive group B streptococci
aIntra Uterine Fetal Death
Serotype distribution of invasive group B streptococci related to clinical manifestation in adults
*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
During the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.
There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).
Serotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa
Early onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410
aIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera
1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
Serotype distribution among invasive group B streptococci
aIntra Uterine Fetal Death
Serotype distribution of invasive group B streptococci related to clinical manifestation in adults
*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
During the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.
Recurrent or reinfections A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas
Patients with more than one invasive isolates of group B streptococci
A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas
Patients with more than one invasive isolates of group B streptococci
Differences between children and adults The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).
The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).
Comparison of the present study with two earlier studies from the same area The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.
Table 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases
Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region
The present study covers a longer time span than the two previous, which explains the increased number of cases
The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.
Table 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases
Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region
The present study covers a longer time span than the two previous, which explains the increased number of cases | Conclusions | In conclusion, this study has shown that there have not been any significant changes of the serotype distribution of invasive GBS infections in this Swedish population since the emergence of serotype V in the late 1990s but a few cases of serotypes VII-IX, which are not being targeted with a vaccine, have emerged in the area. It is therefore important to have an ongoing surveillance of GBS even though a trivalent vaccine would probably lead to much less disease among neonates and infants. | [
"Statistics",
"Ethical approval",
"Neonates and infants",
"Adults",
"Recurrent or reinfections",
"Differences between children and adults",
"Comparison of the present study with two earlier studies from the same area"
] | [
"Fisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.",
"The study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.",
"A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.\nSerotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nSerotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nA total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.",
"There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).\nSerotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa\nEarly onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410\naIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera\n1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nSerotype distribution among invasive group B streptococci\n\naIntra Uterine Fetal Death\nSerotype distribution of invasive group B streptococci related to clinical manifestation in adults\n*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\n\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nDuring the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.",
"A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas\nPatients with more than one invasive isolates of group B streptococci",
"The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).",
"The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.\nTable 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases\nSerotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region\nThe present study covers a longer time span than the two previous, which explains the increased number of cases"
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Statistics",
"Ethical approval",
"Results",
"Neonates and infants",
"Adults",
"Recurrent or reinfections",
"Differences between children and adults",
"Comparison of the present study with two earlier studies from the same area",
"Discussion",
"Conclusions"
] | [
"Group B Streptococci (GBS) remain a leading cause of invasive neonatal infections and an important cause of infections in pregnant women, immune compromised adults and the elderly [1–3]. GBS possess ten distinct capsular polysaccharide (CPS) types: Ia, Ib and II-IX [4]. The principal disease-associated serotypes are currently Ia, Ib, II, III and V [2, 5–7] according to studies from North America and Norway.\nMost centers in Sweden have been using a risk-based prophylaxis strategy which identifies candidates for intra partum antimicrobial prophylaxis (IAP) according to the presence of any of the following risk factors: a previous GBS-infected baby, positive for GBS in urine, delivering at <37 weeks’ gestation, having an intra-partum temperature ≥38.0°, or rupture of membranes for ≥18 h. There is no plan to begin with universal prenatal GBS screening followed by IAP of all GBS carriers as is recommended by the US guidelines from 2002 [8, 9]. Both strategies have dramatically reduced the incidence and mortality among early onset (EO, <7 days after birth) GBS infections, but they have not reduced the incidence of late onset (LO, 7–27 days after birth) or very late onset (VLO, 28 days–4 months after birth) disease [1, 10–13].\nThere are no efficacy studies of GBS vaccines, but most likely protection would be achieved by anticapsular serum antibodies as it is for other bacteria with polysaccharide capsules. The first vaccines against GBS consisted of pure polysaccharides and had disappointingly low immunogenicity. Polysaccharide-protein conjugates are more immunogenic and have been given in phase 1 to 2 clinical trials (including pregnant women with 1 to 5 serotypes. Proteins used have been tetanus toxoid or genetically inactivated diphtheria toxoid [14]. To make the best possible GBS vaccines they must be directed against the polysaccharides of the most common GBS serotypes.\nIt is important to follow the distribution of serotypes since it may differ over time and between populations [3].\nThe main aim of this study was to survey the current serotype distribution of invasive GBS infections in a Swedish population and to compare it with previous studies in the same area to detect any changes over time.",
"Invasive GBS isolates were collected prospectively between 2004 and 2009 from the six bacteriological laboratories, which served all 13 hospitals, in the counties of Västra Götaland and Halland in western Sweden. The mean population of the surveillance area was 1,828,140 which corresponds 20 % of the total population in Sweden (9,128,308 inhabitants). The total number of live births was 127,882 during the study period. (Central Bureau of Statistics, Stockholm, Sweden (http://www.scb.se). The two regions studied include the city of Gothenburg with suburbs, (urban area population about 850,000, cities with populations up to 100,000 inhabitants, small villages and rural areas. There are no major socioeconomic differences within the area studied nor between the area studied and the rest of Sweden.\nAn invasive GBS case was defined as isolation of the organism from a normally sterile site (blood, cerebrospinal fluid and synovial fluid) in a surveillance area resident. No isolates came from pleural, pericardial or peritoneal fluid.\nThe total number of invasive GBS isolates during the time period was 515. Thus of all isolates 410/515 (80 %) were serotyped. The strains which were not serotyped were evenly distributed during the time period and the participating laboratories. Age, date of culture, possible risk factors, and complications, were obtained from medical records and an evaluation was made to see if death was attributed to the GBS infection. One neonate had protected identity, so its records could not be studied. The records of one adult patient could not be found. These two patients are excluded from all parts of the study.\nIsolates were identified as GBS by colony morphology, microscopy following Gram’s stain of smears, and co-agglutination with group-specific reagents (Streptex; Murex Biotech, Dartford, UK). The isolates were stored in a broth at −70 °C until serotyping was performed using the latex agglutination test with type-specific antisera for serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX (Statens Serum Institut, Copenhagen, Denmark), as previously described [15].\nOnly one isolate from each infectious episode was included in the study and it was considered the same episode if it was less than 30 days between the isolates. GBS was isolated from 410 infectious episodes in 398 patients, 11 patients had more than one episode. Each infectious episode is reported as a separate patient.\n Statistics Fisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.\nFisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.\n Ethical approval The study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.\nThe study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.",
"Fisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.",
"The study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.",
" Neonates and infants A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.\nSerotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nSerotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nA total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.\nA total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.\nSerotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nSerotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nA total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.\n Adults There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).\nSerotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa\nEarly onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410\naIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera\n1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nSerotype distribution among invasive group B streptococci\n\naIntra Uterine Fetal Death\nSerotype distribution of invasive group B streptococci related to clinical manifestation in adults\n*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\n\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nDuring the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.\nThere were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).\nSerotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa\nEarly onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410\naIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera\n1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nSerotype distribution among invasive group B streptococci\n\naIntra Uterine Fetal Death\nSerotype distribution of invasive group B streptococci related to clinical manifestation in adults\n*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\n\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nDuring the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.\n Recurrent or reinfections A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas\nPatients with more than one invasive isolates of group B streptococci\nA total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas\nPatients with more than one invasive isolates of group B streptococci\n Differences between children and adults The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).\nThe major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).\n Comparison of the present study with two earlier studies from the same area The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.\nTable 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases\nSerotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region\nThe present study covers a longer time span than the two previous, which explains the increased number of cases\nThe main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.\nTable 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases\nSerotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region\nThe present study covers a longer time span than the two previous, which explains the increased number of cases",
"A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.\nSerotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nSerotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)\nA total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.",
"There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).\nSerotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa\nEarly onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410\naIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera\n1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nSerotype distribution among invasive group B streptococci\n\naIntra Uterine Fetal Death\nSerotype distribution of invasive group B streptococci related to clinical manifestation in adults\n*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes\n\naChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis\nDuring the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.",
"A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas\nPatients with more than one invasive isolates of group B streptococci",
"The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).",
"The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.\nTable 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases\nSerotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region\nThe present study covers a longer time span than the two previous, which explains the increased number of cases",
"The differences in serotype distribution between neonates/infants and adults must be considered in future GBS vaccines. Pregnant women are the obvious target for GBS vaccination in order to protect neonates from early onset disease through passively transferred antibodies, so (at least currently) the most important in a vaccine must be type III followed by serotype Ia while serotype V seems to be of little importance. A GBS vaccine for elderly or patients with severe underlying diseases, in which serotype V infections are the most common, is probably unrealistic. There has been little change in serotype distribution among neonates and infants over the last 30 years, even after the rise of serotype V among adults [5]. Serotype V was first reported in 1985 and soon became the most common serotype among adults in many parts of the world [17–19].\nThe present study showed that five serotypes, Ia, Ib, II, III and V, accounted for 94 % (neonates and infants) and 93 % (adults) of cases with invasive GBS infections. This is consistent with studies from other countries [2, 5, 20, 21]. Serotype III is still the most common serotype among neonates and infants, especially for LO disease [5, 11, 13, 19, 20, 22]. Other studies have shown a dominance of serotype Ia for EO disease [19, 23, 24]. A possible reason for this differences between different parts of the world could be that GBS has similarities with pneumococci. It is well known that serotypes of pneumococci change regionally and over time [25, 26]. This was seen already in the prevaccination era. One example is the increase in pneumococcal serogroup 1 in Northern Europe [27–29]. A reason for this could be that pneumococci have different clones with different virulence within the same serotype. This has been suggested to be the case in increase of pneumococcal serogroup 1. Possibly this may be true also for GBS.\nIt has previously been suggested that a trivalent vaccine has the potential to further reduce the burden of GBS disease and especially LO cases [30]. Regarding our findings a trivalent vaccine (CRM197-conjugated capsular polysaccharides of GBS serotypes Ia, Ib and III), could have had an impact on 80 % (24/30) of the LO cases and 75 % (69/92) of all infants and neonates in our population during the study period. Several studies have shown serotype replacement among pneumococci when pneumococcal conjugate vaccines have been used in large scale for some years [31–33]. Only future can tell if this will occur also among GBS if such vaccines are used extensively.\nSuch a vaccine would have little impact on GBS diseases in adults, e.g. those with severe underlying diseases. Among adults three serotypes, Ia, III and V account for 70 % of cases with invasive GBS infections. Other studies have reported similar findings (64–71.8 %) but with variation in which of the three is the most prevalent [2, 18, 20, 22, 34, 35].\nSome studies have reported an increase in serotype IV among invasive GBS isolates [36, 37]. We did not see any rise of serotype IV in the present study in comparison with the two previous studies done in the same region [16, 17]. Other studies show like ours that these serotypes are nonexistent or very uncommon [22, 23].\nThere were few relations between serotype and clinical manifestations but the study showed that serotypes V and III were found more often isolated from synovial fluid in adults but others have found it to be serotype II [38].",
"In conclusion, this study has shown that there have not been any significant changes of the serotype distribution of invasive GBS infections in this Swedish population since the emergence of serotype V in the late 1990s but a few cases of serotypes VII-IX, which are not being targeted with a vaccine, have emerged in the area. It is therefore important to have an ongoing surveillance of GBS even though a trivalent vaccine would probably lead to much less disease among neonates and infants."
] | [
"introduction",
"materials|methods",
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
"conclusion"
] | [
"Group B Streptococci",
"Serotypes",
"Invasive infections",
"Neonates",
"Streptococcus agalactiae",
"Epidemiology"
] | Background:
Group B Streptococci (GBS) remain a leading cause of invasive neonatal infections and an important cause of infections in pregnant women, immune compromised adults and the elderly [1–3]. GBS possess ten distinct capsular polysaccharide (CPS) types: Ia, Ib and II-IX [4]. The principal disease-associated serotypes are currently Ia, Ib, II, III and V [2, 5–7] according to studies from North America and Norway.
Most centers in Sweden have been using a risk-based prophylaxis strategy which identifies candidates for intra partum antimicrobial prophylaxis (IAP) according to the presence of any of the following risk factors: a previous GBS-infected baby, positive for GBS in urine, delivering at <37 weeks’ gestation, having an intra-partum temperature ≥38.0°, or rupture of membranes for ≥18 h. There is no plan to begin with universal prenatal GBS screening followed by IAP of all GBS carriers as is recommended by the US guidelines from 2002 [8, 9]. Both strategies have dramatically reduced the incidence and mortality among early onset (EO, <7 days after birth) GBS infections, but they have not reduced the incidence of late onset (LO, 7–27 days after birth) or very late onset (VLO, 28 days–4 months after birth) disease [1, 10–13].
There are no efficacy studies of GBS vaccines, but most likely protection would be achieved by anticapsular serum antibodies as it is for other bacteria with polysaccharide capsules. The first vaccines against GBS consisted of pure polysaccharides and had disappointingly low immunogenicity. Polysaccharide-protein conjugates are more immunogenic and have been given in phase 1 to 2 clinical trials (including pregnant women with 1 to 5 serotypes. Proteins used have been tetanus toxoid or genetically inactivated diphtheria toxoid [14]. To make the best possible GBS vaccines they must be directed against the polysaccharides of the most common GBS serotypes.
It is important to follow the distribution of serotypes since it may differ over time and between populations [3].
The main aim of this study was to survey the current serotype distribution of invasive GBS infections in a Swedish population and to compare it with previous studies in the same area to detect any changes over time.
Methods:
Invasive GBS isolates were collected prospectively between 2004 and 2009 from the six bacteriological laboratories, which served all 13 hospitals, in the counties of Västra Götaland and Halland in western Sweden. The mean population of the surveillance area was 1,828,140 which corresponds 20 % of the total population in Sweden (9,128,308 inhabitants). The total number of live births was 127,882 during the study period. (Central Bureau of Statistics, Stockholm, Sweden (http://www.scb.se). The two regions studied include the city of Gothenburg with suburbs, (urban area population about 850,000, cities with populations up to 100,000 inhabitants, small villages and rural areas. There are no major socioeconomic differences within the area studied nor between the area studied and the rest of Sweden.
An invasive GBS case was defined as isolation of the organism from a normally sterile site (blood, cerebrospinal fluid and synovial fluid) in a surveillance area resident. No isolates came from pleural, pericardial or peritoneal fluid.
The total number of invasive GBS isolates during the time period was 515. Thus of all isolates 410/515 (80 %) were serotyped. The strains which were not serotyped were evenly distributed during the time period and the participating laboratories. Age, date of culture, possible risk factors, and complications, were obtained from medical records and an evaluation was made to see if death was attributed to the GBS infection. One neonate had protected identity, so its records could not be studied. The records of one adult patient could not be found. These two patients are excluded from all parts of the study.
Isolates were identified as GBS by colony morphology, microscopy following Gram’s stain of smears, and co-agglutination with group-specific reagents (Streptex; Murex Biotech, Dartford, UK). The isolates were stored in a broth at −70 °C until serotyping was performed using the latex agglutination test with type-specific antisera for serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX (Statens Serum Institut, Copenhagen, Denmark), as previously described [15].
Only one isolate from each infectious episode was included in the study and it was considered the same episode if it was less than 30 days between the isolates. GBS was isolated from 410 infectious episodes in 398 patients, 11 patients had more than one episode. Each infectious episode is reported as a separate patient.
Statistics Fisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.
Fisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.
Ethical approval The study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.
The study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.
Statistics:
Fisher’s exact test (two-tailed) was used for comparisons of proportions (http://graphpad.com/quickcalcs/contingency1.cfm). A p value <0.05 was considered significant.
Ethical approval:
The study was approved by the Ethics Committees of Gothenburg University and Lund University (registration numbers Ö524-03, LU674-02). The committees did not require individual consent, because the clinical data were obtained in retrospect, when many patients and/or relatives could not be found.
Results:
Neonates and infants A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.
Serotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
A total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.
A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.
Serotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
A total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.
Adults There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).
Serotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa
Early onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410
aIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera
1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
Serotype distribution among invasive group B streptococci
aIntra Uterine Fetal Death
Serotype distribution of invasive group B streptococci related to clinical manifestation in adults
*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
During the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.
There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).
Serotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa
Early onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410
aIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera
1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
Serotype distribution among invasive group B streptococci
aIntra Uterine Fetal Death
Serotype distribution of invasive group B streptococci related to clinical manifestation in adults
*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
During the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.
Recurrent or reinfections A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas
Patients with more than one invasive isolates of group B streptococci
A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas
Patients with more than one invasive isolates of group B streptococci
Differences between children and adults The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).
The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).
Comparison of the present study with two earlier studies from the same area The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.
Table 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases
Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region
The present study covers a longer time span than the two previous, which explains the increased number of cases
The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.
Table 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases
Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region
The present study covers a longer time span than the two previous, which explains the increased number of cases
Neonates and infants:
A total of 91 invasive GBS isolates from 91 neonates and infants aged 0–209 days were serotyped, 47 boys and 44 girls. Four were intra uterine fetal deaths (IUFD). Sepsis without a known focus was the most common clinical manifestation among live born neonates and infants with 63 cases (72 %). Meningitis was seen in 12 patients (14 %). Other manifestations were pneumonia (eight patients), skin infection (two patients), urosepsis (one patient) and septic arthritis (one patient). Altogether 33 (38 %) live-born patients were preterm, i.e. gestational age <37 weeks, 20 had EO disease, two had LO disease and 11 had VLO disease.
Serotype III was the most prevalent serotype (48 %) in neonates and infants followed by serotype Ia (18 %) and serotype V (16 %). The serotype distribution compared to clinical manifestations is shown in Fig.1. Serotype III was more common (p = 0.03) in patients with sepsis with no known focus (IUFD not included) than the other serotypes. There were no other significant differences related to clinical manifestations and serotypes.Fig. 1Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
Serotype distribution among live-born neonates and infants (n = 87) with different manifestations. *Other: Pneumonia (8), Erysipelas (2), Urosepsis (1), Septic Arthritis (1)
A total of seven children died because of invasive GBS infection; all had sepsis, three were born full term and four were born prematurely. A total of five were EO, one VLO and one was 122 days old. They had a variety of serotypes with no one dominating.
Adults:
There were 317 adults (median age 73 years (23–103 years) 170 males and 147 females). Underlying medical conditions were documented in 259 (81 %), the most common being cardiovascular disease (n = 114), diabetes (n = 80) and malignant disease (n = 62).
Serotype V was the most prevalent serotype (39 %) followed by serotypes III and Ib (20 and 14 %, respectively) (Table 1). There were a few significant relations between serotype and clinical manifestation. Serotypes V and III were more common among patients with septic arthritis than with any other manifestation (p = 0.008 and p = 0.03 respectively). Otherwise there was no difference in the serotype distribution related to clinical manifestations (Table 2).Table 1Serotype distribution among invasive group B streptococciNeonates and infantsAdultsTotalIUFDa
Early onsetLate and very late onsetSerotypeNo.(%)No.(%)No.(%)No.(%)No.%Ia1(25)12(20)4(13)34(11)51(12)Ib5(9)0(0)45(14)50(12)II3(5)2(7)29(9)34(8)III3(75)24(41)20(67)62(20)109(27)IV1(2)1(3)14(4)16(4)V11(19)1(3)124(39)136(33)VII1(2)0(0)0(0)1(<1)VIII0(0)1(3)3(1)4(1)IX1(2)1(3)7(2)9(2)Total45830318410
aIntra Uterine Fetal DeathTable 2Serotype distribution of invasive group B streptococci related to clinical manifestation in adultsSerotypeManifestationIaIbIIIIIIVVVIIIIXTotal (%)Sepsis, unknown focus11139205320292 (29)Erysipelas1213791302175 (24)Septic arthritis*36382290152 (16)Urosepsis364122120241 (13)Pneumonia1234291022 (7)Endocarditis2315170120 (6)Meningitis100300004 (1)Othera
1221140011 (3)Missing clinical data000001001 (0,3)Total (%)34 (11)45 (14)29 (9)62 (20)14 (4)124 (39)3 (1)7 (2)318*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
Serotype distribution among invasive group B streptococci
aIntra Uterine Fetal Death
Serotype distribution of invasive group B streptococci related to clinical manifestation in adults
*p = 0.008 and p = 0.03 when the proportion of arthritis was caused by serotype V and III respectively was compared with the proportion of arthritis for all other serotypes
aChorioamnionitis, Mediastinitis, Periotinitis, Cholecystitis, Vulvitis
During the study period 38 patients (12 %) died during or within 30 days after the infection, 20 (53 %) had sepsis with unknown focus. Other manifestations were erysipelas (five), pneumonia (four), endocarditis (three), urosepsis (three), meningitis (one), septic arthritis (one), and mediastinitis (one). Only five of the patients who died did not have a known underlying medical condition, three were older than 72 years, one woman at age 53 died in multi organ failure 2 days after diagnosis of sepsis, one woman died at the age of 24 because of pneumonia, she had given birth 17 days prior to death. The serotypes isolated from the patients who died were V (12), III (ten), Ia (five), II (five), Ib (three), IV (two) and IX (one). No difference in serotype distribution could be seen among the fatal infections and in patients who survived.
Recurrent or reinfections:
A total of 11 patients, all adults with a preexisting underlying condition, had more than one episode at intervals from 2 to 23 months. The second infection was caused by a different serotype in six patients and by the same serotype in 5 patients (Table 3). The clinical manifestations varied but were the same in both (or all three) second infections in 8 patients.Table 3Patients with more than one invasive isolates of group B streptococciPatient no.Age (months)SerotypeManifestationTime between infections (months)Underlying condition181IbSeptic arthritis9Prosthetic hip, malignancy81IbSeptic arthritis279VSeptic arthritis3Prosthetic hip79VSeptic arthritis377IIIUrosepsis2Chronic urinary catheter malignancy, dementia77IbUrosepsis473VSepsis, unknown focus4Heart valve operation73VMediastinitis567IaSpondylitis2Spinal stenosis68IaSpondylitis662IIErysipelas10Progressive multiple sclerosis, urostomy63IIErysipelas760VSeptic arthritis8Ischaemic Heart Disease60VEndocarditis861VErysipelas8Malignancy. Died after the skeptical infection62IVSepsis, unknown focus946VUrosepsis23Chronic urinary catheter48IIUrosepsis1033VSeptic arthritis22Chronic renal failure, urostomy34IISeptic arthritis1182VErysipelas10Malignancy83VErysipelas2585IIIErysipelas
Patients with more than one invasive isolates of group B streptococci
Differences between children and adults:
The major difference between children (neonates and infants) and adults was that serotype III was much more common among children (p < 0.0001) and serotype V among adults (p < 0.0001). Serotype Ib was also more common among adults than children (p = 0.029).
Comparison of the present study with two earlier studies from the same area:
The main aim of this study was to survey the serotype distribution of invasive GBS infections and to compare it with previous studies in the same area to detect any changes over time.
Table 4 compares the serotype distribution with results from previous studies in the same region [16, 17]. There was a significant increase of serotype V between the first study (1988–1997) [16] and the second study (1998–2001) (p = 0.007 when serotype V was compared with all other serotypes) [17]. Serotype V has remained on approximately the same high level among adults in the present study. Serotype III has been the most prevalent serotype isolated from neonates and infants in all three studies from south-west Sweden since 1988 [16, 17, present study]. Serotypes Ia, Ib, II, III and IV have shown some fluctuations between the studies, but all differences were non-significant and showed no obvious trends over time. There were a few cases of serotype VII-IX, both in children and adults, which have not previously been seen in this area. Serotype VI has remained absent.Table 4Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same regionNeonates and infantsAdultsBerg et al. [15]Persson et al. [16]Present studyBerg et al. [15]Persson et al. [16]Present studySerotypeNo.(%)No.(%)No.(%)No.(%)No.(%)No.(%)Ia14(18)5(10)17(18)4(6)10(9)34(11)Ib2(3)2(4)5(6)15(23)10(9)45(14)II4(5)1(2)5(6)10(15)7(6)29(9)III48(62)30(60)44(48)19(29)28(25)62(20)IV2(3)1(2)2(2)1(2)8(7)14(4)V7(9)11(22)15(16)14(21)47(42)124(39)VII0(0)0(0)0(0)0(0)0(0)1(1)VIII0(0)0(0)3(1)0(0)0(0)1(1)IX0(0)0(0)7(2)0(0)0(0)2(2)Non-typeable1(1)0(0)0(0)3(5)1(1)0(0)The present study covers a longer time span than the two previous, which explains the increased number of cases
Serotype distribution of invasive group B streptococci isolates in the present study compared with two previous studies in the same region
The present study covers a longer time span than the two previous, which explains the increased number of cases
Discussion:
The differences in serotype distribution between neonates/infants and adults must be considered in future GBS vaccines. Pregnant women are the obvious target for GBS vaccination in order to protect neonates from early onset disease through passively transferred antibodies, so (at least currently) the most important in a vaccine must be type III followed by serotype Ia while serotype V seems to be of little importance. A GBS vaccine for elderly or patients with severe underlying diseases, in which serotype V infections are the most common, is probably unrealistic. There has been little change in serotype distribution among neonates and infants over the last 30 years, even after the rise of serotype V among adults [5]. Serotype V was first reported in 1985 and soon became the most common serotype among adults in many parts of the world [17–19].
The present study showed that five serotypes, Ia, Ib, II, III and V, accounted for 94 % (neonates and infants) and 93 % (adults) of cases with invasive GBS infections. This is consistent with studies from other countries [2, 5, 20, 21]. Serotype III is still the most common serotype among neonates and infants, especially for LO disease [5, 11, 13, 19, 20, 22]. Other studies have shown a dominance of serotype Ia for EO disease [19, 23, 24]. A possible reason for this differences between different parts of the world could be that GBS has similarities with pneumococci. It is well known that serotypes of pneumococci change regionally and over time [25, 26]. This was seen already in the prevaccination era. One example is the increase in pneumococcal serogroup 1 in Northern Europe [27–29]. A reason for this could be that pneumococci have different clones with different virulence within the same serotype. This has been suggested to be the case in increase of pneumococcal serogroup 1. Possibly this may be true also for GBS.
It has previously been suggested that a trivalent vaccine has the potential to further reduce the burden of GBS disease and especially LO cases [30]. Regarding our findings a trivalent vaccine (CRM197-conjugated capsular polysaccharides of GBS serotypes Ia, Ib and III), could have had an impact on 80 % (24/30) of the LO cases and 75 % (69/92) of all infants and neonates in our population during the study period. Several studies have shown serotype replacement among pneumococci when pneumococcal conjugate vaccines have been used in large scale for some years [31–33]. Only future can tell if this will occur also among GBS if such vaccines are used extensively.
Such a vaccine would have little impact on GBS diseases in adults, e.g. those with severe underlying diseases. Among adults three serotypes, Ia, III and V account for 70 % of cases with invasive GBS infections. Other studies have reported similar findings (64–71.8 %) but with variation in which of the three is the most prevalent [2, 18, 20, 22, 34, 35].
Some studies have reported an increase in serotype IV among invasive GBS isolates [36, 37]. We did not see any rise of serotype IV in the present study in comparison with the two previous studies done in the same region [16, 17]. Other studies show like ours that these serotypes are nonexistent or very uncommon [22, 23].
There were few relations between serotype and clinical manifestations but the study showed that serotypes V and III were found more often isolated from synovial fluid in adults but others have found it to be serotype II [38].
Conclusions:
In conclusion, this study has shown that there have not been any significant changes of the serotype distribution of invasive GBS infections in this Swedish population since the emergence of serotype V in the late 1990s but a few cases of serotypes VII-IX, which are not being targeted with a vaccine, have emerged in the area. It is therefore important to have an ongoing surveillance of GBS even though a trivalent vaccine would probably lead to much less disease among neonates and infants.
| Background:
Group B Streptococci (GBS) are the most common neonatal pathogens and infect immunocompromised and elderly individuals. The species has 10 different serotypes. Serotypes have been studied in the south-west area of Sweden in 1988-1997 and 1998-2001. The aim of this study was to study serotypes in the same area from 2004 to 2009.
Methods:
Invasive GBS isolates were collected prospectively from 2004 to 2009 in two counties in western Sweden with a population of 1.8 million, and were serotyped by latex agglutination. Clinical data were obtained from hospital records. During the study period 410 invasive GBS isolates from 398 patients were collected (multiple episodes ≥ 1 month apart). Clinical data were not available for two patients who are excluded. Four isolates were from stillborn neonates, 88 were from live born neonates and infants, and 318 from adults.
Results:
Serotype III was the most common serotype (48%) in neonates and infants followed by serotypes Ia (18%) and V (16%). In adults serotype V (39%) dominated followed by serotypes III (20%) and Ib (14%). There was a significant increase of serotype V in comparison with the first study (1988-1997) but there were no significant changes in the serotype distribution between the present study and the second study (1998-2001). There were a few cases of serotype VI-IX, both in children and adults, not seen in the previous studies. Serotype V was more common among patients with arthritis than with any other manifestation.
Conclusions:
Changes in GBS serotypes occur over time in the same region, which must be considered when GBS vaccines are formulated. | Background:
Group B Streptococci (GBS) remain a leading cause of invasive neonatal infections and an important cause of infections in pregnant women, immune compromised adults and the elderly [1–3]. GBS possess ten distinct capsular polysaccharide (CPS) types: Ia, Ib and II-IX [4]. The principal disease-associated serotypes are currently Ia, Ib, II, III and V [2, 5–7] according to studies from North America and Norway.
Most centers in Sweden have been using a risk-based prophylaxis strategy which identifies candidates for intra partum antimicrobial prophylaxis (IAP) according to the presence of any of the following risk factors: a previous GBS-infected baby, positive for GBS in urine, delivering at <37 weeks’ gestation, having an intra-partum temperature ≥38.0°, or rupture of membranes for ≥18 h. There is no plan to begin with universal prenatal GBS screening followed by IAP of all GBS carriers as is recommended by the US guidelines from 2002 [8, 9]. Both strategies have dramatically reduced the incidence and mortality among early onset (EO, <7 days after birth) GBS infections, but they have not reduced the incidence of late onset (LO, 7–27 days after birth) or very late onset (VLO, 28 days–4 months after birth) disease [1, 10–13].
There are no efficacy studies of GBS vaccines, but most likely protection would be achieved by anticapsular serum antibodies as it is for other bacteria with polysaccharide capsules. The first vaccines against GBS consisted of pure polysaccharides and had disappointingly low immunogenicity. Polysaccharide-protein conjugates are more immunogenic and have been given in phase 1 to 2 clinical trials (including pregnant women with 1 to 5 serotypes. Proteins used have been tetanus toxoid or genetically inactivated diphtheria toxoid [14]. To make the best possible GBS vaccines they must be directed against the polysaccharides of the most common GBS serotypes.
It is important to follow the distribution of serotypes since it may differ over time and between populations [3].
The main aim of this study was to survey the current serotype distribution of invasive GBS infections in a Swedish population and to compare it with previous studies in the same area to detect any changes over time.
Conclusions:
In conclusion, this study has shown that there have not been any significant changes of the serotype distribution of invasive GBS infections in this Swedish population since the emergence of serotype V in the late 1990s but a few cases of serotypes VII-IX, which are not being targeted with a vaccine, have emerged in the area. It is therefore important to have an ongoing surveillance of GBS even though a trivalent vaccine would probably lead to much less disease among neonates and infants. | Background:
Group B Streptococci (GBS) are the most common neonatal pathogens and infect immunocompromised and elderly individuals. The species has 10 different serotypes. Serotypes have been studied in the south-west area of Sweden in 1988-1997 and 1998-2001. The aim of this study was to study serotypes in the same area from 2004 to 2009.
Methods:
Invasive GBS isolates were collected prospectively from 2004 to 2009 in two counties in western Sweden with a population of 1.8 million, and were serotyped by latex agglutination. Clinical data were obtained from hospital records. During the study period 410 invasive GBS isolates from 398 patients were collected (multiple episodes ≥ 1 month apart). Clinical data were not available for two patients who are excluded. Four isolates were from stillborn neonates, 88 were from live born neonates and infants, and 318 from adults.
Results:
Serotype III was the most common serotype (48%) in neonates and infants followed by serotypes Ia (18%) and V (16%). In adults serotype V (39%) dominated followed by serotypes III (20%) and Ib (14%). There was a significant increase of serotype V in comparison with the first study (1988-1997) but there were no significant changes in the serotype distribution between the present study and the second study (1998-2001). There were a few cases of serotype VI-IX, both in children and adults, not seen in the previous studies. Serotype V was more common among patients with arthritis than with any other manifestation.
Conclusions:
Changes in GBS serotypes occur over time in the same region, which must be considered when GBS vaccines are formulated. | 6,472 | 331 | [
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] | null | [CONTENT] Group B Streptococci | Serotypes | Invasive infections | Neonates | Streptococcus agalactiae | Epidemiology [SUMMARY] | null | [CONTENT] Group B Streptococci | Serotypes | Invasive infections | Neonates | Streptococcus agalactiae | Epidemiology [SUMMARY] | [CONTENT] Group B Streptococci | Serotypes | Invasive infections | Neonates | Streptococcus agalactiae | Epidemiology [SUMMARY] | [CONTENT] Group B Streptococci | Serotypes | Invasive infections | Neonates | Streptococcus agalactiae | Epidemiology [SUMMARY] | [CONTENT] Group B Streptococci | Serotypes | Invasive infections | Neonates | Streptococcus agalactiae | Epidemiology [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Female | Hospital Records | Humans | Infant | Infant, Newborn | Latex Fixation Tests | Male | Middle Aged | Prospective Studies | Serogroup | Streptococcal Infections | Streptococcus agalactiae | Sweden | Young Adult [SUMMARY] | null | [CONTENT] Adult | Aged | Aged, 80 and over | Female | Hospital Records | Humans | Infant | Infant, Newborn | Latex Fixation Tests | Male | Middle Aged | Prospective Studies | Serogroup | Streptococcal Infections | Streptococcus agalactiae | Sweden | Young Adult [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Female | Hospital Records | Humans | Infant | Infant, Newborn | Latex Fixation Tests | Male | Middle Aged | Prospective Studies | Serogroup | Streptococcal Infections | Streptococcus agalactiae | Sweden | Young Adult [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Female | Hospital Records | Humans | Infant | Infant, Newborn | Latex Fixation Tests | Male | Middle Aged | Prospective Studies | Serogroup | Streptococcal Infections | Streptococcus agalactiae | Sweden | Young Adult [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Female | Hospital Records | Humans | Infant | Infant, Newborn | Latex Fixation Tests | Male | Middle Aged | Prospective Studies | Serogroup | Streptococcal Infections | Streptococcus agalactiae | Sweden | Young Adult [SUMMARY] | [CONTENT] streptococcineonates infantsadultstotaliufda | isolates group streptococci | birth gbs infections | group streptococcineonates infantsadultstotaliufda | streptococci gbs remain [SUMMARY] | null | [CONTENT] streptococcineonates infantsadultstotaliufda | isolates group streptococci | birth gbs infections | group streptococcineonates infantsadultstotaliufda | streptococci gbs remain [SUMMARY] | [CONTENT] streptococcineonates infantsadultstotaliufda | isolates group streptococci | birth gbs infections | group streptococcineonates infantsadultstotaliufda | streptococci gbs remain [SUMMARY] | [CONTENT] streptococcineonates infantsadultstotaliufda | isolates group streptococci | birth gbs infections | group streptococcineonates infantsadultstotaliufda | streptococci gbs remain [SUMMARY] | [CONTENT] streptococcineonates infantsadultstotaliufda | isolates group streptococci | birth gbs infections | group streptococcineonates infantsadultstotaliufda | streptococci gbs remain [SUMMARY] | [CONTENT] serotype | patients | study | distribution | serotypes | invasive | gbs | iii | adults | clinical [SUMMARY] | null | [CONTENT] serotype | patients | study | distribution | serotypes | invasive | gbs | iii | adults | clinical [SUMMARY] | [CONTENT] serotype | patients | study | distribution | serotypes | invasive | gbs | iii | adults | clinical [SUMMARY] | [CONTENT] serotype | patients | study | distribution | serotypes | invasive | gbs | iii | adults | clinical [SUMMARY] | [CONTENT] serotype | patients | study | distribution | serotypes | invasive | gbs | iii | adults | clinical [SUMMARY] | [CONTENT] gbs | polysaccharide | vaccines | onset | birth | infections | studies | intra partum | according | toxoid [SUMMARY] | null | [CONTENT] serotype | patients | arthritis | distribution | present | 16 | 14 | born | study | manifestations [SUMMARY] | [CONTENT] vaccine | gbs | serotype late 1990s | trivalent vaccine probably lead | vaccine probably | swedish population emergence serotype | swedish population emergence | targeted | targeted vaccine | targeted vaccine emerged [SUMMARY] | [CONTENT] serotype | gbs | patients | adults | study | distribution | serotypes | neonates | studies | invasive [SUMMARY] | [CONTENT] serotype | gbs | patients | adults | study | distribution | serotypes | neonates | studies | invasive [SUMMARY] | [CONTENT] Group B Streptococci | GBS ||| 10 ||| Sweden | 1988-1997 | 1998-2001 ||| from 2004 to 2009 [SUMMARY] | null | [CONTENT] 48% | 18% | V | 16% ||| 39% | 20% | Ib | 14% ||| first | 1988-1997 | second | 1998-2001 ||| VI-IX ||| Serotype V [SUMMARY] | [CONTENT] GBS | GBS [SUMMARY] | [CONTENT] GBS ||| 10 ||| Sweden | 1988-1997 | 1998-2001 ||| from 2004 to 2009 ||| 2004 to 2009 | two | Sweden | 1.8 million ||| ||| 410 | GBS | 398 | ≥ | 1 month ||| two ||| Four | 88 | 318 ||| ||| 48% | 18% | V | 16% ||| 39% | 20% | Ib | 14% ||| first | 1988-1997 | second | 1998-2001 ||| VI-IX ||| 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|||||
Acupotomy by ultrasound-guided versus anatomical guidance in knee osteoarthritis: A protocol for systematic review and meta-analysis. | 36451390 | At present, there is no systematic evaluation on whether ultrasonic-guided acupotomy is more effective compared with anatomical guidance in knee osteoarthritis. We conducted a protocol for systematic review and meta-analysis to provide a method for evaluating the effectiveness and safety of acupotomy by ultrasound-guided technique. | BACKGROUND | An all-round retrieval will be performed in the following electronic journal databases from their inception to October 2022, which comprise PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. The following key words were used on combination with Boolean operators AND or OR: "acupotomy," "ultrasound," "knee osteoarthritis." Two authors completed the quality assessment using the Cochrane Collaborations risk of bias tool. The meta-analysis was conducted using Review Manager 5.3 software from the Cochrane Collaboration (London, UK). | METHODS | The findings of this study will be submitted to peer-reviewed journals for publication. | RESULTS | This systematic review will provide evidence to judge whether acupotomy by ultrasound-guided technique is effective and safe for knee osteoarthritis. | CONCLUSION | [
"Humans",
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] | 9704967 | 1. Introduction | Knee osteoarthritis (OA) is the most prevalent chronic joint disease.[1–3] Cartilage is the central tissue affected by OA and causes subsequent symptoms, including joint pain, stiffness and joint swelling, which diminishes the range of motion.[4,5] It is one of the major causes of deformity, resulting in huge medical expense and poor quality of life. It affects nearly 34% of those aged 65 and older.[6] The number of patients with knee OA has increased in tandem with population aging and it remains a huge healthcare challenge. Currently, no reliable treatment has been confirmed to prevent progression of knee OA. The aim of treatment was to relieve pain and increase functional outcomes.[7,8] Numerous conservative methods for pain management, including modification of daily activities and peri-articular infiltration analgesia have been tested, and the optimal method is currently still under debate.
Acupotomy therapy is widely used in Chinese clinical practice and recommended in the Chinese medicine expert consensus for knee OA.[9] Acupotomy is a type of acupuncture used in traditional Chinese medicine (TCM), and it has both the characteristics of a “needle” in traditional Chinese medicine and a “knife” in Western medicine.[10] The mechanism of acupotomy remains unclear and remains to be explored, but acupotomy can be used to release ligaments, joint sacs, and synovium.[11] Some studies have shown that acupotomy therapy can release adhesions, alter the mechanical balance of the knee joint, improve lymphatic circulation, and reduce abnormal tissue pressures.[12] Acupotomy has been widely used clinically with a satisfactory efficacy. With the development of ultrasound technology, ultrasound-guided acupotomy has shown great value in clinical practice.[13,14] But it is not yet clear that ultrasound-guided acupotomy is effective and safe in knee OA. Therefore, it is important to reevaluate the available evidence to reach a relatively convincing conclusion that acupotomy by ultrasound-guided technique is a better choice than anatomical guidance. | 2. Methods | 2.1. Protocol and registration This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).
This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).
2.2. Ethics We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.
We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.
2.3. Inclusion criteria 2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
2.4. Search strategy Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.
Search strategy of PubMed.
Flow diagram of study selection.
Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.
Search strategy of PubMed.
Flow diagram of study selection.
2.5. Data extraction Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.
Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.
2.6. Risk of bias The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.
The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.
2.7. Statistical analysis We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]
We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20] | null | null | null | null | [
"2.1. Protocol and registration",
"2.2. Ethics",
"2.3. Inclusion criteria",
"2.3.1. Types of studies.",
"2.3.2. Types of patients.",
"2.3.3. Types of interventions.",
"2.3.4. Outcome measures.",
"2.4. Search strategy",
"2.5. Data extraction",
"2.6. Risk of bias",
"2.7. Statistical analysis",
"Author contributions"
] | [
"This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).",
"We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.",
"2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\nAll randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\n2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\nAll patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\n2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\nThe treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\n2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.\nThe primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.",
"All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.",
"All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.",
"The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.",
"The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.",
"Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.\nSearch strategy of PubMed.\nFlow diagram of study selection.",
"Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.",
"The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.",
"We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]",
"Data analysis: Jiantong Wei.\nData collection: Zhi Qian.\nData curation: Jiantong Wei.\nInvestigation methodology: Zhi Qian.\nStudy design: Jun Qian.\nWriting – original draft: Li Wang.\nWriting – review & editing: Jun Qian."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"1. Introduction",
"2. Methods",
"2.1. Protocol and registration",
"2.2. Ethics",
"2.3. Inclusion criteria",
"2.3.1. Types of studies.",
"2.3.2. Types of patients.",
"2.3.3. Types of interventions.",
"2.3.4. Outcome measures.",
"2.4. Search strategy",
"2.5. Data extraction",
"2.6. Risk of bias",
"2.7. Statistical analysis",
"3. Discussion",
"Author contributions"
] | [
"Knee osteoarthritis (OA) is the most prevalent chronic joint disease.[1–3] Cartilage is the central tissue affected by OA and causes subsequent symptoms, including joint pain, stiffness and joint swelling, which diminishes the range of motion.[4,5] It is one of the major causes of deformity, resulting in huge medical expense and poor quality of life. It affects nearly 34% of those aged 65 and older.[6] The number of patients with knee OA has increased in tandem with population aging and it remains a huge healthcare challenge. Currently, no reliable treatment has been confirmed to prevent progression of knee OA. The aim of treatment was to relieve pain and increase functional outcomes.[7,8] Numerous conservative methods for pain management, including modification of daily activities and peri-articular infiltration analgesia have been tested, and the optimal method is currently still under debate.\nAcupotomy therapy is widely used in Chinese clinical practice and recommended in the Chinese medicine expert consensus for knee OA.[9] Acupotomy is a type of acupuncture used in traditional Chinese medicine (TCM), and it has both the characteristics of a “needle” in traditional Chinese medicine and a “knife” in Western medicine.[10] The mechanism of acupotomy remains unclear and remains to be explored, but acupotomy can be used to release ligaments, joint sacs, and synovium.[11] Some studies have shown that acupotomy therapy can release adhesions, alter the mechanical balance of the knee joint, improve lymphatic circulation, and reduce abnormal tissue pressures.[12] Acupotomy has been widely used clinically with a satisfactory efficacy. With the development of ultrasound technology, ultrasound-guided acupotomy has shown great value in clinical practice.[13,14] But it is not yet clear that ultrasound-guided acupotomy is effective and safe in knee OA. Therefore, it is important to reevaluate the available evidence to reach a relatively convincing conclusion that acupotomy by ultrasound-guided technique is a better choice than anatomical guidance.",
"2.1. Protocol and registration This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).\nThis protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).\n2.2. Ethics We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.\nWe will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.\n2.3. Inclusion criteria 2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\nAll randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\n2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\nAll patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\n2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\nThe treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\n2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.\nThe primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.\n2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\nAll randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\n2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\nAll patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\n2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\nThe treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\n2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.\nThe primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.\n2.4. Search strategy Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.\nSearch strategy of PubMed.\nFlow diagram of study selection.\nFollowing databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.\nSearch strategy of PubMed.\nFlow diagram of study selection.\n2.5. Data extraction Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.\nTwo independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.\n2.6. Risk of bias The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.\nThe risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.\n2.7. Statistical analysis We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]\nWe performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]",
"This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).",
"We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.",
"2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\nAll randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.\n2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\nAll patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.\n2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\nThe treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.\n2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.\nThe primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.",
"All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.",
"All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.",
"The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.",
"The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.",
"Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.\nSearch strategy of PubMed.\nFlow diagram of study selection.",
"Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.",
"The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.",
"We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]",
"The purpose of this study is to evaluate whether ultrasonic-guided acupotomy is more effective compared with anatomical guidance. The conclusions drawn from this review may benefit patients who are ready for acupotomy, as well as clinicians. This review has some potential limitations:\ndifferent types of needle knives, ultrasound equipment, and diseases may lead to heterogeneities;\nthe quality of the included studies may be poor, which will decrease the evidence level;\nmost of the studies are reported in Chinese, which may produce language bias.\nAt present, there is no systematic evaluation or research undergoing on this issue. Therefore, it is important to perform a systematic review to reach a relatively convincing conclusion that ultrasound-guided technique is a better choice for acupotomy.",
"Data analysis: Jiantong Wei.\nData collection: Zhi Qian.\nData curation: Jiantong Wei.\nInvestigation methodology: Zhi Qian.\nStudy design: Jun Qian.\nWriting – original draft: Li Wang.\nWriting – review & editing: Jun Qian."
] | [
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"methods",
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"discussion",
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] | [
"knee osteoarthritis",
"pain",
"range of motion",
"ultrasonic-guided acupotomy"
] | 1. Introduction:
Knee osteoarthritis (OA) is the most prevalent chronic joint disease.[1–3] Cartilage is the central tissue affected by OA and causes subsequent symptoms, including joint pain, stiffness and joint swelling, which diminishes the range of motion.[4,5] It is one of the major causes of deformity, resulting in huge medical expense and poor quality of life. It affects nearly 34% of those aged 65 and older.[6] The number of patients with knee OA has increased in tandem with population aging and it remains a huge healthcare challenge. Currently, no reliable treatment has been confirmed to prevent progression of knee OA. The aim of treatment was to relieve pain and increase functional outcomes.[7,8] Numerous conservative methods for pain management, including modification of daily activities and peri-articular infiltration analgesia have been tested, and the optimal method is currently still under debate.
Acupotomy therapy is widely used in Chinese clinical practice and recommended in the Chinese medicine expert consensus for knee OA.[9] Acupotomy is a type of acupuncture used in traditional Chinese medicine (TCM), and it has both the characteristics of a “needle” in traditional Chinese medicine and a “knife” in Western medicine.[10] The mechanism of acupotomy remains unclear and remains to be explored, but acupotomy can be used to release ligaments, joint sacs, and synovium.[11] Some studies have shown that acupotomy therapy can release adhesions, alter the mechanical balance of the knee joint, improve lymphatic circulation, and reduce abnormal tissue pressures.[12] Acupotomy has been widely used clinically with a satisfactory efficacy. With the development of ultrasound technology, ultrasound-guided acupotomy has shown great value in clinical practice.[13,14] But it is not yet clear that ultrasound-guided acupotomy is effective and safe in knee OA. Therefore, it is important to reevaluate the available evidence to reach a relatively convincing conclusion that acupotomy by ultrasound-guided technique is a better choice than anatomical guidance.
2. Methods:
2.1. Protocol and registration This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).
This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).
2.2. Ethics We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.
We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.
2.3. Inclusion criteria 2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
2.4. Search strategy Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.
Search strategy of PubMed.
Flow diagram of study selection.
Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.
Search strategy of PubMed.
Flow diagram of study selection.
2.5. Data extraction Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.
Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.
2.6. Risk of bias The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.
The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.
2.7. Statistical analysis We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]
We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]
2.1. Protocol and registration:
This protocol was drafted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.[15] If there are any adjustments throughout the study, we will fix and update the details in the final report. The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO),[16] registration number (CRD42019145167).
2.2. Ethics:
We will not need individual data of each patient in the research as this is a systematic review. Therefore institutional review board approval and ethics committee is not needed.
2.3. Inclusion criteria:
2.3.1. Types of studies. All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
2.3.2. Types of patients. All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
2.3.3. Types of interventions. The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
2.3.4. Outcome measures. The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
2.3.1. Types of studies.:
All randomized controlled trials (RCTs) comparing acupotomy by ultrasound-guided and anatomical guidance will be included without any restriction to publication status or language. Non-randomized controlled trial and uncontrolled clinical trials will be excluded. Any study with a sample size of <10 people will also be excluded from this review.
2.3.2. Types of patients.:
All patients undergoing acupotomy by the ultrasound-guided technique and anatomical guidance will be included in the trial. All eligible patients will not be restricted by disease, age, sex, race, education, or economic status. Patients who will be decided unsuitable for acupotomy by ultrasound-guided technique, such as patients with fracture and dislocation, space-occupying lesions, cardiovascular and cerebrovascular diseases and other serious diseases will be excluded.
2.3.3. Types of interventions.:
The treatment group will be treated with acupotomy by ultrasound-guided technique (there is no limit on the needle materials, ultrasonic equipment, and course of treatment). The control group will adapt to routine acupotomy. Studies comparing different acupotomy insertion sites or different forms of acupotomy will be excluded.
2.3.4. Outcome measures.:
The primary outcomes were visual analogue score,[17] Western Ontario and McMaster Universities (WOMAC) index[18] and range of motion. Secondary outcomes included quality of life and adverse effects.
2.4. Search strategy:
Following databases will be searched: PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. We will select the eligible studies published up to October, 2022. We adopt the combination of heading terms and free words as a search strategy which is decided by all the reviewers. Search terms: acupotomy, ultrasound, knee osteoarthritis. Taking PubMed as an example, the initial search strategy is shown in Table 1, which will be adjusted according to the specific database. The reference lists of the included studies were also checked for additional studies that were not identified with the database search. The flowchart of this systematic review is shown in Figure 1.
Search strategy of PubMed.
Flow diagram of study selection.
2.5. Data extraction:
Two independent authors will extract the below descriptive information from the included articles: demographic information of patients, such as average age, number of patients, sex ratio and body mass index; study characteristics, such as authors, year of publication, study language, study design, and the average follow-up period; details of interventions and outcome measures. If the data cannot be directly extracted or is missing, we will contact the relevant author to ensure that the information is complete.
2.6. Risk of bias:
The risk of bias assessment of the included studies was performed by two authors independently using the Cochrane Collaborations risk of bias tool.[19] This tool included seven aspects which were sequence generation (selection bias), allocation sequence concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias) and other bias (baseline balance and fund). Additionally, each of the aspects was ranked low risk of bias, high risk of bias, and unclear risk of bias.
2.7. Statistical analysis:
We performed the meta-analysis by Review Manager software version 5.3 from the Cochrane Collaboration (London, UK). Continuous variables were expressed as the weighted mean difference or standardized mean difference and 95% confidence interval. Weighted mean difference was used when data were measured in the same scale and standardized mean difference were used if data were measured using different scales. Heterogeneity among the studies was quantified with the I2 statistic. If I2 > 50% or P < .1, a random-effect model was used to decrease heterogeneity, and the subgroup and sensitivity analysis were performed to explore the sources of heterogeneity; otherwise, heterogeneity was negligible and a fixed-effect model was used. To evaluate publication bias, we perform a funnel plot if the number of included studies is sufficient (>10 articles). A symmetrical funnel plot indicates no possibility of publication bias, while an asymmetrical funnel plot indicates a high possibility of publication bias.[20]
3. Discussion:
The purpose of this study is to evaluate whether ultrasonic-guided acupotomy is more effective compared with anatomical guidance. The conclusions drawn from this review may benefit patients who are ready for acupotomy, as well as clinicians. This review has some potential limitations:
different types of needle knives, ultrasound equipment, and diseases may lead to heterogeneities;
the quality of the included studies may be poor, which will decrease the evidence level;
most of the studies are reported in Chinese, which may produce language bias.
At present, there is no systematic evaluation or research undergoing on this issue. Therefore, it is important to perform a systematic review to reach a relatively convincing conclusion that ultrasound-guided technique is a better choice for acupotomy.
Author contributions:
Data analysis: Jiantong Wei.
Data collection: Zhi Qian.
Data curation: Jiantong Wei.
Investigation methodology: Zhi Qian.
Study design: Jun Qian.
Writing – original draft: Li Wang.
Writing – review & editing: Jun Qian.
| Background:
At present, there is no systematic evaluation on whether ultrasonic-guided acupotomy is more effective compared with anatomical guidance in knee osteoarthritis. We conducted a protocol for systematic review and meta-analysis to provide a method for evaluating the effectiveness and safety of acupotomy by ultrasound-guided technique.
Methods:
An all-round retrieval will be performed in the following electronic journal databases from their inception to October 2022, which comprise PubMed, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journals Database, and China biomedical literature database. The following key words were used on combination with Boolean operators AND or OR: "acupotomy," "ultrasound," "knee osteoarthritis." Two authors completed the quality assessment using the Cochrane Collaborations risk of bias tool. The meta-analysis was conducted using Review Manager 5.3 software from the Cochrane Collaboration (London, UK).
Results:
The findings of this study will be submitted to peer-reviewed journals for publication.
Conclusions:
This systematic review will provide evidence to judge whether acupotomy by ultrasound-guided technique is effective and safe for knee osteoarthritis. | null | null | 4,320 | 223 | [
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] | [
"knee joint improve",
"knee osteoarthritis taking",
"knee oa increased",
"osteoarthritis oa prevalent",
"knee oa acupotomy"
] | null | null | null | [CONTENT] knee osteoarthritis | pain | range of motion | ultrasonic-guided acupotomy [SUMMARY] | [CONTENT] knee osteoarthritis | pain | range of motion | ultrasonic-guided acupotomy [SUMMARY] | null | null | [CONTENT] knee osteoarthritis | pain | range of motion | ultrasonic-guided acupotomy [SUMMARY] | null | [CONTENT] Humans | Osteoarthritis, Knee | Systematic Reviews as Topic | Meta-Analysis as Topic | Acupuncture Therapy | Ultrasonography, Interventional [SUMMARY] | [CONTENT] Humans | Osteoarthritis, Knee | Systematic Reviews as Topic | Meta-Analysis as Topic | Acupuncture Therapy | Ultrasonography, Interventional [SUMMARY] | null | null | [CONTENT] Humans | Osteoarthritis, Knee | Systematic Reviews as Topic | Meta-Analysis as Topic | Acupuncture Therapy | Ultrasonography, Interventional [SUMMARY] | null | [CONTENT] knee joint improve | knee osteoarthritis taking | knee oa increased | osteoarthritis oa prevalent | knee oa acupotomy [SUMMARY] | [CONTENT] knee joint improve | knee osteoarthritis taking | knee oa increased | osteoarthritis oa prevalent | knee oa acupotomy [SUMMARY] | null | null | [CONTENT] knee joint improve | knee osteoarthritis taking | knee oa increased | osteoarthritis oa prevalent | knee oa acupotomy [SUMMARY] | null | [CONTENT] acupotomy | bias | patients | ultrasound | included | guided | ultrasound guided | studies | acupotomy ultrasound | acupotomy ultrasound guided [SUMMARY] | [CONTENT] acupotomy | bias | patients | ultrasound | included | guided | ultrasound guided | studies | acupotomy ultrasound | acupotomy ultrasound guided [SUMMARY] | null | null | [CONTENT] acupotomy | bias | patients | ultrasound | included | guided | ultrasound guided | studies | acupotomy ultrasound | acupotomy ultrasound guided [SUMMARY] | null | [CONTENT] oa | joint | knee | acupotomy | knee oa | medicine | remains | pain | chinese medicine | chinese [SUMMARY] | [CONTENT] bias | acupotomy | patients | excluded | acupotomy ultrasound | included | risk bias | risk | search | acupotomy ultrasound guided [SUMMARY] | null | null | [CONTENT] acupotomy | bias | patients | ultrasound | guided | ultrasound guided | review | excluded | acupotomy ultrasound | acupotomy ultrasound guided [SUMMARY] | null | [CONTENT] ||| [SUMMARY] | [CONTENT] October 2022 | PubMed | MEDLINE | EMBASE | Cochrane Library | China National Knowledge Infrastructure | Wanfang | Chinese Scientific Journals Database | China ||| Boolean ||| Two | Cochrane Collaborations ||| Review | 5.3 | the Cochrane Collaboration | London | UK [SUMMARY] | null | null | [CONTENT] ||| ||| October 2022 | PubMed | MEDLINE | EMBASE | Cochrane Library | China National Knowledge Infrastructure | Wanfang | Chinese Scientific Journals Database | China ||| Boolean ||| Two | Cochrane Collaborations ||| Review | 5.3 | the Cochrane Collaboration | London | UK ||| ||| [SUMMARY] | null |
|||
HPV infection among rural American Indian women and urban white women in South Dakota: an HPV prevalence study. | 21943050 | High-risk strains of human papillomavirus (HPV) cause cervical cancer. American Indian (AI) women in the Northern Plains of the U.S. have significantly higher incidence and mortality rates for cervical cancer than White women in the same geographical area. We compared HPV prevalence, patterns of HPV types, and infection with multiple HPV types in AI and White women living in South Dakota, U.S. | BACKGROUND | We analyzed the HPV status of cervical samples collected in 2006-2008 from women aged 18-65 years who attended two rural AI reservation clinics (n = 235) or an urban clinic in the same area serving mostly White women (n = 246). Data collection occurred before HPV vaccination was available to study participants. HPV DNA was amplified by using the L1 consensus primer system and an HPV Linear Array detection assay to identify HPV types. We used chi-square tests to compare HPV variables, with percentages standardized by age and lifetime number of sexual partners. | METHODS | Compared to White women, AI women were younger (p = 0.01) and reported more sexual partners (p < 0.001). A lower percentage of AI women tested negative for HPV infection compared to Whites (58% [95% CI = 51-65] vs. 77% [95% CI = 71-82]; p < 0.001), and a higher percentage of AI women were infected by oncogenic types (30% [95% CI = 25-36] vs. 16% [95% CI = 11-21]; p = 0.001). Infections among AI women showed a wider variety and very different pattern of HPV types, including a higher prevalence of mixed HPV infections (19% [95% CI = 26-38] vs. 7% [95% CI = 4-11]; p = 0.001). AI women had a higher percentage of HPV infections that were not preventable by HPV vaccination (32% [95% CI = 26-38] vs. 15% [95% CI = 11-21]; p < 0.001). | RESULTS | A higher HPV burden and a different HPV genotyping profile may contribute to the high rate of cervical cancer among AI women. | CONCLUSIONS | [
"Adolescent",
"Adult",
"Aged",
"Female",
"Genotype",
"Humans",
"Indians, North American",
"Middle Aged",
"Papillomaviridae",
"Papillomavirus Infections",
"Prevalence",
"Rural Population",
"South Dakota",
"Urban Population",
"White People",
"Young Adult"
] | 3190376 | Background | Cervical cancer is the second most common cause of cancer-related deaths in women worldwide [1] and a leading cause of cancer mortality among American Indian (AI) women [2,3]. Compared to other U.S. racial and ethnic groups, AI women experience striking health disparities in relation to cervical cancer, including a higher prevalence [4-9], a more rapidly increasing incidence [10,11], and poorer survival [12,13]. The Aberdeen Area Indian Health Service, which encompasses 16 different tribes located in South Dakota, North Dakota, Nebraska, and Iowa, reported an age-adjusted cervical cancer mortality rate of 4.3/100,000 from 1996 to 1998 [14], almost twice the rate reported in the general U.S. population [15]. Our previous work found a high prevalence of human papillomavirus (HPV) infection among AI women residing on reservations in the same geographic region, and suggested that types other than HPV-16 and HPV-18 comprised a substantial proportion of oncogenic HPV infections [16]. In a recent study of more than 9,600 U.S. women, the overall prevalence of high-risk HPV types among AI and Alaska Native women (representing 2% of the total sample) was 25% [17], which is substantially higher than the general U.S. population prevalence of 15% [18].
Infection with HPV, especially HPV-16 and HPV-18, is the most common cause of cervical cancer [19]. Preventing HPV infection is critical to public health efforts to reduce precancerous lesions and cervical cancer incidence and mortality. In 2007, the U.S. Food and Drug Administration approved the first vaccine to prevent infection by the most common oncogenic HPV types present in the general U.S. population; a second HPV vaccine was approved in 2009. Despite striking cervical cancer disparities, little is known about patterns of HPV infection in AI communities that can inform the adequacy of prevention provided for AI women by existing HPV vaccines. We therefore examined women seen in two rural AI reservation clinics and one urban clinic serving primarily White women in the Northern Plains. Our aims were to 1) compare HPV prevalence and patterns between the AI and White clinic samples, and 2) compare proportions of women in each group who were infected by HPV types that can be prevented by existing vaccines. | Methods | Setting This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].
The urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].
Of note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.
Before enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.
This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].
The urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].
Of note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.
Before enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.
Participants Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.
Two of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.
Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.
Two of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.
Oncogenic HPV types All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.
All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.
HPV Vaccines Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported "cross protection" phenomenon [30].
Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported "cross protection" phenomenon [30].
Sample Collection and Assay Methods The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.
After the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.
The DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.
The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.
After the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.
The DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.
Measures We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.
We used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).
We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.
We used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).
Statistical Analysis Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.
For our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.
All analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.
Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.
For our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.
All analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance. | null | null | Conclusions | The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2334/11/252/prepub
| [
"Background",
"Setting",
"Participants",
"Oncogenic HPV types",
"HPV Vaccines",
"Sample Collection and Assay Methods",
"Measures",
"Statistical Analysis",
"Results",
"Discussion",
"Conclusions"
] | [
"Cervical cancer is the second most common cause of cancer-related deaths in women worldwide [1] and a leading cause of cancer mortality among American Indian (AI) women [2,3]. Compared to other U.S. racial and ethnic groups, AI women experience striking health disparities in relation to cervical cancer, including a higher prevalence [4-9], a more rapidly increasing incidence [10,11], and poorer survival [12,13]. The Aberdeen Area Indian Health Service, which encompasses 16 different tribes located in South Dakota, North Dakota, Nebraska, and Iowa, reported an age-adjusted cervical cancer mortality rate of 4.3/100,000 from 1996 to 1998 [14], almost twice the rate reported in the general U.S. population [15]. Our previous work found a high prevalence of human papillomavirus (HPV) infection among AI women residing on reservations in the same geographic region, and suggested that types other than HPV-16 and HPV-18 comprised a substantial proportion of oncogenic HPV infections [16]. In a recent study of more than 9,600 U.S. women, the overall prevalence of high-risk HPV types among AI and Alaska Native women (representing 2% of the total sample) was 25% [17], which is substantially higher than the general U.S. population prevalence of 15% [18].\nInfection with HPV, especially HPV-16 and HPV-18, is the most common cause of cervical cancer [19]. Preventing HPV infection is critical to public health efforts to reduce precancerous lesions and cervical cancer incidence and mortality. In 2007, the U.S. Food and Drug Administration approved the first vaccine to prevent infection by the most common oncogenic HPV types present in the general U.S. population; a second HPV vaccine was approved in 2009. Despite striking cervical cancer disparities, little is known about patterns of HPV infection in AI communities that can inform the adequacy of prevention provided for AI women by existing HPV vaccines. We therefore examined women seen in two rural AI reservation clinics and one urban clinic serving primarily White women in the Northern Plains. Our aims were to 1) compare HPV prevalence and patterns between the AI and White clinic samples, and 2) compare proportions of women in each group who were infected by HPV types that can be prevented by existing vaccines.",
"This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].\nThe urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].\nOf note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.\nBefore enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.",
"Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.\nTwo of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.",
"All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.",
"Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported \"cross protection\" phenomenon [30].",
"The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.\nAfter the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.\nThe DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.",
"We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.\nWe used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).",
"Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.\nFor our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.\nAll analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.",
"We collected cervical samples from 258 AI women (104 and 154 women at Sites 1 and 2, respectively) and from 252 White women at the urban clinic. Participation rates exceeded 90%, with the total number of refusals ranging from four to seven among the three clinic sites. Some faint positive HPV results required adjudication by a third reader, but all positive results were typable. After excluding three women with missing data for HPV status and 26 women with missing data for age or number of sexual partners, data on 481 women (AI n = 235, White n = 246) were available for analysis. The vast majority of study participants were recruited during annual gynecological examinations. All participants had health insurance of some kind. For AI women, healthcare was provided and paid for by the Indian Health Service; the White women were covered by private or government-sponsored insurance (e.g., Medicaid).\nAs shown in Table 1, AI women were typically younger (p = 0.01) and reported a higher lifetime number of sexual partners (p < 0.001) than their White counterparts.\nDistribution of risk factors and HPV prevalence by clinic site\na Prevalence estimates are standardized by age and number of sexual partners; b HPV types 16, 31, 33, 35, 52, 58, and 67; c HPV types 18, 39, 45, 59, 68, and 70; d HPV types 51, 56, 66, 73, 82.\nOne hundred thirteen AI women and 57 White women tested positive for HPV. After standardization for age and number of sexual partners, prevalent HPV infection status differed between the two groups (p < 0.001). Overall HPV infection estimates were 42% for AI women and 23% for White women, with prevalence of multiple HPV infection nearly three times higher in AI women than in White women (19% vs. 7%). AI women also had higher standardized prevalence estimates for infection by Alpha 7, Alpha 9, and other oncogenic types, as well as nearly double the prevalence of infection by any oncogenic HPV type, compared to White women (30% vs. 16%, p < 0.001). In addition, AI and White women exhibited different distributions of HPV infection across age categories (Table 2). Among AI women, prevalent infection decreased from the youngest to the oldest age groups (ptrend < 0.001), whereas no age trend was evident among White women (ptrend = 0.23). Figure 1 displays the prevalence for each of 36 HPV types. Overall, AI women exhibited a wider variety of HPV infections, with seven HPV types detected only in AI women. In contrast, White women showed less variation in prevalent infections, with just one HPV type detected only in White women.\nPrevalence of single and multiple HPV infection by age category for American Indian and White women\nResults are unstandardized and presented as prevalence estimates with 95% CIs.\na Any HPV infection non-parametric test for trend p < 0.001\nb Any HPV infection non-parametric test for trend p = 0.29\nUnstandardized prevalence of each HPV type among American Indian and White women in South Dakota.\nTable 3 shows the percentages of women infected with each of the HPV types prevented by the Gardasil vaccine, standardized for age and number of sexual partners. There was no statistically significant difference in the percentages of women infected by HPV types 6 or 16, but significantly more AI women were infected by HPV-11 (p = 0.03) and HPV-18 (p = 0.02). The two clinic populations differed significantly with regard to the prevalence of infections by HPV types that are not prevented by Gardasil (p < 0.001). The AI group had a strikingly higher percentage of women with HPV infections that would not have been prevented at all by the vaccine (32% vs. 15%), and a higher percentage of women with infections that would have been only partially prevented by the vaccine (5% vs. 2%).\nPrevalence of HPV types prevented by the Gardasil HPV vaccine and hypothetical vaccine protection against prevalent infectionsa\nPrevalence estimates are standardized by age and lifetime number of sexual partners.\na Presumed protection against infection, assuming a counterfactual scenario in which each woman had been vaccinated before HPV exposure.",
"The primary aims of this study were to examine the prevalence of HPV infection and the distribution of HPV genotypes, as well as the potential benefit of HPV vaccination, in two distinct South Dakota populations. We found that the prevalence of HPV infection among AI women was more than twice that of White women in our samples. Because we are extremely confident that none of the study participants had prior Gardasil vaccination, these differences cannot be attributed, even in part, to discrepancies in vaccine administration or uptake. Increasing age was negatively correlated with HPV infection among AI women, whereas no such trend was detected among the White sample. These findings suggest that efforts to prevent HPV infection must be tailored to the disease burden, the specific HPV prevalence patterns, and the educational and social background of the target community.\nWe previously reported that 22% of AI women in a Northern Plains service unit had HPV infections, a prevalence considerably higher than the 7% observed among AI women in New Mexico [4] and comparable to the 21% documented for Alaska Native women [5]. In a recent study of a large and diverse population of U.S. women, Datta and colleagues reported the prevalence of high-risk HPV infection among AI and Alaska Native women as 25% [17]. High-risk types were determined by the Hybrid Capture 2 assay (Digene, Gaithersburg, MD), which returns a positive result in the presence of any of 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 68). Unlike the present study, however, Datta and colleagues did not identify individual HPV types, and their sample included fewer than 180 AI women recruited nationwide.\nThe disparity in HPV prevalence between AI and White women residing in the Northern Plains may contribute to the disproportionately high rate of cervical cancer in the AI population. The Northern Plains incidence rate for cervical cancer in AI women is 11.3 per 100,000, which is 1.5 times higher than in non-Hispanic White women (7.5 per 100,000). Recent publications have documented an incidence of cervical cancer among AI women in South Dakota as high as 16.2/100,000, compared to 6.1/100,000 among non-Hispanic White women [9], and an age-adjusted cervical cancer mortality in the Dakotas nearly twice the national average (4.5 per 100,000 vs. 2.7 per 100,000) [8].\nWe also observed different patterns of HPV infection in our two study populations. Overall, AI women showed more variation in prevalent HPV types, and significantly more AI women were infected by HPV types that would not have been prevented by current HPV vaccines. Previous genotyping studies conducted in predominantly White populations have documented that HPV-16 and HPV-18 are implicated in up to 70% of cervical cancer cases in the U.S and worldwide [32,33], but virtually no AI women participated in these studies. Although vaccination is still in order, our results suggest that the protective benefit conferred by current HPV vaccines might be less for some AI women than for their White counterparts. Of note, recent evidence suggests that despite receiving HPV vaccination, some women still develop cervical cancer, likely unrelated to HPV-16 or HPV-18 [34]. Among AI women in the present study, the diversity of oncogenic types that are not preventable by existing vaccines might further contribute to the high rates of cervical cancer previously observed among AI women in the Northern Plains.\nOur findings have substantial relevance to public health efforts aimed at improving cervical cancer screening among AI women. The \"All Women Count\" cancer screening program funded by the State of South Dakota covers Pap testing for eligible women under the age of 30, but likely misses many women with HPV and cervical dysplasia [35]. Notably, the National Breast and Cervical Cancer Early Detection Program found that AI women more often reported never having a Pap test than their non-AI counterparts, and also had the highest proportion (4.4%) of abnormal Pap tests [36]. Although our findings need to be replicated in other AI communities, they raise the question of whether the Indian Health Service might consider universal HPV screening for selected service units.\nThis study is limited in several ways. First, because the study population was a convenience sample drawn from the local service units, our findings may not be pertinent to all AI women living in the Northern Plains of the U.S., and cannot be generalized to other rural or urban populations. Nevertheless, we had an exceptionally high participation rate, and we believe that our samples are an accurate representation of the patient populations, both AI and White, at all clinic sites. Of note, we have additional, unpublished results from 212 AI women living in Montana (ages 16-65), suggesting an HPV prevalence of 37%, very similar to the 42% prevalence observed in our sample of AIs living in South Dakota.\nSecond, our sample size was relatively small compared to many studies of HPV prevalence. Even so, this is the first study to examine HPV prevalence patterns in a high-risk AI population with a geographically localized White comparison group, and despite the small sample size, we were able to detect statistically significant differences between the groups, with important clinical and public health implications.\nThird, we cannot disentangle the effects of urban residence from race. However, very few White women live on reservations, and only a negligible proportion of AI women were evaluated in the regionally-matched urban clinic, making the selection of samples evenly distributed between race and region virtually impossible.\nFourth, we did not use prior receipt of Gardasil vaccination as an exclusion criterion. Nevertheless, we are confident that our results remain unaffected by this limitation, since data on all White women and many AI women were collected before vaccine approval. Further, the AI women who were sampled in 2007-2008 were older than the target age range for HPV vaccination and had no insurance coverage from the Indian Health Service for this purpose. Even in the unlikely event that a few AI participants had prior vaccination, we would expect any resulting bias to be in the direction of less prevalent infection in this group.\nFifth, the absence of HPV vaccination among study participants prevented us from considering population differences in vaccine uptake; future research should examine this phenomenon more closely in minority communities.\nFinally, we did not consider the additional prevention of HPV-31 provided by the Cervarix vaccine. Nevertheless, only four AI women, and no White women, were infected by HPV-31, so the inclusion of Cervarix in this analysis would have added confusion without altering our conclusions.",
"This is the first study of HPV genotyping in a community-based sample of AI and White women. Our results suggest the existence of substantial differences in HPV prevalence and infection patterns among AI and White populations, highlighting the need for research on HPV in high-risk minority groups. If confirmed by future studies, our results might help to explain the documented cervical cancer disparities experienced by AI women. As it is, our findings underscore the need to evaluate vaccine efficacy in this population."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Setting",
"Participants",
"Oncogenic HPV types",
"HPV Vaccines",
"Sample Collection and Assay Methods",
"Measures",
"Statistical Analysis",
"Results",
"Discussion",
"Conclusions"
] | [
"Cervical cancer is the second most common cause of cancer-related deaths in women worldwide [1] and a leading cause of cancer mortality among American Indian (AI) women [2,3]. Compared to other U.S. racial and ethnic groups, AI women experience striking health disparities in relation to cervical cancer, including a higher prevalence [4-9], a more rapidly increasing incidence [10,11], and poorer survival [12,13]. The Aberdeen Area Indian Health Service, which encompasses 16 different tribes located in South Dakota, North Dakota, Nebraska, and Iowa, reported an age-adjusted cervical cancer mortality rate of 4.3/100,000 from 1996 to 1998 [14], almost twice the rate reported in the general U.S. population [15]. Our previous work found a high prevalence of human papillomavirus (HPV) infection among AI women residing on reservations in the same geographic region, and suggested that types other than HPV-16 and HPV-18 comprised a substantial proportion of oncogenic HPV infections [16]. In a recent study of more than 9,600 U.S. women, the overall prevalence of high-risk HPV types among AI and Alaska Native women (representing 2% of the total sample) was 25% [17], which is substantially higher than the general U.S. population prevalence of 15% [18].\nInfection with HPV, especially HPV-16 and HPV-18, is the most common cause of cervical cancer [19]. Preventing HPV infection is critical to public health efforts to reduce precancerous lesions and cervical cancer incidence and mortality. In 2007, the U.S. Food and Drug Administration approved the first vaccine to prevent infection by the most common oncogenic HPV types present in the general U.S. population; a second HPV vaccine was approved in 2009. Despite striking cervical cancer disparities, little is known about patterns of HPV infection in AI communities that can inform the adequacy of prevention provided for AI women by existing HPV vaccines. We therefore examined women seen in two rural AI reservation clinics and one urban clinic serving primarily White women in the Northern Plains. Our aims were to 1) compare HPV prevalence and patterns between the AI and White clinic samples, and 2) compare proportions of women in each group who were infected by HPV types that can be prevented by existing vaccines.",
" Setting This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].\nThe urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].\nOf note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.\nBefore enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.\nThis study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].\nThe urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].\nOf note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.\nBefore enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.\n Participants Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.\nTwo of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.\nPotential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.\nTwo of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.\n Oncogenic HPV types All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.\nAll HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.\n HPV Vaccines Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported \"cross protection\" phenomenon [30].\nTwo HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported \"cross protection\" phenomenon [30].\n Sample Collection and Assay Methods The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.\nAfter the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.\nThe DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.\nThe study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.\nAfter the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.\nThe DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.\n Measures We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.\nWe used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).\nWe categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.\nWe used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).\n Statistical Analysis Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.\nFor our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.\nAll analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.\nPreliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.\nFor our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.\nAll analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.",
"This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].\nThe urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].\nOf note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.\nBefore enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.",
"Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.\nTwo of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.",
"All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.",
"Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported \"cross protection\" phenomenon [30].",
"The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.\nAfter the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.\nThe DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.",
"We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.\nWe used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).",
"Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.\nFor our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.\nAll analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.",
"We collected cervical samples from 258 AI women (104 and 154 women at Sites 1 and 2, respectively) and from 252 White women at the urban clinic. Participation rates exceeded 90%, with the total number of refusals ranging from four to seven among the three clinic sites. Some faint positive HPV results required adjudication by a third reader, but all positive results were typable. After excluding three women with missing data for HPV status and 26 women with missing data for age or number of sexual partners, data on 481 women (AI n = 235, White n = 246) were available for analysis. The vast majority of study participants were recruited during annual gynecological examinations. All participants had health insurance of some kind. For AI women, healthcare was provided and paid for by the Indian Health Service; the White women were covered by private or government-sponsored insurance (e.g., Medicaid).\nAs shown in Table 1, AI women were typically younger (p = 0.01) and reported a higher lifetime number of sexual partners (p < 0.001) than their White counterparts.\nDistribution of risk factors and HPV prevalence by clinic site\na Prevalence estimates are standardized by age and number of sexual partners; b HPV types 16, 31, 33, 35, 52, 58, and 67; c HPV types 18, 39, 45, 59, 68, and 70; d HPV types 51, 56, 66, 73, 82.\nOne hundred thirteen AI women and 57 White women tested positive for HPV. After standardization for age and number of sexual partners, prevalent HPV infection status differed between the two groups (p < 0.001). Overall HPV infection estimates were 42% for AI women and 23% for White women, with prevalence of multiple HPV infection nearly three times higher in AI women than in White women (19% vs. 7%). AI women also had higher standardized prevalence estimates for infection by Alpha 7, Alpha 9, and other oncogenic types, as well as nearly double the prevalence of infection by any oncogenic HPV type, compared to White women (30% vs. 16%, p < 0.001). In addition, AI and White women exhibited different distributions of HPV infection across age categories (Table 2). Among AI women, prevalent infection decreased from the youngest to the oldest age groups (ptrend < 0.001), whereas no age trend was evident among White women (ptrend = 0.23). Figure 1 displays the prevalence for each of 36 HPV types. Overall, AI women exhibited a wider variety of HPV infections, with seven HPV types detected only in AI women. In contrast, White women showed less variation in prevalent infections, with just one HPV type detected only in White women.\nPrevalence of single and multiple HPV infection by age category for American Indian and White women\nResults are unstandardized and presented as prevalence estimates with 95% CIs.\na Any HPV infection non-parametric test for trend p < 0.001\nb Any HPV infection non-parametric test for trend p = 0.29\nUnstandardized prevalence of each HPV type among American Indian and White women in South Dakota.\nTable 3 shows the percentages of women infected with each of the HPV types prevented by the Gardasil vaccine, standardized for age and number of sexual partners. There was no statistically significant difference in the percentages of women infected by HPV types 6 or 16, but significantly more AI women were infected by HPV-11 (p = 0.03) and HPV-18 (p = 0.02). The two clinic populations differed significantly with regard to the prevalence of infections by HPV types that are not prevented by Gardasil (p < 0.001). The AI group had a strikingly higher percentage of women with HPV infections that would not have been prevented at all by the vaccine (32% vs. 15%), and a higher percentage of women with infections that would have been only partially prevented by the vaccine (5% vs. 2%).\nPrevalence of HPV types prevented by the Gardasil HPV vaccine and hypothetical vaccine protection against prevalent infectionsa\nPrevalence estimates are standardized by age and lifetime number of sexual partners.\na Presumed protection against infection, assuming a counterfactual scenario in which each woman had been vaccinated before HPV exposure.",
"The primary aims of this study were to examine the prevalence of HPV infection and the distribution of HPV genotypes, as well as the potential benefit of HPV vaccination, in two distinct South Dakota populations. We found that the prevalence of HPV infection among AI women was more than twice that of White women in our samples. Because we are extremely confident that none of the study participants had prior Gardasil vaccination, these differences cannot be attributed, even in part, to discrepancies in vaccine administration or uptake. Increasing age was negatively correlated with HPV infection among AI women, whereas no such trend was detected among the White sample. These findings suggest that efforts to prevent HPV infection must be tailored to the disease burden, the specific HPV prevalence patterns, and the educational and social background of the target community.\nWe previously reported that 22% of AI women in a Northern Plains service unit had HPV infections, a prevalence considerably higher than the 7% observed among AI women in New Mexico [4] and comparable to the 21% documented for Alaska Native women [5]. In a recent study of a large and diverse population of U.S. women, Datta and colleagues reported the prevalence of high-risk HPV infection among AI and Alaska Native women as 25% [17]. High-risk types were determined by the Hybrid Capture 2 assay (Digene, Gaithersburg, MD), which returns a positive result in the presence of any of 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 68). Unlike the present study, however, Datta and colleagues did not identify individual HPV types, and their sample included fewer than 180 AI women recruited nationwide.\nThe disparity in HPV prevalence between AI and White women residing in the Northern Plains may contribute to the disproportionately high rate of cervical cancer in the AI population. The Northern Plains incidence rate for cervical cancer in AI women is 11.3 per 100,000, which is 1.5 times higher than in non-Hispanic White women (7.5 per 100,000). Recent publications have documented an incidence of cervical cancer among AI women in South Dakota as high as 16.2/100,000, compared to 6.1/100,000 among non-Hispanic White women [9], and an age-adjusted cervical cancer mortality in the Dakotas nearly twice the national average (4.5 per 100,000 vs. 2.7 per 100,000) [8].\nWe also observed different patterns of HPV infection in our two study populations. Overall, AI women showed more variation in prevalent HPV types, and significantly more AI women were infected by HPV types that would not have been prevented by current HPV vaccines. Previous genotyping studies conducted in predominantly White populations have documented that HPV-16 and HPV-18 are implicated in up to 70% of cervical cancer cases in the U.S and worldwide [32,33], but virtually no AI women participated in these studies. Although vaccination is still in order, our results suggest that the protective benefit conferred by current HPV vaccines might be less for some AI women than for their White counterparts. Of note, recent evidence suggests that despite receiving HPV vaccination, some women still develop cervical cancer, likely unrelated to HPV-16 or HPV-18 [34]. Among AI women in the present study, the diversity of oncogenic types that are not preventable by existing vaccines might further contribute to the high rates of cervical cancer previously observed among AI women in the Northern Plains.\nOur findings have substantial relevance to public health efforts aimed at improving cervical cancer screening among AI women. The \"All Women Count\" cancer screening program funded by the State of South Dakota covers Pap testing for eligible women under the age of 30, but likely misses many women with HPV and cervical dysplasia [35]. Notably, the National Breast and Cervical Cancer Early Detection Program found that AI women more often reported never having a Pap test than their non-AI counterparts, and also had the highest proportion (4.4%) of abnormal Pap tests [36]. Although our findings need to be replicated in other AI communities, they raise the question of whether the Indian Health Service might consider universal HPV screening for selected service units.\nThis study is limited in several ways. First, because the study population was a convenience sample drawn from the local service units, our findings may not be pertinent to all AI women living in the Northern Plains of the U.S., and cannot be generalized to other rural or urban populations. Nevertheless, we had an exceptionally high participation rate, and we believe that our samples are an accurate representation of the patient populations, both AI and White, at all clinic sites. Of note, we have additional, unpublished results from 212 AI women living in Montana (ages 16-65), suggesting an HPV prevalence of 37%, very similar to the 42% prevalence observed in our sample of AIs living in South Dakota.\nSecond, our sample size was relatively small compared to many studies of HPV prevalence. Even so, this is the first study to examine HPV prevalence patterns in a high-risk AI population with a geographically localized White comparison group, and despite the small sample size, we were able to detect statistically significant differences between the groups, with important clinical and public health implications.\nThird, we cannot disentangle the effects of urban residence from race. However, very few White women live on reservations, and only a negligible proportion of AI women were evaluated in the regionally-matched urban clinic, making the selection of samples evenly distributed between race and region virtually impossible.\nFourth, we did not use prior receipt of Gardasil vaccination as an exclusion criterion. Nevertheless, we are confident that our results remain unaffected by this limitation, since data on all White women and many AI women were collected before vaccine approval. Further, the AI women who were sampled in 2007-2008 were older than the target age range for HPV vaccination and had no insurance coverage from the Indian Health Service for this purpose. Even in the unlikely event that a few AI participants had prior vaccination, we would expect any resulting bias to be in the direction of less prevalent infection in this group.\nFifth, the absence of HPV vaccination among study participants prevented us from considering population differences in vaccine uptake; future research should examine this phenomenon more closely in minority communities.\nFinally, we did not consider the additional prevention of HPV-31 provided by the Cervarix vaccine. Nevertheless, only four AI women, and no White women, were infected by HPV-31, so the inclusion of Cervarix in this analysis would have added confusion without altering our conclusions.",
"This is the first study of HPV genotyping in a community-based sample of AI and White women. Our results suggest the existence of substantial differences in HPV prevalence and infection patterns among AI and White populations, highlighting the need for research on HPV in high-risk minority groups. If confirmed by future studies, our results might help to explain the documented cervical cancer disparities experienced by AI women. As it is, our findings underscore the need to evaluate vaccine efficacy in this population."
] | [
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"cervical cancer",
"Pap screening",
"HPV genotypes",
"American Indians",
"health disparities",
"human papillomavirus",
"types"
] | Background:
Cervical cancer is the second most common cause of cancer-related deaths in women worldwide [1] and a leading cause of cancer mortality among American Indian (AI) women [2,3]. Compared to other U.S. racial and ethnic groups, AI women experience striking health disparities in relation to cervical cancer, including a higher prevalence [4-9], a more rapidly increasing incidence [10,11], and poorer survival [12,13]. The Aberdeen Area Indian Health Service, which encompasses 16 different tribes located in South Dakota, North Dakota, Nebraska, and Iowa, reported an age-adjusted cervical cancer mortality rate of 4.3/100,000 from 1996 to 1998 [14], almost twice the rate reported in the general U.S. population [15]. Our previous work found a high prevalence of human papillomavirus (HPV) infection among AI women residing on reservations in the same geographic region, and suggested that types other than HPV-16 and HPV-18 comprised a substantial proportion of oncogenic HPV infections [16]. In a recent study of more than 9,600 U.S. women, the overall prevalence of high-risk HPV types among AI and Alaska Native women (representing 2% of the total sample) was 25% [17], which is substantially higher than the general U.S. population prevalence of 15% [18].
Infection with HPV, especially HPV-16 and HPV-18, is the most common cause of cervical cancer [19]. Preventing HPV infection is critical to public health efforts to reduce precancerous lesions and cervical cancer incidence and mortality. In 2007, the U.S. Food and Drug Administration approved the first vaccine to prevent infection by the most common oncogenic HPV types present in the general U.S. population; a second HPV vaccine was approved in 2009. Despite striking cervical cancer disparities, little is known about patterns of HPV infection in AI communities that can inform the adequacy of prevention provided for AI women by existing HPV vaccines. We therefore examined women seen in two rural AI reservation clinics and one urban clinic serving primarily White women in the Northern Plains. Our aims were to 1) compare HPV prevalence and patterns between the AI and White clinic samples, and 2) compare proportions of women in each group who were infected by HPV types that can be prevented by existing vaccines.
Methods:
Setting This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].
The urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].
Of note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.
Before enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.
This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].
The urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].
Of note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.
Before enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.
Participants Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.
Two of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.
Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.
Two of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.
Oncogenic HPV types All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.
All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.
HPV Vaccines Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported "cross protection" phenomenon [30].
Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported "cross protection" phenomenon [30].
Sample Collection and Assay Methods The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.
After the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.
The DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.
The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.
After the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.
The DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.
Measures We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.
We used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).
We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.
We used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).
Statistical Analysis Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.
For our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.
All analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.
Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.
For our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.
All analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.
Setting:
This study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site [20]. Site 2 had 21,245 tribal members living within reservation boundaries in 2004 [21], and in fiscal year 2002, 3,100 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site. Of note, the high school dropout rate in Site 1 is over 70%, and the teacher turnover rate is 800% that of the U.S. national average [22]. In contrast, in Site 2, the tribe implemented a program that increased graduation rates from 48% to 72% at the main public high school [23].
The urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line [24]. The urban population is predominantly White (92%), with small numbers of Latino (2%), African American (2%), Asian (1%), and AI (2%) residents [25]. Only 5% of adults lack a high school education or equivalent [26].
Of note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.
Before enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.
Participants:
Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) [27], so we are confident that no AI participants had prior HPV vaccination.
Two of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.
Oncogenic HPV types:
All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) [28,29]. Of note, the Alpha 7 and Alpha 9 types are most frequently implicated in cervical cancer cases worldwide, although all types in the Alpha genus have been implicated.
HPV Vaccines:
Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a post hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported "cross protection" phenomenon [30].
Sample Collection and Assay Methods:
The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.
After the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.
The DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study [16]. Briefly, 20 microliters of PCR product were separated on a 2% agarose gel to confirm amplification of internal controls (Β-globin) and the HPV L1 gene. HPV genotyping strips (Roche Diagnostics, Indianapolis, IN) were used to detect 37 high- and low-risk HPV genotypes, including high-risk genotypes that could progress to cervical cancer. Non-oncogenic HPV genotypes included 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 69, 71, 72, 81, 82, 83, 84, IS39, and CP6108. Oncogenic or probably oncogenic HPV genotypes included 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, 70, and 73. Reactions were amplified with a Biorad system according to published instructions. Seventy-five microliters of denatured, biotinylated amplified product were placed with genotyping strips into individual wells of the typing trays and covered with hybridization buffer. After HPV DNA detection, individual strips were rinsed in deionized water and stored in citrate buffer. Interpretation was based on a labeled acetate overlay with lines indicating the position of each probe relative to a reference mark on the strip denoting high- and low-risk types. Two independent investigators interpreted the data, with discrepancies resolved by a third investigator.
Measures:
We categorized age in years (18-24, 25-34, 35-44, 45-54, 55-65) and lifetime number of sexual partners (1, 2-3, 4-5, 6-9, ≥10), after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection (oncogenic HPV types 16, 31, 33, 35, 52, 58, or 67), Alpha 9 infection (oncogenic HPV types 18, 39, 45, 59, 68, or 70), and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 (HPV types 51, 56, 66, 73, or 82). Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.
We used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine (types 6, 11, 16, and 18). First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention (infected only by HPV types not prevented by the vaccine), 3) partial prevention (infected by at least one of HPV types 6, 11, 16, or 18 and at least one other HPV type not prevented by the vaccine), or 4) full prevention (infected only by HPV types 6, 11, 16, or 18).
Statistical Analysis:
Preliminary data analyses indicated that the two AI groups were sufficiently similar in relation to all study variables to combine them into a single sample for this analysis. To examine our first study aim, we calculated percentages to describe the distribution of age, lifetime number of sexual partners, HPV infection status, and oncogenic infection measures separately by clinic population (reservation vs. urban), with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.
For our second study aim, we calculated the percentage of women who were infected by each of the four HPV types (6, 11, 16, and 18) that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention (not infected, infection not prevented by the vaccine, infection partially prevented by the vaccine, infection completely prevented by the vaccine) by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.
All analyses were performed by using Stata version 10 [31]. For chi-square tests, we considered an alpha of 0.05 as the threshold for statistical significance.
Results:
We collected cervical samples from 258 AI women (104 and 154 women at Sites 1 and 2, respectively) and from 252 White women at the urban clinic. Participation rates exceeded 90%, with the total number of refusals ranging from four to seven among the three clinic sites. Some faint positive HPV results required adjudication by a third reader, but all positive results were typable. After excluding three women with missing data for HPV status and 26 women with missing data for age or number of sexual partners, data on 481 women (AI n = 235, White n = 246) were available for analysis. The vast majority of study participants were recruited during annual gynecological examinations. All participants had health insurance of some kind. For AI women, healthcare was provided and paid for by the Indian Health Service; the White women were covered by private or government-sponsored insurance (e.g., Medicaid).
As shown in Table 1, AI women were typically younger (p = 0.01) and reported a higher lifetime number of sexual partners (p < 0.001) than their White counterparts.
Distribution of risk factors and HPV prevalence by clinic site
a Prevalence estimates are standardized by age and number of sexual partners; b HPV types 16, 31, 33, 35, 52, 58, and 67; c HPV types 18, 39, 45, 59, 68, and 70; d HPV types 51, 56, 66, 73, 82.
One hundred thirteen AI women and 57 White women tested positive for HPV. After standardization for age and number of sexual partners, prevalent HPV infection status differed between the two groups (p < 0.001). Overall HPV infection estimates were 42% for AI women and 23% for White women, with prevalence of multiple HPV infection nearly three times higher in AI women than in White women (19% vs. 7%). AI women also had higher standardized prevalence estimates for infection by Alpha 7, Alpha 9, and other oncogenic types, as well as nearly double the prevalence of infection by any oncogenic HPV type, compared to White women (30% vs. 16%, p < 0.001). In addition, AI and White women exhibited different distributions of HPV infection across age categories (Table 2). Among AI women, prevalent infection decreased from the youngest to the oldest age groups (ptrend < 0.001), whereas no age trend was evident among White women (ptrend = 0.23). Figure 1 displays the prevalence for each of 36 HPV types. Overall, AI women exhibited a wider variety of HPV infections, with seven HPV types detected only in AI women. In contrast, White women showed less variation in prevalent infections, with just one HPV type detected only in White women.
Prevalence of single and multiple HPV infection by age category for American Indian and White women
Results are unstandardized and presented as prevalence estimates with 95% CIs.
a Any HPV infection non-parametric test for trend p < 0.001
b Any HPV infection non-parametric test for trend p = 0.29
Unstandardized prevalence of each HPV type among American Indian and White women in South Dakota.
Table 3 shows the percentages of women infected with each of the HPV types prevented by the Gardasil vaccine, standardized for age and number of sexual partners. There was no statistically significant difference in the percentages of women infected by HPV types 6 or 16, but significantly more AI women were infected by HPV-11 (p = 0.03) and HPV-18 (p = 0.02). The two clinic populations differed significantly with regard to the prevalence of infections by HPV types that are not prevented by Gardasil (p < 0.001). The AI group had a strikingly higher percentage of women with HPV infections that would not have been prevented at all by the vaccine (32% vs. 15%), and a higher percentage of women with infections that would have been only partially prevented by the vaccine (5% vs. 2%).
Prevalence of HPV types prevented by the Gardasil HPV vaccine and hypothetical vaccine protection against prevalent infectionsa
Prevalence estimates are standardized by age and lifetime number of sexual partners.
a Presumed protection against infection, assuming a counterfactual scenario in which each woman had been vaccinated before HPV exposure.
Discussion:
The primary aims of this study were to examine the prevalence of HPV infection and the distribution of HPV genotypes, as well as the potential benefit of HPV vaccination, in two distinct South Dakota populations. We found that the prevalence of HPV infection among AI women was more than twice that of White women in our samples. Because we are extremely confident that none of the study participants had prior Gardasil vaccination, these differences cannot be attributed, even in part, to discrepancies in vaccine administration or uptake. Increasing age was negatively correlated with HPV infection among AI women, whereas no such trend was detected among the White sample. These findings suggest that efforts to prevent HPV infection must be tailored to the disease burden, the specific HPV prevalence patterns, and the educational and social background of the target community.
We previously reported that 22% of AI women in a Northern Plains service unit had HPV infections, a prevalence considerably higher than the 7% observed among AI women in New Mexico [4] and comparable to the 21% documented for Alaska Native women [5]. In a recent study of a large and diverse population of U.S. women, Datta and colleagues reported the prevalence of high-risk HPV infection among AI and Alaska Native women as 25% [17]. High-risk types were determined by the Hybrid Capture 2 assay (Digene, Gaithersburg, MD), which returns a positive result in the presence of any of 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 68). Unlike the present study, however, Datta and colleagues did not identify individual HPV types, and their sample included fewer than 180 AI women recruited nationwide.
The disparity in HPV prevalence between AI and White women residing in the Northern Plains may contribute to the disproportionately high rate of cervical cancer in the AI population. The Northern Plains incidence rate for cervical cancer in AI women is 11.3 per 100,000, which is 1.5 times higher than in non-Hispanic White women (7.5 per 100,000). Recent publications have documented an incidence of cervical cancer among AI women in South Dakota as high as 16.2/100,000, compared to 6.1/100,000 among non-Hispanic White women [9], and an age-adjusted cervical cancer mortality in the Dakotas nearly twice the national average (4.5 per 100,000 vs. 2.7 per 100,000) [8].
We also observed different patterns of HPV infection in our two study populations. Overall, AI women showed more variation in prevalent HPV types, and significantly more AI women were infected by HPV types that would not have been prevented by current HPV vaccines. Previous genotyping studies conducted in predominantly White populations have documented that HPV-16 and HPV-18 are implicated in up to 70% of cervical cancer cases in the U.S and worldwide [32,33], but virtually no AI women participated in these studies. Although vaccination is still in order, our results suggest that the protective benefit conferred by current HPV vaccines might be less for some AI women than for their White counterparts. Of note, recent evidence suggests that despite receiving HPV vaccination, some women still develop cervical cancer, likely unrelated to HPV-16 or HPV-18 [34]. Among AI women in the present study, the diversity of oncogenic types that are not preventable by existing vaccines might further contribute to the high rates of cervical cancer previously observed among AI women in the Northern Plains.
Our findings have substantial relevance to public health efforts aimed at improving cervical cancer screening among AI women. The "All Women Count" cancer screening program funded by the State of South Dakota covers Pap testing for eligible women under the age of 30, but likely misses many women with HPV and cervical dysplasia [35]. Notably, the National Breast and Cervical Cancer Early Detection Program found that AI women more often reported never having a Pap test than their non-AI counterparts, and also had the highest proportion (4.4%) of abnormal Pap tests [36]. Although our findings need to be replicated in other AI communities, they raise the question of whether the Indian Health Service might consider universal HPV screening for selected service units.
This study is limited in several ways. First, because the study population was a convenience sample drawn from the local service units, our findings may not be pertinent to all AI women living in the Northern Plains of the U.S., and cannot be generalized to other rural or urban populations. Nevertheless, we had an exceptionally high participation rate, and we believe that our samples are an accurate representation of the patient populations, both AI and White, at all clinic sites. Of note, we have additional, unpublished results from 212 AI women living in Montana (ages 16-65), suggesting an HPV prevalence of 37%, very similar to the 42% prevalence observed in our sample of AIs living in South Dakota.
Second, our sample size was relatively small compared to many studies of HPV prevalence. Even so, this is the first study to examine HPV prevalence patterns in a high-risk AI population with a geographically localized White comparison group, and despite the small sample size, we were able to detect statistically significant differences between the groups, with important clinical and public health implications.
Third, we cannot disentangle the effects of urban residence from race. However, very few White women live on reservations, and only a negligible proportion of AI women were evaluated in the regionally-matched urban clinic, making the selection of samples evenly distributed between race and region virtually impossible.
Fourth, we did not use prior receipt of Gardasil vaccination as an exclusion criterion. Nevertheless, we are confident that our results remain unaffected by this limitation, since data on all White women and many AI women were collected before vaccine approval. Further, the AI women who were sampled in 2007-2008 were older than the target age range for HPV vaccination and had no insurance coverage from the Indian Health Service for this purpose. Even in the unlikely event that a few AI participants had prior vaccination, we would expect any resulting bias to be in the direction of less prevalent infection in this group.
Fifth, the absence of HPV vaccination among study participants prevented us from considering population differences in vaccine uptake; future research should examine this phenomenon more closely in minority communities.
Finally, we did not consider the additional prevention of HPV-31 provided by the Cervarix vaccine. Nevertheless, only four AI women, and no White women, were infected by HPV-31, so the inclusion of Cervarix in this analysis would have added confusion without altering our conclusions.
Conclusions:
This is the first study of HPV genotyping in a community-based sample of AI and White women. Our results suggest the existence of substantial differences in HPV prevalence and infection patterns among AI and White populations, highlighting the need for research on HPV in high-risk minority groups. If confirmed by future studies, our results might help to explain the documented cervical cancer disparities experienced by AI women. As it is, our findings underscore the need to evaluate vaccine efficacy in this population.
| Background:
High-risk strains of human papillomavirus (HPV) cause cervical cancer. American Indian (AI) women in the Northern Plains of the U.S. have significantly higher incidence and mortality rates for cervical cancer than White women in the same geographical area. We compared HPV prevalence, patterns of HPV types, and infection with multiple HPV types in AI and White women living in South Dakota, U.S.
Methods:
We analyzed the HPV status of cervical samples collected in 2006-2008 from women aged 18-65 years who attended two rural AI reservation clinics (n = 235) or an urban clinic in the same area serving mostly White women (n = 246). Data collection occurred before HPV vaccination was available to study participants. HPV DNA was amplified by using the L1 consensus primer system and an HPV Linear Array detection assay to identify HPV types. We used chi-square tests to compare HPV variables, with percentages standardized by age and lifetime number of sexual partners.
Results:
Compared to White women, AI women were younger (p = 0.01) and reported more sexual partners (p < 0.001). A lower percentage of AI women tested negative for HPV infection compared to Whites (58% [95% CI = 51-65] vs. 77% [95% CI = 71-82]; p < 0.001), and a higher percentage of AI women were infected by oncogenic types (30% [95% CI = 25-36] vs. 16% [95% CI = 11-21]; p = 0.001). Infections among AI women showed a wider variety and very different pattern of HPV types, including a higher prevalence of mixed HPV infections (19% [95% CI = 26-38] vs. 7% [95% CI = 4-11]; p = 0.001). AI women had a higher percentage of HPV infections that were not preventable by HPV vaccination (32% [95% CI = 26-38] vs. 15% [95% CI = 11-21]; p < 0.001).
Conclusions:
A higher HPV burden and a different HPV genotyping profile may contribute to the high rate of cervical cancer among AI women. | Background:
Cervical cancer is the second most common cause of cancer-related deaths in women worldwide [1] and a leading cause of cancer mortality among American Indian (AI) women [2,3]. Compared to other U.S. racial and ethnic groups, AI women experience striking health disparities in relation to cervical cancer, including a higher prevalence [4-9], a more rapidly increasing incidence [10,11], and poorer survival [12,13]. The Aberdeen Area Indian Health Service, which encompasses 16 different tribes located in South Dakota, North Dakota, Nebraska, and Iowa, reported an age-adjusted cervical cancer mortality rate of 4.3/100,000 from 1996 to 1998 [14], almost twice the rate reported in the general U.S. population [15]. Our previous work found a high prevalence of human papillomavirus (HPV) infection among AI women residing on reservations in the same geographic region, and suggested that types other than HPV-16 and HPV-18 comprised a substantial proportion of oncogenic HPV infections [16]. In a recent study of more than 9,600 U.S. women, the overall prevalence of high-risk HPV types among AI and Alaska Native women (representing 2% of the total sample) was 25% [17], which is substantially higher than the general U.S. population prevalence of 15% [18].
Infection with HPV, especially HPV-16 and HPV-18, is the most common cause of cervical cancer [19]. Preventing HPV infection is critical to public health efforts to reduce precancerous lesions and cervical cancer incidence and mortality. In 2007, the U.S. Food and Drug Administration approved the first vaccine to prevent infection by the most common oncogenic HPV types present in the general U.S. population; a second HPV vaccine was approved in 2009. Despite striking cervical cancer disparities, little is known about patterns of HPV infection in AI communities that can inform the adequacy of prevention provided for AI women by existing HPV vaccines. We therefore examined women seen in two rural AI reservation clinics and one urban clinic serving primarily White women in the Northern Plains. Our aims were to 1) compare HPV prevalence and patterns between the AI and White clinic samples, and 2) compare proportions of women in each group who were infected by HPV types that can be prevented by existing vaccines.
Conclusions:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2334/11/252/prepub
| Background:
High-risk strains of human papillomavirus (HPV) cause cervical cancer. American Indian (AI) women in the Northern Plains of the U.S. have significantly higher incidence and mortality rates for cervical cancer than White women in the same geographical area. We compared HPV prevalence, patterns of HPV types, and infection with multiple HPV types in AI and White women living in South Dakota, U.S.
Methods:
We analyzed the HPV status of cervical samples collected in 2006-2008 from women aged 18-65 years who attended two rural AI reservation clinics (n = 235) or an urban clinic in the same area serving mostly White women (n = 246). Data collection occurred before HPV vaccination was available to study participants. HPV DNA was amplified by using the L1 consensus primer system and an HPV Linear Array detection assay to identify HPV types. We used chi-square tests to compare HPV variables, with percentages standardized by age and lifetime number of sexual partners.
Results:
Compared to White women, AI women were younger (p = 0.01) and reported more sexual partners (p < 0.001). A lower percentage of AI women tested negative for HPV infection compared to Whites (58% [95% CI = 51-65] vs. 77% [95% CI = 71-82]; p < 0.001), and a higher percentage of AI women were infected by oncogenic types (30% [95% CI = 25-36] vs. 16% [95% CI = 11-21]; p = 0.001). Infections among AI women showed a wider variety and very different pattern of HPV types, including a higher prevalence of mixed HPV infections (19% [95% CI = 26-38] vs. 7% [95% CI = 4-11]; p = 0.001). AI women had a higher percentage of HPV infections that were not preventable by HPV vaccination (32% [95% CI = 26-38] vs. 15% [95% CI = 11-21]; p < 0.001).
Conclusions:
A higher HPV burden and a different HPV genotyping profile may contribute to the high rate of cervical cancer among AI women. | 9,648 | 430 | [
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] | null | [CONTENT] cervical cancer | Pap screening | HPV genotypes | American Indians | health disparities | human papillomavirus | types [SUMMARY] | [CONTENT] cervical cancer | Pap screening | HPV genotypes | American Indians | health disparities | human papillomavirus | types [SUMMARY] | null | [CONTENT] cervical cancer | Pap screening | HPV genotypes | American Indians | health disparities | human papillomavirus | types [SUMMARY] | [CONTENT] cervical cancer | Pap screening | HPV genotypes | American Indians | health disparities | human papillomavirus | types [SUMMARY] | [CONTENT] cervical cancer | Pap screening | HPV genotypes | American Indians | health disparities | human papillomavirus | types [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Female | Genotype | Humans | Indians, North American | Middle Aged | Papillomaviridae | Papillomavirus Infections | Prevalence | Rural Population | South Dakota | Urban Population | White People | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Female | Genotype | Humans | Indians, North American | Middle Aged | Papillomaviridae | Papillomavirus Infections | Prevalence | Rural Population | South Dakota | Urban Population | White People | Young Adult [SUMMARY] | null | [CONTENT] Adolescent | Adult | Aged | Female | Genotype | Humans | Indians, North American | Middle Aged | Papillomaviridae | Papillomavirus Infections | Prevalence | Rural Population | South Dakota | Urban Population | White People | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Female | Genotype | Humans | Indians, North American | Middle Aged | Papillomaviridae | Papillomavirus Infections | Prevalence | Rural Population | South Dakota | Urban Population | White People | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Female | Genotype | Humans | Indians, North American | Middle Aged | Papillomaviridae | Papillomavirus Infections | Prevalence | Rural Population | South Dakota | Urban Population | White People | Young Adult [SUMMARY] | [CONTENT] prevalence infections hpv | women hpv cervical | cervical cancer mortality | prevalence human papillomavirus | cervical cancer disparities [SUMMARY] | [CONTENT] prevalence infections hpv | women hpv cervical | cervical cancer mortality | prevalence human papillomavirus | cervical cancer disparities [SUMMARY] | null | [CONTENT] prevalence infections hpv | women hpv cervical | cervical cancer mortality | prevalence human papillomavirus | cervical cancer disparities [SUMMARY] | [CONTENT] prevalence infections hpv | women hpv cervical | cervical cancer mortality | prevalence human papillomavirus | cervical cancer disparities [SUMMARY] | [CONTENT] prevalence infections hpv | women hpv cervical | cervical cancer mortality | prevalence human papillomavirus | cervical cancer disparities [SUMMARY] | [CONTENT] hpv | women | infection | ai | types | hpv types | study | white | 16 | vaccine [SUMMARY] | [CONTENT] hpv | women | infection | ai | types | hpv types | study | white | 16 | vaccine [SUMMARY] | null | [CONTENT] hpv | women | infection | ai | types | hpv types | study | white | 16 | vaccine [SUMMARY] | [CONTENT] hpv | women | infection | ai | types | hpv types | study | white | 16 | vaccine [SUMMARY] | [CONTENT] hpv | women | infection | ai | types | hpv types | study | white | 16 | vaccine [SUMMARY] | [CONTENT] hpv | women | cancer | ai | cervical cancer | common | general | general population | cervical | prevalence [SUMMARY] | [CONTENT] hpv | infection | types | alpha | hpv types | site | study | women | dna | oncogenic [SUMMARY] | null | [CONTENT] need | results | ai | hpv | ai white | populations highlighting need research | confirmed future | ai white populations highlighting | findings underscore | findings underscore need [SUMMARY] | [CONTENT] hpv | women | infection | ai | types | hpv types | alpha | white | study | ai women [SUMMARY] | [CONTENT] hpv | women | infection | ai | types | hpv types | alpha | white | study | ai women [SUMMARY] | [CONTENT] HPV ||| American | Indian | the Northern Plains | U.S. ||| HPV | HPV | AI | South Dakota | U.S. [SUMMARY] | [CONTENT] HPV | 2006-2008 | 18-65 years | two | AI | 235 | 246 ||| HPV ||| L1 | HPV ||| HPV [SUMMARY] | null | [CONTENT] HPV | HPV | AI [SUMMARY] | [CONTENT] HPV ||| American | Indian | the Northern Plains | U.S. ||| HPV | HPV | AI | South Dakota | U.S. | HPV | 2006-2008 | 18-65 years | two | AI | 235 | 246 ||| HPV ||| L1 | HPV ||| HPV ||| AI | 0.01 ||| AI | HPV | Whites | 58% ||| 95% | CI | 51-65 | 77% ||| 95% | CI | 71 | AI | 30% | 95% | CI | 25 | 16% ||| 95% | CI | 11 | 0.001 ||| Infections | AI | HPV | 19% ||| 95% | CI | 26 | 7% | 95% | CI | 4 | 0.001 ||| AI | HPV | HPV | 32% | 95% | CI | 26 | 15% ||| 95% | CI | 11 ||| HPV | HPV | AI [SUMMARY] | [CONTENT] HPV ||| American | Indian | the Northern Plains | U.S. ||| HPV | HPV | AI | South Dakota | U.S. | HPV | 2006-2008 | 18-65 years | two | AI | 235 | 246 ||| HPV ||| L1 | HPV ||| HPV ||| AI | 0.01 ||| AI | HPV | Whites | 58% ||| 95% | CI | 51-65 | 77% ||| 95% | CI | 71 | AI | 30% | 95% | CI | 25 | 16% ||| 95% | CI | 11 | 0.001 ||| Infections | AI | HPV | 19% ||| 95% | CI | 26 | 7% | 95% | CI | 4 | 0.001 ||| AI | HPV | HPV | 32% | 95% | CI | 26 | 15% ||| 95% | CI | 11 ||| HPV | HPV | AI [SUMMARY] |
|||||
Clinical utility of immunological methods based on the singleplex and multiplex ImmunoCap systems for diagnosis of shrimp allergy. | 33840250 | Levels of specific IgE (sIgE) against allergen components can be assessed using multiplex assays or with highly sensitive, quantitative methods. The aim of this study was to compare the sensitivity and specificity of different immunological methods for diagnosis of shrimp allergy. | BACKGROUND | Twenty patients with positive skin prick tests for frozen tiger shrimp were selected for further examination. Blood samples were taken to assess concentrations of sIgE against the house dust mites Dermatophagoides pteronyssinus and D. farinae, shrimp allergen extract, allergen components Der p 1, Der p 2 and Pan a 1 (ImmunoCap), and the ImmunoCap ISAC 112 panel. | METHODS | All patients had elevated levels of sIgE against shrimp and D pteronyssinus. Eight patients were sensitized to Pen m 1, three patients were sensitized to Pen m 2, and two patients were sensitized to Pen m 4 (ISAC). ImmunoCap ISAC detected shrimp sensitization in 50% of patients. There was a strong correlation between concentrations of sIgE against Pen m1 and Der p 10 detected by ImmunoCap. | RESULTS | The singleplex ImmunoCap system remains the reference diagnostic method, but in the case of shrimp allergy ImmunoCap ISAC provided better insight into patient allergen profiles. | CONCLUSIONS | [
"Allergens",
"Animals",
"Humans",
"Hypersensitivity",
"Immunoglobulin E",
"Pyroglyphidae",
"Skin Tests"
] | 8044572 | Background | Shrimp allergy is an increasing problem in the European population. Symptoms associated with the consumption of shrimp by allergic individuals can range from mild, local reactions to systemic reactions and anaphylactic shock. The main allergen of crustaceans is tropomyosin, which shows high interspecies homology and is also found in house dust mites (HDMs). Other shrimp allergens have also been reported and may be associated with a severe course of sensitization.
HDM allergy is widespread in Europe, including Poland. The largest epidemiological study conducted in Poland, the Epidemiology of Allergic Diseases in Poland (ECAP) study, found that allergic rhinitis was the most prevalent allergic condition affecting both children and adults and was diagnosed in 24% to 30% of the study population.1 Boquete et al.2 indicated that 71% of patients allergic to HDMs also had specific IgE (sIgE) against shrimp, and 55% had increased levels of sIgE against shrimp tropomyosin. Canadian studies demonstrated a high prevalence of allergy to HDMs in 95 patients with confirmed shrimp allergy. In the study population, 86 (90.5%) patients had positive skin prick tests for HDM allergens.3 In a recent Italian study, 9% of 526 patients with HDM allergies also had allergy to shrimp. Patients with shrimp allergies were less frequently hypersensitive to airborne allergens other than HDM than crustacean-tolerant subjects. Only 51% of patients who were tropomyosin sensitized had shrimp allergies, and only 48% of patients who were shrimp sensitized were also sensitized to tropomyosin.4
There are several different methods of diagnosing shrimp allergy. Double blind, placebo-controlled food challenge remains the gold standard for diagnosis of food allergy. Oral food challenges are technically difficult to perform and time consuming. For patients with histories of anaphylaxis, the risks may also outweigh the potential benefits of performing the challenge.
To our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. The aim of the current study was to analyze the clinical utility of different diagnostic methods for patients with shrimp allergy. It was not feasible to use all available diagnostic methods in patients suspected of shrimp allergy. This is the first study to compare the utility of singleplex ImmunoCap using allergen extracts and allergen components with ImmunoCap ISAC in this specific group of patients. Establishing the sensitivity and specificity of different immunological methods and knowledge of their limitations may help clinicians in deciding the best diagnostic approach for patients with suspected shrimp allergies. | Methods | Patients with symptoms of persistent allergic rhinitis were selected for screening. The selection was random and was based on order of visiting the Outpatient Clinic of Allergic Diseases. Patients being treated for serious chronic diseases and patients on medication that could influence the results of the study were excluded.
A detailed interview and physical examination was conducted for each patient. All patients underwent skin prick tests (SPTs) with HDM extracts (Dermatophagoides pteronyssinus and D. farinae; Allergopharma, Hamburg, Germany) and with frozen tiger shrimp purchased from a local eco-shop. Patients with positive SPTs for frozen tiger shrimp and self-declared symptoms of shrimp allergy were selected for further examination. As a control group, patients with negative SPTs for frozen tiger shrimp who declared no symptoms of shrimp allergy were enrolled.
Blood samples were taken from all patients to assess concentrations of sIgE to the HDMs D. pteronyssinus and D. farinae, shrimp allergen extracts (Pandalus borealis, Penaeus monodon, Metapenaeopsis barbata, and Metapenaus joyneri), and the allergen components Der p 1, Der p 2, and Pan a 1. All immunological assays were performed using the highly sensitive immunofluorescent ImmunoCap system (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations of IgE were evaluated as elevated when they exceeded 0.35 kUA/L (ImmunoCap) in accordance with common practice in the field. In all patients we determined levels of sIgE against 112 allergen components using the ImmunoCAP ISAC 112 panel, a semi-quantitative test whose results are reported in ISAC Standardized Units (ISU) and provide indications of sIgE levels within the range of 0.3 to 100 ISU-E. The units used in the ImmunoCap ISAC (ISU-E) were developed especially for this test. The result is shown on a scale of four values for sIgE (indeterminate, <0.3 ISU-E; low, 0.3–0.9 ISU-E; medium, 1–14.9 ISU-E; high, >15 ISU-E). Levels of sIgE in ImmunoCap ISAC tests were considered as elevated when they exceeded 0.3 ISU-E. ImmunoCap ISAC tests included allergen components from D. pteronyssinus (Der p 1, Der p 2, and Der p 10), D. farinae (Der f 1 and Der f 2), and shrimp (Peneus monodon Pen m 1, Pen m 2, and Pen m 3).
Differences between groups were assessed using Mann–Whitney and Kruskal–Wallis tests with Dunn’s post hoc test. Spearman correlations were also calculated. Analyses were conducted using R, version 3.3.1 (www.r-project.org) and MS Excel 365. The study was approved by the local Bioethics Committee (number: 147/2015). All patients provided written informed consent to participate in the study. | Results | A total of 290 patients (176 women and 114 men) were screened and 20 had positive SPTs for frozen tiger shrimp (wheel >3 mm). All 20 patients had histories of allergic reaction after consumption of shrimp. A control group of 13 patients were selected with negative SPTs for frozen tiger shrimp and no symptoms of shrimp allergy. The characteristics of the study population and the results of immunoassays are presented in Table 1. Oral food challenges were not performed for several reasons, including lack of consent, time requirements, challenges in proper performance, and risks to patients with histories of anaphylaxis. All 20 patients reported symptoms following consumption of shrimp. Urticaria was reported by seven patients, anaphylaxis by six patients (including two patients with anaphylactic shock), digestive problems by four patients, and dyspnea by three patients.
General characteristics of the study population.
Data are shown as counts or as means ± standard deviations (ranges).
HDM, house dust mite.
All patients with shrimp allergies had elevated levels of shrimp sIgE by ImmunoCap. In addition, all patients were sensitized to D. pteronyssinus and 18 (90%) were sensitized to D. farinae. ImmunoCap ISAC results for shrimp and HDM allergens are presented in Table 2. Patients with shrimp allergies were sensitized to 72 allergen components by ImmunoCap ISAC. The most common sensitization in patients with shrimp allergy was to Der f 2 (16 patients). In addition, 12 patients were sensitized to Fel d 1, 11 patients were sensitized to Bla g 7 and Cyn d 1, and 10 patients were sensitized to Bet v 1, Cor a 1 and Phl p 1. The sensitization patterns are presented in Appendix Table 1. Among the 20 patients sensitized to shrimp, ImmunoCap ISAC detected elevated levels of sIgE against at least one out of three shrimp allergen components in only 10 patients (50%). In the control group all patients were sensitized to HDM by ImmunoCap, but none were sensitized to shrimp allergen extracts (Table 2).
Results of ImmunoCap ISAC tests.
HDM, house dust mite.
Table 3 shows a comparison between the concentrations of sIgE against allergen components detected by ImmunoCap and levels of sIgE detected by ImmunoCap ISAC. The results showed good concordance. Correlations in the 20 patients studied here were >0.7, but this result should be treated with caution. Although it is an indication that the results of the ImmunoCap ISAC and singleplex ImmunoCap methods were similar, the former method is semi-quantitative and the latter is quantitative. In addition, the units and the source shrimp species of tropomyosin differ between the two assays.
Comparison between concentrations of specific IgE measured using the quantitative ImmunoCap method and the semi-quantitative ImmunoCap ISAC microarray.
For HDM allergen components, ImmunoCap ISAC found that 84.6% patients were sensitized to Der p 1, 88.9% of patients were sensitized to Der p 2, and 90% of patients were sensitized to Der p 10. Only 50% of patients who were Pen m 1-sensitized had elevated levels of sIgE against Pen a 1 by ImmunoCap ISAC. There was a strong correlation between the concentrations of sIgE against Pen m 1 and Der p 10 in ImmunoCap assays (Figure 1).
Strong correlation (0.918, p < 0.001) between levels of IgE specific to Pen m 1 and Der p 10 in ImmunoCap (kU/L).
As shown in Table 1, ImmunoCap revealed that 18 of 20 patients sensitized to allergen extracts of D. pteronyssinus were also sensitized to Der p 2 (90%). All patients sensitized to Der p 1 were also sensitized to Der p 2, but only 10 (50%) were sensitized to HDM tropomyosin Der p 10. | Conclusion | Extract-based singleplex ImmunoCap methods are highly sensitive and widely available. Component resolved diagnosis adds additional information on the nature of sensitization and potential cross reactivity. The results of sIgE levels against corresponding allergen components determined using singleplex ImmunoCap and ImmunoCap ISAC were similar in most cases, with correlations above 0.7. In the case of shrimp allergy, the lack of many important shrimp allergen components, such as hemocyanin, was the probable cause of low detectability rates in molecular analysis (both singleplex and multiplex). The prevalence of HDM sensitization among patients with shrimp allergies was high, as described previously. The concordance of sIgE concentrations against Der p 10 and Pen m 1 between singleplex ImmunoCap and ImmunoCap ISAC was very high. Only 50% of patients with shrimp allergies were sensitized to tropomyosin and a lack of other important shrimp allergen components used in quantitative ImmunoCap ISAC measurements is a serious drawback for diagnosis of shrimp allergy. The results of immunoassays should be interpreted with care and a combination of extract and component-based diagnosis gives optimal insight into patient sensitization patterns.
Numbers of patients sensitized to specific allergen components in ImmunoCap ISAC. Levels of sIgE against allergen components that are not included in the table were not elevated in any patients.
sIgE, specific IgE | [
"Background"
] | [
"Shrimp allergy is an increasing problem in the European population. Symptoms associated with the consumption of shrimp by allergic individuals can range from mild, local reactions to systemic reactions and anaphylactic shock. The main allergen of crustaceans is tropomyosin, which shows high interspecies homology and is also found in house dust mites (HDMs). Other shrimp allergens have also been reported and may be associated with a severe course of sensitization.\nHDM allergy is widespread in Europe, including Poland. The largest epidemiological study conducted in Poland, the Epidemiology of Allergic Diseases in Poland (ECAP) study, found that allergic rhinitis was the most prevalent allergic condition affecting both children and adults and was diagnosed in 24% to 30% of the study population.1 Boquete et al.2 indicated that 71% of patients allergic to HDMs also had specific IgE (sIgE) against shrimp, and 55% had increased levels of sIgE against shrimp tropomyosin. Canadian studies demonstrated a high prevalence of allergy to HDMs in 95 patients with confirmed shrimp allergy. In the study population, 86 (90.5%) patients had positive skin prick tests for HDM allergens.3 In a recent Italian study, 9% of 526 patients with HDM allergies also had allergy to shrimp. Patients with shrimp allergies were less frequently hypersensitive to airborne allergens other than HDM than crustacean-tolerant subjects. Only 51% of patients who were tropomyosin sensitized had shrimp allergies, and only 48% of patients who were shrimp sensitized were also sensitized to tropomyosin.4\nThere are several different methods of diagnosing shrimp allergy. Double blind, placebo-controlled food challenge remains the gold standard for diagnosis of food allergy. Oral food challenges are technically difficult to perform and time consuming. For patients with histories of anaphylaxis, the risks may also outweigh the potential benefits of performing the challenge.\nTo our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. The aim of the current study was to analyze the clinical utility of different diagnostic methods for patients with shrimp allergy. It was not feasible to use all available diagnostic methods in patients suspected of shrimp allergy. This is the first study to compare the utility of singleplex ImmunoCap using allergen extracts and allergen components with ImmunoCap ISAC in this specific group of patients. Establishing the sensitivity and specificity of different immunological methods and knowledge of their limitations may help clinicians in deciding the best diagnostic approach for patients with suspected shrimp allergies."
] | [
null
] | [
"Background",
"Methods",
"Results",
"Discussion",
"Conclusion"
] | [
"Shrimp allergy is an increasing problem in the European population. Symptoms associated with the consumption of shrimp by allergic individuals can range from mild, local reactions to systemic reactions and anaphylactic shock. The main allergen of crustaceans is tropomyosin, which shows high interspecies homology and is also found in house dust mites (HDMs). Other shrimp allergens have also been reported and may be associated with a severe course of sensitization.\nHDM allergy is widespread in Europe, including Poland. The largest epidemiological study conducted in Poland, the Epidemiology of Allergic Diseases in Poland (ECAP) study, found that allergic rhinitis was the most prevalent allergic condition affecting both children and adults and was diagnosed in 24% to 30% of the study population.1 Boquete et al.2 indicated that 71% of patients allergic to HDMs also had specific IgE (sIgE) against shrimp, and 55% had increased levels of sIgE against shrimp tropomyosin. Canadian studies demonstrated a high prevalence of allergy to HDMs in 95 patients with confirmed shrimp allergy. In the study population, 86 (90.5%) patients had positive skin prick tests for HDM allergens.3 In a recent Italian study, 9% of 526 patients with HDM allergies also had allergy to shrimp. Patients with shrimp allergies were less frequently hypersensitive to airborne allergens other than HDM than crustacean-tolerant subjects. Only 51% of patients who were tropomyosin sensitized had shrimp allergies, and only 48% of patients who were shrimp sensitized were also sensitized to tropomyosin.4\nThere are several different methods of diagnosing shrimp allergy. Double blind, placebo-controlled food challenge remains the gold standard for diagnosis of food allergy. Oral food challenges are technically difficult to perform and time consuming. For patients with histories of anaphylaxis, the risks may also outweigh the potential benefits of performing the challenge.\nTo our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. The aim of the current study was to analyze the clinical utility of different diagnostic methods for patients with shrimp allergy. It was not feasible to use all available diagnostic methods in patients suspected of shrimp allergy. This is the first study to compare the utility of singleplex ImmunoCap using allergen extracts and allergen components with ImmunoCap ISAC in this specific group of patients. Establishing the sensitivity and specificity of different immunological methods and knowledge of their limitations may help clinicians in deciding the best diagnostic approach for patients with suspected shrimp allergies.",
"Patients with symptoms of persistent allergic rhinitis were selected for screening. The selection was random and was based on order of visiting the Outpatient Clinic of Allergic Diseases. Patients being treated for serious chronic diseases and patients on medication that could influence the results of the study were excluded.\nA detailed interview and physical examination was conducted for each patient. All patients underwent skin prick tests (SPTs) with HDM extracts (Dermatophagoides pteronyssinus and D. farinae; Allergopharma, Hamburg, Germany) and with frozen tiger shrimp purchased from a local eco-shop. Patients with positive SPTs for frozen tiger shrimp and self-declared symptoms of shrimp allergy were selected for further examination. As a control group, patients with negative SPTs for frozen tiger shrimp who declared no symptoms of shrimp allergy were enrolled.\nBlood samples were taken from all patients to assess concentrations of sIgE to the HDMs D. pteronyssinus and D. farinae, shrimp allergen extracts (Pandalus borealis, Penaeus monodon, Metapenaeopsis barbata, and Metapenaus joyneri), and the allergen components Der p 1, Der p 2, and Pan a 1. All immunological assays were performed using the highly sensitive immunofluorescent ImmunoCap system (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations of IgE were evaluated as elevated when they exceeded 0.35 kUA/L (ImmunoCap) in accordance with common practice in the field. In all patients we determined levels of sIgE against 112 allergen components using the ImmunoCAP ISAC 112 panel, a semi-quantitative test whose results are reported in ISAC Standardized Units (ISU) and provide indications of sIgE levels within the range of 0.3 to 100 ISU-E. The units used in the ImmunoCap ISAC (ISU-E) were developed especially for this test. The result is shown on a scale of four values for sIgE (indeterminate, <0.3 ISU-E; low, 0.3–0.9 ISU-E; medium, 1–14.9 ISU-E; high, >15 ISU-E). Levels of sIgE in ImmunoCap ISAC tests were considered as elevated when they exceeded 0.3 ISU-E. ImmunoCap ISAC tests included allergen components from D. pteronyssinus (Der p 1, Der p 2, and Der p 10), D. farinae (Der f 1 and Der f 2), and shrimp (Peneus monodon Pen m 1, Pen m 2, and Pen m 3).\nDifferences between groups were assessed using Mann–Whitney and Kruskal–Wallis tests with Dunn’s post hoc test. Spearman correlations were also calculated. Analyses were conducted using R, version 3.3.1 (www.r-project.org) and MS Excel 365. The study was approved by the local Bioethics Committee (number: 147/2015). All patients provided written informed consent to participate in the study.",
"A total of 290 patients (176 women and 114 men) were screened and 20 had positive SPTs for frozen tiger shrimp (wheel >3 mm). All 20 patients had histories of allergic reaction after consumption of shrimp. A control group of 13 patients were selected with negative SPTs for frozen tiger shrimp and no symptoms of shrimp allergy. The characteristics of the study population and the results of immunoassays are presented in Table 1. Oral food challenges were not performed for several reasons, including lack of consent, time requirements, challenges in proper performance, and risks to patients with histories of anaphylaxis. All 20 patients reported symptoms following consumption of shrimp. Urticaria was reported by seven patients, anaphylaxis by six patients (including two patients with anaphylactic shock), digestive problems by four patients, and dyspnea by three patients.\nGeneral characteristics of the study population.\nData are shown as counts or as means ± standard deviations (ranges).\nHDM, house dust mite.\nAll patients with shrimp allergies had elevated levels of shrimp sIgE by ImmunoCap. In addition, all patients were sensitized to D. pteronyssinus and 18 (90%) were sensitized to D. farinae. ImmunoCap ISAC results for shrimp and HDM allergens are presented in Table 2. Patients with shrimp allergies were sensitized to 72 allergen components by ImmunoCap ISAC. The most common sensitization in patients with shrimp allergy was to Der f 2 (16 patients). In addition, 12 patients were sensitized to Fel d 1, 11 patients were sensitized to Bla g 7 and Cyn d 1, and 10 patients were sensitized to Bet v 1, Cor a 1 and Phl p 1. The sensitization patterns are presented in Appendix Table 1. Among the 20 patients sensitized to shrimp, ImmunoCap ISAC detected elevated levels of sIgE against at least one out of three shrimp allergen components in only 10 patients (50%). In the control group all patients were sensitized to HDM by ImmunoCap, but none were sensitized to shrimp allergen extracts (Table 2).\nResults of ImmunoCap ISAC tests.\nHDM, house dust mite.\nTable 3 shows a comparison between the concentrations of sIgE against allergen components detected by ImmunoCap and levels of sIgE detected by ImmunoCap ISAC. The results showed good concordance. Correlations in the 20 patients studied here were >0.7, but this result should be treated with caution. Although it is an indication that the results of the ImmunoCap ISAC and singleplex ImmunoCap methods were similar, the former method is semi-quantitative and the latter is quantitative. In addition, the units and the source shrimp species of tropomyosin differ between the two assays.\nComparison between concentrations of specific IgE measured using the quantitative ImmunoCap method and the semi-quantitative ImmunoCap ISAC microarray.\nFor HDM allergen components, ImmunoCap ISAC found that 84.6% patients were sensitized to Der p 1, 88.9% of patients were sensitized to Der p 2, and 90% of patients were sensitized to Der p 10. Only 50% of patients who were Pen m 1-sensitized had elevated levels of sIgE against Pen a 1 by ImmunoCap ISAC. There was a strong correlation between the concentrations of sIgE against Pen m 1 and Der p 10 in ImmunoCap assays (Figure 1).\nStrong correlation (0.918, p < 0.001) between levels of IgE specific to Pen m 1 and Der p 10 in ImmunoCap (kU/L).\nAs shown in Table 1, ImmunoCap revealed that 18 of 20 patients sensitized to allergen extracts of D. pteronyssinus were also sensitized to Der p 2 (90%). All patients sensitized to Der p 1 were also sensitized to Der p 2, but only 10 (50%) were sensitized to HDM tropomyosin Der p 10.",
"Diagnosis of shrimp allergy is based on clinical interview combined with SPTs, prick by prick tests and measurements of sIgE against allergen extracts or allergenic components. The gold standard for diagnosis of food allergy is still double-blind placebo-controlled food challenge. Although this is the reference method for diagnosis of food allergy, it is rarely performed. The main limitations of oral food challenges are that they are time consuming, expensive, technically difficult to perform, and are contraindicated in patients at risk of anaphylaxis.5 In the current study we established a diagnosis of shrimp sensitization on the basis of reported symptoms after eating shrimp, the results of SPTs, and concentrations of sIgE in the blood.\nThere are currently several approaches for diagnosis of food allergy. SPTs or their derivatives (prick by prick tests) are simple, inexpensive, and yield results within minutes. Unfortunately, for food allergens SPTs have limited sensitivity. There are also contraindications to these tests, especially for patients taking certain medications or patients with skin diseases.6\nIn vitro diagnosis of food allergy can be conducted using different approaches. Measuring the concentration of sIgE against food allergen extracts provides good insight into sensitization and often is sufficient to establish a diagnosis. Component resolved diagnosis takes the issue further, allowing assessment of the patient's detailed allergic profile. The concentrations of sIgE against specific proteins that mediate the allergic reaction can be measured using semi-quantitative methods such as micro-array testing or with highly sensitive, quantitative methods (e.g., ImmunoCap). A summary of the currently available methods for immunological diagnosis of shrimp allergy is presented in Table 4.7–10\nCurrently available methods for determining specific IgE levels against shrimp allergens.\nN – natural source of allergen; E – allergen extract; R – recombinant allergen.\nTo our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. Ours is the first study to compare the utility of singleplex ImmunoCap assays using allergen extracts and allergen components and ImmunoCap ISAC in this specific group of patients. The present study focused on hypersensitivity to commercially available shrimp allergen extracts and allergen components in adult patients who had positive SPTs against frozen tiger shrimp and who experienced symptoms after consumption of shrimp. We also assessed the utility of singleplex vs multiplex methods of evaluating sIgE concentrations in this group of patients.\nAmong patients with shrimp allergies, we found a high prevalence of co-sensitization to HDM allergens. All 20 of the patients with shrimp allergies studied here were simultaneously sensitized to D. pteronyssinus and 18 were sensitized to D. farinae. This finding was not unexpected because of potential cross reactivity between shrimp and HDM allergens. Recently Asero et al. 11 emphasized in a review article that in cases of shrimp allergy, other allergens beyond tropomyosin are likely to be cross reactive with HDM and allergy to shrimp may be one factor triggering HDM-related asthma. Other shrimp allergens potentially cross reactive with HDM include arginine kinase (Der p 20), myosin light chain, and high molecular weight allergen, such as hemocyanin and paramyosin.12–15 It is likely that this list of homologous allergens will increase.11\nHDM allergens are relatively well described. In a study by Weghofer et al., over 97% of patients were allergic to a few HDM proteins including cysteine proteases (Der p 1 and Der f 1) and members of the NPC2 family (Der p 2 and Der f 2).16 Tropomyosin is considered a minor allergen of HDMs that triggers allergy in as few as 5.6% of patients demonstrating increased sIgE levels against HDM allergens.17 This finding was confirmed in our study: sensitization to cysteine proteases and members of the NPC2 family were the main causes of HDM sensitization in the patients studied here. Using both singleplex ImmunoCap and ImmunoCap ISAC methods, a limited number of HDM allergen components were available. Nevertheless, sIgE was detected at high prevalence compared with HDM allergen extracts (ImmunoCap).\nImmunoCap ISAC detected that 100% of patients were sensitized to D. pteronyssinus and that 88.9% of patients were sensitized to D. farinae. New multiplex platforms available for component resolved diagnosis (FABER and ALEX) include HDM allergen components (Der p 5, Der p 7, Der p 7, Der p 9 and/or Der p 23), providing additional value to these tests. However, in most cases the evaluation of sIgE against HDM allergen groups 1 and 2, as available in ImmunoCap ISAC, should be sufficient.4 Previously, ImmunoCap ISAC was described as a valuable method for diagnosis of mite allergy. Yadzir et al. compared SPTs with the results of ImmunoCap ISAC. Among 40 patients with positive SPTs against HDM allergen extracts, elevated levels of sIgE against at least one HDM allergen component were detected in 34 patients (85%).18\nPanzer et al. in analyzed the ImmunoCap ISAC results of 1766 patients. In 1255 patients, increased levels of sIgE against at least one allergen component were detected. Sensitization to HDMs (Der p 1 or Der p 2) was detected in 32.7% of patients, and sensitization to tropomyosin (Der p 10) was detected in 1.9% of patients. Overall, allergy to tropomyosin was diagnosed in 2.2% of the study population.19\nThe ImmunoCap ISAC contains three shrimp allergen components, whereas other multiplex methods include only tropomyosin.10 Still, compared with the singleplex ImmunoCap method using shrimp allergen extract, the multiplex ImmunoCap ISAC method showed only a 50% detection rate. This indirectly shows that other shrimp allergen components are responsible for sensitization. It also shows that ImmunoCap ISAC is not sufficient for screening patients for shrimp sensitization.\nThe ImmunoCap ISAC allows better insight into patient sensitization profiles. In our study population, most patients who were shrimp sensitized, as detected by ImmunoCap ISAC, were also sensitized to only one of three shrimp allergen components. There were no patients sensitized to all three allergen components; only two patients were sensitized to Pen m 1 and Pen m 2, and one patient was sensitized to Pen m 1 and Pen m 4. There were many other allergen components sensitizing patients in the study population and polysensitization was an important issue complicating the clinical picture.\nThe relatively low prevalence of tropomyosin sensitization in our study population was consistent with the results of other studies. In 2012, Asero et al. found that only 41% of patients who were shrimp sensitized had elevated levels of sIgE against tropomyosin, whereas the sera of 52% of patients were reactive with a protein of molecular weight >60 kDa. Moreover, IgE reactive with proteins with molecular masses corresponding to arginine kinase (Pen m 2, 40 kDa), calcium-binding sarcoplasm binding protein (Lit v 4, 20 kDa), light myosin chain (Lit v 3, 20 kDa), triphosphate isomerase (Cra c 8, 27 kDa), troponin C (Cra c 6, 17 kDa), and fatty acid binding protein (15 kDa) was rarely observed (13% of patients).12 Girffida et al.20 identified the high molecular weight protein (>60 kDa) as hemocyanin. There are no commercially available methods of determining levels of sIgE against this allergen, but in previous studies it may have been responsible for clinically significant reactions to shrimp, including anaphylactic shock. Hemocyanins are considered to be among the allergens responsible for cross reactivity of HDM allergens.20,21 Recently, Tonomura et al.22 identified another clinically important protein with a molecular weight of 40 kDa as fructose 1,6–bisphosphate aldolase in a case of food dependent, exercise induced anaphylaxis. In 2018, Kimura et al.23 identified a 43-kDa shrimp allergen as a cause of food-dependent, exercise-induced anaphylaxis.\nJohnston at al. investigated the sera of 21 patients who had clinical reactions to shellfish. Most subjects (13/21, 62%) showed positive sIgE to shrimp allergens by ImmunoCap; 43% of patients were sensitized to tropomyosin, while an additional 29% were sensitized to other shrimp allergens including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. The authors highlighted the lack of standardized shrimp allergens and the inadequacy of current extracts for shrimp allergy diagnosis.24\nAn important aspect of this study was to assess the utility of different immunological methods for diagnosis of shrimp allergy. As observed in our previous study, there was high concordance of sIgE against Der p 10 and Pen m 1 (ImmunoCap) (Spearman correlation 0.918, p < 0.001), which may indicate that it is economically sensible to diagnose shrimp allergy using only one of the methods.25 We also found positive correlations between concentrations of sIgE to Der p 1, Der p 2 and Der p 10 by singleplex ImmunoCap and the corresponding allergen components in ImmunoCap ISAC. Not all patients with elevated sIgE according to the singleplex ImmunoCap method were detected, but the detection rate was above 84% in both cases. The detection rate of sIgE against shrimp tropomyosin was lower (50%); however, the ImmunoCap ISAC and singleplex ImmunoCap assays use tropomyosin from different allergen sources (P. monodon vs F. aztecus). Although published data on different immunological assays for diagnosis of shrimp allergy are limited, our data are consistent with previously published research compering the utility of ImmunoCap ISAC vs ImmunoCap assays (either extract based or component based). Griffiths et al. retrospectively analyzed 118 patients to evaluate what testing strategy (SPT, ImmunoCAP, or ISAC) was the most appropriate for diagnosis in a “real-life” clinical setting. Inpatients with nut allergy, the detection rates of SPTs (56%) and ISAC (65%) were lower than those of ImmunoCap (71%). By contrast, ISAC had a higher detection rate (88%) than ImmunoCap (69%) or SPTs (33%) for diagnosis of oral allergy. ImmunoCap results identified all nine patients with anaphylaxis resulting from wheat allergy (100%), whereas ISAC was positive in only six of nine patients (67%). ImmunoCap showed high specificity and sensitivity overall.26\nHuss-Marp et al. compared ImmunoCap ISAC with singleplex ImmunoCap in a 2015 study. A total of 101 patients with grass pollen allergy were compared in terms of sIgE detection rates against extracts vs ImmunoCap ISAC components. The authors identified four possible explanations of discrepancies between ImmunoCap ISAC and singleplex ImmunoCap results: (i) the trigger allergen was not present in the ISAC panel; (ii) the sensitivity of the ISAC is lower than that of singleplex ImmunoCap tests; (iii) the native extract-based antigen is not identical to the recombinant molecular allergen in ISAC; or (iv) the trigger allergen is absent or underrepresented in the allergen extract. In prior studies the positive percent agreement (PPA) and negative percent agreement (NPA) of corresponding allergens between the ISAC test and the extract-based singleplex ImmunoCap results at a cut-off of 0.1 kUA/L varied between 60% and 100% for PPA and 78% and 97% for NPA. At a cut-off of 0.35 kUA/L (as in our study) the PPA for D. pteronyssinus was 61%, but for timothy grass (with seven allergen components in the ImmunoCap ISAC panel: Phl p 1, 2, 4, 5, 6, 7, 11, and 12) it was 100%. Shrimp sensitization was not analyzed.27 In our study the PPA of sIgE against D. pteronyssinus was 100%, but for shrimp allergy the PPA between extract detection and ImmunoCap ISAC was low (50%). The main cause was probably a lack of important shrimp allergen components in ImmunoCap ISAC.\nAlthough in our study, the presence of Der p 1 and Der p 2 was sufficient to diagnose HDM sensitivity in patients, the new ImmunoCap ISACE112i, available in Poland since 2019, contains an additional recombinant HDM allergen component (Der p23). Huang et al. developed a customized microarray based on ImmunoCap ISAC technology that was produced by Phadia Austria GmbH (Vienna, Austria) and contained as many as 13 HDM allergen components (the clinically relevant nDer p 1; rDer p 2, 5, 7, 21, and 23; and Der p 4, 10, 11, 14, 15, 18, and clone 16). Streptavidin-based ImmunoCaps (o212 ImmunoCap, Thermo Fisher Scientific/Phadia) were used to prepare microarrays containing a mixture of Der p 1, 2, 5, 7, 21, and 23. The strongest correlation was identified between extract-based ImmunoCaps and molecular ImmunoCaps containing a mixture of Der p 1, 2, 5,7, 21, and 23. Interestingly, HDM-specific IgE levels determined using the molecular ImmunoCap were considerably higher than those measured using the allergen extract-based ImmunoCap. The authors found that Der p 2, Der p 5, Der p 21, and Der p 23 appeared to be underrepresented in natural allergen extracts. This finding suggested that adding components to ImmunoCap ISAC might increase detection rates of sensitized cases. An interesting suggestion was made that adding recombinant allergen molecules to natural HDM allergen extracts or preparing molecular ImmunoCaps containing the most important allergens could be of benefit in enhancing diagnosis.28",
"Extract-based singleplex ImmunoCap methods are highly sensitive and widely available. Component resolved diagnosis adds additional information on the nature of sensitization and potential cross reactivity. The results of sIgE levels against corresponding allergen components determined using singleplex ImmunoCap and ImmunoCap ISAC were similar in most cases, with correlations above 0.7. In the case of shrimp allergy, the lack of many important shrimp allergen components, such as hemocyanin, was the probable cause of low detectability rates in molecular analysis (both singleplex and multiplex). The prevalence of HDM sensitization among patients with shrimp allergies was high, as described previously. The concordance of sIgE concentrations against Der p 10 and Pen m 1 between singleplex ImmunoCap and ImmunoCap ISAC was very high. Only 50% of patients with shrimp allergies were sensitized to tropomyosin and a lack of other important shrimp allergen components used in quantitative ImmunoCap ISAC measurements is a serious drawback for diagnosis of shrimp allergy. The results of immunoassays should be interpreted with care and a combination of extract and component-based diagnosis gives optimal insight into patient sensitization patterns.\nNumbers of patients sensitized to specific allergen components in ImmunoCap ISAC. Levels of sIgE against allergen components that are not included in the table were not elevated in any patients.\nsIgE, specific IgE"
] | [
null,
"methods",
"results",
"discussion",
"conclusions"
] | [
"Allergy",
"shrimp",
"sensitization",
"in vitro diagnostics",
"IgE",
"tropomyosin"
] | Background:
Shrimp allergy is an increasing problem in the European population. Symptoms associated with the consumption of shrimp by allergic individuals can range from mild, local reactions to systemic reactions and anaphylactic shock. The main allergen of crustaceans is tropomyosin, which shows high interspecies homology and is also found in house dust mites (HDMs). Other shrimp allergens have also been reported and may be associated with a severe course of sensitization.
HDM allergy is widespread in Europe, including Poland. The largest epidemiological study conducted in Poland, the Epidemiology of Allergic Diseases in Poland (ECAP) study, found that allergic rhinitis was the most prevalent allergic condition affecting both children and adults and was diagnosed in 24% to 30% of the study population.1 Boquete et al.2 indicated that 71% of patients allergic to HDMs also had specific IgE (sIgE) against shrimp, and 55% had increased levels of sIgE against shrimp tropomyosin. Canadian studies demonstrated a high prevalence of allergy to HDMs in 95 patients with confirmed shrimp allergy. In the study population, 86 (90.5%) patients had positive skin prick tests for HDM allergens.3 In a recent Italian study, 9% of 526 patients with HDM allergies also had allergy to shrimp. Patients with shrimp allergies were less frequently hypersensitive to airborne allergens other than HDM than crustacean-tolerant subjects. Only 51% of patients who were tropomyosin sensitized had shrimp allergies, and only 48% of patients who were shrimp sensitized were also sensitized to tropomyosin.4
There are several different methods of diagnosing shrimp allergy. Double blind, placebo-controlled food challenge remains the gold standard for diagnosis of food allergy. Oral food challenges are technically difficult to perform and time consuming. For patients with histories of anaphylaxis, the risks may also outweigh the potential benefits of performing the challenge.
To our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. The aim of the current study was to analyze the clinical utility of different diagnostic methods for patients with shrimp allergy. It was not feasible to use all available diagnostic methods in patients suspected of shrimp allergy. This is the first study to compare the utility of singleplex ImmunoCap using allergen extracts and allergen components with ImmunoCap ISAC in this specific group of patients. Establishing the sensitivity and specificity of different immunological methods and knowledge of their limitations may help clinicians in deciding the best diagnostic approach for patients with suspected shrimp allergies.
Methods:
Patients with symptoms of persistent allergic rhinitis were selected for screening. The selection was random and was based on order of visiting the Outpatient Clinic of Allergic Diseases. Patients being treated for serious chronic diseases and patients on medication that could influence the results of the study were excluded.
A detailed interview and physical examination was conducted for each patient. All patients underwent skin prick tests (SPTs) with HDM extracts (Dermatophagoides pteronyssinus and D. farinae; Allergopharma, Hamburg, Germany) and with frozen tiger shrimp purchased from a local eco-shop. Patients with positive SPTs for frozen tiger shrimp and self-declared symptoms of shrimp allergy were selected for further examination. As a control group, patients with negative SPTs for frozen tiger shrimp who declared no symptoms of shrimp allergy were enrolled.
Blood samples were taken from all patients to assess concentrations of sIgE to the HDMs D. pteronyssinus and D. farinae, shrimp allergen extracts (Pandalus borealis, Penaeus monodon, Metapenaeopsis barbata, and Metapenaus joyneri), and the allergen components Der p 1, Der p 2, and Pan a 1. All immunological assays were performed using the highly sensitive immunofluorescent ImmunoCap system (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations of IgE were evaluated as elevated when they exceeded 0.35 kUA/L (ImmunoCap) in accordance with common practice in the field. In all patients we determined levels of sIgE against 112 allergen components using the ImmunoCAP ISAC 112 panel, a semi-quantitative test whose results are reported in ISAC Standardized Units (ISU) and provide indications of sIgE levels within the range of 0.3 to 100 ISU-E. The units used in the ImmunoCap ISAC (ISU-E) were developed especially for this test. The result is shown on a scale of four values for sIgE (indeterminate, <0.3 ISU-E; low, 0.3–0.9 ISU-E; medium, 1–14.9 ISU-E; high, >15 ISU-E). Levels of sIgE in ImmunoCap ISAC tests were considered as elevated when they exceeded 0.3 ISU-E. ImmunoCap ISAC tests included allergen components from D. pteronyssinus (Der p 1, Der p 2, and Der p 10), D. farinae (Der f 1 and Der f 2), and shrimp (Peneus monodon Pen m 1, Pen m 2, and Pen m 3).
Differences between groups were assessed using Mann–Whitney and Kruskal–Wallis tests with Dunn’s post hoc test. Spearman correlations were also calculated. Analyses were conducted using R, version 3.3.1 (www.r-project.org) and MS Excel 365. The study was approved by the local Bioethics Committee (number: 147/2015). All patients provided written informed consent to participate in the study.
Results:
A total of 290 patients (176 women and 114 men) were screened and 20 had positive SPTs for frozen tiger shrimp (wheel >3 mm). All 20 patients had histories of allergic reaction after consumption of shrimp. A control group of 13 patients were selected with negative SPTs for frozen tiger shrimp and no symptoms of shrimp allergy. The characteristics of the study population and the results of immunoassays are presented in Table 1. Oral food challenges were not performed for several reasons, including lack of consent, time requirements, challenges in proper performance, and risks to patients with histories of anaphylaxis. All 20 patients reported symptoms following consumption of shrimp. Urticaria was reported by seven patients, anaphylaxis by six patients (including two patients with anaphylactic shock), digestive problems by four patients, and dyspnea by three patients.
General characteristics of the study population.
Data are shown as counts or as means ± standard deviations (ranges).
HDM, house dust mite.
All patients with shrimp allergies had elevated levels of shrimp sIgE by ImmunoCap. In addition, all patients were sensitized to D. pteronyssinus and 18 (90%) were sensitized to D. farinae. ImmunoCap ISAC results for shrimp and HDM allergens are presented in Table 2. Patients with shrimp allergies were sensitized to 72 allergen components by ImmunoCap ISAC. The most common sensitization in patients with shrimp allergy was to Der f 2 (16 patients). In addition, 12 patients were sensitized to Fel d 1, 11 patients were sensitized to Bla g 7 and Cyn d 1, and 10 patients were sensitized to Bet v 1, Cor a 1 and Phl p 1. The sensitization patterns are presented in Appendix Table 1. Among the 20 patients sensitized to shrimp, ImmunoCap ISAC detected elevated levels of sIgE against at least one out of three shrimp allergen components in only 10 patients (50%). In the control group all patients were sensitized to HDM by ImmunoCap, but none were sensitized to shrimp allergen extracts (Table 2).
Results of ImmunoCap ISAC tests.
HDM, house dust mite.
Table 3 shows a comparison between the concentrations of sIgE against allergen components detected by ImmunoCap and levels of sIgE detected by ImmunoCap ISAC. The results showed good concordance. Correlations in the 20 patients studied here were >0.7, but this result should be treated with caution. Although it is an indication that the results of the ImmunoCap ISAC and singleplex ImmunoCap methods were similar, the former method is semi-quantitative and the latter is quantitative. In addition, the units and the source shrimp species of tropomyosin differ between the two assays.
Comparison between concentrations of specific IgE measured using the quantitative ImmunoCap method and the semi-quantitative ImmunoCap ISAC microarray.
For HDM allergen components, ImmunoCap ISAC found that 84.6% patients were sensitized to Der p 1, 88.9% of patients were sensitized to Der p 2, and 90% of patients were sensitized to Der p 10. Only 50% of patients who were Pen m 1-sensitized had elevated levels of sIgE against Pen a 1 by ImmunoCap ISAC. There was a strong correlation between the concentrations of sIgE against Pen m 1 and Der p 10 in ImmunoCap assays (Figure 1).
Strong correlation (0.918, p < 0.001) between levels of IgE specific to Pen m 1 and Der p 10 in ImmunoCap (kU/L).
As shown in Table 1, ImmunoCap revealed that 18 of 20 patients sensitized to allergen extracts of D. pteronyssinus were also sensitized to Der p 2 (90%). All patients sensitized to Der p 1 were also sensitized to Der p 2, but only 10 (50%) were sensitized to HDM tropomyosin Der p 10.
Discussion:
Diagnosis of shrimp allergy is based on clinical interview combined with SPTs, prick by prick tests and measurements of sIgE against allergen extracts or allergenic components. The gold standard for diagnosis of food allergy is still double-blind placebo-controlled food challenge. Although this is the reference method for diagnosis of food allergy, it is rarely performed. The main limitations of oral food challenges are that they are time consuming, expensive, technically difficult to perform, and are contraindicated in patients at risk of anaphylaxis.5 In the current study we established a diagnosis of shrimp sensitization on the basis of reported symptoms after eating shrimp, the results of SPTs, and concentrations of sIgE in the blood.
There are currently several approaches for diagnosis of food allergy. SPTs or their derivatives (prick by prick tests) are simple, inexpensive, and yield results within minutes. Unfortunately, for food allergens SPTs have limited sensitivity. There are also contraindications to these tests, especially for patients taking certain medications or patients with skin diseases.6
In vitro diagnosis of food allergy can be conducted using different approaches. Measuring the concentration of sIgE against food allergen extracts provides good insight into sensitization and often is sufficient to establish a diagnosis. Component resolved diagnosis takes the issue further, allowing assessment of the patient's detailed allergic profile. The concentrations of sIgE against specific proteins that mediate the allergic reaction can be measured using semi-quantitative methods such as micro-array testing or with highly sensitive, quantitative methods (e.g., ImmunoCap). A summary of the currently available methods for immunological diagnosis of shrimp allergy is presented in Table 4.7–10
Currently available methods for determining specific IgE levels against shrimp allergens.
N – natural source of allergen; E – allergen extract; R – recombinant allergen.
To our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. Ours is the first study to compare the utility of singleplex ImmunoCap assays using allergen extracts and allergen components and ImmunoCap ISAC in this specific group of patients. The present study focused on hypersensitivity to commercially available shrimp allergen extracts and allergen components in adult patients who had positive SPTs against frozen tiger shrimp and who experienced symptoms after consumption of shrimp. We also assessed the utility of singleplex vs multiplex methods of evaluating sIgE concentrations in this group of patients.
Among patients with shrimp allergies, we found a high prevalence of co-sensitization to HDM allergens. All 20 of the patients with shrimp allergies studied here were simultaneously sensitized to D. pteronyssinus and 18 were sensitized to D. farinae. This finding was not unexpected because of potential cross reactivity between shrimp and HDM allergens. Recently Asero et al. 11 emphasized in a review article that in cases of shrimp allergy, other allergens beyond tropomyosin are likely to be cross reactive with HDM and allergy to shrimp may be one factor triggering HDM-related asthma. Other shrimp allergens potentially cross reactive with HDM include arginine kinase (Der p 20), myosin light chain, and high molecular weight allergen, such as hemocyanin and paramyosin.12–15 It is likely that this list of homologous allergens will increase.11
HDM allergens are relatively well described. In a study by Weghofer et al., over 97% of patients were allergic to a few HDM proteins including cysteine proteases (Der p 1 and Der f 1) and members of the NPC2 family (Der p 2 and Der f 2).16 Tropomyosin is considered a minor allergen of HDMs that triggers allergy in as few as 5.6% of patients demonstrating increased sIgE levels against HDM allergens.17 This finding was confirmed in our study: sensitization to cysteine proteases and members of the NPC2 family were the main causes of HDM sensitization in the patients studied here. Using both singleplex ImmunoCap and ImmunoCap ISAC methods, a limited number of HDM allergen components were available. Nevertheless, sIgE was detected at high prevalence compared with HDM allergen extracts (ImmunoCap).
ImmunoCap ISAC detected that 100% of patients were sensitized to D. pteronyssinus and that 88.9% of patients were sensitized to D. farinae. New multiplex platforms available for component resolved diagnosis (FABER and ALEX) include HDM allergen components (Der p 5, Der p 7, Der p 7, Der p 9 and/or Der p 23), providing additional value to these tests. However, in most cases the evaluation of sIgE against HDM allergen groups 1 and 2, as available in ImmunoCap ISAC, should be sufficient.4 Previously, ImmunoCap ISAC was described as a valuable method for diagnosis of mite allergy. Yadzir et al. compared SPTs with the results of ImmunoCap ISAC. Among 40 patients with positive SPTs against HDM allergen extracts, elevated levels of sIgE against at least one HDM allergen component were detected in 34 patients (85%).18
Panzer et al. in analyzed the ImmunoCap ISAC results of 1766 patients. In 1255 patients, increased levels of sIgE against at least one allergen component were detected. Sensitization to HDMs (Der p 1 or Der p 2) was detected in 32.7% of patients, and sensitization to tropomyosin (Der p 10) was detected in 1.9% of patients. Overall, allergy to tropomyosin was diagnosed in 2.2% of the study population.19
The ImmunoCap ISAC contains three shrimp allergen components, whereas other multiplex methods include only tropomyosin.10 Still, compared with the singleplex ImmunoCap method using shrimp allergen extract, the multiplex ImmunoCap ISAC method showed only a 50% detection rate. This indirectly shows that other shrimp allergen components are responsible for sensitization. It also shows that ImmunoCap ISAC is not sufficient for screening patients for shrimp sensitization.
The ImmunoCap ISAC allows better insight into patient sensitization profiles. In our study population, most patients who were shrimp sensitized, as detected by ImmunoCap ISAC, were also sensitized to only one of three shrimp allergen components. There were no patients sensitized to all three allergen components; only two patients were sensitized to Pen m 1 and Pen m 2, and one patient was sensitized to Pen m 1 and Pen m 4. There were many other allergen components sensitizing patients in the study population and polysensitization was an important issue complicating the clinical picture.
The relatively low prevalence of tropomyosin sensitization in our study population was consistent with the results of other studies. In 2012, Asero et al. found that only 41% of patients who were shrimp sensitized had elevated levels of sIgE against tropomyosin, whereas the sera of 52% of patients were reactive with a protein of molecular weight >60 kDa. Moreover, IgE reactive with proteins with molecular masses corresponding to arginine kinase (Pen m 2, 40 kDa), calcium-binding sarcoplasm binding protein (Lit v 4, 20 kDa), light myosin chain (Lit v 3, 20 kDa), triphosphate isomerase (Cra c 8, 27 kDa), troponin C (Cra c 6, 17 kDa), and fatty acid binding protein (15 kDa) was rarely observed (13% of patients).12 Girffida et al.20 identified the high molecular weight protein (>60 kDa) as hemocyanin. There are no commercially available methods of determining levels of sIgE against this allergen, but in previous studies it may have been responsible for clinically significant reactions to shrimp, including anaphylactic shock. Hemocyanins are considered to be among the allergens responsible for cross reactivity of HDM allergens.20,21 Recently, Tonomura et al.22 identified another clinically important protein with a molecular weight of 40 kDa as fructose 1,6–bisphosphate aldolase in a case of food dependent, exercise induced anaphylaxis. In 2018, Kimura et al.23 identified a 43-kDa shrimp allergen as a cause of food-dependent, exercise-induced anaphylaxis.
Johnston at al. investigated the sera of 21 patients who had clinical reactions to shellfish. Most subjects (13/21, 62%) showed positive sIgE to shrimp allergens by ImmunoCap; 43% of patients were sensitized to tropomyosin, while an additional 29% were sensitized to other shrimp allergens including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. The authors highlighted the lack of standardized shrimp allergens and the inadequacy of current extracts for shrimp allergy diagnosis.24
An important aspect of this study was to assess the utility of different immunological methods for diagnosis of shrimp allergy. As observed in our previous study, there was high concordance of sIgE against Der p 10 and Pen m 1 (ImmunoCap) (Spearman correlation 0.918, p < 0.001), which may indicate that it is economically sensible to diagnose shrimp allergy using only one of the methods.25 We also found positive correlations between concentrations of sIgE to Der p 1, Der p 2 and Der p 10 by singleplex ImmunoCap and the corresponding allergen components in ImmunoCap ISAC. Not all patients with elevated sIgE according to the singleplex ImmunoCap method were detected, but the detection rate was above 84% in both cases. The detection rate of sIgE against shrimp tropomyosin was lower (50%); however, the ImmunoCap ISAC and singleplex ImmunoCap assays use tropomyosin from different allergen sources (P. monodon vs F. aztecus). Although published data on different immunological assays for diagnosis of shrimp allergy are limited, our data are consistent with previously published research compering the utility of ImmunoCap ISAC vs ImmunoCap assays (either extract based or component based). Griffiths et al. retrospectively analyzed 118 patients to evaluate what testing strategy (SPT, ImmunoCAP, or ISAC) was the most appropriate for diagnosis in a “real-life” clinical setting. Inpatients with nut allergy, the detection rates of SPTs (56%) and ISAC (65%) were lower than those of ImmunoCap (71%). By contrast, ISAC had a higher detection rate (88%) than ImmunoCap (69%) or SPTs (33%) for diagnosis of oral allergy. ImmunoCap results identified all nine patients with anaphylaxis resulting from wheat allergy (100%), whereas ISAC was positive in only six of nine patients (67%). ImmunoCap showed high specificity and sensitivity overall.26
Huss-Marp et al. compared ImmunoCap ISAC with singleplex ImmunoCap in a 2015 study. A total of 101 patients with grass pollen allergy were compared in terms of sIgE detection rates against extracts vs ImmunoCap ISAC components. The authors identified four possible explanations of discrepancies between ImmunoCap ISAC and singleplex ImmunoCap results: (i) the trigger allergen was not present in the ISAC panel; (ii) the sensitivity of the ISAC is lower than that of singleplex ImmunoCap tests; (iii) the native extract-based antigen is not identical to the recombinant molecular allergen in ISAC; or (iv) the trigger allergen is absent or underrepresented in the allergen extract. In prior studies the positive percent agreement (PPA) and negative percent agreement (NPA) of corresponding allergens between the ISAC test and the extract-based singleplex ImmunoCap results at a cut-off of 0.1 kUA/L varied between 60% and 100% for PPA and 78% and 97% for NPA. At a cut-off of 0.35 kUA/L (as in our study) the PPA for D. pteronyssinus was 61%, but for timothy grass (with seven allergen components in the ImmunoCap ISAC panel: Phl p 1, 2, 4, 5, 6, 7, 11, and 12) it was 100%. Shrimp sensitization was not analyzed.27 In our study the PPA of sIgE against D. pteronyssinus was 100%, but for shrimp allergy the PPA between extract detection and ImmunoCap ISAC was low (50%). The main cause was probably a lack of important shrimp allergen components in ImmunoCap ISAC.
Although in our study, the presence of Der p 1 and Der p 2 was sufficient to diagnose HDM sensitivity in patients, the new ImmunoCap ISACE112i, available in Poland since 2019, contains an additional recombinant HDM allergen component (Der p23). Huang et al. developed a customized microarray based on ImmunoCap ISAC technology that was produced by Phadia Austria GmbH (Vienna, Austria) and contained as many as 13 HDM allergen components (the clinically relevant nDer p 1; rDer p 2, 5, 7, 21, and 23; and Der p 4, 10, 11, 14, 15, 18, and clone 16). Streptavidin-based ImmunoCaps (o212 ImmunoCap, Thermo Fisher Scientific/Phadia) were used to prepare microarrays containing a mixture of Der p 1, 2, 5, 7, 21, and 23. The strongest correlation was identified between extract-based ImmunoCaps and molecular ImmunoCaps containing a mixture of Der p 1, 2, 5,7, 21, and 23. Interestingly, HDM-specific IgE levels determined using the molecular ImmunoCap were considerably higher than those measured using the allergen extract-based ImmunoCap. The authors found that Der p 2, Der p 5, Der p 21, and Der p 23 appeared to be underrepresented in natural allergen extracts. This finding suggested that adding components to ImmunoCap ISAC might increase detection rates of sensitized cases. An interesting suggestion was made that adding recombinant allergen molecules to natural HDM allergen extracts or preparing molecular ImmunoCaps containing the most important allergens could be of benefit in enhancing diagnosis.28
Conclusion:
Extract-based singleplex ImmunoCap methods are highly sensitive and widely available. Component resolved diagnosis adds additional information on the nature of sensitization and potential cross reactivity. The results of sIgE levels against corresponding allergen components determined using singleplex ImmunoCap and ImmunoCap ISAC were similar in most cases, with correlations above 0.7. In the case of shrimp allergy, the lack of many important shrimp allergen components, such as hemocyanin, was the probable cause of low detectability rates in molecular analysis (both singleplex and multiplex). The prevalence of HDM sensitization among patients with shrimp allergies was high, as described previously. The concordance of sIgE concentrations against Der p 10 and Pen m 1 between singleplex ImmunoCap and ImmunoCap ISAC was very high. Only 50% of patients with shrimp allergies were sensitized to tropomyosin and a lack of other important shrimp allergen components used in quantitative ImmunoCap ISAC measurements is a serious drawback for diagnosis of shrimp allergy. The results of immunoassays should be interpreted with care and a combination of extract and component-based diagnosis gives optimal insight into patient sensitization patterns.
Numbers of patients sensitized to specific allergen components in ImmunoCap ISAC. Levels of sIgE against allergen components that are not included in the table were not elevated in any patients.
sIgE, specific IgE
| Background:
Levels of specific IgE (sIgE) against allergen components can be assessed using multiplex assays or with highly sensitive, quantitative methods. The aim of this study was to compare the sensitivity and specificity of different immunological methods for diagnosis of shrimp allergy.
Methods:
Twenty patients with positive skin prick tests for frozen tiger shrimp were selected for further examination. Blood samples were taken to assess concentrations of sIgE against the house dust mites Dermatophagoides pteronyssinus and D. farinae, shrimp allergen extract, allergen components Der p 1, Der p 2 and Pan a 1 (ImmunoCap), and the ImmunoCap ISAC 112 panel.
Results:
All patients had elevated levels of sIgE against shrimp and D pteronyssinus. Eight patients were sensitized to Pen m 1, three patients were sensitized to Pen m 2, and two patients were sensitized to Pen m 4 (ISAC). ImmunoCap ISAC detected shrimp sensitization in 50% of patients. There was a strong correlation between concentrations of sIgE against Pen m1 and Der p 10 detected by ImmunoCap.
Conclusions:
The singleplex ImmunoCap system remains the reference diagnostic method, but in the case of shrimp allergy ImmunoCap ISAC provided better insight into patient allergen profiles. | Background:
Shrimp allergy is an increasing problem in the European population. Symptoms associated with the consumption of shrimp by allergic individuals can range from mild, local reactions to systemic reactions and anaphylactic shock. The main allergen of crustaceans is tropomyosin, which shows high interspecies homology and is also found in house dust mites (HDMs). Other shrimp allergens have also been reported and may be associated with a severe course of sensitization.
HDM allergy is widespread in Europe, including Poland. The largest epidemiological study conducted in Poland, the Epidemiology of Allergic Diseases in Poland (ECAP) study, found that allergic rhinitis was the most prevalent allergic condition affecting both children and adults and was diagnosed in 24% to 30% of the study population.1 Boquete et al.2 indicated that 71% of patients allergic to HDMs also had specific IgE (sIgE) against shrimp, and 55% had increased levels of sIgE against shrimp tropomyosin. Canadian studies demonstrated a high prevalence of allergy to HDMs in 95 patients with confirmed shrimp allergy. In the study population, 86 (90.5%) patients had positive skin prick tests for HDM allergens.3 In a recent Italian study, 9% of 526 patients with HDM allergies also had allergy to shrimp. Patients with shrimp allergies were less frequently hypersensitive to airborne allergens other than HDM than crustacean-tolerant subjects. Only 51% of patients who were tropomyosin sensitized had shrimp allergies, and only 48% of patients who were shrimp sensitized were also sensitized to tropomyosin.4
There are several different methods of diagnosing shrimp allergy. Double blind, placebo-controlled food challenge remains the gold standard for diagnosis of food allergy. Oral food challenges are technically difficult to perform and time consuming. For patients with histories of anaphylaxis, the risks may also outweigh the potential benefits of performing the challenge.
To our knowledge, only a few studies have addressed the clinical relevance of component-resolved diagnosis of shrimp sensitization. The aim of the current study was to analyze the clinical utility of different diagnostic methods for patients with shrimp allergy. It was not feasible to use all available diagnostic methods in patients suspected of shrimp allergy. This is the first study to compare the utility of singleplex ImmunoCap using allergen extracts and allergen components with ImmunoCap ISAC in this specific group of patients. Establishing the sensitivity and specificity of different immunological methods and knowledge of their limitations may help clinicians in deciding the best diagnostic approach for patients with suspected shrimp allergies.
Conclusion:
Extract-based singleplex ImmunoCap methods are highly sensitive and widely available. Component resolved diagnosis adds additional information on the nature of sensitization and potential cross reactivity. The results of sIgE levels against corresponding allergen components determined using singleplex ImmunoCap and ImmunoCap ISAC were similar in most cases, with correlations above 0.7. In the case of shrimp allergy, the lack of many important shrimp allergen components, such as hemocyanin, was the probable cause of low detectability rates in molecular analysis (both singleplex and multiplex). The prevalence of HDM sensitization among patients with shrimp allergies was high, as described previously. The concordance of sIgE concentrations against Der p 10 and Pen m 1 between singleplex ImmunoCap and ImmunoCap ISAC was very high. Only 50% of patients with shrimp allergies were sensitized to tropomyosin and a lack of other important shrimp allergen components used in quantitative ImmunoCap ISAC measurements is a serious drawback for diagnosis of shrimp allergy. The results of immunoassays should be interpreted with care and a combination of extract and component-based diagnosis gives optimal insight into patient sensitization patterns.
Numbers of patients sensitized to specific allergen components in ImmunoCap ISAC. Levels of sIgE against allergen components that are not included in the table were not elevated in any patients.
sIgE, specific IgE | Background:
Levels of specific IgE (sIgE) against allergen components can be assessed using multiplex assays or with highly sensitive, quantitative methods. The aim of this study was to compare the sensitivity and specificity of different immunological methods for diagnosis of shrimp allergy.
Methods:
Twenty patients with positive skin prick tests for frozen tiger shrimp were selected for further examination. Blood samples were taken to assess concentrations of sIgE against the house dust mites Dermatophagoides pteronyssinus and D. farinae, shrimp allergen extract, allergen components Der p 1, Der p 2 and Pan a 1 (ImmunoCap), and the ImmunoCap ISAC 112 panel.
Results:
All patients had elevated levels of sIgE against shrimp and D pteronyssinus. Eight patients were sensitized to Pen m 1, three patients were sensitized to Pen m 2, and two patients were sensitized to Pen m 4 (ISAC). ImmunoCap ISAC detected shrimp sensitization in 50% of patients. There was a strong correlation between concentrations of sIgE against Pen m1 and Der p 10 detected by ImmunoCap.
Conclusions:
The singleplex ImmunoCap system remains the reference diagnostic method, but in the case of shrimp allergy ImmunoCap ISAC provided better insight into patient allergen profiles. | 4,432 | 228 | [
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||||||
IPNV with high and low virulence: host immune responses and viral mutations during infection. | 21827718 | Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described. | BACKGROUND | In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney. | METHODS | Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates). | RESULTS | Mortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties. | CONCLUSIONS | [
"Animals",
"Birnaviridae Infections",
"Fish Diseases",
"Gene Expression Profiling",
"Infectious pancreatic necrosis virus",
"Kidney",
"Microarray Analysis",
"Mutation",
"RNA, Viral",
"Real-Time Polymerase Chain Reaction",
"Salmo salar",
"Survival Analysis",
"Virulence",
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] | 3169513 | Background | Despite vaccination programs, outbreaks of infectious pancreatic necrosis disease (IPN) are frequent in farmed salmon fry and post-smolts. Mortality rates observed in outbreaks vary considerably and have in part been ascribed to the inherited differences in susceptibility of the host [1-3]. Environmental stress [4-7] and the viral strains [8] also influence mortality. Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods [9,10]. The production of virus may increase under stress, and carriers can shed the virus and infect surrounding fish [11].
The IPN virus (IPNV) is a bi-segmented double-stranded RNA (dsRNA) virus in the family Birnaviridae encoding 5 viral proteins. Segment B encodes the RNA-dependent RNA polymerase VP1. Segment A encodes a polyprotein which is cotranslationally cleaved by the viral encoded serine-lysine protease (VP4) releasing the proteins pVP2 and VP3 [12,13]. pVP2 is further processed by host cell proteases to form the mature outer capsid protein VP2 [14], which is the most abundant virus protein and contains the antigenic regions responsible for induction of neutralizing antibodies in the host [15]. VP3 is the inner structural protein, which bound to dsRNA constitutes the ribonucleoprotein core structure [16]. Additionally VP3 is shown to bind VP1 and to self-associate strongly, indicating that it is a matrix protein [17]. An alternative open reading frame (ORF) on Segment A encodes the small, arginine-rich, non-structural protein VP5. The biological function of IPNV VP5 remains to be determined.
The molecular basis of IPNV virulence and its interplay with host antiviral mechanisms are not fully understood. Sano and co-workers [18] were the first to suggest that the virulence of IPNV is associated with Segment A. Several studies using nucleotide sequence analyses have confirmed this and have shown that the VP2 residues 217 and 221 are the major determinant of virulence of IPNV serotype Sp strains. In addition, position 247 was seen as highly variable [19]. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221, while the strains containing Thr221 are almost avirulent, irrespective of the residue at position 217. IPNV isolates also differ in properties related to replication rate and the ability to cause persistent infections. These characteristics can be attributed to the same amino acids as those determining the virulence [8]. Although some of the factors behind these mechanisms are known there are still many questions to be answered.
During viral infections the initial response of the immune system is the induction of type I interferons (IFN), which mediate antiviral and immunomodulatory activity. In Atlantic salmon three different subtypes of type I IFN have been identified: IFN-a, b and c [20]. IFN-a1 and c are both expressed in head kidney and are induced by poly I:C [20]. IFN-a1 has been shown to provide protection against IPNV in salmonid cells [21,22]. The generation of anti-viral responses during infections requires a rapid viral sensing by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) on the cell-surface or within endosomes recognize single-stranded RNA (ssRNA) and dsRNA [23], while the helicases RIG-I and MDA5 recognize ssRNA and dsRNA in the cytosol [24]. Additionally, dsRNA are recognized by PKR [25]. A number of PRRs have been identified in Atlantic salmon including RIG-I [26], MDA5 (GenBank: EG820831), PKR (GenBank: EF523422), TLR3 [20], TLR8 [27], TLR9 [28] and TLR22 (GenBank: CAJ80696[29] and FM206383). The latter is a dsRNA-specific PRR found exclusively in lower vertebrates [30]. Several studies have shown strong activation of immune genes upon challenge with highly virulent IPNV isolates [31,32], and type I IFNs and the IFN-inducible Mx gene were among the most highly up-regulated genes. However, it remains to find, which PRRs are required for the induction of a systemic type I IFN response during IPNV infections.
Little is known about the relationship between the host responses and the virulence of different IPNV isolates. The latter can be associated with either down-regulation or excessive stimulation of innate immunity. Studies of IPNV infected cell-lines [33,34] have shown inhibition of IFN signaling. In the current work we have assessed immune gene expression changes during an experimental challenge of salmon post-smolts with both a virulent and an avirulent IPNV field strain. In addition to quantitative real-time RT PCR (qPCR) analyses of selected genes we used cDNA microarray, which expanded the repertoire of genes.
The two IPNV isolates used in this study were originally collected from field outbreaks of IPN with 32% and 5% reported mortality, respectively. Initial characterization of the isolates in our lab showed that they both contained the high virulent motif Thr217 Ala221 Thr247 in VP2 defined by Santi et al [19]. A previous bath challenge performed by our group included these isolates, and indicated that they differed substantially in virulence levels reflecting the field mortalities (unpublished). Sequencing of VP2 after this challenge confirmed the Thr217 Ala221 motif in both isolates. However, propagation of the isolates in cell-cultures before the challenge experiment reported here changed the isolate with the initial low mortality to contain a Thr217 Thr221 Thr247 motif (low virulent motif). Santi et al [19] suggested that the Ala-to-Thr substitution at position 221 in VP2 is a molecular determinant for the establishment of a persistent IPNV infection. Thus, we have analyzed the IPNV VP2 nucleotide composition in virus recovered from fish head kidney during the infection. Comparing sequences to the input strains could provide information about the rate and character of virus changes in vivo. | Methods | Virus isolates Two IPNV isolates, referred to as NFH-Ar and NFH-El, collected from field outbreaks of IPN in 2004 were used in this study. Veterinarians had diagnosed IPN based on clinical observations and by use of an agglutination assay (Phadebact coating kit, Boule Diagnostics AB). The NFH-Ar isolate, obtained from an IPN outbreak in Frøya, Norway in 2004, was reported to give 32% mortality, while the NFH-El, obtained from an IPN outbreak in Alta, Norway in 2004, was reported to give 5% mortality. Head kidney samples collected during these outbreaks were sent to our lab and tissue homogenates were made and inoculated on CHSE-214 cells for propagation and sequencing. For NFH-Ar a second, and for NFH-El a third cell-culture passage of the virus strain was used in the present experiment. The input strains were sequenced before challenge.
Two IPNV isolates, referred to as NFH-Ar and NFH-El, collected from field outbreaks of IPN in 2004 were used in this study. Veterinarians had diagnosed IPN based on clinical observations and by use of an agglutination assay (Phadebact coating kit, Boule Diagnostics AB). The NFH-Ar isolate, obtained from an IPN outbreak in Frøya, Norway in 2004, was reported to give 32% mortality, while the NFH-El, obtained from an IPN outbreak in Alta, Norway in 2004, was reported to give 5% mortality. Head kidney samples collected during these outbreaks were sent to our lab and tissue homogenates were made and inoculated on CHSE-214 cells for propagation and sequencing. For NFH-Ar a second, and for NFH-El a third cell-culture passage of the virus strain was used in the present experiment. The input strains were sequenced before challenge.
IPNV challenge of Atlantic salmon The challenge was carried out at Tromsø Aquaculture Research Station (Tromsø, Norway). Non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen IPNV sensitive (Aquagen, Kyrksæterøra, Norway), with an average size of 51 g, was used for the challenge two days after transfer to seawater. Bath challenge was performed as described by Johansen and Sommer [53] using an infectious dose of TCID50/ml = 5 × 105 of the field isolates NFH-Ar and NFH-El. Two parallel tanks supplied with 200 L of 10°C seawater were used for each isolate. Each tank contained 70 fish and a separate tank was used for 70 uninfected control fish. The fish were fed daily on commercial feed. The experiment was terminated 30 days after challenge.
The challenge was carried out at Tromsø Aquaculture Research Station (Tromsø, Norway). Non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen IPNV sensitive (Aquagen, Kyrksæterøra, Norway), with an average size of 51 g, was used for the challenge two days after transfer to seawater. Bath challenge was performed as described by Johansen and Sommer [53] using an infectious dose of TCID50/ml = 5 × 105 of the field isolates NFH-Ar and NFH-El. Two parallel tanks supplied with 200 L of 10°C seawater were used for each isolate. Each tank contained 70 fish and a separate tank was used for 70 uninfected control fish. The fish were fed daily on commercial feed. The experiment was terminated 30 days after challenge.
Sampling Mortality was monitored throughout the experiment by counting dead fish. An IPNV rapid agglutination kit (Phadebact coating kit, Boule Diagnostics AB) was used on head kidney samples from dead fish to verify IPN as the cause of death. Percent cumulative mortality in infected fish was calculated compared to control fish using GraphPad Prism 4.0 tool for statistical analyses. Sampling was performed on surviving fish. Pancreatic tissue and head kidney were aseptically removed from 4 fish in each tank at 6, 13 and 29 d p.i. Pancreatic tissue was stored in L-15 medium on ice until frozen at -80°C, whereas head kidney samples were stored in RNAlater® (Ambion) at -80°C.
Mortality was monitored throughout the experiment by counting dead fish. An IPNV rapid agglutination kit (Phadebact coating kit, Boule Diagnostics AB) was used on head kidney samples from dead fish to verify IPN as the cause of death. Percent cumulative mortality in infected fish was calculated compared to control fish using GraphPad Prism 4.0 tool for statistical analyses. Sampling was performed on surviving fish. Pancreatic tissue and head kidney were aseptically removed from 4 fish in each tank at 6, 13 and 29 d p.i. Pancreatic tissue was stored in L-15 medium on ice until frozen at -80°C, whereas head kidney samples were stored in RNAlater® (Ambion) at -80°C.
Quantification of IPNV by titration Pancreatic tissue was homogenized using an Ultra Thurrax T25 basic crusher (IKA-WERKE). Homogenized tissue was diluted to 5% in Eagle's minimum essential medium (EMEM) (Gibco) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 2 mM L-glutamine and 1% non-essential amino acids, before centrifuged at 15000 × g for 5 min at 4°C. Homogenates were inoculated onto CHSE-214 cells in 96 wells plates at a starting concentration of 0.5% (w/v). Individual viral titers were determined by end-point titration using 10-fold dilutions and 2 replicates and calculated by the TCID50 method [54].
Pancreatic tissue was homogenized using an Ultra Thurrax T25 basic crusher (IKA-WERKE). Homogenized tissue was diluted to 5% in Eagle's minimum essential medium (EMEM) (Gibco) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 2 mM L-glutamine and 1% non-essential amino acids, before centrifuged at 15000 × g for 5 min at 4°C. Homogenates were inoculated onto CHSE-214 cells in 96 wells plates at a starting concentration of 0.5% (w/v). Individual viral titers were determined by end-point titration using 10-fold dilutions and 2 replicates and calculated by the TCID50 method [54].
RNA isolation and cDNA synthesis For analyses of cellular and viral gene expression total RNA from head kidney sampled from 8 challenged and 4 control fish at each time point, was isolated using a combination of Trizol and PureLink RNA Kit (Invitrogen). RNA quality was assessed on an agarose gel and the quantity determined by NanoDrop 2000 spectrophotometry (Thermo Scientific). No RNA degradation was observed. After isolation, the RNA was DNase treated applying TURBO DNase (Ambion). cDNA was synthesized in a 25 μl reaction from 200 ng DNase treated total RNA primed with random hexamers (TaqMan Reverse Transcription Reagents kit, Applied Biosystems). The manufacturer's protocol was followed. For isolation of viral RNA after passage in cell culture QIAamp Viral RNA Mini Kit (Qiagen) was used according to the manufacturer's instructions.
For analyses of cellular and viral gene expression total RNA from head kidney sampled from 8 challenged and 4 control fish at each time point, was isolated using a combination of Trizol and PureLink RNA Kit (Invitrogen). RNA quality was assessed on an agarose gel and the quantity determined by NanoDrop 2000 spectrophotometry (Thermo Scientific). No RNA degradation was observed. After isolation, the RNA was DNase treated applying TURBO DNase (Ambion). cDNA was synthesized in a 25 μl reaction from 200 ng DNase treated total RNA primed with random hexamers (TaqMan Reverse Transcription Reagents kit, Applied Biosystems). The manufacturer's protocol was followed. For isolation of viral RNA after passage in cell culture QIAamp Viral RNA Mini Kit (Qiagen) was used according to the manufacturer's instructions.
Sequencing of virus isolates Each virus isolate was sequenced from virus derived material under the following conditions; origin (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 1× in cell culture), input (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 2-3× in cell culture), during infection(head kidney from day 13 (NFH-Ar) or day 29 (NFH-El) infected fish, 8 pooled individuals from each group). Primers specific for each of the genes encoding the five IPNV proteins were used to amplify the individual genes (from head kidney cDNA pooled from 8 fish or cDNA obtained from head kidney virus homogenates from 2 fish passaged in cell culture) in a PCR reaction using Pfu Turbo Hotstart DNA polymerase (Stratagene). The PCR-products were sequenced in both directions using the same primers and BigDye3.1 chemistry and a 3100 gene analyzer. The sequences were aligned with BioEdit 7.0.5 [55] and DnaSP V5 [56] was used for evaluation of mutation rates and search for HVR.
Each virus isolate was sequenced from virus derived material under the following conditions; origin (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 1× in cell culture), input (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 2-3× in cell culture), during infection(head kidney from day 13 (NFH-Ar) or day 29 (NFH-El) infected fish, 8 pooled individuals from each group). Primers specific for each of the genes encoding the five IPNV proteins were used to amplify the individual genes (from head kidney cDNA pooled from 8 fish or cDNA obtained from head kidney virus homogenates from 2 fish passaged in cell culture) in a PCR reaction using Pfu Turbo Hotstart DNA polymerase (Stratagene). The PCR-products were sequenced in both directions using the same primers and BigDye3.1 chemistry and a 3100 gene analyzer. The sequences were aligned with BioEdit 7.0.5 [55] and DnaSP V5 [56] was used for evaluation of mutation rates and search for HVR.
Microarray analyses Microarray analyses were performed at 13 d p.i. on head kidney samples of salmon infected with isolates NFH-Ar and NFH-El (5 individuals from each group, one microarray per individual). The salmonid fish microarray (SFA 2.0 or immunochip, Geo Omnibus GPL6154) contains 1800 unique clones printed each in 6 spot replicates. Pooled samples of uninfected salmon (equal amounts of RNA, n = 4) were used as a common reference. Test and reference RNA (10 μg) were labeled with the fluorescent dyes Cy5-dUTP and Cy3-dUTP respectively (Amersham Pharmacia), which were incorporated in cDNA using the SuperScript™ Indirect cDNA Labeling System (Invitrogen). Synthesis of cDNA was performed at 46°C for 3 hours in a 23 μl reaction volume, following RNA degradation with 2.5 M NaOH at 37°C for 15 min and alkaline neutralization with 2 M Hepes. Labeled cDNA was combined and purified with Microcon YM30 (Millipore). Microarray slides were pre-treated with 1% BSA fraction V, 5× SSC and 0.1% SDS for 30 min at 50°C and then washed with 2× SSC (3 min) followed by 0.2× SSC (3 min) at room temperature and hybridized over-night at 60°C in a cocktail containing 1.3× Denhardt's, 3× SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), 0.06 × SSC (2 min) and 0.06 × SSC (1 min). Scanning was performed with GenePix 4100A and images were processed with GenePix 6.0 (Molecular Devices). The spots were filtered by criterion (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analyses and genes with less than 3 high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. Statistical analyses were performed in two stages. First, technical accuracy was assessed by difference of log2-ER from zero in six spot replicates and genes with significant changes (Student's t-test, p < 0.05) in at least 3 of 5 individuals per group were selected. Next, analysis of biological replicates was carried out and differential expression was assessed by the mean fold change (> 1.5) and difference from control (one sample t-test, p < 0.05).
Microarray analyses were performed at 13 d p.i. on head kidney samples of salmon infected with isolates NFH-Ar and NFH-El (5 individuals from each group, one microarray per individual). The salmonid fish microarray (SFA 2.0 or immunochip, Geo Omnibus GPL6154) contains 1800 unique clones printed each in 6 spot replicates. Pooled samples of uninfected salmon (equal amounts of RNA, n = 4) were used as a common reference. Test and reference RNA (10 μg) were labeled with the fluorescent dyes Cy5-dUTP and Cy3-dUTP respectively (Amersham Pharmacia), which were incorporated in cDNA using the SuperScript™ Indirect cDNA Labeling System (Invitrogen). Synthesis of cDNA was performed at 46°C for 3 hours in a 23 μl reaction volume, following RNA degradation with 2.5 M NaOH at 37°C for 15 min and alkaline neutralization with 2 M Hepes. Labeled cDNA was combined and purified with Microcon YM30 (Millipore). Microarray slides were pre-treated with 1% BSA fraction V, 5× SSC and 0.1% SDS for 30 min at 50°C and then washed with 2× SSC (3 min) followed by 0.2× SSC (3 min) at room temperature and hybridized over-night at 60°C in a cocktail containing 1.3× Denhardt's, 3× SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), 0.06 × SSC (2 min) and 0.06 × SSC (1 min). Scanning was performed with GenePix 4100A and images were processed with GenePix 6.0 (Molecular Devices). The spots were filtered by criterion (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analyses and genes with less than 3 high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. Statistical analyses were performed in two stages. First, technical accuracy was assessed by difference of log2-ER from zero in six spot replicates and genes with significant changes (Student's t-test, p < 0.05) in at least 3 of 5 individuals per group were selected. Next, analysis of biological replicates was carried out and differential expression was assessed by the mean fold change (> 1.5) and difference from control (one sample t-test, p < 0.05).
Quantitative real-time RT PCR (qPCR) analyses PCR primers used with Sybr Green assay were designed using Vector NTI (Invitrogen) and synthesized by Invitrogen (table 3). The amplicon lengths set to be between 50 and 200 bases were checked on 1.5% agarose gel. TaqMan assays employing a hydrolysis probe were designed using Assays-by-design (Applied Biosystems) and synthesized by Applied Biosystems. PCR efficiency was calculated from tenfold serial dilutions of cDNA for each assay in triplicates. qPCR assays were conducted using 2× SYBR® Green Master Mix (Roche Diagnostics) in an optimized 12 μl reaction volume, using 1:10 diluted cDNA, with primer concentrations of 0.4-0.6 μM. PCR was performed in duplicate in 96-well optical plates on Light Cycler 480 (Roche Diagnostics) under the following conditions: 95°C for 5 min (pre-incubation), 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec (amplification), 95°C for 5 sec, and 65°C for 1 min (melting curve). Forty cycles were performed. Assays employing a hydrolysis probe was conducted in a 20 μl reaction using 2.5 μl 1:10 fold diluted cDNA as template and 2× TaqMan Fast Universal PCR Master mix (Applied Biosystems). The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) and the amplification profile was: 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. PCR analyses of the SybrGreen assays for RIG-I, MDA5 and PKR at 13 and 29 d p.i. was also performed at the ABI Prism 7500 FAST Cycler. Relative expression of mRNA was evaluated by ΔΔCT, adjusted for PCR efficiency and normalized against the elongation factor (EF1AB). IPNV VP2 was undetected in control samples and at 6 d p.i., in order to calculate the ΔΔCT values the average of the NFH-El infected fish sampled at 29 d p.i was used as calibrator. Results were analyzed with ANOVA followed with Newman - Keuls test (p < 0.05). (See Table 1 for primers and probes used in the qPCR assays).
Primers and probes used for qPCR analyses
PCR primers used with Sybr Green assay were designed using Vector NTI (Invitrogen) and synthesized by Invitrogen (table 3). The amplicon lengths set to be between 50 and 200 bases were checked on 1.5% agarose gel. TaqMan assays employing a hydrolysis probe were designed using Assays-by-design (Applied Biosystems) and synthesized by Applied Biosystems. PCR efficiency was calculated from tenfold serial dilutions of cDNA for each assay in triplicates. qPCR assays were conducted using 2× SYBR® Green Master Mix (Roche Diagnostics) in an optimized 12 μl reaction volume, using 1:10 diluted cDNA, with primer concentrations of 0.4-0.6 μM. PCR was performed in duplicate in 96-well optical plates on Light Cycler 480 (Roche Diagnostics) under the following conditions: 95°C for 5 min (pre-incubation), 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec (amplification), 95°C for 5 sec, and 65°C for 1 min (melting curve). Forty cycles were performed. Assays employing a hydrolysis probe was conducted in a 20 μl reaction using 2.5 μl 1:10 fold diluted cDNA as template and 2× TaqMan Fast Universal PCR Master mix (Applied Biosystems). The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) and the amplification profile was: 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. PCR analyses of the SybrGreen assays for RIG-I, MDA5 and PKR at 13 and 29 d p.i. was also performed at the ABI Prism 7500 FAST Cycler. Relative expression of mRNA was evaluated by ΔΔCT, adjusted for PCR efficiency and normalized against the elongation factor (EF1AB). IPNV VP2 was undetected in control samples and at 6 d p.i., in order to calculate the ΔΔCT values the average of the NFH-El infected fish sampled at 29 d p.i was used as calibrator. Results were analyzed with ANOVA followed with Newman - Keuls test (p < 0.05). (See Table 1 for primers and probes used in the qPCR assays).
Primers and probes used for qPCR analyses | null | null | Conclusions | AS performed most of the sequencing, assisted with the challenge experiment and quantification of virus, in addition he drafted the manuscript together with IS. qPCR analyses were performed by IS and GT. ME and BNF performed the challenge experiment and some of the sequencing and virus quantification. The microarray analyzes were done by SMJ and GT. AK contributed to this work with gene expression and sequence data and drafting the manuscript. JBJ designed the experiments, analyzed data and contributed to finalizing the manuscript. All authors read and approved the final manuscript. | [
"Background",
"Results",
"Two isolates of IPNV gave divergent mortality rates in a bath challenge",
"Virus quantification in pancreas and head kidney",
"Sequence comparison of the viral genes",
"Gene expression",
"Discussion",
"Conclusions"
] | [
"Despite vaccination programs, outbreaks of infectious pancreatic necrosis disease (IPN) are frequent in farmed salmon fry and post-smolts. Mortality rates observed in outbreaks vary considerably and have in part been ascribed to the inherited differences in susceptibility of the host [1-3]. Environmental stress [4-7] and the viral strains [8] also influence mortality. Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods [9,10]. The production of virus may increase under stress, and carriers can shed the virus and infect surrounding fish [11].\nThe IPN virus (IPNV) is a bi-segmented double-stranded RNA (dsRNA) virus in the family Birnaviridae encoding 5 viral proteins. Segment B encodes the RNA-dependent RNA polymerase VP1. Segment A encodes a polyprotein which is cotranslationally cleaved by the viral encoded serine-lysine protease (VP4) releasing the proteins pVP2 and VP3 [12,13]. pVP2 is further processed by host cell proteases to form the mature outer capsid protein VP2 [14], which is the most abundant virus protein and contains the antigenic regions responsible for induction of neutralizing antibodies in the host [15]. VP3 is the inner structural protein, which bound to dsRNA constitutes the ribonucleoprotein core structure [16]. Additionally VP3 is shown to bind VP1 and to self-associate strongly, indicating that it is a matrix protein [17]. An alternative open reading frame (ORF) on Segment A encodes the small, arginine-rich, non-structural protein VP5. The biological function of IPNV VP5 remains to be determined.\nThe molecular basis of IPNV virulence and its interplay with host antiviral mechanisms are not fully understood. Sano and co-workers [18] were the first to suggest that the virulence of IPNV is associated with Segment A. Several studies using nucleotide sequence analyses have confirmed this and have shown that the VP2 residues 217 and 221 are the major determinant of virulence of IPNV serotype Sp strains. In addition, position 247 was seen as highly variable [19]. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221, while the strains containing Thr221 are almost avirulent, irrespective of the residue at position 217. IPNV isolates also differ in properties related to replication rate and the ability to cause persistent infections. These characteristics can be attributed to the same amino acids as those determining the virulence [8]. Although some of the factors behind these mechanisms are known there are still many questions to be answered.\nDuring viral infections the initial response of the immune system is the induction of type I interferons (IFN), which mediate antiviral and immunomodulatory activity. In Atlantic salmon three different subtypes of type I IFN have been identified: IFN-a, b and c [20]. IFN-a1 and c are both expressed in head kidney and are induced by poly I:C [20]. IFN-a1 has been shown to provide protection against IPNV in salmonid cells [21,22]. The generation of anti-viral responses during infections requires a rapid viral sensing by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) on the cell-surface or within endosomes recognize single-stranded RNA (ssRNA) and dsRNA [23], while the helicases RIG-I and MDA5 recognize ssRNA and dsRNA in the cytosol [24]. Additionally, dsRNA are recognized by PKR [25]. A number of PRRs have been identified in Atlantic salmon including RIG-I [26], MDA5 (GenBank: EG820831), PKR (GenBank: EF523422), TLR3 [20], TLR8 [27], TLR9 [28] and TLR22 (GenBank: CAJ80696[29] and FM206383). The latter is a dsRNA-specific PRR found exclusively in lower vertebrates [30]. Several studies have shown strong activation of immune genes upon challenge with highly virulent IPNV isolates [31,32], and type I IFNs and the IFN-inducible Mx gene were among the most highly up-regulated genes. However, it remains to find, which PRRs are required for the induction of a systemic type I IFN response during IPNV infections.\nLittle is known about the relationship between the host responses and the virulence of different IPNV isolates. The latter can be associated with either down-regulation or excessive stimulation of innate immunity. Studies of IPNV infected cell-lines [33,34] have shown inhibition of IFN signaling. In the current work we have assessed immune gene expression changes during an experimental challenge of salmon post-smolts with both a virulent and an avirulent IPNV field strain. In addition to quantitative real-time RT PCR (qPCR) analyses of selected genes we used cDNA microarray, which expanded the repertoire of genes.\nThe two IPNV isolates used in this study were originally collected from field outbreaks of IPN with 32% and 5% reported mortality, respectively. Initial characterization of the isolates in our lab showed that they both contained the high virulent motif Thr217 Ala221 Thr247 in VP2 defined by Santi et al [19]. A previous bath challenge performed by our group included these isolates, and indicated that they differed substantially in virulence levels reflecting the field mortalities (unpublished). Sequencing of VP2 after this challenge confirmed the Thr217 Ala221 motif in both isolates. However, propagation of the isolates in cell-cultures before the challenge experiment reported here changed the isolate with the initial low mortality to contain a Thr217 Thr221 Thr247 motif (low virulent motif). Santi et al [19] suggested that the Ala-to-Thr substitution at position 221 in VP2 is a molecular determinant for the establishment of a persistent IPNV infection. Thus, we have analyzed the IPNV VP2 nucleotide composition in virus recovered from fish head kidney during the infection. Comparing sequences to the input strains could provide information about the rate and character of virus changes in vivo.",
" Two isolates of IPNV gave divergent mortality rates in a bath challenge Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.\nCumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.\nBefore initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.\nCumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.\n Virus quantification in pancreas and head kidney The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.\nThe levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)\nThe pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.\nThe levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)\n Sequence comparison of the viral genes The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).\nMutation rates in IPNV genome\nThe highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).\nSequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.\nThe sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).\nMutation rates in IPNV genome\nThe highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).\nSequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.\n Gene expression Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.\nDifferentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.\nData are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.\nRegulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.\nThe expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.\nThe antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).\nVariations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.\nMicroarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.\nDifferentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.\nData are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.\nRegulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.\nThe expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.\nThe antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).\nVariations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.",
"Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.\nCumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.",
"The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.\nThe levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)",
"The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).\nMutation rates in IPNV genome\nThe highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).\nSequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.",
"Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.\nDifferentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.\nData are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.\nRegulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.\nThe expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.\nThe antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).\nVariations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.",
"IPNV isolates have been reported to vary considerably in their virulence and pathogenicity for Atlantic salmon [18,19]. Previous studies have indicated that a combination of IPNV virulence and host pathogen interactions determines the outcome of IPNV infections. Occurrence of multiple strains indicates rapid changes of the virus but little is known about the rate and character of mutations. It also remains undefined if susceptibility of salmon to IPNV could be associated with low or excessive immune responses. In this study two Norwegian field isolates of IPNV with marked difference in mortality were applied for experimental infection of Atlantic salmon. For the first time the sequence changes and immune responses to low and high virulence strain were compared within a single challenge test.\nPrevious studies have identified the outer capsid protein VP2 as the main determinant of IPNV virulence as it comprises all the neutralizing epitopes and cell attachment sites that determine host or cell specificity [15]. The amino acid signature associated with virulence of different strains is also identified within the VP2 region [8,19,39]. The sequences of the two isolates in this study were determined before and after the challenge. Initially NFH-Ar had Thr217-Ala221-Thr247 while NFH-El had Thr217-Thr221-Thr247, implying high and low virulence of NFH-Ar and NFH-El respectively (Figure 3A). Thus the results from this study are in accordance with previously reports of the virulence motifs within VP2 [19]. Rapid changes were seen during passage in cell culture and also during infection in the fish. After the challenge both isolates acquired Pro217-Ala221-Ala247 motif in VP2 associated with the moderate to low virulence [19]. Sequence analyses revealed a remarkably high rate of non-synonymous substitutions in the HVR containing the virulence motif. Commonly, high Ka/Ks ratios point to the divergent selection meaning that sequence changes increase the rate of reproduction. However in this study we observed accumulation of mutations that correlated with a reduction in virus proliferation, thus being favorable for the fish. End of mortality in NFH-Ar group coincided with the loss of the high virulence motif. To explain this finding, virus modification as the host's defense mechanisms can be hypothesized. Virus editing is a rapidly expanding research area. At present, the best studied actors are adenosine deaminases (ADARs) that target regions of dsRNA, converting adenosine (A) to inosine (I) resulting in an A to guanosine (G) change after second strand synthesis [40]. ADARs target mRNAs, transposable elements and RNA viruses' genomes. In mammals, several families of viruses show A to G mutations thought to be caused by ADARs [41]. The induction of ADAR in the NFH-Ar infected fish observed in the microarray in this study might indicate a possible role for ADAR in editing IPNV. Most of the changes observed in our study, disregarding whether synonymous or non-synonymous, were from A to G or from T to C (or vice versa) associated with deamination (results not shown). However, whether the mutations in IPNV detected in this study are caused by salmon ADARs is an interesting question that needs to be addressed in future studies.\nThe two virus isolates used in this study showed greater discrepancies in the VP5 region than previously described isolates. The change of VP5 into a shorter protein in the NFH-Ar infected fish might imply a more benign virus since the longer 15.2 kDa VP5 protein was shown to have a potential antagonistic effect on the IFN response by inhibiting IFN-induced expression from the Mx promoter [34] and might thereby benefit the viral replication. However, the domains responsible for this function have not been mapped and can still be present in the shorter 12.1 kDa version. An early stop-codon located in NFH-El leaves this isolate with a severely truncated form of VP5, only 28 amino acids long, and to our knowledge such a mutation has not been reported in other surveys of IPNV field isolates. It has been suggested that VP5 has an anti-apoptotic function, which is probably not essential for the virulence or persistence of the virus [42,43]. Functional significance of the observed changes in VP5 remained unclear. The amino acid sequences of VP1 ORF were identical between the two isolates throughout the study and VP4 were subject to very few mutations.\nThe mortality rates associated with the structural differences described above may be linked to the ability of virus to invade and replicate within the host cells and/or the scale and character of immune responses. Association between mortality and the virus titers was obvious (Figure 1). NFH-El characterized with low replication was avirulent while NFH-Ar infection was fatal at high rate of proliferation at 13 d p.i. To assess the immune responses, we used expression profiling with microarray and qPCR analyses of genes with well-established roles (IFNs, Mx and PRRs) and both approaches produced similar results. The expression levels of VRGs were apparently mirroring the viral titers and expression levels of the IPNV VP2. At 13 d p.i. VRG were induced in salmon infected with NFH-Ar but not with NFH-El consistently with the difference of virus titers. Up-regulation of VRG in NFH-E1 infected fish at 29 d p.i. was in line with the slight increase of virus titer and did not affect mortality. It is likely that the slower replication and concomitant slower spread of the NFH-El strain may allow time for a systemic induction of the host anti-viral system, including adaptive responses.\nThe IFN system is believed to have a crucial role in the first line of defense against virus infections, and in vitro studies have demonstrated that IPNV replication in cell cultures is efficiently inhibited by salmon IFN-a1 [21,22]. Additionally, injection of synthetic IFN-inducers like CpG and poly I:C induce protection against IPNV in Atlantic salmon [44]. In this study IFN-a1 was induced by both viral strains and major up-regulation was seen in the IFN-a1 dependent gene Mx. IFN-c, was not induced and even slightly down-regulated at 29 d p.i. for NFH-Ar. IFN-c is suggested to have a separate regulation from a and b and can be produced by a different cell population than IFN-a1 [20]. IFN-b was not detected in this study or showed consistently low expression levels (data not shown). Despite the suggested role of type I IFN in restraining virus production, results in our lab has demonstrated that IFN-a1 does not completely inhibit IPNV growth but causes a delay in viral protein synthesis [34]. Furthermore, our data suggest that IPNV-encoded proteins may be involved in weakening of IFN signaling [34]. As a result high levels of viral proteins may impair the activity of IFN-induced genes, thus the higher replication rate of NFH-Ar compared to NFH-El may cause a more potent IFN-antagonizing effect of the NFH-Ar strain. However when interpreting the results from live pathogen challenges, it is important to keep in mind the complexity of such studies, where it is not straight forward whether an observed response is a strategy employed by the virus for its own benefit or a response by the host to control the virus.\nAlthough innate immunity by itself represents a powerful system to combat viral invaders, many infections can only be cleared in combination with adaptive immunity. In this regard type I IFNs are known to promote the adaptive arm including both T cell mediated cellular responses and antibody production [45,46]. It is likely that the reduction of virus titers by 29 d p.i. could be associated with the onset of adaptive immune responses. Unlike the genes implicated in the innate immunity, sIgM showed no expression changes at 13 d p.i. and was slightly induced at 29 d p.i.\nResults of this study added knowledge to the understanding of the immune responses after IPNV infections. Expression of a panel of PRRs was assessed including several recently identified genes. TLR8, 9 RIG-I and MDA5 showed up-regulation and followed the same trend as other immune genes. Earlier we observed a modest increase of TLR8 and 9 expression during stimulation and infection [27,28]. TLR22 is reported to recognize dsRNA in pufferfish and when over-expressed it induces type I IFN expression upon IPNV infection, which suggests a possible role for TLR22 in protection against IPNV [30]. However, TLR22 was not induced by IPNV in this study. Unexpectedly, PKR, an IFN-inducible gene, was down-regulated at all time points except 13 d p.i. (Figure 4A). This was in contrast to Mx, another IFN-inducible gene and IFN-a1, which were up-regulated. Functional studies of salmon PKR have to our knowledge not been reported, however flounder PKR was up-regulated both in vitro and in vivo by a negative single stranded RNA virus (SMRV), which also induced Mx expression in vitro [47]. PRRs, PKR and MyD88 were down-regulated at 6 d p.i. in both study groups. This could be explained by migration of leukocytes expressing the PRRs from the head-kidney into the bloodstream at early time-points of the infection.\nIFN-γ was together with Mx the most highly induced immune gene in this study, and high levels of IFN-γ has been reported in other IPNV challenge experiments [31]. IFN-γ is regarded as a typical Th1 cytokine which bridges the innate and adaptive immune responses. Fish IFN-γ share several functional properties with mammalian IFN-γ including macrophage activation [48-50] and rainbow trout IFN-γ is shown to signal through STAT1 [50,51]. Recently, we have observed antiviral activity against IPNV by IFN-γ, although the effect was not as pronounced as described for IFN-a1 [52]. Like in mammals, fish IFN-γ plays diverse roles in different facets of the immune system, and the increased levels of IFN-γ upon IPNV challenge detected here suggest anti-IPNV activity.",
"Our study aimed at investigation of different mortality associated with two IPNV isolates. High incidence of death of NFH-Ar infected salmon was in line with the initial presence of the virulence motif in VP2 and higher virus titers in the head kidney and pancreas compared to NHF-El. PPRs, IFNs and ISGs were induced to a higher magnitude by the high virulent strain NFH-Ar and correlated with the viral load in the head kidney. Subsequent decrease of virus titers and mortality could be due to the sequence changes in the hyper variable regions of IPNV together with the development of acquired immunity in fish."
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Results",
"Two isolates of IPNV gave divergent mortality rates in a bath challenge",
"Virus quantification in pancreas and head kidney",
"Sequence comparison of the viral genes",
"Gene expression",
"Discussion",
"Conclusions",
"Methods",
"Supplementary Material"
] | [
"Despite vaccination programs, outbreaks of infectious pancreatic necrosis disease (IPN) are frequent in farmed salmon fry and post-smolts. Mortality rates observed in outbreaks vary considerably and have in part been ascribed to the inherited differences in susceptibility of the host [1-3]. Environmental stress [4-7] and the viral strains [8] also influence mortality. Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods [9,10]. The production of virus may increase under stress, and carriers can shed the virus and infect surrounding fish [11].\nThe IPN virus (IPNV) is a bi-segmented double-stranded RNA (dsRNA) virus in the family Birnaviridae encoding 5 viral proteins. Segment B encodes the RNA-dependent RNA polymerase VP1. Segment A encodes a polyprotein which is cotranslationally cleaved by the viral encoded serine-lysine protease (VP4) releasing the proteins pVP2 and VP3 [12,13]. pVP2 is further processed by host cell proteases to form the mature outer capsid protein VP2 [14], which is the most abundant virus protein and contains the antigenic regions responsible for induction of neutralizing antibodies in the host [15]. VP3 is the inner structural protein, which bound to dsRNA constitutes the ribonucleoprotein core structure [16]. Additionally VP3 is shown to bind VP1 and to self-associate strongly, indicating that it is a matrix protein [17]. An alternative open reading frame (ORF) on Segment A encodes the small, arginine-rich, non-structural protein VP5. The biological function of IPNV VP5 remains to be determined.\nThe molecular basis of IPNV virulence and its interplay with host antiviral mechanisms are not fully understood. Sano and co-workers [18] were the first to suggest that the virulence of IPNV is associated with Segment A. Several studies using nucleotide sequence analyses have confirmed this and have shown that the VP2 residues 217 and 221 are the major determinant of virulence of IPNV serotype Sp strains. In addition, position 247 was seen as highly variable [19]. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221, while the strains containing Thr221 are almost avirulent, irrespective of the residue at position 217. IPNV isolates also differ in properties related to replication rate and the ability to cause persistent infections. These characteristics can be attributed to the same amino acids as those determining the virulence [8]. Although some of the factors behind these mechanisms are known there are still many questions to be answered.\nDuring viral infections the initial response of the immune system is the induction of type I interferons (IFN), which mediate antiviral and immunomodulatory activity. In Atlantic salmon three different subtypes of type I IFN have been identified: IFN-a, b and c [20]. IFN-a1 and c are both expressed in head kidney and are induced by poly I:C [20]. IFN-a1 has been shown to provide protection against IPNV in salmonid cells [21,22]. The generation of anti-viral responses during infections requires a rapid viral sensing by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) on the cell-surface or within endosomes recognize single-stranded RNA (ssRNA) and dsRNA [23], while the helicases RIG-I and MDA5 recognize ssRNA and dsRNA in the cytosol [24]. Additionally, dsRNA are recognized by PKR [25]. A number of PRRs have been identified in Atlantic salmon including RIG-I [26], MDA5 (GenBank: EG820831), PKR (GenBank: EF523422), TLR3 [20], TLR8 [27], TLR9 [28] and TLR22 (GenBank: CAJ80696[29] and FM206383). The latter is a dsRNA-specific PRR found exclusively in lower vertebrates [30]. Several studies have shown strong activation of immune genes upon challenge with highly virulent IPNV isolates [31,32], and type I IFNs and the IFN-inducible Mx gene were among the most highly up-regulated genes. However, it remains to find, which PRRs are required for the induction of a systemic type I IFN response during IPNV infections.\nLittle is known about the relationship between the host responses and the virulence of different IPNV isolates. The latter can be associated with either down-regulation or excessive stimulation of innate immunity. Studies of IPNV infected cell-lines [33,34] have shown inhibition of IFN signaling. In the current work we have assessed immune gene expression changes during an experimental challenge of salmon post-smolts with both a virulent and an avirulent IPNV field strain. In addition to quantitative real-time RT PCR (qPCR) analyses of selected genes we used cDNA microarray, which expanded the repertoire of genes.\nThe two IPNV isolates used in this study were originally collected from field outbreaks of IPN with 32% and 5% reported mortality, respectively. Initial characterization of the isolates in our lab showed that they both contained the high virulent motif Thr217 Ala221 Thr247 in VP2 defined by Santi et al [19]. A previous bath challenge performed by our group included these isolates, and indicated that they differed substantially in virulence levels reflecting the field mortalities (unpublished). Sequencing of VP2 after this challenge confirmed the Thr217 Ala221 motif in both isolates. However, propagation of the isolates in cell-cultures before the challenge experiment reported here changed the isolate with the initial low mortality to contain a Thr217 Thr221 Thr247 motif (low virulent motif). Santi et al [19] suggested that the Ala-to-Thr substitution at position 221 in VP2 is a molecular determinant for the establishment of a persistent IPNV infection. Thus, we have analyzed the IPNV VP2 nucleotide composition in virus recovered from fish head kidney during the infection. Comparing sequences to the input strains could provide information about the rate and character of virus changes in vivo.",
" Two isolates of IPNV gave divergent mortality rates in a bath challenge Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.\nCumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.\nBefore initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.\nCumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.\n Virus quantification in pancreas and head kidney The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.\nThe levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)\nThe pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.\nThe levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)\n Sequence comparison of the viral genes The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).\nMutation rates in IPNV genome\nThe highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).\nSequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.\nThe sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).\nMutation rates in IPNV genome\nThe highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).\nSequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.\n Gene expression Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.\nDifferentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.\nData are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.\nRegulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.\nThe expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.\nThe antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).\nVariations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.\nMicroarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.\nDifferentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.\nData are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.\nRegulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.\nThe expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.\nThe antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).\nVariations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.",
"Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.\nCumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.",
"The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.\nThe levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)",
"The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).\nMutation rates in IPNV genome\nThe highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).\nSequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.",
"Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.\nDifferentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.\nData are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.\nRegulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.\nThe expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.\nThe antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).\nVariations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.",
"IPNV isolates have been reported to vary considerably in their virulence and pathogenicity for Atlantic salmon [18,19]. Previous studies have indicated that a combination of IPNV virulence and host pathogen interactions determines the outcome of IPNV infections. Occurrence of multiple strains indicates rapid changes of the virus but little is known about the rate and character of mutations. It also remains undefined if susceptibility of salmon to IPNV could be associated with low or excessive immune responses. In this study two Norwegian field isolates of IPNV with marked difference in mortality were applied for experimental infection of Atlantic salmon. For the first time the sequence changes and immune responses to low and high virulence strain were compared within a single challenge test.\nPrevious studies have identified the outer capsid protein VP2 as the main determinant of IPNV virulence as it comprises all the neutralizing epitopes and cell attachment sites that determine host or cell specificity [15]. The amino acid signature associated with virulence of different strains is also identified within the VP2 region [8,19,39]. The sequences of the two isolates in this study were determined before and after the challenge. Initially NFH-Ar had Thr217-Ala221-Thr247 while NFH-El had Thr217-Thr221-Thr247, implying high and low virulence of NFH-Ar and NFH-El respectively (Figure 3A). Thus the results from this study are in accordance with previously reports of the virulence motifs within VP2 [19]. Rapid changes were seen during passage in cell culture and also during infection in the fish. After the challenge both isolates acquired Pro217-Ala221-Ala247 motif in VP2 associated with the moderate to low virulence [19]. Sequence analyses revealed a remarkably high rate of non-synonymous substitutions in the HVR containing the virulence motif. Commonly, high Ka/Ks ratios point to the divergent selection meaning that sequence changes increase the rate of reproduction. However in this study we observed accumulation of mutations that correlated with a reduction in virus proliferation, thus being favorable for the fish. End of mortality in NFH-Ar group coincided with the loss of the high virulence motif. To explain this finding, virus modification as the host's defense mechanisms can be hypothesized. Virus editing is a rapidly expanding research area. At present, the best studied actors are adenosine deaminases (ADARs) that target regions of dsRNA, converting adenosine (A) to inosine (I) resulting in an A to guanosine (G) change after second strand synthesis [40]. ADARs target mRNAs, transposable elements and RNA viruses' genomes. In mammals, several families of viruses show A to G mutations thought to be caused by ADARs [41]. The induction of ADAR in the NFH-Ar infected fish observed in the microarray in this study might indicate a possible role for ADAR in editing IPNV. Most of the changes observed in our study, disregarding whether synonymous or non-synonymous, were from A to G or from T to C (or vice versa) associated with deamination (results not shown). However, whether the mutations in IPNV detected in this study are caused by salmon ADARs is an interesting question that needs to be addressed in future studies.\nThe two virus isolates used in this study showed greater discrepancies in the VP5 region than previously described isolates. The change of VP5 into a shorter protein in the NFH-Ar infected fish might imply a more benign virus since the longer 15.2 kDa VP5 protein was shown to have a potential antagonistic effect on the IFN response by inhibiting IFN-induced expression from the Mx promoter [34] and might thereby benefit the viral replication. However, the domains responsible for this function have not been mapped and can still be present in the shorter 12.1 kDa version. An early stop-codon located in NFH-El leaves this isolate with a severely truncated form of VP5, only 28 amino acids long, and to our knowledge such a mutation has not been reported in other surveys of IPNV field isolates. It has been suggested that VP5 has an anti-apoptotic function, which is probably not essential for the virulence or persistence of the virus [42,43]. Functional significance of the observed changes in VP5 remained unclear. The amino acid sequences of VP1 ORF were identical between the two isolates throughout the study and VP4 were subject to very few mutations.\nThe mortality rates associated with the structural differences described above may be linked to the ability of virus to invade and replicate within the host cells and/or the scale and character of immune responses. Association between mortality and the virus titers was obvious (Figure 1). NFH-El characterized with low replication was avirulent while NFH-Ar infection was fatal at high rate of proliferation at 13 d p.i. To assess the immune responses, we used expression profiling with microarray and qPCR analyses of genes with well-established roles (IFNs, Mx and PRRs) and both approaches produced similar results. The expression levels of VRGs were apparently mirroring the viral titers and expression levels of the IPNV VP2. At 13 d p.i. VRG were induced in salmon infected with NFH-Ar but not with NFH-El consistently with the difference of virus titers. Up-regulation of VRG in NFH-E1 infected fish at 29 d p.i. was in line with the slight increase of virus titer and did not affect mortality. It is likely that the slower replication and concomitant slower spread of the NFH-El strain may allow time for a systemic induction of the host anti-viral system, including adaptive responses.\nThe IFN system is believed to have a crucial role in the first line of defense against virus infections, and in vitro studies have demonstrated that IPNV replication in cell cultures is efficiently inhibited by salmon IFN-a1 [21,22]. Additionally, injection of synthetic IFN-inducers like CpG and poly I:C induce protection against IPNV in Atlantic salmon [44]. In this study IFN-a1 was induced by both viral strains and major up-regulation was seen in the IFN-a1 dependent gene Mx. IFN-c, was not induced and even slightly down-regulated at 29 d p.i. for NFH-Ar. IFN-c is suggested to have a separate regulation from a and b and can be produced by a different cell population than IFN-a1 [20]. IFN-b was not detected in this study or showed consistently low expression levels (data not shown). Despite the suggested role of type I IFN in restraining virus production, results in our lab has demonstrated that IFN-a1 does not completely inhibit IPNV growth but causes a delay in viral protein synthesis [34]. Furthermore, our data suggest that IPNV-encoded proteins may be involved in weakening of IFN signaling [34]. As a result high levels of viral proteins may impair the activity of IFN-induced genes, thus the higher replication rate of NFH-Ar compared to NFH-El may cause a more potent IFN-antagonizing effect of the NFH-Ar strain. However when interpreting the results from live pathogen challenges, it is important to keep in mind the complexity of such studies, where it is not straight forward whether an observed response is a strategy employed by the virus for its own benefit or a response by the host to control the virus.\nAlthough innate immunity by itself represents a powerful system to combat viral invaders, many infections can only be cleared in combination with adaptive immunity. In this regard type I IFNs are known to promote the adaptive arm including both T cell mediated cellular responses and antibody production [45,46]. It is likely that the reduction of virus titers by 29 d p.i. could be associated with the onset of adaptive immune responses. Unlike the genes implicated in the innate immunity, sIgM showed no expression changes at 13 d p.i. and was slightly induced at 29 d p.i.\nResults of this study added knowledge to the understanding of the immune responses after IPNV infections. Expression of a panel of PRRs was assessed including several recently identified genes. TLR8, 9 RIG-I and MDA5 showed up-regulation and followed the same trend as other immune genes. Earlier we observed a modest increase of TLR8 and 9 expression during stimulation and infection [27,28]. TLR22 is reported to recognize dsRNA in pufferfish and when over-expressed it induces type I IFN expression upon IPNV infection, which suggests a possible role for TLR22 in protection against IPNV [30]. However, TLR22 was not induced by IPNV in this study. Unexpectedly, PKR, an IFN-inducible gene, was down-regulated at all time points except 13 d p.i. (Figure 4A). This was in contrast to Mx, another IFN-inducible gene and IFN-a1, which were up-regulated. Functional studies of salmon PKR have to our knowledge not been reported, however flounder PKR was up-regulated both in vitro and in vivo by a negative single stranded RNA virus (SMRV), which also induced Mx expression in vitro [47]. PRRs, PKR and MyD88 were down-regulated at 6 d p.i. in both study groups. This could be explained by migration of leukocytes expressing the PRRs from the head-kidney into the bloodstream at early time-points of the infection.\nIFN-γ was together with Mx the most highly induced immune gene in this study, and high levels of IFN-γ has been reported in other IPNV challenge experiments [31]. IFN-γ is regarded as a typical Th1 cytokine which bridges the innate and adaptive immune responses. Fish IFN-γ share several functional properties with mammalian IFN-γ including macrophage activation [48-50] and rainbow trout IFN-γ is shown to signal through STAT1 [50,51]. Recently, we have observed antiviral activity against IPNV by IFN-γ, although the effect was not as pronounced as described for IFN-a1 [52]. Like in mammals, fish IFN-γ plays diverse roles in different facets of the immune system, and the increased levels of IFN-γ upon IPNV challenge detected here suggest anti-IPNV activity.",
"Our study aimed at investigation of different mortality associated with two IPNV isolates. High incidence of death of NFH-Ar infected salmon was in line with the initial presence of the virulence motif in VP2 and higher virus titers in the head kidney and pancreas compared to NHF-El. PPRs, IFNs and ISGs were induced to a higher magnitude by the high virulent strain NFH-Ar and correlated with the viral load in the head kidney. Subsequent decrease of virus titers and mortality could be due to the sequence changes in the hyper variable regions of IPNV together with the development of acquired immunity in fish.",
" Virus isolates Two IPNV isolates, referred to as NFH-Ar and NFH-El, collected from field outbreaks of IPN in 2004 were used in this study. Veterinarians had diagnosed IPN based on clinical observations and by use of an agglutination assay (Phadebact coating kit, Boule Diagnostics AB). The NFH-Ar isolate, obtained from an IPN outbreak in Frøya, Norway in 2004, was reported to give 32% mortality, while the NFH-El, obtained from an IPN outbreak in Alta, Norway in 2004, was reported to give 5% mortality. Head kidney samples collected during these outbreaks were sent to our lab and tissue homogenates were made and inoculated on CHSE-214 cells for propagation and sequencing. For NFH-Ar a second, and for NFH-El a third cell-culture passage of the virus strain was used in the present experiment. The input strains were sequenced before challenge.\nTwo IPNV isolates, referred to as NFH-Ar and NFH-El, collected from field outbreaks of IPN in 2004 were used in this study. Veterinarians had diagnosed IPN based on clinical observations and by use of an agglutination assay (Phadebact coating kit, Boule Diagnostics AB). The NFH-Ar isolate, obtained from an IPN outbreak in Frøya, Norway in 2004, was reported to give 32% mortality, while the NFH-El, obtained from an IPN outbreak in Alta, Norway in 2004, was reported to give 5% mortality. Head kidney samples collected during these outbreaks were sent to our lab and tissue homogenates were made and inoculated on CHSE-214 cells for propagation and sequencing. For NFH-Ar a second, and for NFH-El a third cell-culture passage of the virus strain was used in the present experiment. The input strains were sequenced before challenge.\n IPNV challenge of Atlantic salmon The challenge was carried out at Tromsø Aquaculture Research Station (Tromsø, Norway). Non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen IPNV sensitive (Aquagen, Kyrksæterøra, Norway), with an average size of 51 g, was used for the challenge two days after transfer to seawater. Bath challenge was performed as described by Johansen and Sommer [53] using an infectious dose of TCID50/ml = 5 × 105 of the field isolates NFH-Ar and NFH-El. Two parallel tanks supplied with 200 L of 10°C seawater were used for each isolate. Each tank contained 70 fish and a separate tank was used for 70 uninfected control fish. The fish were fed daily on commercial feed. The experiment was terminated 30 days after challenge.\nThe challenge was carried out at Tromsø Aquaculture Research Station (Tromsø, Norway). Non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen IPNV sensitive (Aquagen, Kyrksæterøra, Norway), with an average size of 51 g, was used for the challenge two days after transfer to seawater. Bath challenge was performed as described by Johansen and Sommer [53] using an infectious dose of TCID50/ml = 5 × 105 of the field isolates NFH-Ar and NFH-El. Two parallel tanks supplied with 200 L of 10°C seawater were used for each isolate. Each tank contained 70 fish and a separate tank was used for 70 uninfected control fish. The fish were fed daily on commercial feed. The experiment was terminated 30 days after challenge.\n Sampling Mortality was monitored throughout the experiment by counting dead fish. An IPNV rapid agglutination kit (Phadebact coating kit, Boule Diagnostics AB) was used on head kidney samples from dead fish to verify IPN as the cause of death. Percent cumulative mortality in infected fish was calculated compared to control fish using GraphPad Prism 4.0 tool for statistical analyses. Sampling was performed on surviving fish. Pancreatic tissue and head kidney were aseptically removed from 4 fish in each tank at 6, 13 and 29 d p.i. Pancreatic tissue was stored in L-15 medium on ice until frozen at -80°C, whereas head kidney samples were stored in RNAlater® (Ambion) at -80°C.\nMortality was monitored throughout the experiment by counting dead fish. An IPNV rapid agglutination kit (Phadebact coating kit, Boule Diagnostics AB) was used on head kidney samples from dead fish to verify IPN as the cause of death. Percent cumulative mortality in infected fish was calculated compared to control fish using GraphPad Prism 4.0 tool for statistical analyses. Sampling was performed on surviving fish. Pancreatic tissue and head kidney were aseptically removed from 4 fish in each tank at 6, 13 and 29 d p.i. Pancreatic tissue was stored in L-15 medium on ice until frozen at -80°C, whereas head kidney samples were stored in RNAlater® (Ambion) at -80°C.\n Quantification of IPNV by titration Pancreatic tissue was homogenized using an Ultra Thurrax T25 basic crusher (IKA-WERKE). Homogenized tissue was diluted to 5% in Eagle's minimum essential medium (EMEM) (Gibco) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 2 mM L-glutamine and 1% non-essential amino acids, before centrifuged at 15000 × g for 5 min at 4°C. Homogenates were inoculated onto CHSE-214 cells in 96 wells plates at a starting concentration of 0.5% (w/v). Individual viral titers were determined by end-point titration using 10-fold dilutions and 2 replicates and calculated by the TCID50 method [54].\nPancreatic tissue was homogenized using an Ultra Thurrax T25 basic crusher (IKA-WERKE). Homogenized tissue was diluted to 5% in Eagle's minimum essential medium (EMEM) (Gibco) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 2 mM L-glutamine and 1% non-essential amino acids, before centrifuged at 15000 × g for 5 min at 4°C. Homogenates were inoculated onto CHSE-214 cells in 96 wells plates at a starting concentration of 0.5% (w/v). Individual viral titers were determined by end-point titration using 10-fold dilutions and 2 replicates and calculated by the TCID50 method [54].\n RNA isolation and cDNA synthesis For analyses of cellular and viral gene expression total RNA from head kidney sampled from 8 challenged and 4 control fish at each time point, was isolated using a combination of Trizol and PureLink RNA Kit (Invitrogen). RNA quality was assessed on an agarose gel and the quantity determined by NanoDrop 2000 spectrophotometry (Thermo Scientific). No RNA degradation was observed. After isolation, the RNA was DNase treated applying TURBO DNase (Ambion). cDNA was synthesized in a 25 μl reaction from 200 ng DNase treated total RNA primed with random hexamers (TaqMan Reverse Transcription Reagents kit, Applied Biosystems). The manufacturer's protocol was followed. For isolation of viral RNA after passage in cell culture QIAamp Viral RNA Mini Kit (Qiagen) was used according to the manufacturer's instructions.\nFor analyses of cellular and viral gene expression total RNA from head kidney sampled from 8 challenged and 4 control fish at each time point, was isolated using a combination of Trizol and PureLink RNA Kit (Invitrogen). RNA quality was assessed on an agarose gel and the quantity determined by NanoDrop 2000 spectrophotometry (Thermo Scientific). No RNA degradation was observed. After isolation, the RNA was DNase treated applying TURBO DNase (Ambion). cDNA was synthesized in a 25 μl reaction from 200 ng DNase treated total RNA primed with random hexamers (TaqMan Reverse Transcription Reagents kit, Applied Biosystems). The manufacturer's protocol was followed. For isolation of viral RNA after passage in cell culture QIAamp Viral RNA Mini Kit (Qiagen) was used according to the manufacturer's instructions.\n Sequencing of virus isolates Each virus isolate was sequenced from virus derived material under the following conditions; origin (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 1× in cell culture), input (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 2-3× in cell culture), during infection(head kidney from day 13 (NFH-Ar) or day 29 (NFH-El) infected fish, 8 pooled individuals from each group). Primers specific for each of the genes encoding the five IPNV proteins were used to amplify the individual genes (from head kidney cDNA pooled from 8 fish or cDNA obtained from head kidney virus homogenates from 2 fish passaged in cell culture) in a PCR reaction using Pfu Turbo Hotstart DNA polymerase (Stratagene). The PCR-products were sequenced in both directions using the same primers and BigDye3.1 chemistry and a 3100 gene analyzer. The sequences were aligned with BioEdit 7.0.5 [55] and DnaSP V5 [56] was used for evaluation of mutation rates and search for HVR.\nEach virus isolate was sequenced from virus derived material under the following conditions; origin (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 1× in cell culture), input (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 2-3× in cell culture), during infection(head kidney from day 13 (NFH-Ar) or day 29 (NFH-El) infected fish, 8 pooled individuals from each group). Primers specific for each of the genes encoding the five IPNV proteins were used to amplify the individual genes (from head kidney cDNA pooled from 8 fish or cDNA obtained from head kidney virus homogenates from 2 fish passaged in cell culture) in a PCR reaction using Pfu Turbo Hotstart DNA polymerase (Stratagene). The PCR-products were sequenced in both directions using the same primers and BigDye3.1 chemistry and a 3100 gene analyzer. The sequences were aligned with BioEdit 7.0.5 [55] and DnaSP V5 [56] was used for evaluation of mutation rates and search for HVR.\n Microarray analyses Microarray analyses were performed at 13 d p.i. on head kidney samples of salmon infected with isolates NFH-Ar and NFH-El (5 individuals from each group, one microarray per individual). The salmonid fish microarray (SFA 2.0 or immunochip, Geo Omnibus GPL6154) contains 1800 unique clones printed each in 6 spot replicates. Pooled samples of uninfected salmon (equal amounts of RNA, n = 4) were used as a common reference. Test and reference RNA (10 μg) were labeled with the fluorescent dyes Cy5-dUTP and Cy3-dUTP respectively (Amersham Pharmacia), which were incorporated in cDNA using the SuperScript™ Indirect cDNA Labeling System (Invitrogen). Synthesis of cDNA was performed at 46°C for 3 hours in a 23 μl reaction volume, following RNA degradation with 2.5 M NaOH at 37°C for 15 min and alkaline neutralization with 2 M Hepes. Labeled cDNA was combined and purified with Microcon YM30 (Millipore). Microarray slides were pre-treated with 1% BSA fraction V, 5× SSC and 0.1% SDS for 30 min at 50°C and then washed with 2× SSC (3 min) followed by 0.2× SSC (3 min) at room temperature and hybridized over-night at 60°C in a cocktail containing 1.3× Denhardt's, 3× SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), 0.06 × SSC (2 min) and 0.06 × SSC (1 min). Scanning was performed with GenePix 4100A and images were processed with GenePix 6.0 (Molecular Devices). The spots were filtered by criterion (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analyses and genes with less than 3 high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. Statistical analyses were performed in two stages. First, technical accuracy was assessed by difference of log2-ER from zero in six spot replicates and genes with significant changes (Student's t-test, p < 0.05) in at least 3 of 5 individuals per group were selected. Next, analysis of biological replicates was carried out and differential expression was assessed by the mean fold change (> 1.5) and difference from control (one sample t-test, p < 0.05).\nMicroarray analyses were performed at 13 d p.i. on head kidney samples of salmon infected with isolates NFH-Ar and NFH-El (5 individuals from each group, one microarray per individual). The salmonid fish microarray (SFA 2.0 or immunochip, Geo Omnibus GPL6154) contains 1800 unique clones printed each in 6 spot replicates. Pooled samples of uninfected salmon (equal amounts of RNA, n = 4) were used as a common reference. Test and reference RNA (10 μg) were labeled with the fluorescent dyes Cy5-dUTP and Cy3-dUTP respectively (Amersham Pharmacia), which were incorporated in cDNA using the SuperScript™ Indirect cDNA Labeling System (Invitrogen). Synthesis of cDNA was performed at 46°C for 3 hours in a 23 μl reaction volume, following RNA degradation with 2.5 M NaOH at 37°C for 15 min and alkaline neutralization with 2 M Hepes. Labeled cDNA was combined and purified with Microcon YM30 (Millipore). Microarray slides were pre-treated with 1% BSA fraction V, 5× SSC and 0.1% SDS for 30 min at 50°C and then washed with 2× SSC (3 min) followed by 0.2× SSC (3 min) at room temperature and hybridized over-night at 60°C in a cocktail containing 1.3× Denhardt's, 3× SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), 0.06 × SSC (2 min) and 0.06 × SSC (1 min). Scanning was performed with GenePix 4100A and images were processed with GenePix 6.0 (Molecular Devices). The spots were filtered by criterion (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analyses and genes with less than 3 high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. Statistical analyses were performed in two stages. First, technical accuracy was assessed by difference of log2-ER from zero in six spot replicates and genes with significant changes (Student's t-test, p < 0.05) in at least 3 of 5 individuals per group were selected. Next, analysis of biological replicates was carried out and differential expression was assessed by the mean fold change (> 1.5) and difference from control (one sample t-test, p < 0.05).\n Quantitative real-time RT PCR (qPCR) analyses PCR primers used with Sybr Green assay were designed using Vector NTI (Invitrogen) and synthesized by Invitrogen (table 3). The amplicon lengths set to be between 50 and 200 bases were checked on 1.5% agarose gel. TaqMan assays employing a hydrolysis probe were designed using Assays-by-design (Applied Biosystems) and synthesized by Applied Biosystems. PCR efficiency was calculated from tenfold serial dilutions of cDNA for each assay in triplicates. qPCR assays were conducted using 2× SYBR® Green Master Mix (Roche Diagnostics) in an optimized 12 μl reaction volume, using 1:10 diluted cDNA, with primer concentrations of 0.4-0.6 μM. PCR was performed in duplicate in 96-well optical plates on Light Cycler 480 (Roche Diagnostics) under the following conditions: 95°C for 5 min (pre-incubation), 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec (amplification), 95°C for 5 sec, and 65°C for 1 min (melting curve). Forty cycles were performed. Assays employing a hydrolysis probe was conducted in a 20 μl reaction using 2.5 μl 1:10 fold diluted cDNA as template and 2× TaqMan Fast Universal PCR Master mix (Applied Biosystems). The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) and the amplification profile was: 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. PCR analyses of the SybrGreen assays for RIG-I, MDA5 and PKR at 13 and 29 d p.i. was also performed at the ABI Prism 7500 FAST Cycler. Relative expression of mRNA was evaluated by ΔΔCT, adjusted for PCR efficiency and normalized against the elongation factor (EF1AB). IPNV VP2 was undetected in control samples and at 6 d p.i., in order to calculate the ΔΔCT values the average of the NFH-El infected fish sampled at 29 d p.i was used as calibrator. Results were analyzed with ANOVA followed with Newman - Keuls test (p < 0.05). (See Table 1 for primers and probes used in the qPCR assays).\nPrimers and probes used for qPCR analyses\nPCR primers used with Sybr Green assay were designed using Vector NTI (Invitrogen) and synthesized by Invitrogen (table 3). The amplicon lengths set to be between 50 and 200 bases were checked on 1.5% agarose gel. TaqMan assays employing a hydrolysis probe were designed using Assays-by-design (Applied Biosystems) and synthesized by Applied Biosystems. PCR efficiency was calculated from tenfold serial dilutions of cDNA for each assay in triplicates. qPCR assays were conducted using 2× SYBR® Green Master Mix (Roche Diagnostics) in an optimized 12 μl reaction volume, using 1:10 diluted cDNA, with primer concentrations of 0.4-0.6 μM. PCR was performed in duplicate in 96-well optical plates on Light Cycler 480 (Roche Diagnostics) under the following conditions: 95°C for 5 min (pre-incubation), 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec (amplification), 95°C for 5 sec, and 65°C for 1 min (melting curve). Forty cycles were performed. Assays employing a hydrolysis probe was conducted in a 20 μl reaction using 2.5 μl 1:10 fold diluted cDNA as template and 2× TaqMan Fast Universal PCR Master mix (Applied Biosystems). The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) and the amplification profile was: 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. PCR analyses of the SybrGreen assays for RIG-I, MDA5 and PKR at 13 and 29 d p.i. was also performed at the ABI Prism 7500 FAST Cycler. Relative expression of mRNA was evaluated by ΔΔCT, adjusted for PCR efficiency and normalized against the elongation factor (EF1AB). IPNV VP2 was undetected in control samples and at 6 d p.i., in order to calculate the ΔΔCT values the average of the NFH-El infected fish sampled at 29 d p.i was used as calibrator. Results were analyzed with ANOVA followed with Newman - Keuls test (p < 0.05). (See Table 1 for primers and probes used in the qPCR assays).\nPrimers and probes used for qPCR analyses",
"Differentially expressed genes. The genes were selected as described in Methods. The data provided represent the log2 (Expression ratios) in IPNV infected salmon and control.\nClick here for file"
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] | [
"IPNV",
"birnavirus",
"virulence",
"immune response",
"fish",
"non-synonymous changes"
] | Background:
Despite vaccination programs, outbreaks of infectious pancreatic necrosis disease (IPN) are frequent in farmed salmon fry and post-smolts. Mortality rates observed in outbreaks vary considerably and have in part been ascribed to the inherited differences in susceptibility of the host [1-3]. Environmental stress [4-7] and the viral strains [8] also influence mortality. Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods [9,10]. The production of virus may increase under stress, and carriers can shed the virus and infect surrounding fish [11].
The IPN virus (IPNV) is a bi-segmented double-stranded RNA (dsRNA) virus in the family Birnaviridae encoding 5 viral proteins. Segment B encodes the RNA-dependent RNA polymerase VP1. Segment A encodes a polyprotein which is cotranslationally cleaved by the viral encoded serine-lysine protease (VP4) releasing the proteins pVP2 and VP3 [12,13]. pVP2 is further processed by host cell proteases to form the mature outer capsid protein VP2 [14], which is the most abundant virus protein and contains the antigenic regions responsible for induction of neutralizing antibodies in the host [15]. VP3 is the inner structural protein, which bound to dsRNA constitutes the ribonucleoprotein core structure [16]. Additionally VP3 is shown to bind VP1 and to self-associate strongly, indicating that it is a matrix protein [17]. An alternative open reading frame (ORF) on Segment A encodes the small, arginine-rich, non-structural protein VP5. The biological function of IPNV VP5 remains to be determined.
The molecular basis of IPNV virulence and its interplay with host antiviral mechanisms are not fully understood. Sano and co-workers [18] were the first to suggest that the virulence of IPNV is associated with Segment A. Several studies using nucleotide sequence analyses have confirmed this and have shown that the VP2 residues 217 and 221 are the major determinant of virulence of IPNV serotype Sp strains. In addition, position 247 was seen as highly variable [19]. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221, while the strains containing Thr221 are almost avirulent, irrespective of the residue at position 217. IPNV isolates also differ in properties related to replication rate and the ability to cause persistent infections. These characteristics can be attributed to the same amino acids as those determining the virulence [8]. Although some of the factors behind these mechanisms are known there are still many questions to be answered.
During viral infections the initial response of the immune system is the induction of type I interferons (IFN), which mediate antiviral and immunomodulatory activity. In Atlantic salmon three different subtypes of type I IFN have been identified: IFN-a, b and c [20]. IFN-a1 and c are both expressed in head kidney and are induced by poly I:C [20]. IFN-a1 has been shown to provide protection against IPNV in salmonid cells [21,22]. The generation of anti-viral responses during infections requires a rapid viral sensing by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) on the cell-surface or within endosomes recognize single-stranded RNA (ssRNA) and dsRNA [23], while the helicases RIG-I and MDA5 recognize ssRNA and dsRNA in the cytosol [24]. Additionally, dsRNA are recognized by PKR [25]. A number of PRRs have been identified in Atlantic salmon including RIG-I [26], MDA5 (GenBank: EG820831), PKR (GenBank: EF523422), TLR3 [20], TLR8 [27], TLR9 [28] and TLR22 (GenBank: CAJ80696[29] and FM206383). The latter is a dsRNA-specific PRR found exclusively in lower vertebrates [30]. Several studies have shown strong activation of immune genes upon challenge with highly virulent IPNV isolates [31,32], and type I IFNs and the IFN-inducible Mx gene were among the most highly up-regulated genes. However, it remains to find, which PRRs are required for the induction of a systemic type I IFN response during IPNV infections.
Little is known about the relationship between the host responses and the virulence of different IPNV isolates. The latter can be associated with either down-regulation or excessive stimulation of innate immunity. Studies of IPNV infected cell-lines [33,34] have shown inhibition of IFN signaling. In the current work we have assessed immune gene expression changes during an experimental challenge of salmon post-smolts with both a virulent and an avirulent IPNV field strain. In addition to quantitative real-time RT PCR (qPCR) analyses of selected genes we used cDNA microarray, which expanded the repertoire of genes.
The two IPNV isolates used in this study were originally collected from field outbreaks of IPN with 32% and 5% reported mortality, respectively. Initial characterization of the isolates in our lab showed that they both contained the high virulent motif Thr217 Ala221 Thr247 in VP2 defined by Santi et al [19]. A previous bath challenge performed by our group included these isolates, and indicated that they differed substantially in virulence levels reflecting the field mortalities (unpublished). Sequencing of VP2 after this challenge confirmed the Thr217 Ala221 motif in both isolates. However, propagation of the isolates in cell-cultures before the challenge experiment reported here changed the isolate with the initial low mortality to contain a Thr217 Thr221 Thr247 motif (low virulent motif). Santi et al [19] suggested that the Ala-to-Thr substitution at position 221 in VP2 is a molecular determinant for the establishment of a persistent IPNV infection. Thus, we have analyzed the IPNV VP2 nucleotide composition in virus recovered from fish head kidney during the infection. Comparing sequences to the input strains could provide information about the rate and character of virus changes in vivo.
Results:
Two isolates of IPNV gave divergent mortality rates in a bath challenge Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.
Cumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.
Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.
Cumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.
Virus quantification in pancreas and head kidney The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.
The levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)
The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.
The levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)
Sequence comparison of the viral genes The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).
Mutation rates in IPNV genome
The highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).
Sequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.
The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).
Mutation rates in IPNV genome
The highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).
Sequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.
Gene expression Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.
Differentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.
Data are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.
Regulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.
The expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.
The antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).
Variations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.
Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.
Differentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.
Data are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.
Regulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.
The expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.
The antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).
Variations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.
Two isolates of IPNV gave divergent mortality rates in a bath challenge:
Before initiation of this challenge, head kidney from 10 fish was sampled and checked for the presence of IPNV by carrier tests in pools of 3 individuals. The lower detection limit was 0.88 virus particles g-1 tissue. No IPNV positive pools were found. In the bath challenge study, mortality caused by IPNV was initiated 12 days post infection (d p.i.) with the NFH-Ar isolate and there was a rapid increase in mortality until day 21. The experiment was terminated 30 days post IPNV challenge which was one week after the fish in both groups had ceased to die. At that point, fish infected with the NFH-Ar isolate showed far higher cumulative mortality (38.1%, average of two tanks) compared to the NFH-El isolate which gave only 1.6% cumulative mortality on average (Figure 1A). No mortality was observed among the control fish.
Cumulative mortality and presence of IPN virus in pancreas and head kidney of Atlantic salmon challenged with two different IPNV strains. A. Percent cumulative mortality is significantly higher in fish infected with NFH-Ar (tank I and tank II indicated with dark and light blue, respectively) than with NFH-El (two tanks indicated with orange or yellow) after bath challenge. B. Viral titers in pancreas from 3-4 fish in duplicate tanks at day 6, day 13 and day 29 post challenge, measured by TCID50/g tissue. C. IPNV VP2 transcript levels relative to levels of NFH-El 29 d.p.i in head kidney measured by qPCR. D. IPNVVP2 Ct-values measured in head kidney from individual fish at day 13 and day 29.
Virus quantification in pancreas and head kidney:
The pancreatic tissue was used for quantification of virus as it is one of the primary target organs for IPNV replication. The viral titer was determined as infectious particles per gram tissue from 6 fish infected with each isolate at day 6, and from 8 fish at day 13 and 29. Viral titers increased for both isolates from day 6 to day 13, although for NFH-El (low-mortality groups) the replication was slower compared to NFH-Ar (high-mortality group). A further increase in titer from day 13 to day 29 was observed for NFH-El, whereas NFH-Ar showed decreased titers at 29 d p.i. (Figure 1B). The number of NFH-Ar infectious particles was 3.4 × 103/g in average at day 6, increased to 2.2 × 108 at day 13 and dropped to 8.5 × 105 by the third sampling at day 29. Whereas the number of infectious particles for the NFH-El isolate was slightly higher at the first sampling (average 1.6 × 104/g), only increasing to 3.3 × 104 at day 13 and further leaps to 6.4 × 107 at day 29. The individual variation was greater in the groups infected with NFH-El than the NFH-Ar infected groups.
The levels of IPNV VP2 RNA in the head kidney of NFH-Ar and NFH-El challenged fish were quantified by qPCR during the time-course. We calculated the relative quantity of VP2 by comparing the expression levels in the different samples to NFH-El sampled at 29 d.p.i. VP2 RNA was detected neither in the control fish nor in the two infected groups at 6 d p.i. The qPCR results for other time-points were overall in agreement with the viral titers determined in pancreas samples from the infected fish. The group infected with NFH-Ar had prevalence of 100% (7/7) at day 13 p.i. with an average 1000 fold higher VP2 RNA load to NFH-El 29 d p.i; while at day 29 p.i. the prevalence was 57% (4/7) and VP2 RNA levels were approximately 7 fold higher compared to NFH-El 29 d p.i. (Figure 1C, D). For NFH-El a decrease in IPNV VP2 RNA levels was observed from 13 d p.i. to 29 d p.i. in head kidney. At day 13 d p.i. the prevalence was 100% (8/8) for NFH-El (2 fold higher virus load than 29 d p.i.), while at day 29 it had decreased to 37.5% (3/8)
Sequence comparison of the viral genes:
The sequences encoding the individual viral proteins were determined for each of the isolates to evaluate the rate and character of changes and to search for the differences associated with virulence. The virus isolates were sequenced at several stages of the experiment: initially after the field outbreaks, then after propagating the virus in the CHSE-214 cell-line, before the challenge and finally, in the infected fish. The rate of sequence changes was lowest in VP4 (7.4 sites/kb) being 2-2.7 times greater in other genes (Table 1). VP1 showed a higher rate of synonymous (silent) substitutions (Ka/Ks = 0.03). For VP2 and VP3 the non-synonymous (amino-acid replacement) nucleotide substitution rate was much higher (one order of magnitude greater in VP2 and VP3), and its distribution along the sequences strongly suggested presence of hyper variable regions (HVR). Importantly, the first of two HVRs of VP2 with the highest level of Ka/Ks (Figure 2) coincided with the region that is regarded as a determinant of virulence [8,19]. All mutations in VP5 produced premature stop codons. The deduced amino acid sequences for VP2 and VP5 from the two isolates were aligned to each other and to the reference strain N1 before and after the challenge (Figure 3A, B, C and 3D). After sampling from the original field outbreaks (which were reported to give 32% mortality for the NFH-Ar and 5% mortality for the NFH-El isolate) the isolates were passaged once in CHSE-214 cells before sequence analyses. The VP2 sequences revealed a Thr217-Ala221-Thr247 motif in both isolates (origin) (Figure 3A, B and 3C). Originally, sequencing of VP5 NFH-Ar gave an ORF resulting in a 133 amino acid protein (15.2 kDa predicted), whereas VP5 from NFH-El had a premature stop-codon resulting in a very short ORF, encoding only 28 amino acid residues (3.3 kDa predicted) (Figure 3A and 3D). In the course of this study sequence changes in VP2 and VP5 accumulated continuously, during passage in culture and infection in the fish (Figure 3A). In order to obtain sufficient amount of virus to perform the bath challenge, the isolates were passaged additional times in CHSE-214 cells (2x for NFH-Ar and 3x and NFH-El). Sequencing of this material (challenge input) revealed a change into a Thr217-Thr221-Thr247 motif in VP2 for the NFH-El and a partial shift from Ala221 to Thr221 in NFH-Ar (Figure 3A, B and 3C). Additionally a partial shift from an Arg (R) in position 106 of VP5 to a stop-codon in this position was observed in NFH-Ar (Figure 3A and 3D). After the challenge viral RNA/cDNA from the head kidney of infected fish (n = 8) was sequenced and the VP2 motifs changed into Pro217-Ala221-Ala247 in both isolates during the infection process in the fish (Figure 3A, B and 3C). VP5 in NFH-Ar acquired a stop-codon in position 106, resulting in a 12.1 kDa protein, whereas NFH-El maintained it's short (3.3 kDa) VP5 (Figure 3A and 3D).
Mutation rates in IPNV genome
The highest level of sequence changes in VP2 coincided with the region that is regarded as a determinant of virulence. A. A sliding window plot of mutation frequencies, and B. Ka/Ks ratio in VP2 (window length - 50 sites, step size - 10 sites).
Sequence analyses of IPNV segment A. A. A schematic presentation of the IPNV genome segment A which encode VP2, VP4, VP3 and VP5. Positions of divergent amino acid are indicated, and the sequences of VP2 and VP5 from the two field isolates NFH-Ar and NFH-El are compared to the non-virulent lab-strain N1 before and after the challenge experiment. B. Sequencing chromatograms showing that amino acids in positions 217, 221 and 247 of VP2 were changing rapidly in cell culture and in challenged fish. The PCR-products sequenced from the NFH-Ar strain changed partially from a TAT motif found in the originally infected fish (origin) to a TTT motif after propagation in CHSE-214 cells for the challenge experiment (input). Virus isolated from the fish after the challenge had a PAA motif (HK d13). C. The NFH-El strain changed from a TAT found in the original material (origin) to a TTT motif after three passages in CHSE-214 cells (input). Virus isolated from the fish after this challenge (HK d29) had a PAA motif like seen in the NFH-Ar challenged fish. D. The ORF of VP5for the NFH-Ar strain changed from a 133 amino acid peptide to a 105 amino acid peptide when a stop codon was introduced in position 106 after the challenge. The NFH-El strain initially had a very short VP5 ORF, only 28 amino acids long, which was maintained after the challenge.
Gene expression:
Microarray analyses revealed 211 differentially expressed genes, of which 81 and 130 genes responded to both isolates or to only NFH-Ar respectively; we did not find genes that were regulated exclusively in the NFH-El infected salmon. Both isolates affected genes within different functional groups. Equal down-regulation was observed in genes encoding motor proteins (myosins, tropomyosins, troponins), plasma proteins (plasminogen precursor, coagulation factor X, angiotensin I converting enzyme) and a suite of enzymes involved in xenobiotic metabolism and detoxification (Additional file 1). Similar changes were seen in immune related genes implicated in different processes (Table 2). It is worth noticing that genes involved in biosynthesis of eicosanoids, the inflammatory regulators of lipid origin and several complement factors are down-regulated. At the same time the virus responsive genes (VRG) were up-regulated only in fish infected with NFH-Ar. Some VRGs have well established roles in antiviral responses: antigen presentation (beta-2 microglobulin, MHCI, proteasome subunit), signal transduction downstream of IFN (Jak, STAT) and editing viral RNA (double-stranded RNA-specific adenosine deaminase - ADAR); several more genes are known as IFN-dependent. This group also includes genes with undetermined functions, which however have shown consistent induction in other virus infection studies that used the same microarray [35,36] or the new oligonucleotide platform [37]; these are galectins and galectin binding protein, a tripartite motif protein and SRK2-like tyrosine kinase. Six immune genes were chosen for verification of microarray analyses and results produced with two independent methods were in a good agreement (Figure 4A). At 13 d p.i. significant up-regulation was observed only in salmon challenged with NFH-Ar. At 29 d p.i. expression changes were smaller with no difference between the isolates. The greatest expression changes were seen in SRK2.
Differentially expressed immune genes between IPNV isolates, as assessed by microarray analyses of head kidney at 13 d p.i.
Data are log2-ER ± SE. Significant difference between the study groups (t-test, p < 0.05) is indicated with italics.
Regulation of immune pathways in response to infection with the two IPNV isolates analyzed by qPCR. Data are mean fold change in head kidney transcript levels +/- SE from infected fish relative to controls calculated by the ΔΔCT method, adjusted for PCR efficiency and normalized against the elongation factor. A. Validation of microarray results by qPCR on selected immune genes presented in Table 2 (n = 6). B. Expression of pattern recognition receptors, MyD88 and PKR analyzed in 8 fish per strain at each time point. C. Expression of genes involved in innate immunity/IFN signaling and sIgM analyzed in 8 fish per strain at each time point.
The expression of selected immune genes not presented on the microarray was analyzed by qPCR (Figure 4B, C). The TLR8, 9, 22, RIG-I, MDA5 and PKR are implicated in viral detection [30,38] and MyD88 is a TLR adaptor signaling molecule [27]. Interestingly, all these nucleic acid recognition molecules were slightly down-regulated at 6 d p.i. in both infected groups (Figure 4B). At the later time-points, TLR22 did not show difference between the infected fish and controls. PKR was down-regulated in the infected fish both at day 6 and day 29 p.i., while a modest but significant (p < 0.05) increase in PKR levels could be detected at day 13 p.i for the NFH-El isolate. The remaining PRRs tested had expression profiles that were similar to those observed in other VRG (Figure 4A, C). TLR8 was up-regulated in NFH-Ar infected fish at both time-points which was significant (p < 0.05) for 13 d p.i, while expression change of TLR9 was significant (p < 0.05) at 29 d p.i. The cytoplasmic RNA sensors RIG-I and MDA5 showed 5-fold induction at 13 d p.i. in the NFH-Ar group which was significant (p < 0.05) for RIG-I. MDA5 also showed a large increase at 29 d p.i. in two individuals infected with NFH-El.
The antiviral genes Mx and IFN-γ (type II IFN) were the most highly induced transcripts in the study, and the NFH-Ar infected fish showed the greatest expression changes at 13 d p.i.; 280-fold for Mx and a significant (p < 0.05) 13-fold for IFN-γ (Figure 4C). Their expression levels decreased at 29 d p.i. to 13- and 3-fold up-regulation respectively (Figure 4C). In the NFH-El group Mx and IFN-γ showed highest expression at day 29 p.i. with 29- and 5-fold up-regulation respectively. As illustrated in Figure 5, exemplified by Mx and IFN-γ, there were variations in immune gene expression between individuals in both groups. Nevertheless, for most individuals the immune gene expression mirrored the virus levels, both the IPNV titers in pancreas and VP2 transcript levels in head kidney. Three type I IFN genes had different expression profiles during the challenge. While IFN-c did not respond to infection, IFN-a1 showed a moderate up-regulation both at day 13 (3.5-fold) and day 29 p.i. (2.5-fold) in NFH-Ar group, and at day 29 p.i. in the NFH-El group (5.9 fold). Expression of IFN-b in the head kidney was consistently low or undetected; however, an induction was observed at 13 d p.i. in three of eight NFH-Ar-infected fish (results not shown). Secreted IgM was slightly up-regulated at 29 d p.i for both isolates at similar levels (about 3-fold).
Variations in immune gene expression and virus levels between individuals. Transcript levels of Mx, IFN-γ and IPNV VP2 in head kidney and titers of IPNV in pancreas are shown at individual levels for the two IPNV isolates. The Mx and IFN-γ transcript levels are compared to uninfected control fish, while IPNV VP2 transcripts are compared to the NFH-El group 29d.p.i, IPNV titers were calculated by end point titration and results show TCID50/g tissue. For some individuals the transcript levels were undetected and are thus not presented.
Discussion:
IPNV isolates have been reported to vary considerably in their virulence and pathogenicity for Atlantic salmon [18,19]. Previous studies have indicated that a combination of IPNV virulence and host pathogen interactions determines the outcome of IPNV infections. Occurrence of multiple strains indicates rapid changes of the virus but little is known about the rate and character of mutations. It also remains undefined if susceptibility of salmon to IPNV could be associated with low or excessive immune responses. In this study two Norwegian field isolates of IPNV with marked difference in mortality were applied for experimental infection of Atlantic salmon. For the first time the sequence changes and immune responses to low and high virulence strain were compared within a single challenge test.
Previous studies have identified the outer capsid protein VP2 as the main determinant of IPNV virulence as it comprises all the neutralizing epitopes and cell attachment sites that determine host or cell specificity [15]. The amino acid signature associated with virulence of different strains is also identified within the VP2 region [8,19,39]. The sequences of the two isolates in this study were determined before and after the challenge. Initially NFH-Ar had Thr217-Ala221-Thr247 while NFH-El had Thr217-Thr221-Thr247, implying high and low virulence of NFH-Ar and NFH-El respectively (Figure 3A). Thus the results from this study are in accordance with previously reports of the virulence motifs within VP2 [19]. Rapid changes were seen during passage in cell culture and also during infection in the fish. After the challenge both isolates acquired Pro217-Ala221-Ala247 motif in VP2 associated with the moderate to low virulence [19]. Sequence analyses revealed a remarkably high rate of non-synonymous substitutions in the HVR containing the virulence motif. Commonly, high Ka/Ks ratios point to the divergent selection meaning that sequence changes increase the rate of reproduction. However in this study we observed accumulation of mutations that correlated with a reduction in virus proliferation, thus being favorable for the fish. End of mortality in NFH-Ar group coincided with the loss of the high virulence motif. To explain this finding, virus modification as the host's defense mechanisms can be hypothesized. Virus editing is a rapidly expanding research area. At present, the best studied actors are adenosine deaminases (ADARs) that target regions of dsRNA, converting adenosine (A) to inosine (I) resulting in an A to guanosine (G) change after second strand synthesis [40]. ADARs target mRNAs, transposable elements and RNA viruses' genomes. In mammals, several families of viruses show A to G mutations thought to be caused by ADARs [41]. The induction of ADAR in the NFH-Ar infected fish observed in the microarray in this study might indicate a possible role for ADAR in editing IPNV. Most of the changes observed in our study, disregarding whether synonymous or non-synonymous, were from A to G or from T to C (or vice versa) associated with deamination (results not shown). However, whether the mutations in IPNV detected in this study are caused by salmon ADARs is an interesting question that needs to be addressed in future studies.
The two virus isolates used in this study showed greater discrepancies in the VP5 region than previously described isolates. The change of VP5 into a shorter protein in the NFH-Ar infected fish might imply a more benign virus since the longer 15.2 kDa VP5 protein was shown to have a potential antagonistic effect on the IFN response by inhibiting IFN-induced expression from the Mx promoter [34] and might thereby benefit the viral replication. However, the domains responsible for this function have not been mapped and can still be present in the shorter 12.1 kDa version. An early stop-codon located in NFH-El leaves this isolate with a severely truncated form of VP5, only 28 amino acids long, and to our knowledge such a mutation has not been reported in other surveys of IPNV field isolates. It has been suggested that VP5 has an anti-apoptotic function, which is probably not essential for the virulence or persistence of the virus [42,43]. Functional significance of the observed changes in VP5 remained unclear. The amino acid sequences of VP1 ORF were identical between the two isolates throughout the study and VP4 were subject to very few mutations.
The mortality rates associated with the structural differences described above may be linked to the ability of virus to invade and replicate within the host cells and/or the scale and character of immune responses. Association between mortality and the virus titers was obvious (Figure 1). NFH-El characterized with low replication was avirulent while NFH-Ar infection was fatal at high rate of proliferation at 13 d p.i. To assess the immune responses, we used expression profiling with microarray and qPCR analyses of genes with well-established roles (IFNs, Mx and PRRs) and both approaches produced similar results. The expression levels of VRGs were apparently mirroring the viral titers and expression levels of the IPNV VP2. At 13 d p.i. VRG were induced in salmon infected with NFH-Ar but not with NFH-El consistently with the difference of virus titers. Up-regulation of VRG in NFH-E1 infected fish at 29 d p.i. was in line with the slight increase of virus titer and did not affect mortality. It is likely that the slower replication and concomitant slower spread of the NFH-El strain may allow time for a systemic induction of the host anti-viral system, including adaptive responses.
The IFN system is believed to have a crucial role in the first line of defense against virus infections, and in vitro studies have demonstrated that IPNV replication in cell cultures is efficiently inhibited by salmon IFN-a1 [21,22]. Additionally, injection of synthetic IFN-inducers like CpG and poly I:C induce protection against IPNV in Atlantic salmon [44]. In this study IFN-a1 was induced by both viral strains and major up-regulation was seen in the IFN-a1 dependent gene Mx. IFN-c, was not induced and even slightly down-regulated at 29 d p.i. for NFH-Ar. IFN-c is suggested to have a separate regulation from a and b and can be produced by a different cell population than IFN-a1 [20]. IFN-b was not detected in this study or showed consistently low expression levels (data not shown). Despite the suggested role of type I IFN in restraining virus production, results in our lab has demonstrated that IFN-a1 does not completely inhibit IPNV growth but causes a delay in viral protein synthesis [34]. Furthermore, our data suggest that IPNV-encoded proteins may be involved in weakening of IFN signaling [34]. As a result high levels of viral proteins may impair the activity of IFN-induced genes, thus the higher replication rate of NFH-Ar compared to NFH-El may cause a more potent IFN-antagonizing effect of the NFH-Ar strain. However when interpreting the results from live pathogen challenges, it is important to keep in mind the complexity of such studies, where it is not straight forward whether an observed response is a strategy employed by the virus for its own benefit or a response by the host to control the virus.
Although innate immunity by itself represents a powerful system to combat viral invaders, many infections can only be cleared in combination with adaptive immunity. In this regard type I IFNs are known to promote the adaptive arm including both T cell mediated cellular responses and antibody production [45,46]. It is likely that the reduction of virus titers by 29 d p.i. could be associated with the onset of adaptive immune responses. Unlike the genes implicated in the innate immunity, sIgM showed no expression changes at 13 d p.i. and was slightly induced at 29 d p.i.
Results of this study added knowledge to the understanding of the immune responses after IPNV infections. Expression of a panel of PRRs was assessed including several recently identified genes. TLR8, 9 RIG-I and MDA5 showed up-regulation and followed the same trend as other immune genes. Earlier we observed a modest increase of TLR8 and 9 expression during stimulation and infection [27,28]. TLR22 is reported to recognize dsRNA in pufferfish and when over-expressed it induces type I IFN expression upon IPNV infection, which suggests a possible role for TLR22 in protection against IPNV [30]. However, TLR22 was not induced by IPNV in this study. Unexpectedly, PKR, an IFN-inducible gene, was down-regulated at all time points except 13 d p.i. (Figure 4A). This was in contrast to Mx, another IFN-inducible gene and IFN-a1, which were up-regulated. Functional studies of salmon PKR have to our knowledge not been reported, however flounder PKR was up-regulated both in vitro and in vivo by a negative single stranded RNA virus (SMRV), which also induced Mx expression in vitro [47]. PRRs, PKR and MyD88 were down-regulated at 6 d p.i. in both study groups. This could be explained by migration of leukocytes expressing the PRRs from the head-kidney into the bloodstream at early time-points of the infection.
IFN-γ was together with Mx the most highly induced immune gene in this study, and high levels of IFN-γ has been reported in other IPNV challenge experiments [31]. IFN-γ is regarded as a typical Th1 cytokine which bridges the innate and adaptive immune responses. Fish IFN-γ share several functional properties with mammalian IFN-γ including macrophage activation [48-50] and rainbow trout IFN-γ is shown to signal through STAT1 [50,51]. Recently, we have observed antiviral activity against IPNV by IFN-γ, although the effect was not as pronounced as described for IFN-a1 [52]. Like in mammals, fish IFN-γ plays diverse roles in different facets of the immune system, and the increased levels of IFN-γ upon IPNV challenge detected here suggest anti-IPNV activity.
Conclusions:
Our study aimed at investigation of different mortality associated with two IPNV isolates. High incidence of death of NFH-Ar infected salmon was in line with the initial presence of the virulence motif in VP2 and higher virus titers in the head kidney and pancreas compared to NHF-El. PPRs, IFNs and ISGs were induced to a higher magnitude by the high virulent strain NFH-Ar and correlated with the viral load in the head kidney. Subsequent decrease of virus titers and mortality could be due to the sequence changes in the hyper variable regions of IPNV together with the development of acquired immunity in fish.
Methods:
Virus isolates Two IPNV isolates, referred to as NFH-Ar and NFH-El, collected from field outbreaks of IPN in 2004 were used in this study. Veterinarians had diagnosed IPN based on clinical observations and by use of an agglutination assay (Phadebact coating kit, Boule Diagnostics AB). The NFH-Ar isolate, obtained from an IPN outbreak in Frøya, Norway in 2004, was reported to give 32% mortality, while the NFH-El, obtained from an IPN outbreak in Alta, Norway in 2004, was reported to give 5% mortality. Head kidney samples collected during these outbreaks were sent to our lab and tissue homogenates were made and inoculated on CHSE-214 cells for propagation and sequencing. For NFH-Ar a second, and for NFH-El a third cell-culture passage of the virus strain was used in the present experiment. The input strains were sequenced before challenge.
Two IPNV isolates, referred to as NFH-Ar and NFH-El, collected from field outbreaks of IPN in 2004 were used in this study. Veterinarians had diagnosed IPN based on clinical observations and by use of an agglutination assay (Phadebact coating kit, Boule Diagnostics AB). The NFH-Ar isolate, obtained from an IPN outbreak in Frøya, Norway in 2004, was reported to give 32% mortality, while the NFH-El, obtained from an IPN outbreak in Alta, Norway in 2004, was reported to give 5% mortality. Head kidney samples collected during these outbreaks were sent to our lab and tissue homogenates were made and inoculated on CHSE-214 cells for propagation and sequencing. For NFH-Ar a second, and for NFH-El a third cell-culture passage of the virus strain was used in the present experiment. The input strains were sequenced before challenge.
IPNV challenge of Atlantic salmon The challenge was carried out at Tromsø Aquaculture Research Station (Tromsø, Norway). Non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen IPNV sensitive (Aquagen, Kyrksæterøra, Norway), with an average size of 51 g, was used for the challenge two days after transfer to seawater. Bath challenge was performed as described by Johansen and Sommer [53] using an infectious dose of TCID50/ml = 5 × 105 of the field isolates NFH-Ar and NFH-El. Two parallel tanks supplied with 200 L of 10°C seawater were used for each isolate. Each tank contained 70 fish and a separate tank was used for 70 uninfected control fish. The fish were fed daily on commercial feed. The experiment was terminated 30 days after challenge.
The challenge was carried out at Tromsø Aquaculture Research Station (Tromsø, Norway). Non-vaccinated Atlantic salmon, Salmo salar L., strain Aquagen IPNV sensitive (Aquagen, Kyrksæterøra, Norway), with an average size of 51 g, was used for the challenge two days after transfer to seawater. Bath challenge was performed as described by Johansen and Sommer [53] using an infectious dose of TCID50/ml = 5 × 105 of the field isolates NFH-Ar and NFH-El. Two parallel tanks supplied with 200 L of 10°C seawater were used for each isolate. Each tank contained 70 fish and a separate tank was used for 70 uninfected control fish. The fish were fed daily on commercial feed. The experiment was terminated 30 days after challenge.
Sampling Mortality was monitored throughout the experiment by counting dead fish. An IPNV rapid agglutination kit (Phadebact coating kit, Boule Diagnostics AB) was used on head kidney samples from dead fish to verify IPN as the cause of death. Percent cumulative mortality in infected fish was calculated compared to control fish using GraphPad Prism 4.0 tool for statistical analyses. Sampling was performed on surviving fish. Pancreatic tissue and head kidney were aseptically removed from 4 fish in each tank at 6, 13 and 29 d p.i. Pancreatic tissue was stored in L-15 medium on ice until frozen at -80°C, whereas head kidney samples were stored in RNAlater® (Ambion) at -80°C.
Mortality was monitored throughout the experiment by counting dead fish. An IPNV rapid agglutination kit (Phadebact coating kit, Boule Diagnostics AB) was used on head kidney samples from dead fish to verify IPN as the cause of death. Percent cumulative mortality in infected fish was calculated compared to control fish using GraphPad Prism 4.0 tool for statistical analyses. Sampling was performed on surviving fish. Pancreatic tissue and head kidney were aseptically removed from 4 fish in each tank at 6, 13 and 29 d p.i. Pancreatic tissue was stored in L-15 medium on ice until frozen at -80°C, whereas head kidney samples were stored in RNAlater® (Ambion) at -80°C.
Quantification of IPNV by titration Pancreatic tissue was homogenized using an Ultra Thurrax T25 basic crusher (IKA-WERKE). Homogenized tissue was diluted to 5% in Eagle's minimum essential medium (EMEM) (Gibco) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 2 mM L-glutamine and 1% non-essential amino acids, before centrifuged at 15000 × g for 5 min at 4°C. Homogenates were inoculated onto CHSE-214 cells in 96 wells plates at a starting concentration of 0.5% (w/v). Individual viral titers were determined by end-point titration using 10-fold dilutions and 2 replicates and calculated by the TCID50 method [54].
Pancreatic tissue was homogenized using an Ultra Thurrax T25 basic crusher (IKA-WERKE). Homogenized tissue was diluted to 5% in Eagle's minimum essential medium (EMEM) (Gibco) supplemented with 100 μg/ml streptomycin, 60 μg/ml penicillin, 2 mM L-glutamine and 1% non-essential amino acids, before centrifuged at 15000 × g for 5 min at 4°C. Homogenates were inoculated onto CHSE-214 cells in 96 wells plates at a starting concentration of 0.5% (w/v). Individual viral titers were determined by end-point titration using 10-fold dilutions and 2 replicates and calculated by the TCID50 method [54].
RNA isolation and cDNA synthesis For analyses of cellular and viral gene expression total RNA from head kidney sampled from 8 challenged and 4 control fish at each time point, was isolated using a combination of Trizol and PureLink RNA Kit (Invitrogen). RNA quality was assessed on an agarose gel and the quantity determined by NanoDrop 2000 spectrophotometry (Thermo Scientific). No RNA degradation was observed. After isolation, the RNA was DNase treated applying TURBO DNase (Ambion). cDNA was synthesized in a 25 μl reaction from 200 ng DNase treated total RNA primed with random hexamers (TaqMan Reverse Transcription Reagents kit, Applied Biosystems). The manufacturer's protocol was followed. For isolation of viral RNA after passage in cell culture QIAamp Viral RNA Mini Kit (Qiagen) was used according to the manufacturer's instructions.
For analyses of cellular and viral gene expression total RNA from head kidney sampled from 8 challenged and 4 control fish at each time point, was isolated using a combination of Trizol and PureLink RNA Kit (Invitrogen). RNA quality was assessed on an agarose gel and the quantity determined by NanoDrop 2000 spectrophotometry (Thermo Scientific). No RNA degradation was observed. After isolation, the RNA was DNase treated applying TURBO DNase (Ambion). cDNA was synthesized in a 25 μl reaction from 200 ng DNase treated total RNA primed with random hexamers (TaqMan Reverse Transcription Reagents kit, Applied Biosystems). The manufacturer's protocol was followed. For isolation of viral RNA after passage in cell culture QIAamp Viral RNA Mini Kit (Qiagen) was used according to the manufacturer's instructions.
Sequencing of virus isolates Each virus isolate was sequenced from virus derived material under the following conditions; origin (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 1× in cell culture), input (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 2-3× in cell culture), during infection(head kidney from day 13 (NFH-Ar) or day 29 (NFH-El) infected fish, 8 pooled individuals from each group). Primers specific for each of the genes encoding the five IPNV proteins were used to amplify the individual genes (from head kidney cDNA pooled from 8 fish or cDNA obtained from head kidney virus homogenates from 2 fish passaged in cell culture) in a PCR reaction using Pfu Turbo Hotstart DNA polymerase (Stratagene). The PCR-products were sequenced in both directions using the same primers and BigDye3.1 chemistry and a 3100 gene analyzer. The sequences were aligned with BioEdit 7.0.5 [55] and DnaSP V5 [56] was used for evaluation of mutation rates and search for HVR.
Each virus isolate was sequenced from virus derived material under the following conditions; origin (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 1× in cell culture), input (virus homogenates from head kidney pooled from 2 fish in field outbreak passaged 2-3× in cell culture), during infection(head kidney from day 13 (NFH-Ar) or day 29 (NFH-El) infected fish, 8 pooled individuals from each group). Primers specific for each of the genes encoding the five IPNV proteins were used to amplify the individual genes (from head kidney cDNA pooled from 8 fish or cDNA obtained from head kidney virus homogenates from 2 fish passaged in cell culture) in a PCR reaction using Pfu Turbo Hotstart DNA polymerase (Stratagene). The PCR-products were sequenced in both directions using the same primers and BigDye3.1 chemistry and a 3100 gene analyzer. The sequences were aligned with BioEdit 7.0.5 [55] and DnaSP V5 [56] was used for evaluation of mutation rates and search for HVR.
Microarray analyses Microarray analyses were performed at 13 d p.i. on head kidney samples of salmon infected with isolates NFH-Ar and NFH-El (5 individuals from each group, one microarray per individual). The salmonid fish microarray (SFA 2.0 or immunochip, Geo Omnibus GPL6154) contains 1800 unique clones printed each in 6 spot replicates. Pooled samples of uninfected salmon (equal amounts of RNA, n = 4) were used as a common reference. Test and reference RNA (10 μg) were labeled with the fluorescent dyes Cy5-dUTP and Cy3-dUTP respectively (Amersham Pharmacia), which were incorporated in cDNA using the SuperScript™ Indirect cDNA Labeling System (Invitrogen). Synthesis of cDNA was performed at 46°C for 3 hours in a 23 μl reaction volume, following RNA degradation with 2.5 M NaOH at 37°C for 15 min and alkaline neutralization with 2 M Hepes. Labeled cDNA was combined and purified with Microcon YM30 (Millipore). Microarray slides were pre-treated with 1% BSA fraction V, 5× SSC and 0.1% SDS for 30 min at 50°C and then washed with 2× SSC (3 min) followed by 0.2× SSC (3 min) at room temperature and hybridized over-night at 60°C in a cocktail containing 1.3× Denhardt's, 3× SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), 0.06 × SSC (2 min) and 0.06 × SSC (1 min). Scanning was performed with GenePix 4100A and images were processed with GenePix 6.0 (Molecular Devices). The spots were filtered by criterion (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analyses and genes with less than 3 high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. Statistical analyses were performed in two stages. First, technical accuracy was assessed by difference of log2-ER from zero in six spot replicates and genes with significant changes (Student's t-test, p < 0.05) in at least 3 of 5 individuals per group were selected. Next, analysis of biological replicates was carried out and differential expression was assessed by the mean fold change (> 1.5) and difference from control (one sample t-test, p < 0.05).
Microarray analyses were performed at 13 d p.i. on head kidney samples of salmon infected with isolates NFH-Ar and NFH-El (5 individuals from each group, one microarray per individual). The salmonid fish microarray (SFA 2.0 or immunochip, Geo Omnibus GPL6154) contains 1800 unique clones printed each in 6 spot replicates. Pooled samples of uninfected salmon (equal amounts of RNA, n = 4) were used as a common reference. Test and reference RNA (10 μg) were labeled with the fluorescent dyes Cy5-dUTP and Cy3-dUTP respectively (Amersham Pharmacia), which were incorporated in cDNA using the SuperScript™ Indirect cDNA Labeling System (Invitrogen). Synthesis of cDNA was performed at 46°C for 3 hours in a 23 μl reaction volume, following RNA degradation with 2.5 M NaOH at 37°C for 15 min and alkaline neutralization with 2 M Hepes. Labeled cDNA was combined and purified with Microcon YM30 (Millipore). Microarray slides were pre-treated with 1% BSA fraction V, 5× SSC and 0.1% SDS for 30 min at 50°C and then washed with 2× SSC (3 min) followed by 0.2× SSC (3 min) at room temperature and hybridized over-night at 60°C in a cocktail containing 1.3× Denhardt's, 3× SSC, 0.3% SDS, 0.67 μg/μl polyadenylate and 1.4 μg/μl yeast tRNA. After hybridization slides were washed at room temperature in 0.5 × SSC and 0.1% SDS (15 min), 0.5 × SSC and 0.01% SDS (15 min), 0.06 × SSC (2 min) and 0.06 × SSC (1 min). Scanning was performed with GenePix 4100A and images were processed with GenePix 6.0 (Molecular Devices). The spots were filtered by criterion (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. Low quality spots were excluded from analyses and genes with less than 3 high quality spots on a slide were discarded. After subtraction of median background from median signal intensities, the expression ratios (ER) were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. Statistical analyses were performed in two stages. First, technical accuracy was assessed by difference of log2-ER from zero in six spot replicates and genes with significant changes (Student's t-test, p < 0.05) in at least 3 of 5 individuals per group were selected. Next, analysis of biological replicates was carried out and differential expression was assessed by the mean fold change (> 1.5) and difference from control (one sample t-test, p < 0.05).
Quantitative real-time RT PCR (qPCR) analyses PCR primers used with Sybr Green assay were designed using Vector NTI (Invitrogen) and synthesized by Invitrogen (table 3). The amplicon lengths set to be between 50 and 200 bases were checked on 1.5% agarose gel. TaqMan assays employing a hydrolysis probe were designed using Assays-by-design (Applied Biosystems) and synthesized by Applied Biosystems. PCR efficiency was calculated from tenfold serial dilutions of cDNA for each assay in triplicates. qPCR assays were conducted using 2× SYBR® Green Master Mix (Roche Diagnostics) in an optimized 12 μl reaction volume, using 1:10 diluted cDNA, with primer concentrations of 0.4-0.6 μM. PCR was performed in duplicate in 96-well optical plates on Light Cycler 480 (Roche Diagnostics) under the following conditions: 95°C for 5 min (pre-incubation), 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec (amplification), 95°C for 5 sec, and 65°C for 1 min (melting curve). Forty cycles were performed. Assays employing a hydrolysis probe was conducted in a 20 μl reaction using 2.5 μl 1:10 fold diluted cDNA as template and 2× TaqMan Fast Universal PCR Master mix (Applied Biosystems). The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) and the amplification profile was: 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. PCR analyses of the SybrGreen assays for RIG-I, MDA5 and PKR at 13 and 29 d p.i. was also performed at the ABI Prism 7500 FAST Cycler. Relative expression of mRNA was evaluated by ΔΔCT, adjusted for PCR efficiency and normalized against the elongation factor (EF1AB). IPNV VP2 was undetected in control samples and at 6 d p.i., in order to calculate the ΔΔCT values the average of the NFH-El infected fish sampled at 29 d p.i was used as calibrator. Results were analyzed with ANOVA followed with Newman - Keuls test (p < 0.05). (See Table 1 for primers and probes used in the qPCR assays).
Primers and probes used for qPCR analyses
PCR primers used with Sybr Green assay were designed using Vector NTI (Invitrogen) and synthesized by Invitrogen (table 3). The amplicon lengths set to be between 50 and 200 bases were checked on 1.5% agarose gel. TaqMan assays employing a hydrolysis probe were designed using Assays-by-design (Applied Biosystems) and synthesized by Applied Biosystems. PCR efficiency was calculated from tenfold serial dilutions of cDNA for each assay in triplicates. qPCR assays were conducted using 2× SYBR® Green Master Mix (Roche Diagnostics) in an optimized 12 μl reaction volume, using 1:10 diluted cDNA, with primer concentrations of 0.4-0.6 μM. PCR was performed in duplicate in 96-well optical plates on Light Cycler 480 (Roche Diagnostics) under the following conditions: 95°C for 5 min (pre-incubation), 95°C for 5 sec, 60°C for 15 sec, 72°C for 15 sec (amplification), 95°C for 5 sec, and 65°C for 1 min (melting curve). Forty cycles were performed. Assays employing a hydrolysis probe was conducted in a 20 μl reaction using 2.5 μl 1:10 fold diluted cDNA as template and 2× TaqMan Fast Universal PCR Master mix (Applied Biosystems). The expression of mRNA was measured in an ABI Prism 7500 FAST Cycler (Applied Biosystems) and the amplification profile was: 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. PCR analyses of the SybrGreen assays for RIG-I, MDA5 and PKR at 13 and 29 d p.i. was also performed at the ABI Prism 7500 FAST Cycler. Relative expression of mRNA was evaluated by ΔΔCT, adjusted for PCR efficiency and normalized against the elongation factor (EF1AB). IPNV VP2 was undetected in control samples and at 6 d p.i., in order to calculate the ΔΔCT values the average of the NFH-El infected fish sampled at 29 d p.i was used as calibrator. Results were analyzed with ANOVA followed with Newman - Keuls test (p < 0.05). (See Table 1 for primers and probes used in the qPCR assays).
Primers and probes used for qPCR analyses
Supplementary Material:
Differentially expressed genes. The genes were selected as described in Methods. The data provided represent the log2 (Expression ratios) in IPNV infected salmon and control.
Click here for file
| Background:
Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described.
Methods:
In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney.
Results:
Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates).
Conclusions:
Mortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties. | Background:
Despite vaccination programs, outbreaks of infectious pancreatic necrosis disease (IPN) are frequent in farmed salmon fry and post-smolts. Mortality rates observed in outbreaks vary considerably and have in part been ascribed to the inherited differences in susceptibility of the host [1-3]. Environmental stress [4-7] and the viral strains [8] also influence mortality. Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods [9,10]. The production of virus may increase under stress, and carriers can shed the virus and infect surrounding fish [11].
The IPN virus (IPNV) is a bi-segmented double-stranded RNA (dsRNA) virus in the family Birnaviridae encoding 5 viral proteins. Segment B encodes the RNA-dependent RNA polymerase VP1. Segment A encodes a polyprotein which is cotranslationally cleaved by the viral encoded serine-lysine protease (VP4) releasing the proteins pVP2 and VP3 [12,13]. pVP2 is further processed by host cell proteases to form the mature outer capsid protein VP2 [14], which is the most abundant virus protein and contains the antigenic regions responsible for induction of neutralizing antibodies in the host [15]. VP3 is the inner structural protein, which bound to dsRNA constitutes the ribonucleoprotein core structure [16]. Additionally VP3 is shown to bind VP1 and to self-associate strongly, indicating that it is a matrix protein [17]. An alternative open reading frame (ORF) on Segment A encodes the small, arginine-rich, non-structural protein VP5. The biological function of IPNV VP5 remains to be determined.
The molecular basis of IPNV virulence and its interplay with host antiviral mechanisms are not fully understood. Sano and co-workers [18] were the first to suggest that the virulence of IPNV is associated with Segment A. Several studies using nucleotide sequence analyses have confirmed this and have shown that the VP2 residues 217 and 221 are the major determinant of virulence of IPNV serotype Sp strains. In addition, position 247 was seen as highly variable [19]. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221, while the strains containing Thr221 are almost avirulent, irrespective of the residue at position 217. IPNV isolates also differ in properties related to replication rate and the ability to cause persistent infections. These characteristics can be attributed to the same amino acids as those determining the virulence [8]. Although some of the factors behind these mechanisms are known there are still many questions to be answered.
During viral infections the initial response of the immune system is the induction of type I interferons (IFN), which mediate antiviral and immunomodulatory activity. In Atlantic salmon three different subtypes of type I IFN have been identified: IFN-a, b and c [20]. IFN-a1 and c are both expressed in head kidney and are induced by poly I:C [20]. IFN-a1 has been shown to provide protection against IPNV in salmonid cells [21,22]. The generation of anti-viral responses during infections requires a rapid viral sensing by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) on the cell-surface or within endosomes recognize single-stranded RNA (ssRNA) and dsRNA [23], while the helicases RIG-I and MDA5 recognize ssRNA and dsRNA in the cytosol [24]. Additionally, dsRNA are recognized by PKR [25]. A number of PRRs have been identified in Atlantic salmon including RIG-I [26], MDA5 (GenBank: EG820831), PKR (GenBank: EF523422), TLR3 [20], TLR8 [27], TLR9 [28] and TLR22 (GenBank: CAJ80696[29] and FM206383). The latter is a dsRNA-specific PRR found exclusively in lower vertebrates [30]. Several studies have shown strong activation of immune genes upon challenge with highly virulent IPNV isolates [31,32], and type I IFNs and the IFN-inducible Mx gene were among the most highly up-regulated genes. However, it remains to find, which PRRs are required for the induction of a systemic type I IFN response during IPNV infections.
Little is known about the relationship between the host responses and the virulence of different IPNV isolates. The latter can be associated with either down-regulation or excessive stimulation of innate immunity. Studies of IPNV infected cell-lines [33,34] have shown inhibition of IFN signaling. In the current work we have assessed immune gene expression changes during an experimental challenge of salmon post-smolts with both a virulent and an avirulent IPNV field strain. In addition to quantitative real-time RT PCR (qPCR) analyses of selected genes we used cDNA microarray, which expanded the repertoire of genes.
The two IPNV isolates used in this study were originally collected from field outbreaks of IPN with 32% and 5% reported mortality, respectively. Initial characterization of the isolates in our lab showed that they both contained the high virulent motif Thr217 Ala221 Thr247 in VP2 defined by Santi et al [19]. A previous bath challenge performed by our group included these isolates, and indicated that they differed substantially in virulence levels reflecting the field mortalities (unpublished). Sequencing of VP2 after this challenge confirmed the Thr217 Ala221 motif in both isolates. However, propagation of the isolates in cell-cultures before the challenge experiment reported here changed the isolate with the initial low mortality to contain a Thr217 Thr221 Thr247 motif (low virulent motif). Santi et al [19] suggested that the Ala-to-Thr substitution at position 221 in VP2 is a molecular determinant for the establishment of a persistent IPNV infection. Thus, we have analyzed the IPNV VP2 nucleotide composition in virus recovered from fish head kidney during the infection. Comparing sequences to the input strains could provide information about the rate and character of virus changes in vivo.
Conclusions:
AS performed most of the sequencing, assisted with the challenge experiment and quantification of virus, in addition he drafted the manuscript together with IS. qPCR analyses were performed by IS and GT. ME and BNF performed the challenge experiment and some of the sequencing and virus quantification. The microarray analyzes were done by SMJ and GT. AK contributed to this work with gene expression and sequence data and drafting the manuscript. JBJ designed the experiments, analyzed data and contributed to finalizing the manuscript. All authors read and approved the final manuscript. | Background:
Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described.
Methods:
In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney.
Results:
Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates).
Conclusions:
Mortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties. | 16,034 | 453 | [
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] | null | [CONTENT] IPNV | birnavirus | virulence | immune response | fish | non-synonymous changes [SUMMARY] | [CONTENT] IPNV | birnavirus | virulence | immune response | fish | non-synonymous changes [SUMMARY] | null | [CONTENT] IPNV | birnavirus | virulence | immune response | fish | non-synonymous changes [SUMMARY] | [CONTENT] IPNV | birnavirus | virulence | immune response | fish | non-synonymous changes [SUMMARY] | [CONTENT] IPNV | birnavirus | virulence | immune response | fish | non-synonymous changes [SUMMARY] | [CONTENT] Animals | Birnaviridae Infections | Fish Diseases | Gene Expression Profiling | Infectious pancreatic necrosis virus | Kidney | Microarray Analysis | Mutation | RNA, Viral | Real-Time Polymerase Chain Reaction | Salmo salar | Survival Analysis | Virulence | Virus Replication [SUMMARY] | [CONTENT] Animals | Birnaviridae Infections | Fish Diseases | Gene Expression Profiling | Infectious pancreatic necrosis virus | Kidney | Microarray Analysis | Mutation | RNA, Viral | Real-Time Polymerase Chain Reaction | Salmo salar | Survival Analysis | Virulence | Virus Replication [SUMMARY] | null | [CONTENT] Animals | Birnaviridae Infections | Fish Diseases | Gene Expression Profiling | Infectious pancreatic necrosis virus | Kidney | Microarray Analysis | Mutation | RNA, Viral | Real-Time Polymerase Chain Reaction | Salmo salar | Survival Analysis | Virulence | Virus Replication [SUMMARY] | [CONTENT] Animals | Birnaviridae Infections | Fish Diseases | Gene Expression Profiling | Infectious pancreatic necrosis virus | Kidney | Microarray Analysis | Mutation | RNA, Viral | Real-Time Polymerase Chain Reaction | Salmo salar | Survival Analysis | Virulence | Virus Replication [SUMMARY] | [CONTENT] Animals | Birnaviridae Infections | Fish Diseases | Gene Expression Profiling | Infectious pancreatic necrosis virus | Kidney | Microarray Analysis | Mutation | RNA, Viral | Real-Time Polymerase Chain Reaction | Salmo salar | Survival Analysis | Virulence | Virus Replication [SUMMARY] | [CONTENT] protection ipnv salmonid | salmon infected | ipnv atlantic salmon | ipnv salmonid cells | pathogenicity atlantic salmon [SUMMARY] | [CONTENT] protection ipnv salmonid | salmon infected | ipnv atlantic salmon | ipnv salmonid cells | pathogenicity atlantic salmon [SUMMARY] | null | [CONTENT] protection ipnv salmonid | salmon infected | ipnv atlantic salmon | ipnv salmonid cells | pathogenicity atlantic salmon [SUMMARY] | [CONTENT] protection ipnv salmonid | salmon infected | ipnv atlantic salmon | ipnv salmonid cells | pathogenicity atlantic salmon [SUMMARY] | [CONTENT] protection ipnv salmonid | salmon infected | ipnv atlantic salmon | ipnv salmonid cells | pathogenicity atlantic salmon [SUMMARY] | [CONTENT] nfh | fish | ipnv | nfh ar | ar | el | nfh el | 29 | day | vp2 [SUMMARY] | [CONTENT] nfh | fish | ipnv | nfh ar | ar | el | nfh el | 29 | day | vp2 [SUMMARY] | null | [CONTENT] nfh | fish | ipnv | nfh ar | ar | el | nfh el | 29 | day | vp2 [SUMMARY] | [CONTENT] nfh | fish | ipnv | nfh ar | ar | el | nfh el | 29 | day | vp2 [SUMMARY] | [CONTENT] nfh | fish | ipnv | nfh ar | ar | el | nfh el | 29 | day | vp2 [SUMMARY] | [CONTENT] ipnv | ifn | dsrna | host | virulence | isolates | infections | virulent | shown | virus [SUMMARY] | [CONTENT] min | performed | ssc | μl | sec | cdna | fish | assays | kit | nfh [SUMMARY] | null | [CONTENT] virus titers | higher | high | head kidney subsequent decrease | viral load | isolates high incidence | isolates high incidence death | mortality sequence changes | mortality sequence | mortality associated ipnv isolates [SUMMARY] | [CONTENT] nfh | day | ifn | fish | ipnv | nfh el | ar | nfh ar | el | 29 [SUMMARY] | [CONTENT] nfh | day | ifn | fish | ipnv | nfh el | ar | nfh ar | el | 29 [SUMMARY] | [CONTENT] IPNV | Birnaviridae ||| IPNV ||| HVR ||| ||| [SUMMARY] | [CONTENT] two | IPNV | NFH-Ar | NFH-El ||| Atlantic ||| qPCR [SUMMARY] | null | [CONTENT] ||| ||| [SUMMARY] | [CONTENT] IPNV | Birnaviridae ||| IPNV ||| HVR ||| ||| ||| two | IPNV | NFH-Ar | NFH-El ||| Atlantic ||| qPCR ||| two | 13 days | more than 4 | NFH-Ar | NFH-El | five | IPNV | Ka ||| HRV | VP3 ||| ||| NFH-Ar | NFH-El ||| ||| ||| IFN | MDA-5 | TLR8 | 9 | MyD88 | 9 | 13 ||| NFH-Ar | 29 d p.i ||| ||| ||| ||| [SUMMARY] | [CONTENT] IPNV | Birnaviridae ||| IPNV ||| HVR ||| ||| ||| two | IPNV | NFH-Ar | NFH-El ||| Atlantic ||| qPCR ||| two | 13 days | more than 4 | NFH-Ar | NFH-El | five | IPNV | Ka ||| HRV | VP3 ||| ||| NFH-Ar | NFH-El ||| ||| ||| IFN | MDA-5 | TLR8 | 9 | MyD88 | 9 | 13 ||| NFH-Ar | 29 d p.i ||| ||| ||| ||| [SUMMARY] |
|||||
Retrospective observational study of diagnostic accuracy of the Xpert® MTB/RIF assay on fiberoptic bronchoscopy sampling for early diagnosis of smear-negative or sputum-scarce patients with suspected tuberculosis. | 25115239 | Fiberoptic bronchoscopy (FOB) is a useful diagnosis tool in low-burden countries for patients with suspected pulmonary tuberculosis (TB) who are smear-negative or sputum-scarce. This study sought to determine the accuracy of the Xpert® MTB/RIF (XP) assay using FOB samples. | BACKGROUND | We retrospectively reviewed clinical, radiological, and microbiological characteristics of 175 TB-suspected patients requiring diagnostic FOB (bronchial aspirate or bronchoalveolar lavage) with XP assay. Polymerase chain reaction (PCR) and smear microscopy (SM) performances were first compared to culture, then to the final diagnosis, established based on clinical or radiological evolution when cultures were negative. | METHODS | Of the total 162 included patients, 30 (18.5%) had a final diagnosis of pulmonary TB, with positive cultures reported in 23. As compared to culture, sensitivity and specificity values were 80.0% and 98.6% for the XP assay, and 25.0% and 95.8% for SM, respectively. As compared to final diagnosis, the corresponding performance values were 60.0% and 100.0% for the XP assay, and 16.7% and 95.5% for SM, respectively. The sensitivity of the XP assay was significantly higher than that of SM in both cases (p=0.003 and p=0.001). Concerning the final diagnosis, both XP assay and culture sensitivities were similar (60% vs. 66.7%). PCR assay enabled pulmonary TB to be diagnosed earlier in 13 more cases, compared to SM. | RESULTS | Our study has confirmed the clinical benefits provided by XP assay compared to SM for the early diagnosis of suspected pulmonary TB cases requiring FOB, on per procedure samples, especially in a low TB-burden country. | CONCLUSION | [
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"Bronchoscopy",
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"Predictive Value of Tests",
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] | 4137109 | Background | Tuberculosis (TB) still constitutes a major health problem worldwide, with an 8.8% incidence and 1.3% mortality rate reported in 2010
[1]. In the same year, TB incidence was reported at 8.1 cases per 100,000 in France
[2], defining it as a low TB-burden country. It is worth noting that 73% of TB cases were pulmonary infections, with almost 50% producing a negative smear microscopy (SM)
[2].
By detecting active pulmonary TB early, an appropriate treatment can be initiated, and disease transmission can then be preemptively blocked. Some patients presenting with active pulmonary TB may, however, exhibit negative sputum acid-fast bacilli (AFB) smears. In developed countries, fiberoptic bronchoscopy (FOB) is considered a good option for these cases that pose a diagnostic challenge
[3], although SM is still exhibiting low sensitivity on FOB samples, with 5-35% on bronchial aspirates (BA) and 10-30% on bronchoalveolar lavages (BAL)
[4-9]. Furthermore, while mycobacterial culture remains the gold standard for laboratory diagnosis of TB, it requires 2–6 weeks to confirm a diagnosis. This results in delays in initiating appropriate treatment while waiting for this confirmation, except for cases where there is strong enough clinical suspicion to initiate a presumptive anti-TB therapy.
Several polymerase chain reaction- (PCR-) based molecular methods have recently been developed for early TB diagnosis and rapid detection of drug resistance from clinical specimens
[10-14]. The Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one of these methods, and consists of a hemi-nested real-time PCR test that simultaneously identifies Mycobacterium tuberculosis and detects rifampicin resistance, as a surrogate of multidrug resistance (MDR), directly from clinical specimens. This assay requires less than 2 hours, and its key advantage over other PCR methods is that it is a fully-automated process, designed to run on the GeneXpert Dx system (Cepheid). This system incorporates DNA extraction, often considered the critical step
[15], along with real-time PCR amplification and detection in a single hands-free process, thus acting as a real “lab-on-chip” device.
Since December 2010, WHO has recommended the Xpert® MTB/RIF assay as a bona fide follow-on test due to its high-quality performance
[16], compared to microscopy, whenever MDR-TB or HIV are of lesser concern, and especially in cases of smear-negative specimens
[17]. This conditional recommendation does, in fact, exclusively concern sputum samples
[17], whereas no specific recommendations for FOB samples have yet been formulated. Finally, there have only been two recent studies that have assessed the Xpert® MTB/RIF assay performance using FOB samples for TB diagnosis in high TB-burden countries
[18,19].
This study sought to evaluate the clinical value of the Xpert® MTB/RIF assay using FOB samples for an early diagnosis of pulmonary TB in either patients with negative sputum AFB smears or those who could not produce an expectorated sputum sample. Patients were treated in a French university hospital in a low TB-burden region (5.9 cases per 100,000 in 2010). | Methods | Study population We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.
Our institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.
We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.
Our institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.
Bronchoscopic procedures Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.
Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.
Microbiological diagnosis All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.
For the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)
[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).
For the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin
[21,22].
All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.
For the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)
[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).
For the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin
[21,22].
Pulmonary TB diagnosis The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.
The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.
Ethical considerations According to the WHO recommendations on the Xpert® MTB/RIF assay
[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).
According to the WHO recommendations on the Xpert® MTB/RIF assay
[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).
Statistical analysis Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA). | Results | Patient characteristics From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.
The principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table
1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table
1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.
Demographic, clinical, and radiological characteristics of the 162 included patients
aGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.
bNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).
cIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.
dIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.
Xpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure
1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure
1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table
1.
Flow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.
From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.
The principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table
1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table
1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.
Demographic, clinical, and radiological characteristics of the 162 included patients
aGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.
bNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).
cIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.
dIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.
Xpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure
1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure
1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table
1.
Flow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.
Performances of microbiological methods In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table
2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table
2).
Performances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis
ap-values were calculated using the McNemar’s test.
bp-value between Xpert®MTB/RIFassay vs. smear microscopy.
c
p-value between Xpert®MTB/RIFassay vs. culture.
d
p-value between culture vs. smear microscopy.
When considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table
2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).
The Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table
3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.
Gain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay
aNTM: Nontuberculous mycobacteria.
bTB: Tuberculosis.
In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table
2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table
2).
Performances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis
ap-values were calculated using the McNemar’s test.
bp-value between Xpert®MTB/RIFassay vs. smear microscopy.
c
p-value between Xpert®MTB/RIFassay vs. culture.
d
p-value between culture vs. smear microscopy.
When considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table
2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).
The Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table
3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.
Gain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay
aNTM: Nontuberculous mycobacteria.
bTB: Tuberculosis. | Conclusion | In summary, our study confirms the clinical usefulness of the Xpert® MTB/RIF assay, compared to SM, for the early diagnosis of suspected pulmonary TB requiring FOB, performed on per procedure samples. This is a reproducible commercial assay, and should especially be considered as a relevant option in low TB-burden countries. | [
"Background",
"Study population",
"Bronchoscopic procedures",
"Microbiological diagnosis",
"Pulmonary TB diagnosis",
"Ethical considerations",
"Statistical analysis",
"Patient characteristics",
"Performances of microbiological methods",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Tuberculosis (TB) still constitutes a major health problem worldwide, with an 8.8% incidence and 1.3% mortality rate reported in 2010\n[1]. In the same year, TB incidence was reported at 8.1 cases per 100,000 in France\n[2], defining it as a low TB-burden country. It is worth noting that 73% of TB cases were pulmonary infections, with almost 50% producing a negative smear microscopy (SM)\n[2].\nBy detecting active pulmonary TB early, an appropriate treatment can be initiated, and disease transmission can then be preemptively blocked. Some patients presenting with active pulmonary TB may, however, exhibit negative sputum acid-fast bacilli (AFB) smears. In developed countries, fiberoptic bronchoscopy (FOB) is considered a good option for these cases that pose a diagnostic challenge\n[3], although SM is still exhibiting low sensitivity on FOB samples, with 5-35% on bronchial aspirates (BA) and 10-30% on bronchoalveolar lavages (BAL)\n[4-9]. Furthermore, while mycobacterial culture remains the gold standard for laboratory diagnosis of TB, it requires 2–6 weeks to confirm a diagnosis. This results in delays in initiating appropriate treatment while waiting for this confirmation, except for cases where there is strong enough clinical suspicion to initiate a presumptive anti-TB therapy.\nSeveral polymerase chain reaction- (PCR-) based molecular methods have recently been developed for early TB diagnosis and rapid detection of drug resistance from clinical specimens\n[10-14]. The Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one of these methods, and consists of a hemi-nested real-time PCR test that simultaneously identifies Mycobacterium tuberculosis and detects rifampicin resistance, as a surrogate of multidrug resistance (MDR), directly from clinical specimens. This assay requires less than 2 hours, and its key advantage over other PCR methods is that it is a fully-automated process, designed to run on the GeneXpert Dx system (Cepheid). This system incorporates DNA extraction, often considered the critical step\n[15], along with real-time PCR amplification and detection in a single hands-free process, thus acting as a real “lab-on-chip” device.\nSince December 2010, WHO has recommended the Xpert® MTB/RIF assay as a bona fide follow-on test due to its high-quality performance\n[16], compared to microscopy, whenever MDR-TB or HIV are of lesser concern, and especially in cases of smear-negative specimens\n[17]. This conditional recommendation does, in fact, exclusively concern sputum samples\n[17], whereas no specific recommendations for FOB samples have yet been formulated. Finally, there have only been two recent studies that have assessed the Xpert® MTB/RIF assay performance using FOB samples for TB diagnosis in high TB-burden countries\n[18,19].\nThis study sought to evaluate the clinical value of the Xpert® MTB/RIF assay using FOB samples for an early diagnosis of pulmonary TB in either patients with negative sputum AFB smears or those who could not produce an expectorated sputum sample. Patients were treated in a French university hospital in a low TB-burden region (5.9 cases per 100,000 in 2010).",
"We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.\nOur institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.",
"Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.",
"All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.\nFor the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)\n[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).\nFor the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin\n[21,22].",
"The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.",
"According to the WHO recommendations on the Xpert® MTB/RIF assay\n[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).",
"Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).",
"From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.\nThe principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table \n1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table \n1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.\nDemographic, clinical, and radiological characteristics of the 162 included patients\naGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.\nbNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).\ncIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.\ndIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.\nXpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure \n1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure \n1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table \n1.\nFlow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.",
"In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table \n2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table \n2).\nPerformances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis\nap-values were calculated using the McNemar’s test.\nbp-value between Xpert®MTB/RIFassay vs. smear microscopy.\n\nc\np-value between Xpert®MTB/RIFassay vs. culture.\n\nd\np-value between culture vs. smear microscopy.\nWhen considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table \n2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).\nThe Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table \n3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.\nGain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay\naNTM: Nontuberculous mycobacteria.\nbTB: Tuberculosis.",
"The authors declare that they have no competing interests.",
"Study conception and design: PLP, VC, GZ, and EB. Acquisition of data: PLP, RM, KC and YO. Drafting of manuscript: PLP, VC, GZ, and EB. Analysis and interpretation of data: BM. Critical revision: VC, GZ, and EB. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2466/14/137/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Study population",
"Bronchoscopic procedures",
"Microbiological diagnosis",
"Pulmonary TB diagnosis",
"Ethical considerations",
"Statistical analysis",
"Results",
"Patient characteristics",
"Performances of microbiological methods",
"Discussion",
"Conclusion",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Tuberculosis (TB) still constitutes a major health problem worldwide, with an 8.8% incidence and 1.3% mortality rate reported in 2010\n[1]. In the same year, TB incidence was reported at 8.1 cases per 100,000 in France\n[2], defining it as a low TB-burden country. It is worth noting that 73% of TB cases were pulmonary infections, with almost 50% producing a negative smear microscopy (SM)\n[2].\nBy detecting active pulmonary TB early, an appropriate treatment can be initiated, and disease transmission can then be preemptively blocked. Some patients presenting with active pulmonary TB may, however, exhibit negative sputum acid-fast bacilli (AFB) smears. In developed countries, fiberoptic bronchoscopy (FOB) is considered a good option for these cases that pose a diagnostic challenge\n[3], although SM is still exhibiting low sensitivity on FOB samples, with 5-35% on bronchial aspirates (BA) and 10-30% on bronchoalveolar lavages (BAL)\n[4-9]. Furthermore, while mycobacterial culture remains the gold standard for laboratory diagnosis of TB, it requires 2–6 weeks to confirm a diagnosis. This results in delays in initiating appropriate treatment while waiting for this confirmation, except for cases where there is strong enough clinical suspicion to initiate a presumptive anti-TB therapy.\nSeveral polymerase chain reaction- (PCR-) based molecular methods have recently been developed for early TB diagnosis and rapid detection of drug resistance from clinical specimens\n[10-14]. The Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one of these methods, and consists of a hemi-nested real-time PCR test that simultaneously identifies Mycobacterium tuberculosis and detects rifampicin resistance, as a surrogate of multidrug resistance (MDR), directly from clinical specimens. This assay requires less than 2 hours, and its key advantage over other PCR methods is that it is a fully-automated process, designed to run on the GeneXpert Dx system (Cepheid). This system incorporates DNA extraction, often considered the critical step\n[15], along with real-time PCR amplification and detection in a single hands-free process, thus acting as a real “lab-on-chip” device.\nSince December 2010, WHO has recommended the Xpert® MTB/RIF assay as a bona fide follow-on test due to its high-quality performance\n[16], compared to microscopy, whenever MDR-TB or HIV are of lesser concern, and especially in cases of smear-negative specimens\n[17]. This conditional recommendation does, in fact, exclusively concern sputum samples\n[17], whereas no specific recommendations for FOB samples have yet been formulated. Finally, there have only been two recent studies that have assessed the Xpert® MTB/RIF assay performance using FOB samples for TB diagnosis in high TB-burden countries\n[18,19].\nThis study sought to evaluate the clinical value of the Xpert® MTB/RIF assay using FOB samples for an early diagnosis of pulmonary TB in either patients with negative sputum AFB smears or those who could not produce an expectorated sputum sample. Patients were treated in a French university hospital in a low TB-burden region (5.9 cases per 100,000 in 2010).",
" Study population We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.\nOur institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.\nWe retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.\nOur institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.\n Bronchoscopic procedures Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.\nBronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.\n Microbiological diagnosis All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.\nFor the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)\n[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).\nFor the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin\n[21,22].\nAll samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.\nFor the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)\n[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).\nFor the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin\n[21,22].\n Pulmonary TB diagnosis The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.\nThe final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.\n Ethical considerations According to the WHO recommendations on the Xpert® MTB/RIF assay\n[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).\nAccording to the WHO recommendations on the Xpert® MTB/RIF assay\n[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).\n Statistical analysis Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).\nSensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).",
"We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.\nOur institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.",
"Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.",
"All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.\nFor the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)\n[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).\nFor the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin\n[21,22].",
"The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.",
"According to the WHO recommendations on the Xpert® MTB/RIF assay\n[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).",
"Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).",
" Patient characteristics From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.\nThe principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table \n1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table \n1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.\nDemographic, clinical, and radiological characteristics of the 162 included patients\naGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.\nbNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).\ncIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.\ndIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.\nXpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure \n1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure \n1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table \n1.\nFlow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.\nFrom October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.\nThe principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table \n1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table \n1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.\nDemographic, clinical, and radiological characteristics of the 162 included patients\naGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.\nbNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).\ncIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.\ndIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.\nXpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure \n1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure \n1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table \n1.\nFlow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.\n Performances of microbiological methods In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table \n2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table \n2).\nPerformances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis\nap-values were calculated using the McNemar’s test.\nbp-value between Xpert®MTB/RIFassay vs. smear microscopy.\n\nc\np-value between Xpert®MTB/RIFassay vs. culture.\n\nd\np-value between culture vs. smear microscopy.\nWhen considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table \n2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).\nThe Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table \n3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.\nGain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay\naNTM: Nontuberculous mycobacteria.\nbTB: Tuberculosis.\nIn comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table \n2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table \n2).\nPerformances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis\nap-values were calculated using the McNemar’s test.\nbp-value between Xpert®MTB/RIFassay vs. smear microscopy.\n\nc\np-value between Xpert®MTB/RIFassay vs. culture.\n\nd\np-value between culture vs. smear microscopy.\nWhen considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table \n2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).\nThe Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table \n3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.\nGain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay\naNTM: Nontuberculous mycobacteria.\nbTB: Tuberculosis.",
"From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.\nThe principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table \n1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table \n1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.\nDemographic, clinical, and radiological characteristics of the 162 included patients\naGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.\nbNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).\ncIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.\ndIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.\nXpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure \n1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure \n1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table \n1.\nFlow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.",
"In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table \n2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table \n2).\nPerformances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis\nap-values were calculated using the McNemar’s test.\nbp-value between Xpert®MTB/RIFassay vs. smear microscopy.\n\nc\np-value between Xpert®MTB/RIFassay vs. culture.\n\nd\np-value between culture vs. smear microscopy.\nWhen considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table \n2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).\nThe Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table \n3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.\nGain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay\naNTM: Nontuberculous mycobacteria.\nbTB: Tuberculosis.",
"FOB constitutes an interesting alternative for TB diagnosis in smear-negative or sputum-scarce patients, especially in developed countries where this procedure is widely available. Given that WHO recommendations on the Xpert® MTB/RIF assay only pertain to sputum samples\n[17], further investigation must be conducted regarding the use of this PCR on FOB samples. To the best of our knowledge, there has been no publication specifically describing the clinical interest and accuracy of the Xpert® MTB/RIF assay using FOB samples for TB diagnosis in a low-prevalence country.\nOur study produced results in line with previous reports on respiratory samples\n[16,23-31], demonstrating that the Xpert® MTB/RIF assay significantly outperformed SM on FOB samples (sensitivities: 80.0% vs. 25.0%; p = 0.003) in a low TB-prevalence area. The sensitivity of the PCR assay was found to be lower when it was calculated relative to the final diagnosis, yet still remained significantly higher than that achieved with SM (60.0% vs. 16.7%; p = 0.001). Our findings revealed similar performances to those reported by two recently published studies, which had previously explored the clinical usefulness of the Xpert® MTB/RIF assay on FOB samples, although this was conducted in high TB-prevalence areas\n[18,19]. In the Lee et al. retrospective study, 132 patients were recruited in a single South-Korean center. The study reported sensitivity and specificity values (relative to the culture) of 81.6% and 100.0% for the Xpert® MTB/RIF assay, compared to 13.2% and 98.8% for SM, respectively\n[18]. Theron et al. prospectively included 154 patients in a South-African single-center study and specifically analyzed BAL samples. The resulting sensitivity and specificity values compared to the culture were 92.6% and 96.0% for the Xpert® MTB/RIF assay, and 57.7% and 99.3% for SM, respectively\n[19].\nWe can therefore confirm that for suspected TB requiring bronchoscopic procedure, the Xpert® MTB/RIF assay is a potential alternative to SM using FOB samples. In our study, PCR allowed for early pulmonary TB diagnosis to be established, enabling appropriate treatment to be started early for 13 more patients as compared to SM (72%) (p < 0.001; Table \n3). In these patients, the definitive TB diagnosis was secondarily confirmed either by culture or clinical evolution under specific antimicrobial therapy. These findings were similar to those of the Theron et al. study (>80%)\n[19], and are of particular significance in a low TB-incidence area, where there is a higher proportion of alternative diagnosis, such as lung cancer or pyogenic bacterial pneumonia.\nOur study objectives did not include assessing the value of the GeneXpert® assay in detecting rifampicin resistance on FOB samples, since the incidence of such resistance was expected to be low, as seen in previous studies\n[18,19]. The unique case of MDR-TB was, however, unambiguously detected, and all PCR results were corroborated by those that were phenotypically determined.\nOur study presented some limitations. Firstly, this was a retrospective study, despite all Xpert® MTB/RIF assays being performed on fresh FOB samples. Secondly, it was a single-center study. Finally the PCR assays were not systematically performed on the same FOB sample for all patients (BA or BAL). A prospective study should be designed with systematized and parallel FOB sampling (BA, BAL). This would provide a more proficient way of testing the Xpert® MTB/RIF assay in different types of per-endoscopic respiratory samples, an endpoint that we were unable to analyze due to our retrospective design. Cost-effectiveness studies should also be conducted in order to specify the rational use of the Xpert® MTB/RIF assay on FOB samples in novel diagnostic algorithms, including this PCR assay in well-resourced countries.",
"In summary, our study confirms the clinical usefulness of the Xpert® MTB/RIF assay, compared to SM, for the early diagnosis of suspected pulmonary TB requiring FOB, performed on per procedure samples. This is a reproducible commercial assay, and should especially be considered as a relevant option in low TB-burden countries.",
"The authors declare that they have no competing interests.",
"Study conception and design: PLP, VC, GZ, and EB. Acquisition of data: PLP, RM, KC and YO. Drafting of manuscript: PLP, VC, GZ, and EB. Analysis and interpretation of data: BM. Critical revision: VC, GZ, and EB. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2466/14/137/prepub\n"
] | [
null,
"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
"discussion",
"conclusions",
null,
null,
null
] | [
"Tuberculosis diagnosis",
"Fiberoptic bronchoscopy",
"Xpert® MTB/RIF assay",
"Smear microscopy"
] | Background:
Tuberculosis (TB) still constitutes a major health problem worldwide, with an 8.8% incidence and 1.3% mortality rate reported in 2010
[1]. In the same year, TB incidence was reported at 8.1 cases per 100,000 in France
[2], defining it as a low TB-burden country. It is worth noting that 73% of TB cases were pulmonary infections, with almost 50% producing a negative smear microscopy (SM)
[2].
By detecting active pulmonary TB early, an appropriate treatment can be initiated, and disease transmission can then be preemptively blocked. Some patients presenting with active pulmonary TB may, however, exhibit negative sputum acid-fast bacilli (AFB) smears. In developed countries, fiberoptic bronchoscopy (FOB) is considered a good option for these cases that pose a diagnostic challenge
[3], although SM is still exhibiting low sensitivity on FOB samples, with 5-35% on bronchial aspirates (BA) and 10-30% on bronchoalveolar lavages (BAL)
[4-9]. Furthermore, while mycobacterial culture remains the gold standard for laboratory diagnosis of TB, it requires 2–6 weeks to confirm a diagnosis. This results in delays in initiating appropriate treatment while waiting for this confirmation, except for cases where there is strong enough clinical suspicion to initiate a presumptive anti-TB therapy.
Several polymerase chain reaction- (PCR-) based molecular methods have recently been developed for early TB diagnosis and rapid detection of drug resistance from clinical specimens
[10-14]. The Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one of these methods, and consists of a hemi-nested real-time PCR test that simultaneously identifies Mycobacterium tuberculosis and detects rifampicin resistance, as a surrogate of multidrug resistance (MDR), directly from clinical specimens. This assay requires less than 2 hours, and its key advantage over other PCR methods is that it is a fully-automated process, designed to run on the GeneXpert Dx system (Cepheid). This system incorporates DNA extraction, often considered the critical step
[15], along with real-time PCR amplification and detection in a single hands-free process, thus acting as a real “lab-on-chip” device.
Since December 2010, WHO has recommended the Xpert® MTB/RIF assay as a bona fide follow-on test due to its high-quality performance
[16], compared to microscopy, whenever MDR-TB or HIV are of lesser concern, and especially in cases of smear-negative specimens
[17]. This conditional recommendation does, in fact, exclusively concern sputum samples
[17], whereas no specific recommendations for FOB samples have yet been formulated. Finally, there have only been two recent studies that have assessed the Xpert® MTB/RIF assay performance using FOB samples for TB diagnosis in high TB-burden countries
[18,19].
This study sought to evaluate the clinical value of the Xpert® MTB/RIF assay using FOB samples for an early diagnosis of pulmonary TB in either patients with negative sputum AFB smears or those who could not produce an expectorated sputum sample. Patients were treated in a French university hospital in a low TB-burden region (5.9 cases per 100,000 in 2010).
Methods:
Study population We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.
Our institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.
We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.
Our institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.
Bronchoscopic procedures Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.
Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.
Microbiological diagnosis All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.
For the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)
[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).
For the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin
[21,22].
All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.
For the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)
[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).
For the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin
[21,22].
Pulmonary TB diagnosis The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.
The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.
Ethical considerations According to the WHO recommendations on the Xpert® MTB/RIF assay
[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).
According to the WHO recommendations on the Xpert® MTB/RIF assay
[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).
Statistical analysis Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).
Study population:
We retrospectively reviewed the medical records of patients with suspected TB requiring a diagnostic FOB at the Caen University Hospital (Basse-Normandie region, North-Western France) from October 2009 to April 2013. TB was suspected based on clinical features (e.g., cough, hemoptysis, fever, asthenia, loss of weight, and night sweats) or radiological features (e.g., nodule, pneumonia, excavation, and pleurisy). All included patients either produced a negative sputum AFB SM prior to FOB procedure or were unable to produce sputum.
Our institution, a regional center of reference for TB diagnosis and treatment, has adopted the Xpert® MTB/RIF assay since 2009. Patients were included in the ongoing retrospective survey if one or more Xpert® MTB/RIF assays had been performed on their FOB samples, in addition to SM and mycobacterial culture procedures. Samples were excluded if culture results were unavailable or if the Xpert® MTB/RIF assay produced invalid results (see below). Patients who had previously received anti-TB drugs were also excluded from analysis.
Bronchoscopic procedures:
Bronchoscopy was performed using a flexible fiberscope of either 4.9 mm (model BF-P180, Olympus Optical) or 5.1 mm in diameter (model F1-16RB, Pentax) by trained lung specialists who first inspected the bronchial tree and then collected BA or BAL specimens. The type of sample was chosen at the specialist’s discretion and depending on the patient’s tolerance of the procedure. The lung section sample was generally chosen based on chest X-ray or CT-scan abnormalities. BA samples were obtained by the aspiration of pure bronchial secretions or following the instillation of 20-50 mL isotonic saline solution. For the BAL samples, 100-150 mL isotonic saline solution was instilled by 50 mL aliquots in a lung segment, and then aspirated.
Microbiological diagnosis:
All samples were digested, decontaminated by means of N-acetyl-cysteine-2% NaOH, concentrated by centrifugation (at 3,500 rpm for 20 min), and tested with SM, culture (used as the reference technique), and PCR.
For the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification). Each slide was observed for 5 min, corresponding to 200 fields examined. Sample aliquots of 500 μL and 200 μL were inoculated in an MGIT liquid medium (BD Diagnostics, Le Pont-de-Claix, France) or on Coletsos slants (Bio-Rad, Marnes-la-Coquette, France), respectively. Liquid cultures were automatically monitored by the BACTEC MGIT 960 system (BD Diagnostics) for up to 6 weeks, while solid media were studied for up to 12 weeks. Positive cultures were those with the presence of M. tuberculosis confirmed by means of the TB Ag MPT64 Rapid® assay (Standard Diagnostics, Yongin, South Korea), and rifampicin susceptibility was tested using the MGIT 960 SIRE kit (BD Diagnostics)
[20]. The different species of nontuberculous mycobacteria (NTM) were identified using the GenoType® Mycobacterium CM/AS (HainLifescience, Nehren, Germany).
For the Xpert® MTB/RIF assay, a 500 μL aliquot was poured into a single-use disposable cartridge that was placed into the GeneXpert™ Dx module, with the results produced in less than 2 hours. Each PCR run comprised an internal control for sample processing (DNA extraction) and PCR validity (presence of inhibitors), with positive and negative controls tested every day. The system automatically interpreted all results from measured fluorescent signals, with embedded calculation algorithms, into the following categories: invalid, if PCR inhibitors were detected with amplification failure; negative or positive. If positive, the strain was categorized as susceptible or resistant to rifampicin
[21,22].
Pulmonary TB diagnosis:
The final diagnosis of active pulmonary TB was primarily based on the M. tuberculosis culture taken from a respiratory specimen. Additional cases were classified as definitive TB, taking into account both the clinical symptoms and histological/radiological findings compatible with active pulmonary TB, as well as improvement observed with anti-TB specific therapy.
Ethical considerations:
According to the WHO recommendations on the Xpert® MTB/RIF assay
[16], the use of this diagnosis TB test has been routinely implemented in our University hospital. According to French laws, a formal agreement from an ethics committee is not required for retrospective collection of data dealing with usual standard medical care (ref: law n°2004-806 from August, 9th 2004, modified by government ordinance n°2066-477 from April 26th 2006, article R1121-3, Journal officiel de la République Française). All collected data from the charts of the Microbiology Department were anonymous and therefore complied with the restrictive requirements of the Commision Nationale de l’Informatique et des Libertés (CNIL), the organization that ensures the application of data privacy laws in France. Moreover, the study protocol was evaluated and approved by the institutional review board of the French Society for Respiratory Medecine (“Société de Pneumologie de Langue Française”) for observational studies (CEPRO). Finally, all patients were informed of our TB diagnostic strategy, received written information about the FOB procedure and gave there oral consent (as recommended by the French Society of Respiratory Diseases).
Statistical analysis:
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, along with the corresponding 95% confidence intervals. Sensitivity and specificity values were compared by means of the McNemar’s test. A p-value inferior to 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS Statistics 20.0 software (Armonk, NY, USA).
Results:
Patient characteristics From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.
The principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table
1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table
1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.
Demographic, clinical, and radiological characteristics of the 162 included patients
aGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.
bNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).
cIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.
dIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.
Xpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure
1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure
1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table
1.
Flow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.
From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.
The principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table
1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table
1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.
Demographic, clinical, and radiological characteristics of the 162 included patients
aGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.
bNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).
cIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.
dIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.
Xpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure
1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure
1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table
1.
Flow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.
Performances of microbiological methods In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table
2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table
2).
Performances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis
ap-values were calculated using the McNemar’s test.
bp-value between Xpert®MTB/RIFassay vs. smear microscopy.
c
p-value between Xpert®MTB/RIFassay vs. culture.
d
p-value between culture vs. smear microscopy.
When considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table
2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).
The Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table
3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.
Gain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay
aNTM: Nontuberculous mycobacteria.
bTB: Tuberculosis.
In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table
2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table
2).
Performances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis
ap-values were calculated using the McNemar’s test.
bp-value between Xpert®MTB/RIFassay vs. smear microscopy.
c
p-value between Xpert®MTB/RIFassay vs. culture.
d
p-value between culture vs. smear microscopy.
When considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table
2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).
The Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table
3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.
Gain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay
aNTM: Nontuberculous mycobacteria.
bTB: Tuberculosis.
Patient characteristics:
From October 2009 to April 2013, 175 consecutive patients presenting with suspected active pulmonary TB underwent FOB with an Xpert® MTB/RIF assay using respiratory samples. A total of 13 samples from 13 different patients were excluded from our study for the following reasons: 1) two due to PCR failure; 2) 10 due to unavailable culture; 3) one due to recent anti-TB treatment. A total of 229 FOB samples taken from 162 patients (median age: 54 years; male/female ratio: 1.7) were included.
The principal clinical symptoms were cough (51.9%) and general symptoms, such as asthenia, loss of appetite, and loss of weight for half of the patients (Table
1). The most common lesions detected by chest imaging were nodules (CT-scan: 60.5%) and pneumonia (CT-scan: 30.2%) (Table
1). It should be noted that 18, 9, and 80 patients had 1, 2, and 3 sputum samples, respectively, that were collected before FOB, with the number of Xpert® MTB/RIF assays performed on the sample ranging from 0 to 3 for each patient (0: 110 patients; 1: 21; 2: 22; 3: 9). No sputum could be collected before the FOB procedure for 55 patients (34%) due to their inability to expectorate sputum.
Demographic, clinical, and radiological characteristics of the 162 included patients
aGeneral symptoms: asthenia and/or loss of appetite and/or loss of weight.
bNTM: Nontuberculous mycobacteria (including four M. avium, two M. xenopi, one M. szulgai, one M. malmoense, and one other).
cIncluding 24 infectious pneumonia, two lung abscesses, and two invasive aspergillosis.
dIncluding eight non-infectious pneumonia, five acute pulmonary edema, five idiopathic hemoptysis, two interstitial lung disease exacerbations, one pulmonary infarction, one intra-alveolar hemorrhage, and one inflammatory pseudo-tumor lung.
Xpert® MTB/RIF assay was performed on BA (n = 48), BAL (n = 47), or a BA/BAL mix (n = 67) (Figure
1). Out of the 162 patients, 30 TB cases were finally diagnosed, corresponding to a prevalence of 18.5% (Figure
1). Of these cases, positive cultures were reported on FOB and sputum samples for 20 and 3, respectively, including one TB case caused by “Mycobacterium canetti”. For the remaining seven patients, diagnosis was based on clinical and histological/radiological features, with favorable evolution under anti-TB therapy serving as confirmation. The final diagnoses for the other patients are detailed in Table
1.
Flow diagram of patients included in the study. BA: bronchial aspirate; BAL: bronchoalveolar lavage; SM: smear microscopy; GX: Xpert®MTB/RIF assay; NTM: nontuberculous mycobacteria; TB: tuberculosis.
Performances of microbiological methods:
In comparison with culture used as reference, the overall sensitivity, specificity, PPV, and NPVs for the Xpert® MTB/RIF assay were 80.0% (95% CI: 57.8-92.5), 98.6% (95% CI: 94.7-99.9), 88.9% (95% CI: 66.0-98.1), and 97.2% (95% CI: 92.8-99.2), respectively. The corresponding values for SM were recorded as 25.0% (95% CI: 10.8-47.3), 95.8% (95% CI: 90.9-98.3), 45.5% (95% CI: 21.3-72.0), and 90.1% (95% CI: 84.2-94.0), respectively (Table
2). It is interesting to note that the sensitivity of the Xpert® MTB/RIF assay was found to be significantly higher than that of SM (p = 0.003; Table
2).
Performances of the Xpert®MTB/RIF assay, smear microscopy, and culture using FOB samples for the diagnosis of pulmonary tuberculosis
ap-values were calculated using the McNemar’s test.
bp-value between Xpert®MTB/RIFassay vs. smear microscopy.
c
p-value between Xpert®MTB/RIFassay vs. culture.
d
p-value between culture vs. smear microscopy.
When considered relative to the final diagnosis, the performances of both sensitivity (60%; 95% CI: 42.3-75.4) and specificity (100%; 95% CI: 96.6-100.0) of the Xpert® MTB/RIF assay were also shown to be significantly higher than those of SM (16.7%; 95% CI: 6.9 to 34.0; 95.5%; 95% CI: 90.2 to 98.1) (p = 0.001 and p = 0.041) (Table
2). PPV and NPV were 100.0% (95% CI: 79.3-100.0) and 91.7% (95% CI: 85.9-95.3) for the Xpert® MTB/RIF assay, respectively, while these values were measured at 45.5% (95% CI: 21.3-72.0) and 83.4% (95% CI: 76.6-88.6) for SM, respectively. It is worth noting that all six samples with a positive SM and a negative PCR assay were seen to grow an NTM species. Regarding the final diagnosis, no significant difference was observed between Xpert® MTB/RIF assay and culture sensitivities (60%; 95% CI: 42.3-75.4 vs. 66.7%; 95% CI: 48.7-80.9; p = 0.683).
The Xpert® MTB/RIF assay also enabled earlier TB diagnosis and treatment for 13 more patients than was possible with SM (Table
3). In addition, the Xpert® MTB/RIF assay led to the early detection of one MDR-TB case in our series, which was subsequently confirmed using phenotypic tests.
Gain in early pulmonary tuberculosis diagnosis with the Xpert® MTB/RIF assay
aNTM: Nontuberculous mycobacteria.
bTB: Tuberculosis.
Discussion:
FOB constitutes an interesting alternative for TB diagnosis in smear-negative or sputum-scarce patients, especially in developed countries where this procedure is widely available. Given that WHO recommendations on the Xpert® MTB/RIF assay only pertain to sputum samples
[17], further investigation must be conducted regarding the use of this PCR on FOB samples. To the best of our knowledge, there has been no publication specifically describing the clinical interest and accuracy of the Xpert® MTB/RIF assay using FOB samples for TB diagnosis in a low-prevalence country.
Our study produced results in line with previous reports on respiratory samples
[16,23-31], demonstrating that the Xpert® MTB/RIF assay significantly outperformed SM on FOB samples (sensitivities: 80.0% vs. 25.0%; p = 0.003) in a low TB-prevalence area. The sensitivity of the PCR assay was found to be lower when it was calculated relative to the final diagnosis, yet still remained significantly higher than that achieved with SM (60.0% vs. 16.7%; p = 0.001). Our findings revealed similar performances to those reported by two recently published studies, which had previously explored the clinical usefulness of the Xpert® MTB/RIF assay on FOB samples, although this was conducted in high TB-prevalence areas
[18,19]. In the Lee et al. retrospective study, 132 patients were recruited in a single South-Korean center. The study reported sensitivity and specificity values (relative to the culture) of 81.6% and 100.0% for the Xpert® MTB/RIF assay, compared to 13.2% and 98.8% for SM, respectively
[18]. Theron et al. prospectively included 154 patients in a South-African single-center study and specifically analyzed BAL samples. The resulting sensitivity and specificity values compared to the culture were 92.6% and 96.0% for the Xpert® MTB/RIF assay, and 57.7% and 99.3% for SM, respectively
[19].
We can therefore confirm that for suspected TB requiring bronchoscopic procedure, the Xpert® MTB/RIF assay is a potential alternative to SM using FOB samples. In our study, PCR allowed for early pulmonary TB diagnosis to be established, enabling appropriate treatment to be started early for 13 more patients as compared to SM (72%) (p < 0.001; Table
3). In these patients, the definitive TB diagnosis was secondarily confirmed either by culture or clinical evolution under specific antimicrobial therapy. These findings were similar to those of the Theron et al. study (>80%)
[19], and are of particular significance in a low TB-incidence area, where there is a higher proportion of alternative diagnosis, such as lung cancer or pyogenic bacterial pneumonia.
Our study objectives did not include assessing the value of the GeneXpert® assay in detecting rifampicin resistance on FOB samples, since the incidence of such resistance was expected to be low, as seen in previous studies
[18,19]. The unique case of MDR-TB was, however, unambiguously detected, and all PCR results were corroborated by those that were phenotypically determined.
Our study presented some limitations. Firstly, this was a retrospective study, despite all Xpert® MTB/RIF assays being performed on fresh FOB samples. Secondly, it was a single-center study. Finally the PCR assays were not systematically performed on the same FOB sample for all patients (BA or BAL). A prospective study should be designed with systematized and parallel FOB sampling (BA, BAL). This would provide a more proficient way of testing the Xpert® MTB/RIF assay in different types of per-endoscopic respiratory samples, an endpoint that we were unable to analyze due to our retrospective design. Cost-effectiveness studies should also be conducted in order to specify the rational use of the Xpert® MTB/RIF assay on FOB samples in novel diagnostic algorithms, including this PCR assay in well-resourced countries.
Conclusion:
In summary, our study confirms the clinical usefulness of the Xpert® MTB/RIF assay, compared to SM, for the early diagnosis of suspected pulmonary TB requiring FOB, performed on per procedure samples. This is a reproducible commercial assay, and should especially be considered as a relevant option in low TB-burden countries.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
Study conception and design: PLP, VC, GZ, and EB. Acquisition of data: PLP, RM, KC and YO. Drafting of manuscript: PLP, VC, GZ, and EB. Analysis and interpretation of data: BM. Critical revision: VC, GZ, and EB. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2466/14/137/prepub
| Background:
Fiberoptic bronchoscopy (FOB) is a useful diagnosis tool in low-burden countries for patients with suspected pulmonary tuberculosis (TB) who are smear-negative or sputum-scarce. This study sought to determine the accuracy of the Xpert® MTB/RIF (XP) assay using FOB samples.
Methods:
We retrospectively reviewed clinical, radiological, and microbiological characteristics of 175 TB-suspected patients requiring diagnostic FOB (bronchial aspirate or bronchoalveolar lavage) with XP assay. Polymerase chain reaction (PCR) and smear microscopy (SM) performances were first compared to culture, then to the final diagnosis, established based on clinical or radiological evolution when cultures were negative.
Results:
Of the total 162 included patients, 30 (18.5%) had a final diagnosis of pulmonary TB, with positive cultures reported in 23. As compared to culture, sensitivity and specificity values were 80.0% and 98.6% for the XP assay, and 25.0% and 95.8% for SM, respectively. As compared to final diagnosis, the corresponding performance values were 60.0% and 100.0% for the XP assay, and 16.7% and 95.5% for SM, respectively. The sensitivity of the XP assay was significantly higher than that of SM in both cases (p=0.003 and p=0.001). Concerning the final diagnosis, both XP assay and culture sensitivities were similar (60% vs. 66.7%). PCR assay enabled pulmonary TB to be diagnosed earlier in 13 more cases, compared to SM.
Conclusions:
Our study has confirmed the clinical benefits provided by XP assay compared to SM for the early diagnosis of suspected pulmonary TB cases requiring FOB, on per procedure samples, especially in a low TB-burden country. | Background:
Tuberculosis (TB) still constitutes a major health problem worldwide, with an 8.8% incidence and 1.3% mortality rate reported in 2010
[1]. In the same year, TB incidence was reported at 8.1 cases per 100,000 in France
[2], defining it as a low TB-burden country. It is worth noting that 73% of TB cases were pulmonary infections, with almost 50% producing a negative smear microscopy (SM)
[2].
By detecting active pulmonary TB early, an appropriate treatment can be initiated, and disease transmission can then be preemptively blocked. Some patients presenting with active pulmonary TB may, however, exhibit negative sputum acid-fast bacilli (AFB) smears. In developed countries, fiberoptic bronchoscopy (FOB) is considered a good option for these cases that pose a diagnostic challenge
[3], although SM is still exhibiting low sensitivity on FOB samples, with 5-35% on bronchial aspirates (BA) and 10-30% on bronchoalveolar lavages (BAL)
[4-9]. Furthermore, while mycobacterial culture remains the gold standard for laboratory diagnosis of TB, it requires 2–6 weeks to confirm a diagnosis. This results in delays in initiating appropriate treatment while waiting for this confirmation, except for cases where there is strong enough clinical suspicion to initiate a presumptive anti-TB therapy.
Several polymerase chain reaction- (PCR-) based molecular methods have recently been developed for early TB diagnosis and rapid detection of drug resistance from clinical specimens
[10-14]. The Xpert® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one of these methods, and consists of a hemi-nested real-time PCR test that simultaneously identifies Mycobacterium tuberculosis and detects rifampicin resistance, as a surrogate of multidrug resistance (MDR), directly from clinical specimens. This assay requires less than 2 hours, and its key advantage over other PCR methods is that it is a fully-automated process, designed to run on the GeneXpert Dx system (Cepheid). This system incorporates DNA extraction, often considered the critical step
[15], along with real-time PCR amplification and detection in a single hands-free process, thus acting as a real “lab-on-chip” device.
Since December 2010, WHO has recommended the Xpert® MTB/RIF assay as a bona fide follow-on test due to its high-quality performance
[16], compared to microscopy, whenever MDR-TB or HIV are of lesser concern, and especially in cases of smear-negative specimens
[17]. This conditional recommendation does, in fact, exclusively concern sputum samples
[17], whereas no specific recommendations for FOB samples have yet been formulated. Finally, there have only been two recent studies that have assessed the Xpert® MTB/RIF assay performance using FOB samples for TB diagnosis in high TB-burden countries
[18,19].
This study sought to evaluate the clinical value of the Xpert® MTB/RIF assay using FOB samples for an early diagnosis of pulmonary TB in either patients with negative sputum AFB smears or those who could not produce an expectorated sputum sample. Patients were treated in a French university hospital in a low TB-burden region (5.9 cases per 100,000 in 2010).
Conclusion:
In summary, our study confirms the clinical usefulness of the Xpert® MTB/RIF assay, compared to SM, for the early diagnosis of suspected pulmonary TB requiring FOB, performed on per procedure samples. This is a reproducible commercial assay, and should especially be considered as a relevant option in low TB-burden countries. | Background:
Fiberoptic bronchoscopy (FOB) is a useful diagnosis tool in low-burden countries for patients with suspected pulmonary tuberculosis (TB) who are smear-negative or sputum-scarce. This study sought to determine the accuracy of the Xpert® MTB/RIF (XP) assay using FOB samples.
Methods:
We retrospectively reviewed clinical, radiological, and microbiological characteristics of 175 TB-suspected patients requiring diagnostic FOB (bronchial aspirate or bronchoalveolar lavage) with XP assay. Polymerase chain reaction (PCR) and smear microscopy (SM) performances were first compared to culture, then to the final diagnosis, established based on clinical or radiological evolution when cultures were negative.
Results:
Of the total 162 included patients, 30 (18.5%) had a final diagnosis of pulmonary TB, with positive cultures reported in 23. As compared to culture, sensitivity and specificity values were 80.0% and 98.6% for the XP assay, and 25.0% and 95.8% for SM, respectively. As compared to final diagnosis, the corresponding performance values were 60.0% and 100.0% for the XP assay, and 16.7% and 95.5% for SM, respectively. The sensitivity of the XP assay was significantly higher than that of SM in both cases (p=0.003 and p=0.001). Concerning the final diagnosis, both XP assay and culture sensitivities were similar (60% vs. 66.7%). PCR assay enabled pulmonary TB to be diagnosed earlier in 13 more cases, compared to SM.
Conclusions:
Our study has confirmed the clinical benefits provided by XP assay compared to SM for the early diagnosis of suspected pulmonary TB cases requiring FOB, on per procedure samples, especially in a low TB-burden country. | 8,457 | 329 | [
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] | [CONTENT] Tuberculosis diagnosis | Fiberoptic bronchoscopy | Xpert® MTB/RIF assay | Smear microscopy [SUMMARY] | [CONTENT] Tuberculosis diagnosis | Fiberoptic bronchoscopy | Xpert® MTB/RIF assay | Smear microscopy [SUMMARY] | [CONTENT] Tuberculosis diagnosis | Fiberoptic bronchoscopy | Xpert® MTB/RIF assay | Smear microscopy [SUMMARY] | [CONTENT] Tuberculosis diagnosis | Fiberoptic bronchoscopy | Xpert® MTB/RIF assay | Smear microscopy [SUMMARY] | [CONTENT] Tuberculosis diagnosis | Fiberoptic bronchoscopy | Xpert® MTB/RIF assay | Smear microscopy [SUMMARY] | [CONTENT] Tuberculosis diagnosis | Fiberoptic bronchoscopy | Xpert® MTB/RIF assay | Smear microscopy [SUMMARY] | [CONTENT] Adult | Aged | Bronchoalveolar Lavage Fluid | Bronchoscopy | Early Diagnosis | Female | Humans | Male | Microscopy | Middle Aged | Molecular Diagnostic Techniques | Predictive Value of Tests | Real-Time Polymerase Chain Reaction | Retrospective Studies | Sputum | Tuberculosis, Multidrug-Resistant | Tuberculosis, Pulmonary [SUMMARY] | [CONTENT] Adult | Aged | Bronchoalveolar Lavage Fluid | Bronchoscopy | Early Diagnosis | Female | Humans | Male | Microscopy | Middle Aged | Molecular Diagnostic Techniques | Predictive Value of Tests | Real-Time Polymerase Chain Reaction | Retrospective Studies | Sputum | Tuberculosis, Multidrug-Resistant | Tuberculosis, Pulmonary [SUMMARY] | [CONTENT] Adult | Aged | Bronchoalveolar Lavage Fluid | Bronchoscopy | Early Diagnosis | Female | Humans | Male | Microscopy | Middle Aged | Molecular Diagnostic Techniques | Predictive Value of Tests | Real-Time Polymerase Chain Reaction | Retrospective Studies | Sputum | Tuberculosis, Multidrug-Resistant | Tuberculosis, Pulmonary [SUMMARY] | [CONTENT] Adult | Aged | Bronchoalveolar Lavage Fluid | Bronchoscopy | Early Diagnosis | Female | Humans | Male | Microscopy | Middle Aged | Molecular Diagnostic Techniques | Predictive Value of Tests | Real-Time Polymerase Chain Reaction | Retrospective Studies | Sputum | Tuberculosis, Multidrug-Resistant | Tuberculosis, Pulmonary [SUMMARY] | [CONTENT] Adult | Aged | Bronchoalveolar Lavage Fluid | Bronchoscopy | Early Diagnosis | Female | Humans | Male | Microscopy | Middle Aged | Molecular Diagnostic Techniques | Predictive Value of Tests | Real-Time Polymerase Chain Reaction | Retrospective Studies | Sputum | Tuberculosis, Multidrug-Resistant | Tuberculosis, Pulmonary [SUMMARY] | [CONTENT] Adult | Aged | Bronchoalveolar Lavage Fluid | Bronchoscopy | Early Diagnosis | Female | Humans | Male | Microscopy | Middle Aged | Molecular Diagnostic Techniques | Predictive Value of Tests | Real-Time Polymerase Chain Reaction | Retrospective Studies | Sputum | Tuberculosis, Multidrug-Resistant | Tuberculosis, Pulmonary [SUMMARY] | [CONTENT] tb requiring bronchoscopic | tb tuberculosis performances | mycobacterium tuberculosis detects | diagnosis pulmonary tuberculosis | tb diagnostic strategy [SUMMARY] | [CONTENT] tb requiring bronchoscopic | tb tuberculosis performances | mycobacterium tuberculosis detects | diagnosis pulmonary tuberculosis | tb diagnostic strategy [SUMMARY] | [CONTENT] tb requiring bronchoscopic | tb tuberculosis performances | mycobacterium tuberculosis detects | diagnosis pulmonary tuberculosis | tb diagnostic strategy [SUMMARY] | [CONTENT] tb requiring bronchoscopic | tb tuberculosis performances | mycobacterium tuberculosis detects | diagnosis pulmonary tuberculosis | tb diagnostic strategy [SUMMARY] | [CONTENT] tb requiring bronchoscopic | tb tuberculosis performances | mycobacterium tuberculosis detects | diagnosis pulmonary tuberculosis | tb diagnostic strategy [SUMMARY] | [CONTENT] tb requiring bronchoscopic | tb tuberculosis performances | mycobacterium tuberculosis detects | diagnosis pulmonary tuberculosis | tb diagnostic strategy [SUMMARY] | [CONTENT] tb | xpert | xpert mtb | mtb | assay | rif | xpert mtb rif | mtb rif | 95 | patients [SUMMARY] | [CONTENT] tb | xpert | xpert mtb | mtb | assay | rif | xpert mtb rif | mtb rif | 95 | patients [SUMMARY] | [CONTENT] tb | xpert | xpert mtb | mtb | assay | rif | xpert mtb rif | mtb rif | 95 | patients [SUMMARY] | [CONTENT] tb | xpert | xpert mtb | mtb | assay | rif | xpert mtb rif | mtb rif | 95 | patients [SUMMARY] | [CONTENT] tb | xpert | xpert mtb | mtb | assay | rif | xpert mtb rif | mtb rif | 95 | patients [SUMMARY] | [CONTENT] tb | xpert | xpert mtb | mtb | assay | rif | xpert mtb rif | mtb rif | 95 | patients [SUMMARY] | [CONTENT] tb | cases | 2010 | real | burden | methods | tb burden | resistance | fob | sputum [SUMMARY] | [CONTENT] tb | de | diagnostics | positive | 500 | bd | tested | bd diagnostics | ml | μl [SUMMARY] | [CONTENT] ci | 95 ci | 95 | xpert mtb | mtb | xpert | patients | xpert mtb rif | mtb rif | assay [SUMMARY] | [CONTENT] procedure samples reproducible | assay especially | relevant option | relevant option low | relevant option low tb | option low tb burden | option low tb | assay especially considered relevant | assay especially considered | option low [SUMMARY] | [CONTENT] tb | assay | 95 | xpert mtb | xpert | mtb | patients | 95 ci | ci | xpert mtb rif [SUMMARY] | [CONTENT] tb | assay | 95 | xpert mtb | xpert | mtb | patients | 95 ci | ci | xpert mtb rif [SUMMARY] | [CONTENT] FOB | TB ||| FOB [SUMMARY] | [CONTENT] 175 | FOB | XP ||| PCR | SM [SUMMARY] | [CONTENT] 162 | 30 | 18.5% | TB | 23 ||| 80.0% | 98.6% | XP | 25.0% | 95.8% | SM ||| 60.0% | 100.0% | XP | 16.7% | 95.5% | SM ||| XP | SM ||| XP | 60% | 66.7% ||| PCR | 13 | SM [SUMMARY] | [CONTENT] XP | SM | TB | FOB [SUMMARY] | [CONTENT] FOB | TB ||| FOB ||| 175 | FOB | XP ||| PCR | SM ||| 162 | 30 | 18.5% | TB | 23 ||| 80.0% | 98.6% | XP | 25.0% | 95.8% | SM ||| 60.0% | 100.0% | XP | 16.7% | 95.5% | SM ||| XP | SM ||| XP | 60% | 66.7% ||| PCR | 13 | SM ||| XP | SM | TB | FOB [SUMMARY] | [CONTENT] FOB | TB ||| FOB ||| 175 | FOB | XP ||| PCR | SM ||| 162 | 30 | 18.5% | TB | 23 ||| 80.0% | 98.6% | XP | 25.0% | 95.8% | SM ||| 60.0% | 100.0% | XP | 16.7% | 95.5% | SM ||| XP | SM ||| XP | 60% | 66.7% ||| PCR | 13 | SM ||| XP | SM | TB | FOB [SUMMARY] |
||||||
A Novel Precise Optical Navigation System for Craniomaxillofacial Surgery Registered With an Occlusal Splint. | 34260445 | An augmented reality tool allows visual tracking of real anatomical structures and superimposing virtual images, so it can be used for navigation of important structures during surgery. | BACKGROUND | Ten beagle dogs were selected and a three-dimensional model was established through computed tomography scanning, dental model making, and laser scanning, and then registration was performed according to the tooth marking points. The bilateral mandibular osteotomy was performed on Beagle dogs under navigation system based on the occlusal splint. The left side was taken to compare the deviation between the preoperative plan and the surgical results, and the accuracy of distance and angle and the stability of the system were analyzed. | METHODS | The average position deviation between the preoperative design and intraoperative navigation was: 0.01 ± 0.73 mm on the lateral height of the mandibular ramus, 0.26 ± 0.57 mm on the inner height of the mandibular ramus, and 0.20 ± 0.51 mm on the osteotomy length. The average angle deviation is 0.94° ± 1.38° on the angle between the mandibular osteotomy plane and ramus plane and 0.66° ± 0.97° on the angle of the retained mandibular angle. And most of the data showed good consistency. | RESULTS | In summary, the accuracy of the system can meet clinical requirements and can be used as a useful tool to improve the accuracy of craniomaxillofacial surgery. | CONCLUSIONS | [
"Animals",
"Augmented Reality",
"Dogs",
"Imaging, Three-Dimensional",
"Mandible",
"Mandibular Osteotomy",
"Occlusal Splints",
"Osteotomy",
"Surgery, Computer-Assisted"
] | 8694255 | null | null | null | null | RESULTS | The mean distance deviation between the preoperative design and postoperative result was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of the retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Fig. 7, Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery.
Judging from the scatter plot of the preoperative design and postoperative results of each index, most of the data showed good consistency and showed a significantly higher correlation coefficient: 0.933 (A), 0.927 (B), 0.961 (C), and 0.913 (E) (Fig. 8, Supplementary Digital Content, Table 2). However, the consistency of the Retained Mandibular Angle data does not show satisfactory consistency, and the correlation coefficient was 0.283 (D) (Fig. 8, Supplementary Digital Content, Table 2). | CONCLUSIONS | This article uses an optical NS to simulate the MASO of experimental animals and verifies the comparison between the results of the osteotomy plane-guided mandibular osteotomy in the AR NS and the preoperative design. The multi-line and multi-angle measurement data are consistent, and there is no statistical difference. Therefore, the use of this registration method can ensure the accuracy of craniofacial surgery, and operating according to the enhanced display of the bone CP can ensure the safety of the osteotomy, and lay the foundation for the application of craniofacial surgery in the future. Although some data show unsatisfactory consistency, this is due to the large difference in the contour and internal structure of the mandible between dogs and humans. In summary, the AR NS established in this experiment explores a simple and easy registration method, and its high-precision navigation performance can meet the actual needs of craniomaxillofacial surgery. This makes the application of the optical surgical NS in actual surgery a step forward. | [
"Three-Dimensional Data Acquisition and Preoperative Planning",
"Fabrication, Scanning, and Registration of Occlusal Splints",
"Intraoperative Navigation",
"Statistical Analysis",
"Navigation",
"Augmented Reality",
"Registration and Accuracy",
"Consistency",
"Limitation and Prospects"
] | [
"Under natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).\nThe three-dimensional image of the mandible.\nPreoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.",
"After planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).\nFabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.\nThe 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.\nScanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.",
"Perform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.\nRegistration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.\nThe cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.",
"In terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).\nMeasurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.\nThe scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.",
"Surgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.",
"Traditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:\nAll navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;\nBecause the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;\nAll surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.\nSpatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.",
"For NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.\nThe parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.\nIn this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.",
"Then, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.\nBesides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.",
"The disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.\nAlso, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.\nThe video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.\nIn this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"MATERIALS AND METHODS",
"Three-Dimensional Data Acquisition and Preoperative Planning",
"Fabrication, Scanning, and Registration of Occlusal Splints",
"Intraoperative Navigation",
"Statistical Analysis",
"RESULTS",
"DISCUSSION",
"Navigation",
"Augmented Reality",
"Registration and Accuracy",
"Consistency",
"Limitation and Prospects",
"CONCLUSIONS",
"Supplementary Material",
"Supplementary Material"
] | [
"In this study, 10 beagle dogs were randomly selected as our model. According to the designed CP, MASO can be carried out in animal experiments. Based on AR technology, the mandibular section, and virtual image can be superimposed on the mandible of the beagle for navigation surgery. Finally, we measure the relevant indicators in the 3D image of the left mandible and perform statistical analysis on the original data before and after the operation. All animal experiments were approved by the Institutional Animal Care and Use Committee and complied with relevant guidelines and regulations.\nThree-Dimensional Data Acquisition and Preoperative Planning Under natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).\nThe three-dimensional image of the mandible.\nPreoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.\nUnder natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).\nThe three-dimensional image of the mandible.\nPreoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.\nFabrication, Scanning, and Registration of Occlusal Splints After planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).\nFabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.\nThe 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.\nScanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.\nAfter planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).\nFabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.\nThe 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.\nScanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.\nIntraoperative Navigation Perform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.\nRegistration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.\nThe cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.\nPerform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.\nRegistration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.\nThe cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.\nStatistical Analysis In terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).\nMeasurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.\nThe scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.\nIn terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).\nMeasurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.\nThe scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.",
"Under natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).\nThe three-dimensional image of the mandible.\nPreoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.",
"After planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).\nFabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.\nThe 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.\nScanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.",
"Perform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.\nRegistration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.\nThe cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.",
"In terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).\nMeasurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.\nThe scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.",
"The mean distance deviation between the preoperative design and postoperative result was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of the retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Fig. 7, Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery.\nJudging from the scatter plot of the preoperative design and postoperative results of each index, most of the data showed good consistency and showed a significantly higher correlation coefficient: 0.933 (A), 0.927 (B), 0.961 (C), and 0.913 (E) (Fig. 8, Supplementary Digital Content, Table 2). However, the consistency of the Retained Mandibular Angle data does not show satisfactory consistency, and the correlation coefficient was 0.283 (D) (Fig. 8, Supplementary Digital Content, Table 2).",
"Navigation Surgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.\nSurgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.\nAugmented Reality Traditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:\nAll navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;\nBecause the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;\nAll surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.\nSpatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.\nTraditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:\nAll navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;\nBecause the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;\nAll surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.\nSpatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.\nRegistration and Accuracy For NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.\nThe parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.\nIn this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.\nFor NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.\nThe parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.\nIn this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.\nConsistency Then, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.\nBesides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.\nThen, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.\nBesides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.\nLimitation and Prospects The disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.\nAlso, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.\nThe video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.\nIn this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS.\nThe disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.\nAlso, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.\nThe video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.\nIn this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS.",
"Surgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.",
"Traditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:\nAll navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;\nBecause the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;\nAll surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.\nSpatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.",
"For NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.\nThe parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.\nIn this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.",
"Then, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.\nBesides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.",
"The disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.\nAlso, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.\nThe video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.\nIn this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS.",
"This article uses an optical NS to simulate the MASO of experimental animals and verifies the comparison between the results of the osteotomy plane-guided mandibular osteotomy in the AR NS and the preoperative design. The multi-line and multi-angle measurement data are consistent, and there is no statistical difference. Therefore, the use of this registration method can ensure the accuracy of craniofacial surgery, and operating according to the enhanced display of the bone CP can ensure the safety of the osteotomy, and lay the foundation for the application of craniofacial surgery in the future. Although some data show unsatisfactory consistency, this is due to the large difference in the contour and internal structure of the mandible between dogs and humans. In summary, the AR NS established in this experiment explores a simple and easy registration method, and its high-precision navigation performance can meet the actual needs of craniomaxillofacial surgery. This makes the application of the optical surgical NS in actual surgery a step forward.",
"",
""
] | [
"materials|methods",
null,
null,
null,
null,
"results",
"discussion",
null,
null,
null,
null,
null,
"conclusion",
"supplementary-material",
"supplementary-material"
] | [
"Cranio-maxillofacial surgery",
"image-guided surgery",
"occlusal splint",
"optical navigation system"
] | MATERIALS AND METHODS:
In this study, 10 beagle dogs were randomly selected as our model. According to the designed CP, MASO can be carried out in animal experiments. Based on AR technology, the mandibular section, and virtual image can be superimposed on the mandible of the beagle for navigation surgery. Finally, we measure the relevant indicators in the 3D image of the left mandible and perform statistical analysis on the original data before and after the operation. All animal experiments were approved by the Institutional Animal Care and Use Committee and complied with relevant guidelines and regulations.
Three-Dimensional Data Acquisition and Preoperative Planning Under natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).
The three-dimensional image of the mandible.
Preoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.
Under natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).
The three-dimensional image of the mandible.
Preoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.
Fabrication, Scanning, and Registration of Occlusal Splints After planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).
Fabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.
The 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.
Scanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.
After planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).
Fabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.
The 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.
Scanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.
Intraoperative Navigation Perform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.
Registration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.
The cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.
Perform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.
Registration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.
The cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.
Statistical Analysis In terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).
Measurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.
The scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.
In terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).
Measurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.
The scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.
Three-Dimensional Data Acquisition and Preoperative Planning:
Under natural occlusion, all selected animals underwent a 3D spiral CT scan of the skull. Preoperative thin-slice (0.5 mm) CT scan (GE LightSpeed 16, Milwaukee, WI) and digital imaging of medical image data have been imported into Mimics 18.0 (Materialize Company, Leuven, Belgium) software to create a 3D image of the mandible through regional growth (Fig. 1). According to the different thresholds of skeletal tissue and nerves, the IANs are separated layer by layer and reconstructed in 3D. We can obtain 3D digital models of the mandible and the IANs from the mental foramen to the mandibular foramen, and design the CPs according to the clear position and shape of the nerves. The 3D information of the mandibular CPs and the IANs is output as an STL file (Fig. 2).
The three-dimensional image of the mandible.
Preoperative design. (A) Raw 3D data of mandible. (B) Marking the inferior alveolar nerves (IANs) and cutting planes. (C) Front perspective view of the osteotomy plan. (D) Simulated 3D image after osteotomy. 3D, three-dimensional.
Fabrication, Scanning, and Registration of Occlusal Splints:
After planning the navigation information, we need to register the virtual image in the real world. The fiducial mark needs to serve as a common reference between the virtual image data and the real world and to maintain a stable relationship with the mandible. The best way to track and register the AR system is to mark it on the board. Then, we use an occlusal splint to connect the marker and the jaw and stabilize the relationship between them. Specifically, using the mold of the mandibular teeth of the Beagle as a reference, a self-curing plastic material was used to make occlusal splints on both canines and the middle 6 teeth. In a stable occlusal relationship, the plaster and the splint are firmly combined. Then connect the tracking marker and occlusal splint in a personalized posture, which is collectively referred to as a marker complex (MC) (Fig. 3).
Fabrication of marker complex. (A) Dental mold. (B) Occlusal splint. (C) Marker complex (MC). (D) Firm combination of dental mold and MC.
The 3D data is obtained after 3D laser scanning the MC and dental model, and then input these data to the graphics workstation, and fitted with the CT data of the mandibular model together through at least 3 landmarks (usually prominent sharp points or tooth pit). At the same time, the 3D information of the mandibular CPs and IANs is combined with the MC and the mandible and then imported into 3ds Max software (Autodesk, San Rafael, CA) in STL format together. Adjust the position and direction of the virtual image to ensure that the center point of the marked point overlaps with the coordinate origin system, and then export all the information of different materials to the WRL file format (Fig. 4). The 3D images of the preoperative mandible and osteotomy plan were imported into AR Toolkit software (ARToolworks, Seattle, WA) to verify the registration effect.
Scanning and registration of occlusal splints. (A) 3D laser scanning of the marker complex. (B) Calibration the center point in the 3ds Max software. 3D, three-dimensional.
Intraoperative Navigation:
Perform intravenous anesthesia, skin preparation, disinfection, prepare a work towel for the beagle, and separate the skin and tissue layers until the mandibular angle is completely exposed. The sterilized MC is firmly worn on the corresponding teeth of the beagle, and then the image is adjusted in AR Toolkit software (ARToolworks) to achieve accurate position, appropriate color contrast, and transparency. Finally, all surgical plans are displayed in the surgical area in real-time through a helmet-mounted display (nVisor ST60, NVIS, Sunnyvale, California, USA). Under the guidance of the predesigned CP displayed in the optical NS based on AR technology, MASO was able to operate (Fig. 5). Collect and keep the cut bone pieces, and then suture the incision layer by layer after hemostasis (Fig. 6). A week later, after performing a CT scan of the postoperative beagle, the image data was imported into Mimics 18.0 software to create a 3D model of the mandible.
Registration and surgery. (A) Intraoperative registration. (B) Operating the mandibular angle split osteotomy (MASO) under the guidance of the predesigned cutting plane displayed in the optical navigation system (NS) based on AR technology. AR, augmented reality.
The cut bone blocks. (A) Front view of the bilateral cut bones. (B) Side view of the bilateral cut bones.
Statistical Analysis:
In terms of data analysis, in the preoperative and postoperative measurements of the 10 groups of mandibular models, the left data was selected to mark many anatomical landmarks, and statistical analysis was performed by defining different line segments, planes, and angles (Fig. 7). Specifically, we marked condyle (A1), coracoid (A2), osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), medial mandibular osteotomy point (B3), and gonion (C). Then, we marked the mandibular ramus plane (A1A2C) as yellow and the osteotomy plane (B1B2B3) as red. Finally, we selected and defined the line segment A1B3 as the inner plate height, the line segment A1B1 as the outer plate height, and the line segment the length of B1B2 was osteotomy length, ∠A1B1B2 was the retained mandibular angle, and the angle between plane A1A2C and plane B1B2B3 was osteotomy angle. Undergoing the statistics of inner plate height, outer plate height, osteotomy length, retained mandibular angle, and osteotomy angle for designed osteotomy, respectively (preoperative) (Fig. 7) and the actual osteotomy (postoperative), respectively, the statistical results are shown in Supplementary Digital Content, Table 1. Statistical software (SPSS 26; IBM Corp, Armonk, NY) was used to perform a paired t-test on the mandibular measurement data, and all measurement data were expressed as mean ± standard deviation. Finally, we made a scatter plot for the preoperative design and postoperative results of each index, and analyzed the correlation coefficient and consistency of each data (Fig. 8, Supplementary Digital Content, Table 2).
Measurement of different line segments, planes, and angles. (A1) Condyle. (A2) Coracoid. (B1) Osteotomy point of the mandibular trailing edge. (B2) Osteotomy point of the mandibular front edge. (B3) Medial mandibular osteotomy point. (C) Gonion. (A1A2C) Mandibular ramus plane (yellow). (B1B2B3) Osteotomy plane (red). (A1B3) Inner plate height. (A1B1) Outer plate height. (B1B2) Osteotomy length. (∠A1B1B2) Retained mandibular angle. (A1A2C across with B1B2B3) Osteotomy angle.
The scatter plot of the preoperative design and the postoperative results. (A) Inner plate height. (B) Outer plate height. (C) Osteotomy length. (D) Retained mandibular angle. (E) Osteotomy angle.
RESULTS:
The mean distance deviation between the preoperative design and postoperative result was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of the retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Fig. 7, Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery.
Judging from the scatter plot of the preoperative design and postoperative results of each index, most of the data showed good consistency and showed a significantly higher correlation coefficient: 0.933 (A), 0.927 (B), 0.961 (C), and 0.913 (E) (Fig. 8, Supplementary Digital Content, Table 2). However, the consistency of the Retained Mandibular Angle data does not show satisfactory consistency, and the correlation coefficient was 0.283 (D) (Fig. 8, Supplementary Digital Content, Table 2).
DISCUSSION:
Navigation Surgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.
Surgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.
Augmented Reality Traditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:
All navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;
Because the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;
All surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.
Spatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.
Traditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:
All navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;
Because the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;
All surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.
Spatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.
Registration and Accuracy For NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.
The parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.
In this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.
For NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.
The parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.
In this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.
Consistency Then, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.
Besides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.
Then, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.
Besides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.
Limitation and Prospects The disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.
Also, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.
The video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.
In this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS.
The disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.
Also, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.
The video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.
In this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS.
Navigation:
Surgical navigation, also known as a computer-assisted surgery (CAS), has rapidly emerged in several surgical disciplines. It is one of the most impressive surgical advances in the past 30 years. It has realized the use of real-time 3D image guidance during surgery. Navigation has not only become a tool to improve medical and health14 but also used as a research tool.15 The use of CAS has expanded the limited visual field of doctors and updated the concept of surgery and surgical instruments. By introducing guided images during surgery, surgery accuracy can be effectively improved, surgery time can be shortened, and surgery complications can be reduced. Due to the complex anatomical structure of the craniomaxillofacial region, CAS seems to be particularly applicable in the field of craniomaxillofacial surgery and has gradually gained recognition in attempts and explorations.16,17 In craniomaxillofacial contour reconstruction surgery, tumor resection and reconstruction surgery, trauma surgery, and orthognathic surgery, It is necessary to reconstruct the functional and aesthetic anatomy by resetting displaced bone pieces, by cutting abnormal bone contours, or by placing various implants.18–21 It is very important to accurately determine the expected result of a surgical correction or the intended position of the implant before surgery. However, in many cases, it is difficult to transmit complex visual planning information to the actual surgical site only by imagination.19,20,22–24 Cranio-maxillofacial surgery requires reliable protection of key anatomical structures and accurate osteotomy,23,25–27 which makes it one of the most promising areas for image-guided surgery.21,28 However, the actual use of CAS is subject to many restrictions and is still in the experimental stage.
Augmented Reality:
Traditional optical navigation uses surgical probes to track external markers and present images point-to-point on the screen.29 However, this navigation technology has the following shortcomings:
All navigation information is displayed on a separate display, and the doctor has to constantly switch the field of view between the virtual image and the patient's anatomy during the operation;
Because the display is flat, therefore, the depth of the two-dimensional image must be used to reflect the 3D space position, which increases the difficulty of operation in actual navigation;
All surgical instruments used for navigation must be equipped with positioning and tracking devices, and there must be more than 3 positioning tracking devices to reflect them.
Spatial location. However, not all surgical instruments can meet the installation requirements, because in the mandibular osteotomy, the narrow surgical area is likely to cause the positioning tracking device to be blocked, thereby affecting the positioning of the equipment, so it can only be used for specific operations and specific surgical instruments. However, in the AR NS, virtual images and real objects will be reflected in the NS through the mode of visual fusion.30 When the surgeon wears a head-mounted display, the virtual image can cover the wearer's field of vision, so the wearer does not need to master hand-eye coordination skills. And by unifying the 3D model, the actual position of the patient, and the real-time position of the surgical instrument in the space for registration, the NS can collect and display the position of the surgical instrument in the space in real-time. This is achieved with the help of the positioning function. Under the guidance of AR-based NS, bone resection and reconstruction in complex anatomical areas (such as the head and neck area) may be more accurate and effective, and at the same time, it can reduce the risk of nerve and blood vessel damage.31 Due to these advantages, AR-based NSs seem to be more suitable for craniomaxillofacial surgery.
Registration and Accuracy:
For NSs, accuracy is the most important measurement index, because it directly affects the results of the operation. The key to precise navigation is how to accurately match the marker with the human body during the operation, and how to accurately reproduce the positional relationship during the preoperative registration process. Image registration based on external features is the most commonly used method in surgical NSs, requiring markers to be visible in preoperative images, so it is easy to be detected during intraoperative registration.32 Registration methods can be divided into 2 types: intrusive registration and nonintrusive registration. Invasive markers include 3D frame-based markers and titanium screw implant-based markers. Noninvasive fiducial markers can be divided into 3 types: adhesive markers, dental instruments, and anatomical markers (such as skin33 and bone). Most of the literature supports that the gold standard for high-precision registration is a stable marker (such as a titanium screw) marked directly on the bone tissue.34,35 This is because the possibility of relative movement between the skull and the screw is relatively small, so it has high application accuracy.
The parameters of the registration method based on external features can be solved without complicated optimization algorithms, and the accuracy is reliable, and it is relatively easy to achieve fast and fully automatic registration.36 But it is invasive, brings great discomfort to the patient, and limits the doctor's operation during the operation. Major craniofacial surgery involves the facial skull and brain skull. Except for the mandible, the other skulls and maxilla are seamlessly connected, and the teeth and maxilla are also firmly integrated into one. Moreover, the irregular shape of the teeth makes it easy to form a mosaic state with other materials. With this, the occlusal splint can provide a noninvasive, high-precision registration method, because it can be stably installed on the teeth with bony structures.32,37,38 Therefore, we use the occlusal relationship of the teeth to make the fiducial markers tightly integrated with the skull. Cutting et al39 studied the use of sharp bony marks for registration in AR systems, that is, through the bite plate positioning mark, the final control error is less than 1.5 mm, and the rotation error of any axial position is <3°. Tsuji et al40 reported using tooth model data for registration, with the front teeth and the left and right second molars as the standard, and the registration error can be controlled within 1 mm, with a maximum of 1.02 mm. Zhu et al6 applied the early occlusal bracket of this navigation mode for registration, and the position error reached 0.96 ± 0.51 mm. Recently, Wang et al41 used an intraoral 3D scanning instrument to obtain a patient's tooth shape model and then registered the tooth model into a customized stereo camera system. Finally, through a simulated mandibular experiment, it was found that the average error of target overlay registration was less than 0.50 mm and the registration time was less than 0.5 seconds.
In this study, we simulated the tooth structure of experimental animals with dental molds. And the braces of the first 6 jaw teeth made of self-curing plastics act as splints, which can be accurately fixed to the solid model of Beagles teeth with good reproducibility. The mean distance deviation between the preoperative design and intraoperative navigation was: 0.26 ± 0.57 mm on the inner plate height, 0.01 ± 0.73 mm on the outer plate height, and 0.20 ± 0.51 mm on the osteotomy length. The angular deviation was: 0.94° ± 1.38° on the angle of retained mandibular angle and 0.66° ± 0.97° on the osteotomy angle (the angle between the mandibular ramus plane and the osteotomy plane) (Supplementary Digital Content, Table 1). Supplementary Digital Content, Table 1 shows that each set of data has a P value > 0.05, so the error between the data was not statistically significant, indicating that the higher accuracy of the system meets the accuracy requirements of the surgery. Nevertheless, the position of the osteotomy point of the mandibular trailing edge (B1), osteotomy point of the mandibular front edge (B2), and medial mandibular osteotomy point (B3) change greatly, because these points have no clear anatomical marks on the mandible of the Beagle. Therefore, the distance deviation on the Inner plate height and the outer plate height have poor stability in animal experiments, but the system performs relatively satisfactorily in clinical applications, because human maxillofacial bones have clear anatomical markings and aesthetic standards, and the surgeon has a clear plan for the length and angle of the osteotomy.
Consistency:
Then, we analyzed the consistency between preoperative design and postoperative results and found that all other indicators showed higher correlation coefficients: 0.933, 0.927, 0.961, and 0.913, except for the angle of retained mandibular angle (0.283) (Supplementary Digital Content, Table 2). This is because the angle that retains the mandibular angle, that is, the angle between A1B1 and B1B2 (∠A1B1B2) is only determined by the positions of point A1, point B1, and point B2. The anatomical positions of point A1 (condylar point) and point B2 (posterior point of the dog's fourth premolar, corresponding to the posterior point of the human second premolar) are relatively easy to determine. The determination of the position of point B1 (the new mandibular angle) is relatively subjective in animal experiments, so the numerical difference between groups is relatively large, so the correlation coefficient is significantly lower than that of other measurement indicators. However, this did not significantly affect the error of each measurement index, nor did it reduce the overall accuracy of the NS.
Besides, point B1 corresponds to the intersection of the depression groove plane and the ascending branch of the mandible in the human body, and the position is relatively fixed and easier to determine. Therefore, we believe that the consistency between groups that preserves the angle of the mandibular angle will perform well in the human body. The consistency of other measurement indicators affected by point B1 (such as the length of A1B1, that is, the height of the mandibular outer plate) will be better, and the overall stability of the system will be better.
Limitation and Prospects:
The disadvantage of this study is that only the preoperative design and postoperative results of Beagle mandibular osteotomy were compared. There was no randomized controlled experiment, such as a comparative study with traditional surgical methods. Mainly because the traditional surgical method only relies on the operator's operating experience, there is no quantitative design plan before surgery. Moreover, the postoperative evaluation method only has symmetry and aesthetics of appearance, so it is impossible to objectively evaluate the errors of traditional surgical methods.
Also, due to the lack of randomized controlled trials, there is no time record of traditional surgical methods, time cost comparison with navigation methods, and statistical analysis of related data. However, as the familiarity with the preoperative design software and process improves, the overall time of osteotomy under each navigation is gradually shortened. We speculate that the magnitude of time reduction may gradually decrease, or even no longer be shortened in a large number of applications in the future, and the preoperative preparation time will be stabilized at a time level higher than that of traditional surgical methods. Nevertheless, the precise and real-time guidance of the NS for the intraoperative operation will greatly avoid the time-consuming and economic cost of the traditional method of visual evaluation and repeated correction.
The video detection method adopted in this paper is to use the tooth model to simulate the real canine teeth during the operation, use the marker bracket to fix the spatial relationship between the marker and the model, and perform registration before the operation (Fig. 8). When wearing bracket marks for experimental animals in the experiment, the NS can recognize the marks through related programs and overlap the virtual image with the mandible, just as the tooth model overlaps the preoperative plan. The registration process is completed before the operation, without waiting for intraoperative registration, which greatly reduces the operation time.
In this study, an AR NS based on occlusal splint was established in animal experiments. The system has good accuracy, the distance error is less than 1.5 mm, and the angle error is less than 3. During the operation, experimenters can view information about important nerves and blood vessels under the surface of animal skin and bones in real-time. This information is a virtual 3D graphic generated by a computer. By combining virtual information with real images for observation, experimenters can better understand the tissues near the surgical site and reduce surgical risks. The research provides guidance and experience for clinical surgery practice, guarantees the safety of surgery, and provides methods to improve the results of surgery. After the osteotomy, the symmetrical mandible and roughly the same bilateral mandibular angle can be seen, as shown in Figure 5. Besides, there is still much room for improvement in the accuracy of the NS. If we continue to reduce some registration and docking steps, because this can further reduce some data loss and errors, thereby improving the overall accuracy of the surgical NS.
CONCLUSIONS:
This article uses an optical NS to simulate the MASO of experimental animals and verifies the comparison between the results of the osteotomy plane-guided mandibular osteotomy in the AR NS and the preoperative design. The multi-line and multi-angle measurement data are consistent, and there is no statistical difference. Therefore, the use of this registration method can ensure the accuracy of craniofacial surgery, and operating according to the enhanced display of the bone CP can ensure the safety of the osteotomy, and lay the foundation for the application of craniofacial surgery in the future. Although some data show unsatisfactory consistency, this is due to the large difference in the contour and internal structure of the mandible between dogs and humans. In summary, the AR NS established in this experiment explores a simple and easy registration method, and its high-precision navigation performance can meet the actual needs of craniomaxillofacial surgery. This makes the application of the optical surgical NS in actual surgery a step forward.
Supplementary Material:
Supplementary Material:
| Background:
An augmented reality tool allows visual tracking of real anatomical structures and superimposing virtual images, so it can be used for navigation of important structures during surgery.
Methods:
Ten beagle dogs were selected and a three-dimensional model was established through computed tomography scanning, dental model making, and laser scanning, and then registration was performed according to the tooth marking points. The bilateral mandibular osteotomy was performed on Beagle dogs under navigation system based on the occlusal splint. The left side was taken to compare the deviation between the preoperative plan and the surgical results, and the accuracy of distance and angle and the stability of the system were analyzed.
Results:
The average position deviation between the preoperative design and intraoperative navigation was: 0.01 ± 0.73 mm on the lateral height of the mandibular ramus, 0.26 ± 0.57 mm on the inner height of the mandibular ramus, and 0.20 ± 0.51 mm on the osteotomy length. The average angle deviation is 0.94° ± 1.38° on the angle between the mandibular osteotomy plane and ramus plane and 0.66° ± 0.97° on the angle of the retained mandibular angle. And most of the data showed good consistency.
Conclusions:
In summary, the accuracy of the system can meet clinical requirements and can be used as a useful tool to improve the accuracy of craniomaxillofacial surgery. | null | null | 12,085 | 269 | [
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"animals underwent 3d",
"ct scan skull",
"3d images preoperative",
"ct data mandibular",
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] | null | null | null | null | null | [CONTENT] Cranio-maxillofacial surgery | image-guided surgery | occlusal splint | optical navigation system [SUMMARY] | [CONTENT] Cranio-maxillofacial surgery | image-guided surgery | occlusal splint | optical navigation system [SUMMARY] | [CONTENT] Cranio-maxillofacial surgery | image-guided surgery | occlusal splint | optical navigation system [SUMMARY] | null | null | null | [CONTENT] Animals | Augmented Reality | Dogs | Imaging, Three-Dimensional | Mandible | Mandibular Osteotomy | Occlusal Splints | Osteotomy | Surgery, Computer-Assisted [SUMMARY] | [CONTENT] Animals | Augmented Reality | Dogs | Imaging, Three-Dimensional | Mandible | Mandibular Osteotomy | Occlusal Splints | Osteotomy | Surgery, Computer-Assisted [SUMMARY] | [CONTENT] Animals | Augmented Reality | Dogs | Imaging, Three-Dimensional | Mandible | Mandibular Osteotomy | Occlusal Splints | Osteotomy | Surgery, Computer-Assisted [SUMMARY] | null | null | null | [CONTENT] animals underwent 3d | ct scan skull | 3d images preoperative | ct data mandibular | beagle mandibular osteotomy [SUMMARY] | [CONTENT] animals underwent 3d | ct scan skull | 3d images preoperative | ct data mandibular | beagle mandibular osteotomy [SUMMARY] | [CONTENT] animals underwent 3d | ct scan skull | 3d images preoperative | ct data mandibular | beagle mandibular osteotomy [SUMMARY] | null | null | null | [CONTENT] mandibular | osteotomy | angle | registration | surgery | point | surgical | 3d | data | time [SUMMARY] | [CONTENT] mandibular | osteotomy | angle | registration | surgery | point | surgical | 3d | data | time [SUMMARY] | [CONTENT] mandibular | osteotomy | angle | registration | surgery | point | surgical | 3d | data | time [SUMMARY] | null | null | null | [CONTENT] angle | content | content table | supplementary | supplementary digital | supplementary digital content | supplementary digital content table | digital content table | digital content | table [SUMMARY] | [CONTENT] ns | multi | surgery | craniofacial surgery | ensure | registration method | application | craniofacial | difference | ar ns [SUMMARY] | [CONTENT] mandibular | angle | osteotomy | point | surgery | surgical | registration | 3d | data | ns [SUMMARY] | null | null | null | [CONTENT] 0.01 | 0.73 mm | 0.26 ± | 0.57 mm | 0.20 ± | 0.51 mm ||| 0.94 | 1.38 | 0.66 | 0.97 ||| [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] ||| Ten | three ||| Beagle ||| ||| ||| 0.01 | 0.73 mm | 0.26 ± | 0.57 mm | 0.20 ± | 0.51 mm ||| 0.94 | 1.38 | 0.66 | 0.97 ||| ||| [SUMMARY] | null |
|||
The HSP72 stress response of monocytes from patients on haemodialysis is impaired. | 19339340 | Induction of heat shock proteins (HSP), i.e. of the major family member HSP70, is an important cytoprotective-resistance mechanism for monocytes/ macrophages (Mphi). Patients on haemodialysis present with a high infectious morbidity and enhanced carcinoma incidence. Renal insufficiency-related alteration of microbicidal and tumoricidal functions of Mphi, major effectors of the immune system, might promote these diseases. | BACKGROUND | Freshly isolated Mphi from Sprague-Dawley rats 2 weeks after 5/6-nephrectomy and from patients on intermittent haemodialysis (IHD) were stimulated by heat shock (HS) and compared to stimulated Mphi of control rats or healthy volunteers (CTR). Expression of HSP72 (inducible HSP70) was assessed by RT-PCR, and/or flow cytometry. Apoptosis of Mphi was detected by flow cytometry (CD14/annexin V-labelling). | METHODS | In rat Mphi, baseline HSP72 expression was similar in both groups, but its induction was significantly impaired in renal insufficiency (214 +/- 68% less HSP70-mRNA versus CTR, n = 6). In patients, HSF-1-mRNA and HSP72-mRNA/protein response to HS was significantly lower, but not affected by dialysis session itself. In parallel, apoptosis of Mphi of patients was enhanced (+83 +/- 29% constitutive apoptotic Mphi versus CTR, n = 8), and HS-dependent protection from apoptosis with and without serum depletion (48 h depletion: HS, +275 +/- 37% apoptotic Mphi versus CTR, n = 6; full medium: +166 +/- 62% versus CTR, n = 8, P < 0.05) was inferior. | RESULTS | Impaired HSP72 stress response of Mphi in patients on haemodialysis might contribute to the observed immune dysfunction and, therefore, to the increased susceptibility to infection and malignancy. Stress impairment is not restricted to uraemia but is already present in a rat model of chronic kidney disease. | CONCLUSIONS | [
"Adolescent",
"Adult",
"Aged",
"Animals",
"Apoptosis",
"Base Sequence",
"Case-Control Studies",
"DNA Primers",
"DNA-Binding Proteins",
"Disease Models, Animal",
"Female",
"HSP72 Heat-Shock Proteins",
"Heat Shock Transcription Factors",
"Heat-Shock Response",
"Humans",
"In Vitro Techniques",
"Kidney Failure, Chronic",
"Male",
"Middle Aged",
"Monocytes",
"RNA, Messenger",
"Rats",
"Rats, Sprague-Dawley",
"Renal Dialysis",
"Transcription Factors",
"Urea",
"Young Adult"
] | 7107957 | Introduction | Patients on haemodialysis present with a high infectious morbidity, enhanced carcinoma incidence, impaired wound healing and reduced effect of vaccination—diseases that are directly related to the function of the immune system [1–4]. Immunological abnormalities of leukocytes have been described in patients on dialysis [5]. Mphi (monocytes/ macrophages) are major effectors of the immune system with widespread microbicidal and tumoricidal functions that are altered in uraemia [6–8]. Uraemic immunodeficiency is a complication of renal failure reflecting stress, e.g. exerted by as yet poorly defined uraemic toxins, imbalances of antioxidants and pro-oxidants, malnutrition or iron overload, membrane bio-incompatibility and inflammation [9–11]. Therefore, patients with end-stage renal disease (ESRD) suffer from increased oxidative stress, which can only be ineffectively treated by haemodialysis [12–14]. The response of human cells to stress, e.g. heat shock (HS), is a highly conserved adaptive cell response found in all cells in all life forms conferring resistance to different stressors, i.e. increased temperature (fever), nutritional deficiency, oxidative stress or hypoxia [15–17]. The corresponding heat shock proteins (HSPs), like the major stress-inducible family member HSP72 (70-kDa HSP, inducible HSP70, HSPA1A, HSP70-1) [18], can also be found in cells under normal physiological conditions controlling the folding of proteins. Constitutively expressed HSP70 (Hsc70) binds to newly translated immature proteins and prevents premature and improper binding and folding [19–21]. This is vital, since HSP72-deficient animals are susceptible to stress or even not viable [22]. In response to stress, housekeeping activities of HSPs, e.g. degradation of unstable and misfolded proteins, control of regulatory proteins or transport of proteins, switch towards reaction against stress to increase the cell's chance of survival. The cellular injury decreases, either by induction of cytoresistance or by enhancement of repair mechanisms [18]. Thus, HSP72 induction confers protection even against sublethal damage causing apoptosis and is, therefore, referred to as a cellular lifeguard. We and others showed HSP72-dependent injury protection of monocytes [23,24]. Of note, housekeeping functions of HSPs are critically involved, e.g. as potent immune adjuvants, into the innate and adaptive immune response in inflammation, infection and tumour defence [25–30]. In the case of infection, HSP72 induction is even crucial keeping Mphi alive and, therefore, capable for anti-hazardous actions [31–33]. Several factors and pathways have been identified as responsible triggers causing immunodeficiency in patients with ESRD [8,34,35]. But still, the underlying mechanisms determining Mphis’ dysfunction are complex and remain, to a certain extent, unknown. Thus, they are addressed in this study. Having identified a reduced stress-related HSP72 response in Mphi of rats with chronic kidney disease, we used HS, as a simple model of fever/stress, to investigate the capacity of HSP72 induction in Mphi of patients on intermittent haemodialysis (IHD). | Subjects and methods | Subjects We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.
We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.
Animal model Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.
In short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).
Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.
In short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).
Antibodies and reagents Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Real-time PCR primers and Antibodies
Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Real-time PCR primers and Antibodies
Histology Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.
Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.
Isolation and culture of mononuclear cells from peripheral blood Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.
Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.
Flow cytometric detection of inducible HSP70 (HSP72) After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.
After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events. | Results | Animal model Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).
PAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.
Functional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.
Effects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)
Mean values ± SD.
CTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.
*Significantly different to CTR (P < 0.05).
Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).
PAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.
Functional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.
Effects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)
Mean values ± SD.
CTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.
*Significantly different to CTR (P < 0.05).
HS induces HSP72-mRNA in rat Mphi Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).
Impaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).
Impaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Temperature- and time-dependent induction of HSP72 protein in human Mphi To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.
Heat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).
HSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.
Heat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).
HSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).
Consistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).
To exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.
Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).
Consistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).
To exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.
Urea does not impair HSP72-HS response of Mphi In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).
In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).
Impaired HS-dependent protection from apoptosis in Mphi of patients on haemodialysis To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).
To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).
Influence of haemodialysis session on HSP72-protein expression To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).
Urea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).
To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).
Urea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05). | null | null | [
"Subjects",
"Animal model",
"Antibodies and reagents",
"Histology",
"Isolation and culture of mononuclear cells from peripheral blood",
"Flow cytometric detection of inducible HSP70 (HSP72)",
"Western Blot",
"Flow cytometric quantification of apoptosis",
"RT-PCR",
"Statistical analyses",
"Animal model",
"HS induces HSP72-mRNA in rat Mphi",
"Temperature- and time-dependent induction of HSP72 protein in human Mphi",
"Impaired HSP72 response of Mphi of patients on haemodialysis",
"Urea does not impair HSP72-HS response of Mphi",
"Impaired HS-dependent protection from apoptosis in Mphi of patients on haemodialysis",
"Influence of haemodialysis session on HSP72-protein expression"
] | [
"We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.",
"Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.\nIn short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).",
"Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).\nReal-time PCR primers and Antibodies",
"Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.",
"Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.",
"After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.",
"For western blot analyses, proteins were separated by SDS-polyacrylamide (4–20%) electrophoresis, and transferred to a PVDF membrane incubated with blocking agent (Amersham, Freiburg, Germany). After incubation with the primary antibodies anti-HSF-1, or anti-HSP70 and anti-GAPDH for 90 min at room temperature, the secondary HRP antibodies goat anti-rabbit and anti-mouse (1:10 000, DakoCytomation, Glostrup, Denmark) were incubated for another 45 min. For signal development, the blots were covered with chemiluminescent substrate (SuperSignal, Pierce, Bonn, Germany) before exposure.\n Flow cytometric quantification of apoptosis Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.\nForty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.\n RT-PCR Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].\nMessanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].\n Statistical analyses Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.\nData are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.",
"Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.",
"Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].",
"Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.",
"Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).\nPAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.\nFunctional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.\nEffects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)\nMean values ± SD.\nCTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.\n\n*Significantly different to CTR (P < 0.05).",
"Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).\nImpaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).",
"To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.\nHeat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).\nHSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nImpaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).",
"Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).\nConsistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).\nTo exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.",
"In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).",
"To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).",
"To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).\nUrea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05)."
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] | [
"Introduction",
"Subjects and methods",
"Subjects",
"Animal model",
"Antibodies and reagents",
"Histology",
"Isolation and culture of mononuclear cells from peripheral blood",
"Flow cytometric detection of inducible HSP70 (HSP72)",
"Western Blot",
"Flow cytometric quantification of apoptosis",
"RT-PCR",
"Statistical analyses",
"Results",
"Animal model",
"HS induces HSP72-mRNA in rat Mphi",
"Temperature- and time-dependent induction of HSP72 protein in human Mphi",
"Impaired HSP72 response of Mphi of patients on haemodialysis",
"Urea does not impair HSP72-HS response of Mphi",
"Impaired HS-dependent protection from apoptosis in Mphi of patients on haemodialysis",
"Influence of haemodialysis session on HSP72-protein expression",
"Discussion"
] | [
"Patients on haemodialysis present with a high infectious morbidity, enhanced carcinoma incidence, impaired wound healing and reduced effect of vaccination—diseases that are directly related to the function of the immune system [1–4]. Immunological abnormalities of leukocytes have been described in patients on dialysis [5]. Mphi (monocytes/ macrophages) are major effectors of the immune system with widespread microbicidal and tumoricidal functions that are altered in uraemia [6–8]. Uraemic immunodeficiency is a complication of renal failure reflecting stress, e.g. exerted by as yet poorly defined uraemic toxins, imbalances of antioxidants and pro-oxidants, malnutrition or iron overload, membrane bio-incompatibility and inflammation [9–11]. Therefore, patients with end-stage renal disease (ESRD) suffer from increased oxidative stress, which can only be ineffectively treated by haemodialysis [12–14]. The response of human cells to stress, e.g. heat shock (HS), is a highly conserved adaptive cell response found in all cells in all life forms conferring resistance to different stressors, i.e. increased temperature (fever), nutritional deficiency, oxidative stress or hypoxia [15–17]. The corresponding heat shock proteins (HSPs), like the major stress-inducible family member HSP72 (70-kDa HSP, inducible HSP70, HSPA1A, HSP70-1) [18], can also be found in cells under normal physiological conditions controlling the folding of proteins. Constitutively expressed HSP70 (Hsc70) binds to newly translated immature proteins and prevents premature and improper binding and folding [19–21]. This is vital, since HSP72-deficient animals are susceptible to stress or even not viable [22]. In response to stress, housekeeping activities of HSPs, e.g. degradation of unstable and misfolded proteins, control of regulatory proteins or transport of proteins, switch towards reaction against stress to increase the cell's chance of survival. The cellular injury decreases, either by induction of cytoresistance or by enhancement of repair mechanisms [18]. Thus, HSP72 induction confers protection even against sublethal damage causing apoptosis and is, therefore, referred to as a cellular lifeguard. We and others showed HSP72-dependent injury protection of monocytes [23,24]. Of note, housekeeping functions of HSPs are critically involved, e.g. as potent immune adjuvants, into the innate and adaptive immune response in inflammation, infection and tumour defence [25–30]. In the case of infection, HSP72 induction is even crucial keeping Mphi alive and, therefore, capable for anti-hazardous actions [31–33]. Several factors and pathways have been identified as responsible triggers causing immunodeficiency in patients with ESRD [8,34,35]. But still, the underlying mechanisms determining Mphis’ dysfunction are complex and remain, to a certain extent, unknown. Thus, they are addressed in this study. Having identified a reduced stress-related HSP72 response in Mphi of rats with chronic kidney disease, we used HS, as a simple model of fever/stress, to investigate the capacity of HSP72 induction in Mphi of patients on intermittent haemodialysis (IHD).",
" Subjects We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.\nWe enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.\n Animal model Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.\nIn short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).\nEight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.\nIn short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).\n Antibodies and reagents Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).\nReal-time PCR primers and Antibodies\nAntibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).\nReal-time PCR primers and Antibodies\n Histology Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.\nPortions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.\n Isolation and culture of mononuclear cells from peripheral blood Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.\nBlood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.\n Flow cytometric detection of inducible HSP70 (HSP72) After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.\nAfter fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.",
"We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.",
"Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.\nIn short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).",
"Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).\nReal-time PCR primers and Antibodies",
"Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.",
"Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.",
"After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.",
"For western blot analyses, proteins were separated by SDS-polyacrylamide (4–20%) electrophoresis, and transferred to a PVDF membrane incubated with blocking agent (Amersham, Freiburg, Germany). After incubation with the primary antibodies anti-HSF-1, or anti-HSP70 and anti-GAPDH for 90 min at room temperature, the secondary HRP antibodies goat anti-rabbit and anti-mouse (1:10 000, DakoCytomation, Glostrup, Denmark) were incubated for another 45 min. For signal development, the blots were covered with chemiluminescent substrate (SuperSignal, Pierce, Bonn, Germany) before exposure.\n Flow cytometric quantification of apoptosis Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.\nForty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.\n RT-PCR Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].\nMessanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].\n Statistical analyses Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.\nData are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.",
"Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.",
"Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].",
"Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.",
" Animal model Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).\nPAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.\nFunctional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.\nEffects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)\nMean values ± SD.\nCTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.\n\n*Significantly different to CTR (P < 0.05).\nTwo weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).\nPAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.\nFunctional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.\nEffects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)\nMean values ± SD.\nCTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.\n\n*Significantly different to CTR (P < 0.05).\n HS induces HSP72-mRNA in rat Mphi Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).\nImpaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nTwelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).\nImpaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\n Temperature- and time-dependent induction of HSP72 protein in human Mphi To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.\nHeat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).\nHSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nImpaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nTo evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.\nHeat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).\nHSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nImpaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\n Impaired HSP72 response of Mphi of patients on haemodialysis Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).\nConsistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).\nTo exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.\nHaving defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).\nConsistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).\nTo exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.\n Urea does not impair HSP72-HS response of Mphi In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).\nIn the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).\n Impaired HS-dependent protection from apoptosis in Mphi of patients on haemodialysis To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).\nTo assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).\n Influence of haemodialysis session on HSP72-protein expression To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).\nUrea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).\nTo clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).\nUrea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).",
"Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).\nPAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.\nFunctional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.\nEffects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)\nMean values ± SD.\nCTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.\n\n*Significantly different to CTR (P < 0.05).",
"Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).\nImpaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).",
"To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.\nHeat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).\nHSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nImpaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).",
"Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).\nConsistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).\nTo exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.",
"In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).",
"To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).",
"To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).\nUrea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).",
"Uraemic stress-related dysfunction of Mphi is an important contributory factor for the increased incidence of infections and tumours, or the impaired wound healing of patients on haemodialysis [1–4,39]. We herein show that in Mphi of rats with renal insufficiency and in Mphi of patients on IHD, the stress-related HSP72 induction, an important cytoprotective, anti-apoptotic mechanism of cells, is impaired, although baseline expression remains unaffected. Reduced HSP72 expression was accompanied by significant lower mRNA expression of the responsible transcription factor HSF-1 [40] and resulted in reduced cellular resistance to pro-apoptotic stress.\nUpregulation of HSP72 in human monocytes started shortly after HS (Figure 3B) and, therefore, confirmed specific detection of the inducible form of HSP70. We have chosen a high and rather long thermal stress, because lower stimulation was less effective (Figure 3A). Expression of HSP72 declined with time (peak 9–12 h, Figure 3B) but is still, as previously demonstrated, even 60 h later enhanced [23]. The rapid and massive induction of HSP acted strongly anti-apoptotic and allowed survival of monocytes, even when thermal stress with sublethal temperature or stress by serum starvation was applied (low apoptosis and necrosis rates, Figure 7). These results are consistent with previous studies mostly analysing cell lines, which confirmed the role of HSP conferring resistance to physical stress or pro-apoptotic triggers [18,21,41].\nInhibition of constitutive Mphi apoptosis by heat shock (HS, 40 min, 47°C). (A) There was slightly more basal apoptosis in Mphi of patients than in Mphi of controls (48-h cultivation in culture medium containing 5% FCS, 11± 2% versus 6 ± 1% apoptotic Mphi in CTR, n = 8, P < 0.05). HS was capable of downregulating basal apoptosis in patients to a lower extent than in controls (3 ± 2% in CTR versus 8 ± 3% in patients, n = 8, P < 0.05). Forty-eight-hour serum depletion (0.2% FCS) caused apoptosis (29 ± 5% in CTR versus 36 ± 6% in patients, n = 6, P < 0.05). HS downregulated apoptosis in both patients and controls, but controls showed a significantly reduced apoptosis rate when compared to patients (4 ± 3% in CTR versus 15 ± 2% in patients, n = 6, P < 0.05). B and C: one out of at least six-independent FACS-measurements of CD14-receptor expression and annexin V-labelling of phosphatidyl serines. Healthy cells are CD14-positive and annexin V-negative (upper left quadrant in dotplots), whereas apoptotic lose their CD14 receptor and gain annexin V-positive (lower right quadrant in dotplots). In CTR (B), HS effectively inhibited apoptosis (33.1% without HS versus 5.8% with HS). In patients on haemodialysis, inhibition of apoptosis was less effective and apoptosis rate more than twice as high as in CTR (C). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). **Statistical difference to controls (0.2% FCS) (P < 0.05). #Statistical difference to matching group without HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).\nFlow cytometric detection of HSP72-specific signals of monocytes from 27 patients before and of nine patients after 4 h of haemodialysis. Haemodialysis did not change the HSP72 signal intensity significantly (2.8 ± 1.1% versus 3 ± 1.9% HSP72-positive cells, 2 ± 1.7% CTR, n = 27). Data are presented as mean ± SD.\nSeveral attempts were made, to clarify the mechanisms causing immunodeficiency in patients with ESRD. What is known so far is that the mechanism cannot be broken down to a single effector. The pathways are rather complex and not yet fully understood. Interestingly, HSP72 induction acts not only anti-apoptotic but influences Mphis’ phagocytosis capacity and resistance to intracellular infection, too [26,29,42,43]. Memoli et al. stated that the impaired immunocompetence observed in Mphi of patients on IHD results from ‘chronic over-stimulation’ caused by pro-inflammatory cytokines, complement activation and toxins, conducting a decreased resonance against further stimulation (fatigue) [35]. Nevertheless, we could neither detect dialysis session-dependent HSP72 induction (stimulation) nor find elevated baseline HSP72 in patients (Figures 4 and 8) or rats with renal insufficiency (Figure 2). Recently, and in contrast to our data, Calabrese et al. measured elevated HSP70 level in lymphocytes of patients with diabetic nephropathy [44]. As diabetes-related stress and patient's metabolism considerably differ between diabetic and non-diabetic patients and the measurements were performed on EDTA-treated lymphocytes (leading via calcium efflux to pre- and de-activation of cellular pathways), the results may vary. Further, monocytes of patients on peritoneal dialysis exhibit significant HS response after contact with peritoneal dialysis fluid meaning that they are not over-stimulated [45]. Unfortunately, this study lacks the healthy controls.\nHSP70 proteins are potent immune adjuvants released e.g. upon tissue damage regulating the immune-response [26–28,30,33,46]. HSP70 also increases the expression of Toll-like receptor 4 (TLR4) [33,46]. Toll-like receptors are critical for activation of immunocompetent cells [47,48]. Ando et al. described reduced expression of TLR4 in monocytes of uraemic patients contributing to the impaired cytokine response [49]. Taken together, a reduced HSP72 response to stress might cause less effective recognition of harmful antigens and less activation of TLR4-dependent pathways.\nUrea in clinically relevant concentrations (40–200 mg/dl) induces HS response in the short run (0.5–10 h) in cultured neuroblastoma cells. Maddock and Westenfelder charged protein carbamylation for being the trigger [50]. Interestingly, the HS response returned to zero by 48 h assuming some kind of cellular adaptation in the long run. This is in agreement with our data, showing the same baseline amounts of HSP72 in Mphi of healthy rats and those with increased serum BUN (Figure 2), in urea-treated Mphi (Figure 6), as well as in patients and controls (Figures 4 and 5). Consistent with Marzec et al. [45], monocytes from rats with renal insufficiency and from patients on IHD responded to HS but, in the case of HSP72, to a significantly lower extent than the ones from controls (Figures 2, 4, and 5). In contrast, skeletal muscle of chronic dialysis patients in the steady state contains increased amounts of protective HS proteins [51]. Notably, HSP72 expression in long-term urea-incubated Mphi responded to a significant higher extent to HS than controls (Figure 6). A possible explanation for urea-induced HSP72 expression in heated Mphi is that urea-mediated protein destabilization may be enhanced by increased temperature. Since disruption of the protein tertiary structure is the final common pathway of HSP induction, this mechanism could also account for urea-induced HSP72 expression at high temperature [52].\nMalnutrition is frequently present in patients on IHD. Uraemia changes plasma and intracellular amino acid levels further whereas IHD alters protein turnover and amino acid transport kinetics [53]. In critically ill patients, a decreased plasma glutamine level causes an impaired monocyte function leading to substantial immunosuppression [54,55]. Glutamine deprivation increases Mphi’ susceptibility to stress and apoptosis, as well as it reduces their responsiveness to stimuli [55]. Glutamine starving reduces thermoresistance (to fever) of Mphi that is, according to Pollheimer et al., caused by impaired HSP70 response [54]. Thus, one can assume that glutamine depletion contributes to the reduced HSP72-HS response and favours apoptosis as demonstrated in patients’ Mphi (Figure 7). Nevertheless, accelerated efflux of amino acids from the muscle (protein catabolism) helps to stabilize the intracellular concentrations of amino acids during IHD [53]. Further, we performed all experiments in CM containing 2 mM L-glutamine excluding undersupply and effects were already present in the rat model of chronic kidney disease. Hence, glutamine starvation is probably not involved.\nTreatment with erythropoietin induces HSP70 in organs, like heart and kidney, exhibiting anti-apoptotic, cytoprotective effects [56,57]. Recently, Ribeil et al. identified an important mechanism leading to differentiation of human erythroid precursors [58]. They showed caspase-3 activation during both terminal differentiation and (erythropoietin-starvation-induced) apoptosis. Only in the presence of erythropoietin, HSP70 protected erythroid precursors from caspase-mediated proteolysis [58]. As erythropoietin is an integral part of the pharmacological therapy in ESRD, one can speculate that in untreated ESRD patients the HSP70 response might be worse and apoptosis even elevated. In addition, the HSP72 response was already impaired in the rat model excluding medications being the main underlying trigger altering Mphi's stress response.\nOur results demonstrate an impaired HSP72-stress response of Mphi from patients on IHD that is accompanied by a decreased HSF-1 response to HS. It is alarming that this impaired stress response is not necessarily bound to uraemia but is already present in chronic kidney disease as demonstrated in a rat model. Patient's Mphi show enhanced susceptibility to stress, e.g. serum starvation, as shown by increased cellular apoptosis values. Impaired HSP72 response of Mphi might explain in part, why patients on IHD are challenged by higher incidences of infections and tumours or impaired wound healing."
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"apoptosis",
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"human monocytes"
] | Introduction:
Patients on haemodialysis present with a high infectious morbidity, enhanced carcinoma incidence, impaired wound healing and reduced effect of vaccination—diseases that are directly related to the function of the immune system [1–4]. Immunological abnormalities of leukocytes have been described in patients on dialysis [5]. Mphi (monocytes/ macrophages) are major effectors of the immune system with widespread microbicidal and tumoricidal functions that are altered in uraemia [6–8]. Uraemic immunodeficiency is a complication of renal failure reflecting stress, e.g. exerted by as yet poorly defined uraemic toxins, imbalances of antioxidants and pro-oxidants, malnutrition or iron overload, membrane bio-incompatibility and inflammation [9–11]. Therefore, patients with end-stage renal disease (ESRD) suffer from increased oxidative stress, which can only be ineffectively treated by haemodialysis [12–14]. The response of human cells to stress, e.g. heat shock (HS), is a highly conserved adaptive cell response found in all cells in all life forms conferring resistance to different stressors, i.e. increased temperature (fever), nutritional deficiency, oxidative stress or hypoxia [15–17]. The corresponding heat shock proteins (HSPs), like the major stress-inducible family member HSP72 (70-kDa HSP, inducible HSP70, HSPA1A, HSP70-1) [18], can also be found in cells under normal physiological conditions controlling the folding of proteins. Constitutively expressed HSP70 (Hsc70) binds to newly translated immature proteins and prevents premature and improper binding and folding [19–21]. This is vital, since HSP72-deficient animals are susceptible to stress or even not viable [22]. In response to stress, housekeeping activities of HSPs, e.g. degradation of unstable and misfolded proteins, control of regulatory proteins or transport of proteins, switch towards reaction against stress to increase the cell's chance of survival. The cellular injury decreases, either by induction of cytoresistance or by enhancement of repair mechanisms [18]. Thus, HSP72 induction confers protection even against sublethal damage causing apoptosis and is, therefore, referred to as a cellular lifeguard. We and others showed HSP72-dependent injury protection of monocytes [23,24]. Of note, housekeeping functions of HSPs are critically involved, e.g. as potent immune adjuvants, into the innate and adaptive immune response in inflammation, infection and tumour defence [25–30]. In the case of infection, HSP72 induction is even crucial keeping Mphi alive and, therefore, capable for anti-hazardous actions [31–33]. Several factors and pathways have been identified as responsible triggers causing immunodeficiency in patients with ESRD [8,34,35]. But still, the underlying mechanisms determining Mphis’ dysfunction are complex and remain, to a certain extent, unknown. Thus, they are addressed in this study. Having identified a reduced stress-related HSP72 response in Mphi of rats with chronic kidney disease, we used HS, as a simple model of fever/stress, to investigate the capacity of HSP72 induction in Mphi of patients on intermittent haemodialysis (IHD).
Subjects and methods:
Subjects We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.
We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.
Animal model Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.
In short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).
Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.
In short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).
Antibodies and reagents Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Real-time PCR primers and Antibodies
Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Real-time PCR primers and Antibodies
Histology Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.
Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.
Isolation and culture of mononuclear cells from peripheral blood Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.
Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.
Flow cytometric detection of inducible HSP70 (HSP72) After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.
After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.
Subjects:
We enrolled 37 stable patients (20 males) on IHD with mean age of 38 (range 18–76) years chosen at random from the Dialysis Unit of the University Hospital Münster (Münster, Germany). Patients’ Mphi were compared to those of 37 apparently healthy volunteers (23 males) on no medication, who presented no complaints or active pathology with a mean age of 32 (CTR, range 22–59) years. There was no statistically significant difference in age among the groups studied. Patients had haemoglobin levels between 11.5 and 13.5 g/dl, treated with erythropoietin (epoetin alpha: 0–24 000 I.E., median 10 000 I.E./week i.v.) and intravenous iron, and standard heparin anticoagulation. Blood pressure was controlled, whereas 23 patients received a dihydropyridine calcium channel blocker and/or an angiotensin-converting enzyme inhibitor, respectively, to reach normotension. Patients received haemodialysis for 4 h on three occasions each week for more than 12 months, using polysulfone membranes (F8, FMC, Bad Homburg, Germany) and bicarbonate dialysate (32 mmol/l) containing 138 mM Na+, 3.0 mM K+, 1.5 mM Ca2+, 0.5 mM Mg2+, 111 mM Cl− and glucose 1 g/l (SK-F 313/1, FMC). Dialysate flow rate was 500 ml/min and blood flow >250 ml/min. Patients had primary arteriovenous fistulae (radiocephalic or brachiocephalic), and two needles were used for each dialysis session. The renal diagnoses were nephrosclerosis (n = 10), inactive glomerulonephritis (n = 7), interstitial nephritis (n = 5), polycystic kidney disease (n = 3), renal failure related to chronic heart failure (n = 3) and diabetic nephropathy (n = 2), and in seven patients the reason remained undefined. In both groups, the exclusion criteria were as follows: haemodialysis duration <12 months, PTH >400 pg/ml, evidence of infection, neoplasia or liver disease. Patients on antibiotic, antiinflammatory or immunosuppressive drugs were also excluded. Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Münster University Hospital, Münster, Germany.
Animal model:
Eight-week-old male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) with free access to standard rat chow (Ssniff, Soest, Germany) and tap water were randomly assigned to surgical 5/6-nephrectomy (5/6-NX, n = 6) or control group (CTR, n = 6). Experiments were approved by a governmental committee on animal welfare and were performed in accordance with National Animal Protection guidelines.
In short, the 5/6-NX involved midline incision, removal of the right kidney and selective ligation of 2–3 branches of the left renal artery. Surgery was performed under general anaesthesia [ketamine (100 mg/kg)/xylazine (10 mg/kg)]. Body weight (BW) was measured before surgery and 2 weeks after surgery. Serum parameters, blood pressure (tail cuff plethysmography) and total kidney function were recorded after 2 weeks of disease progression. Twenty-four hours before kidney recovery, animals were housed in metabolic cages. Urine and blood samples were analysed for creatinine (enzymatic assay, Creatinine-Pap, Roche Diagnostics, Mannheim, Germany), BUN (urease-GLDH method) and protein (Bradford Blue, BioRad Laboratories, München, Germany).
Antibodies and reagents:
Antibodies were obtained as described in Table 1. Saponin and D-mannitol were purchased from Merck (Darmstadt, Germany). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Real-time PCR primers and Antibodies
Histology:
Portions of kidneys were snap-frozen and fixed in 4% formaldehyde in PBS. Histological changes (e.g. glomerular sclerosis, vascular walls, tubular casts and infiltration) were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff staining.
Isolation and culture of mononuclear cells from peripheral blood:
Blood samples were obtained under sterile conditions in all patients just before or before-and-after the first weekly dialysis session (long interval), respectively. In rats, trunk blood was collected after decapitation 2 weeks after 5/6-NX. Mphi were purified as published before [36]. In short, Mphi were separated from heparinized whole blood by density gradient separation (Ficoll/Hypaque; Biochrom Seromed, Berlin, Germany). Monocytes were further purified by adherence to the culture dishes, resulting in a final purity of >85%. Cells were washed and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 culture medium (CM, PAA Laboratories, Pasching, Austria) supplemented with 2 mM l-glutamine, 50 μg/ml penicillin/streptomycin, 5 mM HEPES and 10 μM mercapto-ethanol. For HS, Mphi (5 × 106/ml) were seeded in preheated CM (temperature as indicated) containing 5% inactivated fetal calf serum (FCS; endotoxin content <0.01 ng/ml) and chemicals as indicated. After 40-min HS, heated CM was replaced by CM (37°C) and cells were further incubated at 37°C until measurement. For experiments with D-mannitol and urea, Mphi were incubated with CM, CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C), and all cells (±HS) were harvested for real-time PCR analysis after 72 h. Differing incubation times or temperatures are given in results or figure legends.
Flow cytometric detection of inducible HSP70 (HSP72):
After fixation with paraformaldehyde (4%, 15 min, 4°C) and washing, cells (5 × 106/ml) were incubated in a staining buffer (1% FCS in a 50 mM HEPES buffer, saponin 0.1%, pH 7.4) for 30 min at 4°C and labelled with mouse anti-human HSP70. After washing, the cells were incubated for 30 min with the secondary FITC-labelled goat anti-mouse-IgG1. Cytofluorometric analysis was performed with a FACScan cytometer (BD Life Science Research) for a total of 10 000 events.
Western Blot:
For western blot analyses, proteins were separated by SDS-polyacrylamide (4–20%) electrophoresis, and transferred to a PVDF membrane incubated with blocking agent (Amersham, Freiburg, Germany). After incubation with the primary antibodies anti-HSF-1, or anti-HSP70 and anti-GAPDH for 90 min at room temperature, the secondary HRP antibodies goat anti-rabbit and anti-mouse (1:10 000, DakoCytomation, Glostrup, Denmark) were incubated for another 45 min. For signal development, the blots were covered with chemiluminescent substrate (SuperSignal, Pierce, Bonn, Germany) before exposure.
Flow cytometric quantification of apoptosis Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.
Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.
RT-PCR Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].
Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].
Statistical analyses Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.
Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.
Flow cytometric quantification of apoptosis:
Forty-eight hour serum depletion (0.2% FCS) was used to render Mphi apoptotic. Simultaneous detection of CD14 expression and annexin V-binding identifies apoptotic monocytes [37]. Mphi were deemed to be apoptotic when CD14 expression was low and annexin V-binding was high. Mphi prepared and treated as described above were double-labelled with PE-conjugated CD14 and annexin V-FITC in a staining buffer (PBS, containing 0.05% FCS and 0.05% sodium azide, pH 7.4) for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specificities were used as controls. The cells were washed after staining and flow cytometry was applied for a total of 10 000 events. Necrosis was excluded by trypan blue staining.
RT-PCR:
Messanger RNA expression profiles for selected genes were validated by real-time PCR. Total RNA (10 μg) from freshly isolated monocytes was used for cDNA synthesis with the high capacity cDNA reverse transcription kit. Real-time PCR was performed using SYBR Green PCR Master Mix on an ABI PRISM 7700 sequence detection system. The specific primer pairs used for amplification are listed in Table 1 (MWG-Biotech, Ebersberg, Germany). All instruments and reagents were purchased from Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2−ΔΔCt method using GAPDH as a housekeeping gene [38].
Statistical analyses:
Data are presented as means ± SD. Data were compared with Student's t-test or ANOVA variance analysis for multiple comparisons using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Significance was inferred at P < 0.05.
Results:
Animal model Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).
PAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.
Functional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.
Effects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)
Mean values ± SD.
CTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.
*Significantly different to CTR (P < 0.05).
Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).
PAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.
Functional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.
Effects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)
Mean values ± SD.
CTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.
*Significantly different to CTR (P < 0.05).
HS induces HSP72-mRNA in rat Mphi Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).
Impaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).
Impaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Temperature- and time-dependent induction of HSP72 protein in human Mphi To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.
Heat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).
HSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.
Heat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).
HSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).
Consistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).
To exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.
Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).
Consistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).
To exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.
Urea does not impair HSP72-HS response of Mphi In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).
In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).
Impaired HS-dependent protection from apoptosis in Mphi of patients on haemodialysis To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).
To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).
Influence of haemodialysis session on HSP72-protein expression To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).
Urea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).
To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).
Urea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).
Animal model:
Two weeks after 5/6-NX, subtotal renal ablation induced adaptations in remnant nephrons, whereas kidneys from controls presented with physiological histology (Figure 1A). Kidneys of 5/6-NX showed glomerular hypertrophy, fibrosis, a mild tubulo-interstitial infiltrate and thickening of the vessels’ muscular layer. Atrophy and detachment of epithelial cells, in addition to luminal proteinaceous casts, were found in proximal tubules (Figure 1B).
PAS staining of a representative kidney from control and from 5/6-nephrectomized rats (5/6-NX) 14 days after surgery. Whereas kidneys of control rats showed physiological histology (A), interstitial fibrosis and infiltration, glomerular sclerosis, tubular atrophy and casts were extensively present in kidneys of 5/6-NX rats (B). Original magnification ×400.
Functional data from measurements performed 2 weeks after 5/6-NX are given in Table 2. Abnormal renal histology in 5/6-NX was accompanied by elevated blood pressure and functional features of renal insufficiency. Creatinine clearance decreased and occurring polyuria was paralleled by significant proteinuria and weight loss. Retention parameters, i.e. serum creatinine and blood urea nitrogen, were increased significantly.
Effects of 5/6-nephrectomy on whole animal functional data (Day 14 after surgery)
Mean values ± SD.
CTR: control; 5/6-NX: 5/6 nephrectomy; BW: body weight; CrCl: creatinine clearance; BUN: blood urea nitrogen. n = 5.
*Significantly different to CTR (P < 0.05).
HS induces HSP72-mRNA in rat Mphi:
Twelve hours after HS, HSP72-mRNA expression of Mphi from rats with renal insufficiency (5/6-NX, n = 6) was compared to those of control rats (CTR, n = 6). Basal mRNA expression showed no difference between Mphi of CTR and 5/6-NX (Figure 2). HS significantly increased monocyte HSP72 in both CTR and 5/6-NX, but to a significantly lower extent in Mphi of 5/6-NX (214 ± 68% less than HS-CTR, n = 6).
Impaired HSP70 response of Mphi from 5/6-NX. HSP72-mRNA response of freshly isolated monocytes from rats 12 h after heat shock (HS, 40 min, 47°C) with (5/6-nehrectomy, 5/6-NX, n = 6) and without (control, n = 6) renal insufficiency. mRNA expression was quantified by real-time PCR. No significant difference was found between Mphi of CTR and 5/6-NX without HS (5/6-NX HSP72-mRNA: 0.83 ± 0.26). HS significantly increased HSP72 (4.56 ± 0.62 fold) in both, Mphi of controls and of 5/6-NX rats (HSP72-mRNA: 2.13 ± 0.68 fold). Results are mean values ± SD. *Statistical difference to controls (basal, P < 0.05). #Statistical difference to basal 5/6-NX (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Temperature- and time-dependent induction of HSP72 protein in human Mphi:
To evaluate the most efficient setting for HSP72 induction (Figure 3A), Mphi isolates from six healthy subjects were exposed (40 min) to different temperatures and then allowed to recover at 37°C for 12 h. Induction was most striking after exposure to 47°C (79 ± 14% HSP72-positive cells versus 3 ± 1% basal, n = 27). To estimate to what extent induced HSP72 remains detectable after HS, samples were exposed to 47°C for 40 min and allowed to recover at 37°C during various time periods. The level of HSP72-positive Mphi increased starting 1 h after HS, with maximal values apparent after 9–12 h of recovery (Figure 3B). As shown in Figure 7 and by trypan blue staining (data not shown), HS even in the applied sublethal range did not evoke injury or apoptosis. Therefore, monocyte viability was maintained most likely by HSP induction.
Heat shock (HS) temperature and time dependently increased HSP72 protein expression in human monocytes. (A) Twelve hours after 40 min HS at given temperatures, monocytes HSP72 protein expression was assessed by FACS-analysis. The number of HSP72-positive monocytes increased with the temperature of HS (42°C: 45 ± 10%, 44°C: 59 ± 15% versus basal: 2 ± 2%, P < 0.05). Maximum values (79 ± 14%) were observed after stimulation at 47°C. Stimulation at 50°C reduced HSP72 response, when compared to 47°C. (B) HSP72 protein expression increased significantly 3 h after stimulation (40 min at 47°C, 40 ± 9% versus 3 ± 2% basal). Six hours after HS 61 ± 9%, 9 h 82 ± 10% and 12 h 79 ± 14% HSP72-positive Mphi were detected. n = 6–27, mean values ± SD. *Statistical difference to the basal HSP72 expression (P < 0.05).
HSP72 (A) and heat shock factor 1 (HSF-1). (B) mRNA response of freshly isolated monocytes from controls and patients 12 h after heat shock. mRNA expression was quantified by real-time PCR. (A) No significant differences were found between basal mRNA expression of HSP72 [patient HSP72-mRNA 0.76 ± 0.37 fold change in times of control (fold), n = 5]. HS significantly increased HSP72 mRNA in both groups, but to a lower value in patients (CTR: 154.2 ± 54.2 versus patient: 68.5 ± 46.8, P < 0.05, n = 5). HSF-1 response to HS was 50% lower in patients, when compared to control (0.55 ± 0.05, P < 0.05, n = 5). In congruence, HSF-1 and HSP72 protein expression of patients after HS was lower than those of CTR (C, exemplary western blot, pooled Mphi, six samples per group). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to basal (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis. One exemplary experiment out of 27 independent experiments is shown in (A, B). No difference of HSP72 protein expression was observed between basal level in Mphi from controls (A, 2.1% HSP72-positive cells) and patients (B, 1.7% HSP72-positive cells). Twelve hours after heat shock (HS, 40 min, 47°C), the HSP72 protein expression is significant lower in Mphi from patients on haemodialysis (B, 60.7%) than in Mphi from controls (A, 94.7%). A summary of HSP72 protein expression measured before and after HS is presented in (C). Basal HSP72 protein expression in Mphi of CTR (2 ± 1% HSP72-positive cells, n = 27) was similar to Mphi of patients (3 ± 1%, n = 27). In contrast, HS led to a significant stronger HSP72 induction in Mphi of CTR than in Mphi of patients (79 ± 3% HSP72-positive Mphi versus 65 ± 3% in patients, n = 27, P < 0.05). To elucidate if there is a posttranscriptional difference in the HSP72 response of CTR and patients, monocytes were incubated with the transcription inhibitor actinomycin D (1 Eg/ml) ± HS or the translation inhibitor cycloheximide (15 Eg/ml) ± HS for 1 h starting at time of HS. Treatment abolished HSP72 induction usually observed 12 h after HS in both groups (actinomycin D: patients: 2 ± 1% HSP72-positive monocytes versus CTR: 3 ± 1%, n = 6, cycloheximide: patients: 13 ± 7% HSP72-positive monocytes versus CTR: 10 ± 5%, n = 3). SSCHeight: side scatter; results are mean values ± SEM. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Impaired HSP72 response of Mphi of patients on haemodialysis:
Having defined optimal HS-stimulation conditions for human Mphi (Figure 3), we tested our initial hypothesis, that the HS response of Mphi from patients on haemodialysis is impaired. Analogously to Mphi from rat with or without renal failure, no differences were found between basal mRNA-expression level of HSP72 from patients and controls (Figure 4A). Nevertheless, after stimulation with HS, we observed significantly increased HSP72 mRNA in Mphi of CTR and patients. In the next step, we analysed the expression of the responsible transcription factor HSF-1 and found a significantly lower expression in HS-stimulated Mphi of patients then in Mphi of CTR (Figure 4B and C).
Consistent with mRNA data, the impaired HSP72-HS response of patients was evident as well, when HSP protein expression was assessed (Figures 4C, 5A and B). Again, no difference was found when basal expression was compared (Figure 5A–C).
To exclude additional posttranscriptional mechanisms possibly involved in the impairment of HSP70 expression of Mphi of patients on haemodialysis, Mphi were incubated either with the transcription inhibitor actinomycin D (1 μg/ml) with or without HS or with the translation inhibitor cycloheximide (15 μg/ml) with or without HS for 1 hour starting at time of HS. Treatment with inhibitors abolished HSP72 induction usually observed 12 h after HS in both groups to the same extent excluding such mechanisms.
Urea does not impair HSP72-HS response of Mphi:
In the next step, we investigated if urea is the responsible trigger for the impaired HS response. To our surprise, incubation with urea significantly increased HSP72 mRNA response to HS, while the osmotic control substance mannitol did not (Figure 6).
Impaired HS-dependent protection from apoptosis in Mphi of patients on haemodialysis:
To assess the impact of the observed impaired HS response of patients’ Mphi on cellular susceptibility, apoptosis was analysed (Figure 7). First, baseline apoptosis rates were quantified. The level of constitutive apoptotic Mphi (after 48-h cultivation in CM containing 5% FCS) was rather low but doubled nearly when Mphi of patients were analysed. Nevertheless, induction of the cytoprotective, anti-apoptotic HS response by HS reduced the apoptosis rate in both, but still, the apoptosis rate of patients’ Mphi was nearly three times higher in patients than in CTR (Figure 7A). Under stress (48-h starvation due to serum depletion), the fraction of apoptotic Mphi significantly increased. HS-treated cells were effectively protected from apoptosis. Again, apoptosis rate of treated CTR was less than a third of the rate of Mphi of patients. One exemplary analysis of stress-induced apoptosis in Mphi is shown in Figure 7B and C (left). After HS, apoptosis remained twice as high in patients’ Mphi as in those of CTR (right).
Influence of haemodialysis session on HSP72-protein expression:
To clarify if haemodialysis session-related stress possibly translates into HSP72 stimulation in patients’ Mphi, HSP72 expression of Mphi freshly isolated from patients before and after haemodialysis was investigated but no difference was observed (Figure 8).
Urea increased HSP72 mRNA response of Mphi to heat shock (HS). Mphi from six healthy controls were incubated with culture medium (CM), CM + 150 mg/dl mannitol or CM + 150 mg/dl urea. HS was performed after 60 h (40 min, 47°C) and all cells (± HS) were harvested for real-time PCR analysis after 72 h. There was no significant difference in baseline HSP70 expression between CTR, mannitol [1.09 ± 0.05 fold change in times of control (fold)] and urea (1.11 ± 0.09 fold) treated Mphi. HS significantly induced HSP72 in all groups with the highest expression level measured in urea-treated Mphi (CTR 9.08 ± 2.5, mannitol 4.53 ± 1.33, urea 52.64 ± 10.63). CTR: control; M: mannitol. Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). #Statistical difference to stimulated controls (P < 0.05).
Discussion:
Uraemic stress-related dysfunction of Mphi is an important contributory factor for the increased incidence of infections and tumours, or the impaired wound healing of patients on haemodialysis [1–4,39]. We herein show that in Mphi of rats with renal insufficiency and in Mphi of patients on IHD, the stress-related HSP72 induction, an important cytoprotective, anti-apoptotic mechanism of cells, is impaired, although baseline expression remains unaffected. Reduced HSP72 expression was accompanied by significant lower mRNA expression of the responsible transcription factor HSF-1 [40] and resulted in reduced cellular resistance to pro-apoptotic stress.
Upregulation of HSP72 in human monocytes started shortly after HS (Figure 3B) and, therefore, confirmed specific detection of the inducible form of HSP70. We have chosen a high and rather long thermal stress, because lower stimulation was less effective (Figure 3A). Expression of HSP72 declined with time (peak 9–12 h, Figure 3B) but is still, as previously demonstrated, even 60 h later enhanced [23]. The rapid and massive induction of HSP acted strongly anti-apoptotic and allowed survival of monocytes, even when thermal stress with sublethal temperature or stress by serum starvation was applied (low apoptosis and necrosis rates, Figure 7). These results are consistent with previous studies mostly analysing cell lines, which confirmed the role of HSP conferring resistance to physical stress or pro-apoptotic triggers [18,21,41].
Inhibition of constitutive Mphi apoptosis by heat shock (HS, 40 min, 47°C). (A) There was slightly more basal apoptosis in Mphi of patients than in Mphi of controls (48-h cultivation in culture medium containing 5% FCS, 11± 2% versus 6 ± 1% apoptotic Mphi in CTR, n = 8, P < 0.05). HS was capable of downregulating basal apoptosis in patients to a lower extent than in controls (3 ± 2% in CTR versus 8 ± 3% in patients, n = 8, P < 0.05). Forty-eight-hour serum depletion (0.2% FCS) caused apoptosis (29 ± 5% in CTR versus 36 ± 6% in patients, n = 6, P < 0.05). HS downregulated apoptosis in both patients and controls, but controls showed a significantly reduced apoptosis rate when compared to patients (4 ± 3% in CTR versus 15 ± 2% in patients, n = 6, P < 0.05). B and C: one out of at least six-independent FACS-measurements of CD14-receptor expression and annexin V-labelling of phosphatidyl serines. Healthy cells are CD14-positive and annexin V-negative (upper left quadrant in dotplots), whereas apoptotic lose their CD14 receptor and gain annexin V-positive (lower right quadrant in dotplots). In CTR (B), HS effectively inhibited apoptosis (33.1% without HS versus 5.8% with HS). In patients on haemodialysis, inhibition of apoptosis was less effective and apoptosis rate more than twice as high as in CTR (C). Results are mean values ± SD. *Statistical difference to controls (basal) (P < 0.05). **Statistical difference to controls (0.2% FCS) (P < 0.05). #Statistical difference to matching group without HS (P < 0.05). §Statistical difference to stimulated controls (P < 0.05).
Flow cytometric detection of HSP72-specific signals of monocytes from 27 patients before and of nine patients after 4 h of haemodialysis. Haemodialysis did not change the HSP72 signal intensity significantly (2.8 ± 1.1% versus 3 ± 1.9% HSP72-positive cells, 2 ± 1.7% CTR, n = 27). Data are presented as mean ± SD.
Several attempts were made, to clarify the mechanisms causing immunodeficiency in patients with ESRD. What is known so far is that the mechanism cannot be broken down to a single effector. The pathways are rather complex and not yet fully understood. Interestingly, HSP72 induction acts not only anti-apoptotic but influences Mphis’ phagocytosis capacity and resistance to intracellular infection, too [26,29,42,43]. Memoli et al. stated that the impaired immunocompetence observed in Mphi of patients on IHD results from ‘chronic over-stimulation’ caused by pro-inflammatory cytokines, complement activation and toxins, conducting a decreased resonance against further stimulation (fatigue) [35]. Nevertheless, we could neither detect dialysis session-dependent HSP72 induction (stimulation) nor find elevated baseline HSP72 in patients (Figures 4 and 8) or rats with renal insufficiency (Figure 2). Recently, and in contrast to our data, Calabrese et al. measured elevated HSP70 level in lymphocytes of patients with diabetic nephropathy [44]. As diabetes-related stress and patient's metabolism considerably differ between diabetic and non-diabetic patients and the measurements were performed on EDTA-treated lymphocytes (leading via calcium efflux to pre- and de-activation of cellular pathways), the results may vary. Further, monocytes of patients on peritoneal dialysis exhibit significant HS response after contact with peritoneal dialysis fluid meaning that they are not over-stimulated [45]. Unfortunately, this study lacks the healthy controls.
HSP70 proteins are potent immune adjuvants released e.g. upon tissue damage regulating the immune-response [26–28,30,33,46]. HSP70 also increases the expression of Toll-like receptor 4 (TLR4) [33,46]. Toll-like receptors are critical for activation of immunocompetent cells [47,48]. Ando et al. described reduced expression of TLR4 in monocytes of uraemic patients contributing to the impaired cytokine response [49]. Taken together, a reduced HSP72 response to stress might cause less effective recognition of harmful antigens and less activation of TLR4-dependent pathways.
Urea in clinically relevant concentrations (40–200 mg/dl) induces HS response in the short run (0.5–10 h) in cultured neuroblastoma cells. Maddock and Westenfelder charged protein carbamylation for being the trigger [50]. Interestingly, the HS response returned to zero by 48 h assuming some kind of cellular adaptation in the long run. This is in agreement with our data, showing the same baseline amounts of HSP72 in Mphi of healthy rats and those with increased serum BUN (Figure 2), in urea-treated Mphi (Figure 6), as well as in patients and controls (Figures 4 and 5). Consistent with Marzec et al. [45], monocytes from rats with renal insufficiency and from patients on IHD responded to HS but, in the case of HSP72, to a significantly lower extent than the ones from controls (Figures 2, 4, and 5). In contrast, skeletal muscle of chronic dialysis patients in the steady state contains increased amounts of protective HS proteins [51]. Notably, HSP72 expression in long-term urea-incubated Mphi responded to a significant higher extent to HS than controls (Figure 6). A possible explanation for urea-induced HSP72 expression in heated Mphi is that urea-mediated protein destabilization may be enhanced by increased temperature. Since disruption of the protein tertiary structure is the final common pathway of HSP induction, this mechanism could also account for urea-induced HSP72 expression at high temperature [52].
Malnutrition is frequently present in patients on IHD. Uraemia changes plasma and intracellular amino acid levels further whereas IHD alters protein turnover and amino acid transport kinetics [53]. In critically ill patients, a decreased plasma glutamine level causes an impaired monocyte function leading to substantial immunosuppression [54,55]. Glutamine deprivation increases Mphi’ susceptibility to stress and apoptosis, as well as it reduces their responsiveness to stimuli [55]. Glutamine starving reduces thermoresistance (to fever) of Mphi that is, according to Pollheimer et al., caused by impaired HSP70 response [54]. Thus, one can assume that glutamine depletion contributes to the reduced HSP72-HS response and favours apoptosis as demonstrated in patients’ Mphi (Figure 7). Nevertheless, accelerated efflux of amino acids from the muscle (protein catabolism) helps to stabilize the intracellular concentrations of amino acids during IHD [53]. Further, we performed all experiments in CM containing 2 mM L-glutamine excluding undersupply and effects were already present in the rat model of chronic kidney disease. Hence, glutamine starvation is probably not involved.
Treatment with erythropoietin induces HSP70 in organs, like heart and kidney, exhibiting anti-apoptotic, cytoprotective effects [56,57]. Recently, Ribeil et al. identified an important mechanism leading to differentiation of human erythroid precursors [58]. They showed caspase-3 activation during both terminal differentiation and (erythropoietin-starvation-induced) apoptosis. Only in the presence of erythropoietin, HSP70 protected erythroid precursors from caspase-mediated proteolysis [58]. As erythropoietin is an integral part of the pharmacological therapy in ESRD, one can speculate that in untreated ESRD patients the HSP70 response might be worse and apoptosis even elevated. In addition, the HSP72 response was already impaired in the rat model excluding medications being the main underlying trigger altering Mphi's stress response.
Our results demonstrate an impaired HSP72-stress response of Mphi from patients on IHD that is accompanied by a decreased HSF-1 response to HS. It is alarming that this impaired stress response is not necessarily bound to uraemia but is already present in chronic kidney disease as demonstrated in a rat model. Patient's Mphi show enhanced susceptibility to stress, e.g. serum starvation, as shown by increased cellular apoptosis values. Impaired HSP72 response of Mphi might explain in part, why patients on IHD are challenged by higher incidences of infections and tumours or impaired wound healing.
| Background:
Induction of heat shock proteins (HSP), i.e. of the major family member HSP70, is an important cytoprotective-resistance mechanism for monocytes/ macrophages (Mphi). Patients on haemodialysis present with a high infectious morbidity and enhanced carcinoma incidence. Renal insufficiency-related alteration of microbicidal and tumoricidal functions of Mphi, major effectors of the immune system, might promote these diseases.
Methods:
Freshly isolated Mphi from Sprague-Dawley rats 2 weeks after 5/6-nephrectomy and from patients on intermittent haemodialysis (IHD) were stimulated by heat shock (HS) and compared to stimulated Mphi of control rats or healthy volunteers (CTR). Expression of HSP72 (inducible HSP70) was assessed by RT-PCR, and/or flow cytometry. Apoptosis of Mphi was detected by flow cytometry (CD14/annexin V-labelling).
Results:
In rat Mphi, baseline HSP72 expression was similar in both groups, but its induction was significantly impaired in renal insufficiency (214 +/- 68% less HSP70-mRNA versus CTR, n = 6). In patients, HSF-1-mRNA and HSP72-mRNA/protein response to HS was significantly lower, but not affected by dialysis session itself. In parallel, apoptosis of Mphi of patients was enhanced (+83 +/- 29% constitutive apoptotic Mphi versus CTR, n = 8), and HS-dependent protection from apoptosis with and without serum depletion (48 h depletion: HS, +275 +/- 37% apoptotic Mphi versus CTR, n = 6; full medium: +166 +/- 62% versus CTR, n = 8, P < 0.05) was inferior.
Conclusions:
Impaired HSP72 stress response of Mphi in patients on haemodialysis might contribute to the observed immune dysfunction and, therefore, to the increased susceptibility to infection and malignancy. Stress impairment is not restricted to uraemia but is already present in a rat model of chronic kidney disease. | null | null | 14,197 | 364 | [
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] | null | null | [CONTENT] apoptosis | haemodialysis | HSP72 | human monocytes [SUMMARY] | [CONTENT] apoptosis | haemodialysis | HSP72 | human monocytes [SUMMARY] | [CONTENT] apoptosis | haemodialysis | HSP72 | human monocytes [SUMMARY] | null | [CONTENT] apoptosis | haemodialysis | HSP72 | human monocytes [SUMMARY] | null | [CONTENT] Adolescent | Adult | Aged | Animals | Apoptosis | Base Sequence | Case-Control Studies | DNA Primers | DNA-Binding Proteins | Disease Models, Animal | Female | HSP72 Heat-Shock Proteins | Heat Shock Transcription Factors | Heat-Shock Response | Humans | In Vitro Techniques | Kidney Failure, Chronic | Male | Middle Aged | Monocytes | RNA, Messenger | Rats | Rats, Sprague-Dawley | Renal Dialysis | Transcription Factors | Urea | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Animals | Apoptosis | Base Sequence | Case-Control Studies | DNA Primers | DNA-Binding Proteins | Disease Models, Animal | Female | HSP72 Heat-Shock Proteins | Heat Shock Transcription Factors | Heat-Shock Response | Humans | In Vitro Techniques | Kidney Failure, Chronic | Male | Middle Aged | Monocytes | RNA, Messenger | Rats | Rats, Sprague-Dawley | Renal Dialysis | Transcription Factors | Urea | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Animals | Apoptosis | Base Sequence | Case-Control Studies | DNA Primers | DNA-Binding Proteins | Disease Models, Animal | Female | HSP72 Heat-Shock Proteins | Heat Shock Transcription Factors | Heat-Shock Response | Humans | In Vitro Techniques | Kidney Failure, Chronic | Male | Middle Aged | Monocytes | RNA, Messenger | Rats | Rats, Sprague-Dawley | Renal Dialysis | Transcription Factors | Urea | Young Adult [SUMMARY] | null | [CONTENT] Adolescent | Adult | Aged | Animals | Apoptosis | Base Sequence | Case-Control Studies | DNA Primers | DNA-Binding Proteins | Disease Models, Animal | Female | HSP72 Heat-Shock Proteins | Heat Shock Transcription Factors | Heat-Shock Response | Humans | In Vitro Techniques | Kidney Failure, Chronic | Male | Middle Aged | Monocytes | RNA, Messenger | Rats | Rats, Sprague-Dawley | Renal Dialysis | Transcription Factors | Urea | Young Adult [SUMMARY] | null | [CONTENT] hs patients haemodialysis | patients haemodialysis impaired | monocytes uraemic | haemodialysis inhibition apoptosis | dialysis mphi monocytes [SUMMARY] | [CONTENT] hs patients haemodialysis | patients haemodialysis impaired | monocytes uraemic | haemodialysis inhibition apoptosis | dialysis mphi monocytes [SUMMARY] | [CONTENT] hs patients haemodialysis | patients haemodialysis impaired | monocytes uraemic | haemodialysis inhibition apoptosis | dialysis mphi monocytes [SUMMARY] | null | [CONTENT] hs patients haemodialysis | patients haemodialysis impaired | monocytes uraemic | haemodialysis inhibition apoptosis | dialysis mphi monocytes [SUMMARY] | null | [CONTENT] mphi | hsp72 | hs | patients | expression | ctr | 05 | response | difference | nx [SUMMARY] | [CONTENT] mphi | hsp72 | hs | patients | expression | ctr | 05 | response | difference | nx [SUMMARY] | [CONTENT] mphi | hsp72 | hs | patients | expression | ctr | 05 | response | difference | nx [SUMMARY] | null | [CONTENT] mphi | hsp72 | hs | patients | expression | ctr | 05 | response | difference | nx [SUMMARY] | null | [CONTENT] stress | proteins | hsp72 | immune | hsps | response | induction | patients | hsp72 induction | oxidative [SUMMARY] | [CONTENT] germany | mm | blood | patients | cm | ml | münster | min | 37 | age [SUMMARY] | [CONTENT] hsp72 | hs | mphi | patients | expression | nx | difference | ctr | basal | 05 [SUMMARY] | null | [CONTENT] hs | mphi | hsp72 | patients | expression | nx | germany | 05 | response | ctr [SUMMARY] | null | [CONTENT] HSP | monocytes/ | Mphi ||| ||| Mphi [SUMMARY] | [CONTENT] Mphi | Sprague-Dawley | 2 weeks | 5/6 | IHD | Mphi | CTR ||| RT-PCR ||| Mphi [SUMMARY] | [CONTENT] Mphi | 214 +/- | 68% | CTR | 6 ||| HSF-1-mRNA ||| Mphi | 29% | Mphi | CTR | 8) | 48 | 37% | Mphi | CTR | 6 | 62% | CTR | 8 [SUMMARY] | null | [CONTENT] HSP | monocytes/ | Mphi ||| ||| Mphi ||| Mphi | Sprague-Dawley | 2 weeks | 5/6 | IHD | Mphi | CTR ||| RT-PCR ||| Mphi ||| ||| Mphi | 214 +/- | 68% | CTR | 6 ||| HSF-1-mRNA ||| Mphi | 29% | Mphi | CTR | 8) | 48 | 37% | Mphi | CTR | 6 | 62% | CTR | 8 ||| Impaired HSP72 | Mphi ||| [SUMMARY] | null |
||||
Prevalence of patent foramen ovale in a consecutive cohort of 261 patients undergoing routine "coronary" 64-multi-detector cardiac computed tomography. | 22347746 | A patent foramen ovale (PFO) is strongly associated with cryptogenic stroke (CS), neurological and other phenomena. The reported prevalence of PFO varies according to the imaging technique used and population studied. | BACKGROUND | Standard coronary imaging protocols were used. PFOs were graded according to the classification of Williamson et al. | METHODS | 261 patients were studied. A PFO was identified in 22.6% (11.5% grade 1 (closed flap), 6.5% grade 2 (open flap) and 4.6% grade 3 (open flap with jet)). A further 6.1% had an atrial septal aneurysm. | RESULTS | The prevalence of all grades of PFO (22.6%) and open flap PFO (11.1% = grade 2 and 3) with this technique compares with 24.3% by trans-oesophageal echocardiography (TOE) and 14.9% by saline contrast echocardiography (SCE). Further comparative studies are required but we believe an open flap PFO or ASA should be identified and recorded during cardiac CT. This approach may identify those at risk of cryptogenic stroke as well as avoid unnecessary tests in stroke patients. | CONCLUSIONS | [
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"Aged",
"Aged, 80 and over",
"Cardiac-Gated Imaging Techniques",
"Contrast Media",
"Coronary Angiography",
"Echocardiography",
"Female",
"Foramen Ovale, Patent",
"Humans",
"Iohexol",
"Ireland",
"Male",
"Middle Aged",
"Prevalence",
"Prospective Studies",
"Tomography, X-Ray Computed"
] | 3229849 | null | null | Methods | The study was approved by the Western HSC Trust Research and Development (Ethics) Chair. Data was collected prospectively on patients attending for 64-MDCT between the 18th June 2009 and 27th February 2010. | null | null | Conclusions | 64-MDCT allows accurate assessment of the IAS during routine “coronary” examination. We found the prevalence of open flap PFO and ASA to be 11.1% and 6.1% respectively in a population of 261 patients undergoing routine 64-MDCT coronary study. Further comparative studies against SCE and TOE are required but we believe open flap PFO and ASA should be clearly identified and recorded during routine coronary CT angiography. This approach may identify those at risk of cryptogenic stroke as well as avoid unnecessary tests in stroke patients.
The authors have no conflict of interest. | [
"Computed Tomography",
"Classification of PFO and ASA",
"Results",
"Discussion",
"Study Limitations",
"Current Guidelines",
"Conclusions"
] | [
"64-MDCT was performed using a Philips Brilliance 64 system (Philips Medical Systems, Eindhoven, Netherlands). Patients were monitored on electrocardiogram (ECG) and a 20FG intravenous cannula placed in an ante-cubital fossa vein. All patients were in sinus rhythm. After heart rate optimisation and sub-lingual administration of 400 micrograms of glyceryl tri-nitrate, non-ionic contrast material (Iohexol, Omnipaque 350, GE Healthcare AS, Norway) was injected into the vein (90 ml at a flow rate of 5.5 ml/sec followed by a saline chaser of 50mls at 5.5 ml/sec). A bolus tracking technique was used to determine when contrast density was optimal for coronary imaging. Scanning was performed during a single breath hold. In patients with a low and stable heart rate (<64 beats per minute), prospective ECG triggering was used for data acquisition; otherwise retrospective ECG gating was used. In all cases, data was obtained at a single time point (75% of the R-R wave interval on ECG). Detector collimation was 64 × 0.625mm with images reconstructed to a slice thickness of 0.6mm. Tube voltage was 120kV with a rotation time of 400 msec. Standard axial images and 2D multiplanar reformations were used for image interpretation. Coronal oblique projections through the IAS were specifically evaluated for the presence of each of the CT criteria for PFO outlined below.",
"Williamson et al. devised a classification system based in part on chart review of 20 patients with PFO who underwent both CT and Trans-oesophageal echocardiography (TOE).1 PFO anatomy on 64-MDCT was classified by 3 criteria:\n\n\n\nA distinct flap at the expected location in the LA (closed flap, Figure 1, panel A).\n\n\nA continuous column of contrast material between the septum primum and septum secundum, connecting the LA and RA (open flap, Figure 1 panel B).\n\n\nAn open flap plus a jet of contrast material from the column into the RA (open flap with jet, Figure 1, panel C).\n\n\n\nA distinct flap at the expected location in the LA (closed flap, Figure 1, panel A).\nA continuous column of contrast material between the septum primum and septum secundum, connecting the LA and RA (open flap, Figure 1 panel B).\nAn open flap plus a jet of contrast material from the column into the RA (open flap with jet, Figure 1, panel C).\nPanel A. Oblique coronal view: hollow arrow demonstrates closed flap (Grade 1 PFO), left atrium =LA, right atrium =RA. Panel B. Oblique coronal view: hollow arrow demonstrates open flap (Grade 2 PFO . Panel C. Oblique coronal view: hollow arrow demonstrates jet from open flap (Grade 3 PFO).\nWhen compared with TOE diagnosis, the positive predictive value of criterion 1 alone was 75%, criteria 1 +2 together was 80% and 1+2+3 together was 100%.1\nASA was defined as a redundant and hyper-mobile portion of the IAS that demonstrated more than 10-mm excursion from the centre line (Figure 2, panel A).9\nPanel A. Axial view with atrial septal aneurysm highlighted by hollow arrow, left atrium =LA, right atrium =RA. Panel B. A small rounded mass is identified over the fossa ovalis with a vascular supply (hollow arrow). This proved to be a left atrial myxoma.\nWe also categorised any potential IAS source of cardio-embolism into medium and high risk according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification.10",
"The study population consisted of 261 consecutive patients of whom 54% were male (mean age 56 years, range 21 to 81 years) and 46% were female (mean age 57 years, range 36 to 81 years). Study quality was sufficient for PFO classification in all cases. Study quality for coronary imaging was graded as excellent in 50%, good in 28%, fair in 16% and poor in 6%. A low dose prospective ECG triggered protocol was used in 38%. Results are displayed in Table 1. Amongst the 261 patients, 59 (22.6%) demonstrated a PFO. This could be subdivided into 30 (11.5%) with a grade 1 PFO, 17 (6.5%) with grade 2 and 12 (4.6%) with grade 3. In total, 29 (11.1%) of patients had an open channel between the atria during the CT scan (grade 2 or 3 PFO).\nNumbers and percentages of patients displaying conditions of the inter-atrial septum.\nTable 1 Legend: PFO = patent foramen ovale. Graded by Williamson classification1.\nAn atrial septal aneurysm was identified in 16 (6.1%) patients but no jet of contrast from left to right atrium was seen in association with this.\nOne patient was seen to have an early (1.1cm diameter) left atrial myxoma with prominent central vasculature (Figure 2, panel B).\nTable 2 lists patients with conditions classified by the TOAST criteria for medium or high risk of cardiac embolism.10 Of note, 17.2% of patients had medium cardio-embolic risk.\nConditions classified under the TOAST criteria for cardioembolic risk. (11)\nTable 2 Legend: PFO = patent foramen ovale. Graded by Williamson classification1.",
"PFO is strongly linked with conditions such as CS, pulmonary embolus and more controversially, migraine with aura.8, 11, 12 Major studies to determine the role of PFO closure devices are underway.13 The reported prevalence of PFO depends however, on the imaging modality used and the population studied. Prevalence based on normal heart autopsy is 27%, whilst SCE and TOE yield figures of 14.9% and 24.3% respectively in normal adults.2, 3, 14\nPrevalence is higher in vulnerable groups; amongst migraineurs in the MIST 1 study, a moderate or large PFO was detected in 37.7% by SCE, whilst 43.9% of young CS patients were demonstrated to have a PFO by TOE.15, 16 TOE is regarded as the gold standard imaging technique for PFO assessment.17, 18 Recently, real time 3D TOE has proved useful in guiding PFO closure device procedures.19\nMoving away from echocardiography, only a handful of studies of PFO anatomy have been performed on 64-MDCT.1, 7, 20, 21\nTwo studies have concentrated on comparing 64-MDCT with TOE. Williamson et al. reviewed 214 charts of patients attending for 64-MDCT and found 20 who had also undergone TOE. Of the six with PFO on TOE, all had grade 1 or higher PFO by the criteria defined in that study.1 Kim et al. retrospectively analysed images from 152 stroke patients who had undergone both TOE and CT. Twenty-six PFOs were identified by TOE with 19 of these patients having a grade 3 (open flap with jet) PFO appearance on CT.20 The authors also noted a “channel-like appearance” of the IAS which corresponded to grade 1 (closed flap) and grade 2 (open flap) in the Williamson et al. paper. Compared with TOE, the two papers combined yield a sensitivity of 67-73%, specificity of 98-100%, positive predictive value of 91-100% and negative predictive value of 85-95% for a grade 3 CT appearance. In a more generalised study, CT was compared with TOE for detection of cardiac sources of embolism in 137 stroke patients.21 Just under a quarter of the patients had PFO, ASA or an atrial septal defect identified by TOE. Overall sensitivity of CT was 89% with a positive predictive value of 100% for all embolic causes. The overall prevalence of all 3 grades of PFO in our study was 22.6%. We agree with previous authors that an open flap with direct communication between atria (grades 2 and 3) is more likely to represent clinically significant PFO, in which case, the prevalence falls to 11.1%.",
"One major problem with assessing PFO by 64-MDCT is that the information is purely anatomical and obtained during a breath hold rather than Valsalva manoeuvre. The functional and flow information obtained with TOE cannot be emulated.\nIt is interesting to note that the prevalence of all 3 grades of PFO at 22.6% is very similar to TOE and autopsy reference data, perhaps some of the closed flaps (11.5%) would open if right atrial pressure was elevated. Unfortunately, the rise in central venous pressure associated with Valsalva manoeuvre or coughing also impedes the passage of intravenous contrast to the heart. Unacceptable respiratory motion artefacts would also be introduced.\nPotentially, cine CT angiography22 could give information about directional flow in a PFO and mobility of ASA, but the Valsalva manoeuvre problem would remain.\nThe point we wish to stress in this paper is that much useful information can be obtained during a routine coronary study without additional measures or high dose protocols.\nThe prevalence of ASA in our study (6.1%) falls within the range seen in TOE studies on non-stroke patients (4-8%) - this figure can rise up to 15 - 28% in stroke populations.23, 24 We did not observe left to right contrast flow through an aneurysm but it is accepted that approximately 33% of adults with ASA also have PFO and ASA is considered a medium risk source of embolus by TOAST criteria.8,9",
"The Society of Cardiovascular Computed Tomography (SCCT) guidelines on reporting studies state that any [non-coronary] abnormalities should be described and that cardiac chamber shunts are a required element of a comprehensive report25 but given that 17.2% of our patients attending for routine coronary CT had a medium cardio-embolic risk (open flap PFO or ASA) perhaps more emphasis should be placed on this requirement.",
"64-MDCT allows accurate assessment of the IAS during routine “coronary” examination. We found the prevalence of open flap PFO and ASA to be 11.1% and 6.1% respectively in a population of 261 patients undergoing routine 64-MDCT coronary study. Further comparative studies against SCE and TOE are required but we believe open flap PFO and ASA should be clearly identified and recorded during routine coronary CT angiography. This approach may identify those at risk of cryptogenic stroke as well as avoid unnecessary tests in stroke patients.\nThe authors have no conflict of interest."
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"Methods",
"Computed Tomography",
"Classification of PFO and ASA",
"Results",
"Discussion",
"Study Limitations",
"Current Guidelines",
"Conclusions"
] | [
"The study was approved by the Western HSC Trust Research and Development (Ethics) Chair. Data was collected prospectively on patients attending for 64-MDCT between the 18th June 2009 and 27th February 2010.",
"64-MDCT was performed using a Philips Brilliance 64 system (Philips Medical Systems, Eindhoven, Netherlands). Patients were monitored on electrocardiogram (ECG) and a 20FG intravenous cannula placed in an ante-cubital fossa vein. All patients were in sinus rhythm. After heart rate optimisation and sub-lingual administration of 400 micrograms of glyceryl tri-nitrate, non-ionic contrast material (Iohexol, Omnipaque 350, GE Healthcare AS, Norway) was injected into the vein (90 ml at a flow rate of 5.5 ml/sec followed by a saline chaser of 50mls at 5.5 ml/sec). A bolus tracking technique was used to determine when contrast density was optimal for coronary imaging. Scanning was performed during a single breath hold. In patients with a low and stable heart rate (<64 beats per minute), prospective ECG triggering was used for data acquisition; otherwise retrospective ECG gating was used. In all cases, data was obtained at a single time point (75% of the R-R wave interval on ECG). Detector collimation was 64 × 0.625mm with images reconstructed to a slice thickness of 0.6mm. Tube voltage was 120kV with a rotation time of 400 msec. Standard axial images and 2D multiplanar reformations were used for image interpretation. Coronal oblique projections through the IAS were specifically evaluated for the presence of each of the CT criteria for PFO outlined below.",
"Williamson et al. devised a classification system based in part on chart review of 20 patients with PFO who underwent both CT and Trans-oesophageal echocardiography (TOE).1 PFO anatomy on 64-MDCT was classified by 3 criteria:\n\n\n\nA distinct flap at the expected location in the LA (closed flap, Figure 1, panel A).\n\n\nA continuous column of contrast material between the septum primum and septum secundum, connecting the LA and RA (open flap, Figure 1 panel B).\n\n\nAn open flap plus a jet of contrast material from the column into the RA (open flap with jet, Figure 1, panel C).\n\n\n\nA distinct flap at the expected location in the LA (closed flap, Figure 1, panel A).\nA continuous column of contrast material between the septum primum and septum secundum, connecting the LA and RA (open flap, Figure 1 panel B).\nAn open flap plus a jet of contrast material from the column into the RA (open flap with jet, Figure 1, panel C).\nPanel A. Oblique coronal view: hollow arrow demonstrates closed flap (Grade 1 PFO), left atrium =LA, right atrium =RA. Panel B. Oblique coronal view: hollow arrow demonstrates open flap (Grade 2 PFO . Panel C. Oblique coronal view: hollow arrow demonstrates jet from open flap (Grade 3 PFO).\nWhen compared with TOE diagnosis, the positive predictive value of criterion 1 alone was 75%, criteria 1 +2 together was 80% and 1+2+3 together was 100%.1\nASA was defined as a redundant and hyper-mobile portion of the IAS that demonstrated more than 10-mm excursion from the centre line (Figure 2, panel A).9\nPanel A. Axial view with atrial septal aneurysm highlighted by hollow arrow, left atrium =LA, right atrium =RA. Panel B. A small rounded mass is identified over the fossa ovalis with a vascular supply (hollow arrow). This proved to be a left atrial myxoma.\nWe also categorised any potential IAS source of cardio-embolism into medium and high risk according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification.10",
"The study population consisted of 261 consecutive patients of whom 54% were male (mean age 56 years, range 21 to 81 years) and 46% were female (mean age 57 years, range 36 to 81 years). Study quality was sufficient for PFO classification in all cases. Study quality for coronary imaging was graded as excellent in 50%, good in 28%, fair in 16% and poor in 6%. A low dose prospective ECG triggered protocol was used in 38%. Results are displayed in Table 1. Amongst the 261 patients, 59 (22.6%) demonstrated a PFO. This could be subdivided into 30 (11.5%) with a grade 1 PFO, 17 (6.5%) with grade 2 and 12 (4.6%) with grade 3. In total, 29 (11.1%) of patients had an open channel between the atria during the CT scan (grade 2 or 3 PFO).\nNumbers and percentages of patients displaying conditions of the inter-atrial septum.\nTable 1 Legend: PFO = patent foramen ovale. Graded by Williamson classification1.\nAn atrial septal aneurysm was identified in 16 (6.1%) patients but no jet of contrast from left to right atrium was seen in association with this.\nOne patient was seen to have an early (1.1cm diameter) left atrial myxoma with prominent central vasculature (Figure 2, panel B).\nTable 2 lists patients with conditions classified by the TOAST criteria for medium or high risk of cardiac embolism.10 Of note, 17.2% of patients had medium cardio-embolic risk.\nConditions classified under the TOAST criteria for cardioembolic risk. (11)\nTable 2 Legend: PFO = patent foramen ovale. Graded by Williamson classification1.",
"PFO is strongly linked with conditions such as CS, pulmonary embolus and more controversially, migraine with aura.8, 11, 12 Major studies to determine the role of PFO closure devices are underway.13 The reported prevalence of PFO depends however, on the imaging modality used and the population studied. Prevalence based on normal heart autopsy is 27%, whilst SCE and TOE yield figures of 14.9% and 24.3% respectively in normal adults.2, 3, 14\nPrevalence is higher in vulnerable groups; amongst migraineurs in the MIST 1 study, a moderate or large PFO was detected in 37.7% by SCE, whilst 43.9% of young CS patients were demonstrated to have a PFO by TOE.15, 16 TOE is regarded as the gold standard imaging technique for PFO assessment.17, 18 Recently, real time 3D TOE has proved useful in guiding PFO closure device procedures.19\nMoving away from echocardiography, only a handful of studies of PFO anatomy have been performed on 64-MDCT.1, 7, 20, 21\nTwo studies have concentrated on comparing 64-MDCT with TOE. Williamson et al. reviewed 214 charts of patients attending for 64-MDCT and found 20 who had also undergone TOE. Of the six with PFO on TOE, all had grade 1 or higher PFO by the criteria defined in that study.1 Kim et al. retrospectively analysed images from 152 stroke patients who had undergone both TOE and CT. Twenty-six PFOs were identified by TOE with 19 of these patients having a grade 3 (open flap with jet) PFO appearance on CT.20 The authors also noted a “channel-like appearance” of the IAS which corresponded to grade 1 (closed flap) and grade 2 (open flap) in the Williamson et al. paper. Compared with TOE, the two papers combined yield a sensitivity of 67-73%, specificity of 98-100%, positive predictive value of 91-100% and negative predictive value of 85-95% for a grade 3 CT appearance. In a more generalised study, CT was compared with TOE for detection of cardiac sources of embolism in 137 stroke patients.21 Just under a quarter of the patients had PFO, ASA or an atrial septal defect identified by TOE. Overall sensitivity of CT was 89% with a positive predictive value of 100% for all embolic causes. The overall prevalence of all 3 grades of PFO in our study was 22.6%. We agree with previous authors that an open flap with direct communication between atria (grades 2 and 3) is more likely to represent clinically significant PFO, in which case, the prevalence falls to 11.1%.",
"One major problem with assessing PFO by 64-MDCT is that the information is purely anatomical and obtained during a breath hold rather than Valsalva manoeuvre. The functional and flow information obtained with TOE cannot be emulated.\nIt is interesting to note that the prevalence of all 3 grades of PFO at 22.6% is very similar to TOE and autopsy reference data, perhaps some of the closed flaps (11.5%) would open if right atrial pressure was elevated. Unfortunately, the rise in central venous pressure associated with Valsalva manoeuvre or coughing also impedes the passage of intravenous contrast to the heart. Unacceptable respiratory motion artefacts would also be introduced.\nPotentially, cine CT angiography22 could give information about directional flow in a PFO and mobility of ASA, but the Valsalva manoeuvre problem would remain.\nThe point we wish to stress in this paper is that much useful information can be obtained during a routine coronary study without additional measures or high dose protocols.\nThe prevalence of ASA in our study (6.1%) falls within the range seen in TOE studies on non-stroke patients (4-8%) - this figure can rise up to 15 - 28% in stroke populations.23, 24 We did not observe left to right contrast flow through an aneurysm but it is accepted that approximately 33% of adults with ASA also have PFO and ASA is considered a medium risk source of embolus by TOAST criteria.8,9",
"The Society of Cardiovascular Computed Tomography (SCCT) guidelines on reporting studies state that any [non-coronary] abnormalities should be described and that cardiac chamber shunts are a required element of a comprehensive report25 but given that 17.2% of our patients attending for routine coronary CT had a medium cardio-embolic risk (open flap PFO or ASA) perhaps more emphasis should be placed on this requirement.",
"64-MDCT allows accurate assessment of the IAS during routine “coronary” examination. We found the prevalence of open flap PFO and ASA to be 11.1% and 6.1% respectively in a population of 261 patients undergoing routine 64-MDCT coronary study. Further comparative studies against SCE and TOE are required but we believe open flap PFO and ASA should be clearly identified and recorded during routine coronary CT angiography. This approach may identify those at risk of cryptogenic stroke as well as avoid unnecessary tests in stroke patients.\nThe authors have no conflict of interest."
] | [
"methods",
null,
null,
null,
null,
null,
null,
null
] | [
"Patent foramen ovale",
"atrial septum",
"cardiac anatomy",
"computed tomographic angiography",
"non-invasive angiography"
] | Methods:
The study was approved by the Western HSC Trust Research and Development (Ethics) Chair. Data was collected prospectively on patients attending for 64-MDCT between the 18th June 2009 and 27th February 2010.
Computed Tomography:
64-MDCT was performed using a Philips Brilliance 64 system (Philips Medical Systems, Eindhoven, Netherlands). Patients were monitored on electrocardiogram (ECG) and a 20FG intravenous cannula placed in an ante-cubital fossa vein. All patients were in sinus rhythm. After heart rate optimisation and sub-lingual administration of 400 micrograms of glyceryl tri-nitrate, non-ionic contrast material (Iohexol, Omnipaque 350, GE Healthcare AS, Norway) was injected into the vein (90 ml at a flow rate of 5.5 ml/sec followed by a saline chaser of 50mls at 5.5 ml/sec). A bolus tracking technique was used to determine when contrast density was optimal for coronary imaging. Scanning was performed during a single breath hold. In patients with a low and stable heart rate (<64 beats per minute), prospective ECG triggering was used for data acquisition; otherwise retrospective ECG gating was used. In all cases, data was obtained at a single time point (75% of the R-R wave interval on ECG). Detector collimation was 64 × 0.625mm with images reconstructed to a slice thickness of 0.6mm. Tube voltage was 120kV with a rotation time of 400 msec. Standard axial images and 2D multiplanar reformations were used for image interpretation. Coronal oblique projections through the IAS were specifically evaluated for the presence of each of the CT criteria for PFO outlined below.
Classification of PFO and ASA:
Williamson et al. devised a classification system based in part on chart review of 20 patients with PFO who underwent both CT and Trans-oesophageal echocardiography (TOE).1 PFO anatomy on 64-MDCT was classified by 3 criteria:
A distinct flap at the expected location in the LA (closed flap, Figure 1, panel A).
A continuous column of contrast material between the septum primum and septum secundum, connecting the LA and RA (open flap, Figure 1 panel B).
An open flap plus a jet of contrast material from the column into the RA (open flap with jet, Figure 1, panel C).
A distinct flap at the expected location in the LA (closed flap, Figure 1, panel A).
A continuous column of contrast material between the septum primum and septum secundum, connecting the LA and RA (open flap, Figure 1 panel B).
An open flap plus a jet of contrast material from the column into the RA (open flap with jet, Figure 1, panel C).
Panel A. Oblique coronal view: hollow arrow demonstrates closed flap (Grade 1 PFO), left atrium =LA, right atrium =RA. Panel B. Oblique coronal view: hollow arrow demonstrates open flap (Grade 2 PFO . Panel C. Oblique coronal view: hollow arrow demonstrates jet from open flap (Grade 3 PFO).
When compared with TOE diagnosis, the positive predictive value of criterion 1 alone was 75%, criteria 1 +2 together was 80% and 1+2+3 together was 100%.1
ASA was defined as a redundant and hyper-mobile portion of the IAS that demonstrated more than 10-mm excursion from the centre line (Figure 2, panel A).9
Panel A. Axial view with atrial septal aneurysm highlighted by hollow arrow, left atrium =LA, right atrium =RA. Panel B. A small rounded mass is identified over the fossa ovalis with a vascular supply (hollow arrow). This proved to be a left atrial myxoma.
We also categorised any potential IAS source of cardio-embolism into medium and high risk according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification.10
Results:
The study population consisted of 261 consecutive patients of whom 54% were male (mean age 56 years, range 21 to 81 years) and 46% were female (mean age 57 years, range 36 to 81 years). Study quality was sufficient for PFO classification in all cases. Study quality for coronary imaging was graded as excellent in 50%, good in 28%, fair in 16% and poor in 6%. A low dose prospective ECG triggered protocol was used in 38%. Results are displayed in Table 1. Amongst the 261 patients, 59 (22.6%) demonstrated a PFO. This could be subdivided into 30 (11.5%) with a grade 1 PFO, 17 (6.5%) with grade 2 and 12 (4.6%) with grade 3. In total, 29 (11.1%) of patients had an open channel between the atria during the CT scan (grade 2 or 3 PFO).
Numbers and percentages of patients displaying conditions of the inter-atrial septum.
Table 1 Legend: PFO = patent foramen ovale. Graded by Williamson classification1.
An atrial septal aneurysm was identified in 16 (6.1%) patients but no jet of contrast from left to right atrium was seen in association with this.
One patient was seen to have an early (1.1cm diameter) left atrial myxoma with prominent central vasculature (Figure 2, panel B).
Table 2 lists patients with conditions classified by the TOAST criteria for medium or high risk of cardiac embolism.10 Of note, 17.2% of patients had medium cardio-embolic risk.
Conditions classified under the TOAST criteria for cardioembolic risk. (11)
Table 2 Legend: PFO = patent foramen ovale. Graded by Williamson classification1.
Discussion:
PFO is strongly linked with conditions such as CS, pulmonary embolus and more controversially, migraine with aura.8, 11, 12 Major studies to determine the role of PFO closure devices are underway.13 The reported prevalence of PFO depends however, on the imaging modality used and the population studied. Prevalence based on normal heart autopsy is 27%, whilst SCE and TOE yield figures of 14.9% and 24.3% respectively in normal adults.2, 3, 14
Prevalence is higher in vulnerable groups; amongst migraineurs in the MIST 1 study, a moderate or large PFO was detected in 37.7% by SCE, whilst 43.9% of young CS patients were demonstrated to have a PFO by TOE.15, 16 TOE is regarded as the gold standard imaging technique for PFO assessment.17, 18 Recently, real time 3D TOE has proved useful in guiding PFO closure device procedures.19
Moving away from echocardiography, only a handful of studies of PFO anatomy have been performed on 64-MDCT.1, 7, 20, 21
Two studies have concentrated on comparing 64-MDCT with TOE. Williamson et al. reviewed 214 charts of patients attending for 64-MDCT and found 20 who had also undergone TOE. Of the six with PFO on TOE, all had grade 1 or higher PFO by the criteria defined in that study.1 Kim et al. retrospectively analysed images from 152 stroke patients who had undergone both TOE and CT. Twenty-six PFOs were identified by TOE with 19 of these patients having a grade 3 (open flap with jet) PFO appearance on CT.20 The authors also noted a “channel-like appearance” of the IAS which corresponded to grade 1 (closed flap) and grade 2 (open flap) in the Williamson et al. paper. Compared with TOE, the two papers combined yield a sensitivity of 67-73%, specificity of 98-100%, positive predictive value of 91-100% and negative predictive value of 85-95% for a grade 3 CT appearance. In a more generalised study, CT was compared with TOE for detection of cardiac sources of embolism in 137 stroke patients.21 Just under a quarter of the patients had PFO, ASA or an atrial septal defect identified by TOE. Overall sensitivity of CT was 89% with a positive predictive value of 100% for all embolic causes. The overall prevalence of all 3 grades of PFO in our study was 22.6%. We agree with previous authors that an open flap with direct communication between atria (grades 2 and 3) is more likely to represent clinically significant PFO, in which case, the prevalence falls to 11.1%.
Study Limitations:
One major problem with assessing PFO by 64-MDCT is that the information is purely anatomical and obtained during a breath hold rather than Valsalva manoeuvre. The functional and flow information obtained with TOE cannot be emulated.
It is interesting to note that the prevalence of all 3 grades of PFO at 22.6% is very similar to TOE and autopsy reference data, perhaps some of the closed flaps (11.5%) would open if right atrial pressure was elevated. Unfortunately, the rise in central venous pressure associated with Valsalva manoeuvre or coughing also impedes the passage of intravenous contrast to the heart. Unacceptable respiratory motion artefacts would also be introduced.
Potentially, cine CT angiography22 could give information about directional flow in a PFO and mobility of ASA, but the Valsalva manoeuvre problem would remain.
The point we wish to stress in this paper is that much useful information can be obtained during a routine coronary study without additional measures or high dose protocols.
The prevalence of ASA in our study (6.1%) falls within the range seen in TOE studies on non-stroke patients (4-8%) - this figure can rise up to 15 - 28% in stroke populations.23, 24 We did not observe left to right contrast flow through an aneurysm but it is accepted that approximately 33% of adults with ASA also have PFO and ASA is considered a medium risk source of embolus by TOAST criteria.8,9
Current Guidelines:
The Society of Cardiovascular Computed Tomography (SCCT) guidelines on reporting studies state that any [non-coronary] abnormalities should be described and that cardiac chamber shunts are a required element of a comprehensive report25 but given that 17.2% of our patients attending for routine coronary CT had a medium cardio-embolic risk (open flap PFO or ASA) perhaps more emphasis should be placed on this requirement.
Conclusions:
64-MDCT allows accurate assessment of the IAS during routine “coronary” examination. We found the prevalence of open flap PFO and ASA to be 11.1% and 6.1% respectively in a population of 261 patients undergoing routine 64-MDCT coronary study. Further comparative studies against SCE and TOE are required but we believe open flap PFO and ASA should be clearly identified and recorded during routine coronary CT angiography. This approach may identify those at risk of cryptogenic stroke as well as avoid unnecessary tests in stroke patients.
The authors have no conflict of interest.
| Background:
A patent foramen ovale (PFO) is strongly associated with cryptogenic stroke (CS), neurological and other phenomena. The reported prevalence of PFO varies according to the imaging technique used and population studied.
Methods:
Standard coronary imaging protocols were used. PFOs were graded according to the classification of Williamson et al.
Results:
261 patients were studied. A PFO was identified in 22.6% (11.5% grade 1 (closed flap), 6.5% grade 2 (open flap) and 4.6% grade 3 (open flap with jet)). A further 6.1% had an atrial septal aneurysm.
Conclusions:
The prevalence of all grades of PFO (22.6%) and open flap PFO (11.1% = grade 2 and 3) with this technique compares with 24.3% by trans-oesophageal echocardiography (TOE) and 14.9% by saline contrast echocardiography (SCE). Further comparative studies are required but we believe an open flap PFO or ASA should be identified and recorded during cardiac CT. This approach may identify those at risk of cryptogenic stroke as well as avoid unnecessary tests in stroke patients. | null | null | 2,042 | 218 | [
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"intravenous contrast heart",
"mdct coronary study"
] | null | null | null | null | [CONTENT] Patent foramen ovale | atrial septum | cardiac anatomy | computed tomographic angiography | non-invasive angiography [SUMMARY] | null | [CONTENT] Patent foramen ovale | atrial septum | cardiac anatomy | computed tomographic angiography | non-invasive angiography [SUMMARY] | [CONTENT] Patent foramen ovale | atrial septum | cardiac anatomy | computed tomographic angiography | non-invasive angiography [SUMMARY] | null | null | [CONTENT] Adult | Aged | Aged, 80 and over | Cardiac-Gated Imaging Techniques | Contrast Media | Coronary Angiography | Echocardiography | Female | Foramen Ovale, Patent | Humans | Iohexol | Ireland | Male | Middle Aged | Prevalence | Prospective Studies | Tomography, X-Ray Computed [SUMMARY] | null | [CONTENT] Adult | Aged | Aged, 80 and over | Cardiac-Gated Imaging Techniques | Contrast Media | Coronary Angiography | Echocardiography | Female | Foramen Ovale, Patent | Humans | Iohexol | Ireland | Male | Middle Aged | Prevalence | Prospective Studies | Tomography, X-Ray Computed [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cardiac-Gated Imaging Techniques | Contrast Media | Coronary Angiography | Echocardiography | Female | Foramen Ovale, Patent | Humans | Iohexol | Ireland | Male | Middle Aged | Prevalence | Prospective Studies | Tomography, X-Ray Computed [SUMMARY] | null | null | [CONTENT] angiography22 information | study quality coronary | fossa vein patients | intravenous contrast heart | mdct coronary study [SUMMARY] | null | [CONTENT] angiography22 information | study quality coronary | fossa vein patients | intravenous contrast heart | mdct coronary study [SUMMARY] | [CONTENT] angiography22 information | study quality coronary | fossa vein patients | intravenous contrast heart | mdct coronary study [SUMMARY] | null | null | [CONTENT] pfo | patients | flap | toe | open | open flap | panel | study | grade | 64 [SUMMARY] | null | [CONTENT] pfo | patients | flap | toe | open | open flap | panel | study | grade | 64 [SUMMARY] | [CONTENT] pfo | patients | flap | toe | open | open flap | panel | study | grade | 64 [SUMMARY] | null | null | [CONTENT] mdct 18th june | 18th june 2009 | 18th | development | february 2010 | february | development ethics | development ethics chair | development ethics chair data | chair [SUMMARY] | null | [CONTENT] routine | coronary | flap pfo asa | flap pfo | open flap pfo asa | open flap pfo | routine coronary | flap | stroke | pfo asa [SUMMARY] | [CONTENT] pfo | flap | patients | toe | open flap | open | 64 | coronary | study | panel [SUMMARY] | null | null | [CONTENT] ||| Williamson et al. [SUMMARY] | null | [CONTENT] PFO | 22.6% | PFO | 11.1% | 2 | 24.3% | TOE | 14.9% | SCE ||| PFO | ASA | CT ||| [SUMMARY] | [CONTENT] PFO | CS ||| PFO ||| ||| Williamson et al. | 261 ||| PFO | 22.6% | 11.5% | 1 | 6.5% | 2 | 4.6% | 3 ||| 6.1% ||| PFO | 22.6% | PFO | 11.1% | 2 | 24.3% | TOE | 14.9% | SCE ||| PFO | ASA | CT ||| [SUMMARY] | null |
|||
Establishing a Customized Guide Plate for Osteotomy in Total Knee Arthroplasty Using Lower-extremity X-ray and Knee Computed Tomography Images. | 26879010 | The conventional method cannot guarantee the precise osteotomies required for a perfect realignment and a better prognosis after total knee arthroplasty (TKA). This study investigated a customized guide plate for osteotomy placement in TKAs with the aid of the statistical shape model technique using weight-bearing lower-extremity X-rays and computed tomography (CT) images of the knee. | BACKGROUND | From October 2014 to June 2015, 42 patients who underwent a TKA in Guizhou Provincial People's Hospital were divided into a guide plate group (GPG, 21 cases) and a traditional surgery group (TSG, 21 cases) using a random number table method. In the GPG group, a guide plate was designed and printed using preoperative three-dimensional measurements to plan and digitally simulate the operation. TSG cases were treated with the conventional method. Outcomes were obtained from the postoperative image examination and short-term follow-up. | METHODS | Operative time was 49.0 ± 10.5 min for GPG, and 62.0 ± 9.7 min in TSG. The coronal femoral angle, coronal tibial angle, posterior tibial slope, and the angle between the posterior condylar osteotomy surface and the surgical transepicondylar axis were 89.2 ± 1.7°, 89.0 ± 1.1°, 6.6 ± 1.4°, and 0.9 ± 0.3° in GPG, and 86.7 ± 2.9°, 87.6 ± 2.1°, 8.9 ± 2.8°, and 1.7 ± 0.8° in TSG, respectively. The Hospital for Special Surgery scores 3 months after surgery were 83.7 ± 18.4 in GPG and 71.5 ± 15.2 in TSG. Statistically significant differences were found between GPG and TSG in all measurements. | RESULTS | A customized guide plate to create an accurate osteotomy in TKAs may be created using lower-extremity X-ray and knee CT images. This allows for shorter operative times and better postoperative alignment than the traditional surgery. Application of the digital guide plate may also result in better short-term outcomes. | CONCLUSIONS | [
"Aged",
"Arthroplasty, Replacement, Knee",
"Female",
"Humans",
"Knee",
"Male",
"Middle Aged",
"Osteotomy",
"Surgery, Computer-Assisted",
"Tomography, X-Ray Computed"
] | 4800837 | INTRODUCTION | Total knee arthroplasty (TKA) is an effective treatment for knee joint diseases such as osteoarthritis and rheumatoid arthritis. The perfect reconstruction of lower limb alignment plays a very important role in the outcomes of knee joint replacement, and can affect both symptom relief and the life time of the prosthesis.[12]
The traditional method for a correctional osteotomy is to use the lower-extremity weight-bearing full-length X-ray to determine the osteotomy location and the angle of the coronal plane. As the accuracy of measuring the joint center with two-dimensional (2D) images cannot be guaranteed, the preoperative osteotomy position and angle planning with these images are imprecise. The angle of the sagittal and transverse plane depends on the surgeon's experience. A prolonged operative time and poor accuracy are inevitable.[3]
With the development of digital technology, the clinical applications of three-dimensional (3D) measurement and operative simulation have gradually increased. In our study, calibrated lower-extremity weight-bearing full-length X-rays and computed tomography (CT) scans of the knee were collected. The statistical shape model (SSM) technique was used to synthesize a 3D model of the knee from the X-ray. A 3D model of the lower limb can be generated with the use of the 3D knee model created using the SSM technique along with CT scans. After the hip and ankle centers were determined on the dual plane X-ray, the relative positional relationship between the two joint centers and the 3D knee model was measured. Using this method, a preoperative analysis can be conducted on the basis of weight-bearing function, avoiding the significant radiation exposure due to CT scan. The preoperative planning, osteotomy simulation, and prosthetic placement were performed. The 3D-printed osteotomy guide plate from the digitally designed model was applied to the TKA. | METHODS | This clinical trial received permission from the Ethics Committee of Guizhou Provincial People's Hospital, and all patients and their families signed informed consents. A total of 42 cases of osteoarthritis in patients who previously underwent a unilateral TKA at Guizhou Provincial People's Hospital from October 2014 to June 2015 were enrolled in our study. Of these patients, 17 were male, and 25 were female. The involved knee was on the left in 20 cases, while 22 were on the right. Average age was 62 ± 7 years. Preoperative mean body mass index was 25.3 ± 6.4 kg/m2. The eligibility criteria included: (1) unilateral TKA; (2) varus deformity of the operative knee; (3) osteoarthritis of the operative knee. Exclusion criteria included: (1) severe comorbidities that may prolong hospitalization time; (2) severe osteoporosis; (3) the patient declined to participate in the research. Patients were divided into two groups: A guide plate group (GPG, 21 cases), and a traditional surgery group (TSG, 21 cases), using the random number table method. In GPG patients, preoperative thin slice CT scans of the knee were obtained using the Siemens 64 row spiral CT (Siemens, Munich, Germany). The scanning voltage was 130 kV, the scanning thickness was 1 mm, and the matrix was 512 × 512. Digital Imaging and Communications in Medicine format CT data were imported into Simpleware 7.2 (Simpleware Ltd., Exeter, UK) to reconstruct the 3D stereolithography format knee model. Calibrated weight-bearing full-length dual plane knee X-ray images were obtained [Figures 1 and 2]. 3D knee models were synthesized from calibrated dual plane images with SSM of the knee and registration.[4567] The automatic registration of the 3D knee model from the CT scan and from the SSM technique can be obtained digitally [Figure 3].
Photo of calibrated leg. (a) Frontal view. (b) Lateral view.
X-ray images of calibrated weight-bearing full-length lower limb. (a) Anteroposterior image. (b) Lateral image.
Registration between the three-dimensional model synthesized from calibrated X-ray and three-dimensional model from computed tomography. (a) The three dimensional diagrammatic sketch of registration. (b) The anteroposterior film after registration. (c) The lateral film after registration.
The hip center (center of the synthesized sphere) and the ankle center (center of the talar articular surface) were determined. Through the position relationship between the centers, points on the calibration device and the X-ray source, the relative spatial relationship between the two central points and the 3D model of the knee joint was determined. The central point of the distal femur was defined as the midpoint of the Whiteside line.[8] The central point of the proximal tibia was defined as the center of the tibial intercondylar eminence. The femoral anatomic axis was defined as the connection between the central point of the distal femur and the medullary cavity center 10 cm above the knee joint space. The femoral mechanical axis was defined as the connection between the central point of the distal femur and the central point of the femoral head. The mechanical axis of the tibia was defined as the connection between the central point of the proximal tibia and the central point of the ankle joint.
Prosthetic models were obtained by 3D scanning the NexGen LPS (Zimmer Biomet, Warsaw, Indiana, USA) with the Laser-RE (Serein Precision Machinery Company, Shenzhen, China).
The femoral distal osteotomy valgus angle was the angle measured between the femoral anatomy and the mechanical axis, while the osteotomy thickness was 9 mm from the lowest point of the medial femoral condyle.
Through the simulated operation, we were able to choose the suitable size of the prosthesis. The osteotomy line of the anterior and posterior condyles was parallel to the surgical transepicondylar axis (STEA) [Figure 4].
Anterior and posterior condylar osteotomy line is parallel to surgical transepicondylar axis.
The proximal tibial osteotomy line was perpendicular to the mechanical axis on the coronal plane, and was 7° posterior slope on the sagittal plane resulting from the design of the prosthesis. The thickness of the proximal tibial osteotomy was 10 mm from the highest point of the lateral tibial condyle.
According to the design above and the operative simulation, the hole position of the nonheaded screw and the size of the prosthesis were determined. The base of the guide plate was reverse engineered in accordance with the shape of the distal femur and proximal tibia. The screw hole and the base were then connected. The digital design was created using Geomagic (3D Systems, Valencia, CA, USA). In the reverse design stage of the base, the more shape characteristic bone surface was selected to be the attachment area for the guide plate to obtain a better match for the installation. The guide plate was 3D-printed with the polylactic acid material using a 3D Printer (3D Systems Company, Rock Hill, South Carolina, USA). After the operative exposure, the guide plates were attached to the bony surface where the articular cartilage was removed to determine the position of the nonheaded screw for the distal femoral, anterior and posterior condylar, and proximal tibial osteotomy devices. All surgeries were performed by a single surgeon [Figure 5]. Postoperative radiographs were obtained to verify the alignment of the reconstruction [Figure 6].
The nonhead screws’ positions were determined according the guide plates in order to locate the osteotomy devices. (a) The guide plate determined the positions of the nonhead screws in the proximal tibia. (b) The nonhead screws determined the positions of the proximal tibial osteotomy divices. (c) The guide plate determined the positions of the nonhead screws in the distal femur. (d)The nonhead screws determined the positions of the anterior and posterior condylar osteotomy divices.
Postoperative coronal femoral/tibial angles and the angle between posterior condylar osteotomy surface and the surgical transepicondylar axis. (a) The postoperative coronal femoral/tibial angles were 90°. (b) The postoperative angle between posterior condylar osteotomy surface and the surgical transepicondylar axis was 0°.
In the TSG, the angle between the femoral anatomic and mechanical axis was measured preoperatively using a weight-bearing full-length lower-extremity X-ray. The valgus angle for the distal femoral osteotomy was chosen using the closest available angle on the surgical instruments. The intramedullary alignment guide was applied to the femur while the extramedullary alignment guide was applied to the tibia. The rotational angle was chosen according to the experience of the surgeon.
Obtained data were statistically analyzed using SPSS 16.0 (IBM, Armonk, NY, USA). A t-test was applied to assess for differences between groups, with P < 0.05 considered significant. | RESULTS | The operation time was 49.0 ± 10.5 min for GPG, and 62.0 ± 9.7 min for TSG. The coronal femoral angle, coronal tibial angle, posterior tibial slope, and the angle between the posterior condylar osteotomy surface and the STEA were 89.2 ± 1.7°, 89.0 ± 1.1°, 6.6 ± 1.4°, and 0.9 ± 0.3° in GPG, and 86.7 ± 2.9°, 87.6 ± 2.1°, 8.9 ± 2.8°, and 1.7 ± 0.8° in TSG, respectively. The Hospital for Special Surgery knee scores 3 months after surgery were 83.7 ± 18.4 after GPG and 71.5 ± 15.2 after TSG, respectively. Statistically significant differences were found between all previously noted comparisons [Table 1].
Comparing the outcomes of the GPG and the TSG
HSS: Hospital for Special Surgery; GPG: Guide plate group; TSG: Traditional surgery group; STEA: Surgical transepicondylar axis. | null | null | [
"Financial support and sponsorship",
"Conflicts of interest"
] | [
"This work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).",
"There are no conflicts of interest."
] | [
null,
null
] | [
"INTRODUCTION",
"METHODS",
"RESULTS",
"DISCUSSION",
"Financial support and sponsorship",
"Conflicts of interest"
] | [
"Total knee arthroplasty (TKA) is an effective treatment for knee joint diseases such as osteoarthritis and rheumatoid arthritis. The perfect reconstruction of lower limb alignment plays a very important role in the outcomes of knee joint replacement, and can affect both symptom relief and the life time of the prosthesis.[12]\nThe traditional method for a correctional osteotomy is to use the lower-extremity weight-bearing full-length X-ray to determine the osteotomy location and the angle of the coronal plane. As the accuracy of measuring the joint center with two-dimensional (2D) images cannot be guaranteed, the preoperative osteotomy position and angle planning with these images are imprecise. The angle of the sagittal and transverse plane depends on the surgeon's experience. A prolonged operative time and poor accuracy are inevitable.[3]\nWith the development of digital technology, the clinical applications of three-dimensional (3D) measurement and operative simulation have gradually increased. In our study, calibrated lower-extremity weight-bearing full-length X-rays and computed tomography (CT) scans of the knee were collected. The statistical shape model (SSM) technique was used to synthesize a 3D model of the knee from the X-ray. A 3D model of the lower limb can be generated with the use of the 3D knee model created using the SSM technique along with CT scans. After the hip and ankle centers were determined on the dual plane X-ray, the relative positional relationship between the two joint centers and the 3D knee model was measured. Using this method, a preoperative analysis can be conducted on the basis of weight-bearing function, avoiding the significant radiation exposure due to CT scan. The preoperative planning, osteotomy simulation, and prosthetic placement were performed. The 3D-printed osteotomy guide plate from the digitally designed model was applied to the TKA.",
"This clinical trial received permission from the Ethics Committee of Guizhou Provincial People's Hospital, and all patients and their families signed informed consents. A total of 42 cases of osteoarthritis in patients who previously underwent a unilateral TKA at Guizhou Provincial People's Hospital from October 2014 to June 2015 were enrolled in our study. Of these patients, 17 were male, and 25 were female. The involved knee was on the left in 20 cases, while 22 were on the right. Average age was 62 ± 7 years. Preoperative mean body mass index was 25.3 ± 6.4 kg/m2. The eligibility criteria included: (1) unilateral TKA; (2) varus deformity of the operative knee; (3) osteoarthritis of the operative knee. Exclusion criteria included: (1) severe comorbidities that may prolong hospitalization time; (2) severe osteoporosis; (3) the patient declined to participate in the research. Patients were divided into two groups: A guide plate group (GPG, 21 cases), and a traditional surgery group (TSG, 21 cases), using the random number table method. In GPG patients, preoperative thin slice CT scans of the knee were obtained using the Siemens 64 row spiral CT (Siemens, Munich, Germany). The scanning voltage was 130 kV, the scanning thickness was 1 mm, and the matrix was 512 × 512. Digital Imaging and Communications in Medicine format CT data were imported into Simpleware 7.2 (Simpleware Ltd., Exeter, UK) to reconstruct the 3D stereolithography format knee model. Calibrated weight-bearing full-length dual plane knee X-ray images were obtained [Figures 1 and 2]. 3D knee models were synthesized from calibrated dual plane images with SSM of the knee and registration.[4567] The automatic registration of the 3D knee model from the CT scan and from the SSM technique can be obtained digitally [Figure 3].\nPhoto of calibrated leg. (a) Frontal view. (b) Lateral view.\nX-ray images of calibrated weight-bearing full-length lower limb. (a) Anteroposterior image. (b) Lateral image.\nRegistration between the three-dimensional model synthesized from calibrated X-ray and three-dimensional model from computed tomography. (a) The three dimensional diagrammatic sketch of registration. (b) The anteroposterior film after registration. (c) The lateral film after registration.\nThe hip center (center of the synthesized sphere) and the ankle center (center of the talar articular surface) were determined. Through the position relationship between the centers, points on the calibration device and the X-ray source, the relative spatial relationship between the two central points and the 3D model of the knee joint was determined. The central point of the distal femur was defined as the midpoint of the Whiteside line.[8] The central point of the proximal tibia was defined as the center of the tibial intercondylar eminence. The femoral anatomic axis was defined as the connection between the central point of the distal femur and the medullary cavity center 10 cm above the knee joint space. The femoral mechanical axis was defined as the connection between the central point of the distal femur and the central point of the femoral head. The mechanical axis of the tibia was defined as the connection between the central point of the proximal tibia and the central point of the ankle joint.\nProsthetic models were obtained by 3D scanning the NexGen LPS (Zimmer Biomet, Warsaw, Indiana, USA) with the Laser-RE (Serein Precision Machinery Company, Shenzhen, China).\nThe femoral distal osteotomy valgus angle was the angle measured between the femoral anatomy and the mechanical axis, while the osteotomy thickness was 9 mm from the lowest point of the medial femoral condyle.\nThrough the simulated operation, we were able to choose the suitable size of the prosthesis. The osteotomy line of the anterior and posterior condyles was parallel to the surgical transepicondylar axis (STEA) [Figure 4].\nAnterior and posterior condylar osteotomy line is parallel to surgical transepicondylar axis.\nThe proximal tibial osteotomy line was perpendicular to the mechanical axis on the coronal plane, and was 7° posterior slope on the sagittal plane resulting from the design of the prosthesis. The thickness of the proximal tibial osteotomy was 10 mm from the highest point of the lateral tibial condyle.\nAccording to the design above and the operative simulation, the hole position of the nonheaded screw and the size of the prosthesis were determined. The base of the guide plate was reverse engineered in accordance with the shape of the distal femur and proximal tibia. The screw hole and the base were then connected. The digital design was created using Geomagic (3D Systems, Valencia, CA, USA). In the reverse design stage of the base, the more shape characteristic bone surface was selected to be the attachment area for the guide plate to obtain a better match for the installation. The guide plate was 3D-printed with the polylactic acid material using a 3D Printer (3D Systems Company, Rock Hill, South Carolina, USA). After the operative exposure, the guide plates were attached to the bony surface where the articular cartilage was removed to determine the position of the nonheaded screw for the distal femoral, anterior and posterior condylar, and proximal tibial osteotomy devices. All surgeries were performed by a single surgeon [Figure 5]. Postoperative radiographs were obtained to verify the alignment of the reconstruction [Figure 6].\nThe nonhead screws’ positions were determined according the guide plates in order to locate the osteotomy devices. (a) The guide plate determined the positions of the nonhead screws in the proximal tibia. (b) The nonhead screws determined the positions of the proximal tibial osteotomy divices. (c) The guide plate determined the positions of the nonhead screws in the distal femur. (d)The nonhead screws determined the positions of the anterior and posterior condylar osteotomy divices.\nPostoperative coronal femoral/tibial angles and the angle between posterior condylar osteotomy surface and the surgical transepicondylar axis. (a) The postoperative coronal femoral/tibial angles were 90°. (b) The postoperative angle between posterior condylar osteotomy surface and the surgical transepicondylar axis was 0°.\nIn the TSG, the angle between the femoral anatomic and mechanical axis was measured preoperatively using a weight-bearing full-length lower-extremity X-ray. The valgus angle for the distal femoral osteotomy was chosen using the closest available angle on the surgical instruments. The intramedullary alignment guide was applied to the femur while the extramedullary alignment guide was applied to the tibia. The rotational angle was chosen according to the experience of the surgeon.\nObtained data were statistically analyzed using SPSS 16.0 (IBM, Armonk, NY, USA). A t-test was applied to assess for differences between groups, with P < 0.05 considered significant.",
"The operation time was 49.0 ± 10.5 min for GPG, and 62.0 ± 9.7 min for TSG. The coronal femoral angle, coronal tibial angle, posterior tibial slope, and the angle between the posterior condylar osteotomy surface and the STEA were 89.2 ± 1.7°, 89.0 ± 1.1°, 6.6 ± 1.4°, and 0.9 ± 0.3° in GPG, and 86.7 ± 2.9°, 87.6 ± 2.1°, 8.9 ± 2.8°, and 1.7 ± 0.8° in TSG, respectively. The Hospital for Special Surgery knee scores 3 months after surgery were 83.7 ± 18.4 after GPG and 71.5 ± 15.2 after TSG, respectively. Statistically significant differences were found between all previously noted comparisons [Table 1].\nComparing the outcomes of the GPG and the TSG\nHSS: Hospital for Special Surgery; GPG: Guide plate group; TSG: Traditional surgery group; STEA: Surgical transepicondylar axis.",
"TKA is an effective treatment for severe knee diseases. An accurate osteotomy and the reconstruction of the normal alignment of the lower limbs are key to obtaining a good curative effect.[12] There is significant anatomic variation of the knee joint, especially in the southwest part of China. The knee joint prosthesis and the matching operative instruments are designed with the anatomic characteristics of the European and American knee joint in mind. There may therefore be a large error when using conventional osteotomy instruments in East Asia, resulting in poor alignment of the lower limb, early component loosening, pain, activity limitation, and other complications. The traditional surgical osteotomy is based on the measurement of the weight-bearing full-length lower-extremity X-ray and the intra/extramedullary alignment guides. There is significant subjectivity when using these measurements. This increases the potential risk of infection, bleeding, and fat embolism in the traditional intramedullary alignment guide-based surgery.[910]\nModern computer technology has developed rapidly, providing a platform for preoperative planning. The orthopedic surgeon can perform computer-assisted precise operative measurements.[111213] Hafez et al.[14] digitally designed and 3D printed the osteotomy guide plate for a TKA for the first time, which can effectively avoid the fat embolisms that often result from the placement of the intramedullary alignment guide. Cai et al.[15] uses digital technology to control the axial alignment of total knee arthroplasties, but they needed a CT of the full-length lower limb. Zhang et al.[16] synthesized a 3D joint model from hip, knee, and ankle CTs and a weight-bearing full-length X-ray. 3D alignment reconstruction and the determination of the femoral prosthetic valgus angle can be made using the preoperative design based on the 3D model of the lower limb. In this study, the measurement and reconstruction of the alignment was conducted under physiologic load to be more consistent with normal biomechanical characteristics. However, it required a thin-cut CT scan of the three joints, which significantly increased the examination cost and the patient's radiation exposure.\nIn this study, the use of advanced 3D synthesis and 2D/3D registration technology allowed for the reconstruction of a weight-bearing 3D model of the lower limb, which was used to achieve an accurate 3D measurement of the coronal, sagittal and transversal plane, and to minimize medical expense and X-ray exposure. Our outcomes suggest that the customized guide plate can increase the accuracy of the operation, shorten operative times, and decrease the complications that can result from intramedullary alignment guide placement.\nIn digital medical studies, the 2D/3D registration of image data is accurate and important.[17181920] With the increased use of low-dose radiation imaging equipment in recent years, 2D/3D registration of X-ray and CT highlights the advantages.[2122] The construction of a 3D bone shape model plays an important role in both surgical navigation and image-based studies of the kinematics of the knee joint in vivo.[232425] In our study, the calibrated dual plane X-ray images were used to synthesize the SSM to conduct the feature-based registration of a 3D model from the CT. Although 2D/3D registration could be obtained by using a single plane X-ray, the accuracy of a single plane X-ray is far inferior to that of a dual plane X-ray.[2627] Li et al.[27] used a dual fluoroscopic imaging system in a kinematic study of the knee joint in vivo. Van de Velde et al.[28] also took the attitude that the 3D model could be registered with a single plane X-ray, but overall accuracy of this approach is much lower than that obtained with the dual plane X-ray. Zhu and Li[29] held the view that in the 2D/3D registration, the accuracy gap in the synthesized distal femur model between single plane and dual plane X-rays is significant, while there is almost no gap between dual plane X-rays and mutual plane X-rays. A number of studies have demonstrated that the irregularities in the 3D model created using a dual plane X-ray can be accepted in the medical field. Zhu et al.[29] introduced a technique to predict the 3D model of the distal femur. The deviation between the predicted 3D model and the model reconstructed from 3D medical images was <0.2 mm. Wang et al.[30] also applied the dual fluoroscopic image matching method in their study of in vivo spinal kinematics. The average positional deviation was 0.2 mm, and the average angular deviation was 0.4°. The repeatability was high. Baka et al.[31] indicated that the deviation between the distal femur model synthesized from dual plane X-rays and the 3D model reconstructed from the CT was <1.68 mm. Zheng et al.[32] confirmed the feasibility of this 2D/3D registration method. They also created the surface shape model from calibrated X-rays to conduct feature-based registration.\nThis study is a preliminary evaluation of a digitally customized osteotomy guide plate for TKA. The limitation of an insufficient sample size in this study may result in unnecessary variation. Future work will also add a cartilage model to the bony knee 3D model to decrease variability and reduce the surgical task of removing the articular cartilage. However, our findings suggest that the indications for a guide plate should be expanded, especially in cases of severe intra-or extra-articular deformity, given its customized and minimally invasive nature.\n Financial support and sponsorship This work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).\nThis work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.",
"This work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).",
"There are no conflicts of interest."
] | [
"intro",
"methods",
"results",
"discussion",
null,
null
] | [
"Digital",
"Guide Plate for Osteotomy",
"Lower-extremity X-ray",
"Statistical Shape Model",
"Total Knee Arthroplasty"
] | INTRODUCTION:
Total knee arthroplasty (TKA) is an effective treatment for knee joint diseases such as osteoarthritis and rheumatoid arthritis. The perfect reconstruction of lower limb alignment plays a very important role in the outcomes of knee joint replacement, and can affect both symptom relief and the life time of the prosthesis.[12]
The traditional method for a correctional osteotomy is to use the lower-extremity weight-bearing full-length X-ray to determine the osteotomy location and the angle of the coronal plane. As the accuracy of measuring the joint center with two-dimensional (2D) images cannot be guaranteed, the preoperative osteotomy position and angle planning with these images are imprecise. The angle of the sagittal and transverse plane depends on the surgeon's experience. A prolonged operative time and poor accuracy are inevitable.[3]
With the development of digital technology, the clinical applications of three-dimensional (3D) measurement and operative simulation have gradually increased. In our study, calibrated lower-extremity weight-bearing full-length X-rays and computed tomography (CT) scans of the knee were collected. The statistical shape model (SSM) technique was used to synthesize a 3D model of the knee from the X-ray. A 3D model of the lower limb can be generated with the use of the 3D knee model created using the SSM technique along with CT scans. After the hip and ankle centers were determined on the dual plane X-ray, the relative positional relationship between the two joint centers and the 3D knee model was measured. Using this method, a preoperative analysis can be conducted on the basis of weight-bearing function, avoiding the significant radiation exposure due to CT scan. The preoperative planning, osteotomy simulation, and prosthetic placement were performed. The 3D-printed osteotomy guide plate from the digitally designed model was applied to the TKA.
METHODS:
This clinical trial received permission from the Ethics Committee of Guizhou Provincial People's Hospital, and all patients and their families signed informed consents. A total of 42 cases of osteoarthritis in patients who previously underwent a unilateral TKA at Guizhou Provincial People's Hospital from October 2014 to June 2015 were enrolled in our study. Of these patients, 17 were male, and 25 were female. The involved knee was on the left in 20 cases, while 22 were on the right. Average age was 62 ± 7 years. Preoperative mean body mass index was 25.3 ± 6.4 kg/m2. The eligibility criteria included: (1) unilateral TKA; (2) varus deformity of the operative knee; (3) osteoarthritis of the operative knee. Exclusion criteria included: (1) severe comorbidities that may prolong hospitalization time; (2) severe osteoporosis; (3) the patient declined to participate in the research. Patients were divided into two groups: A guide plate group (GPG, 21 cases), and a traditional surgery group (TSG, 21 cases), using the random number table method. In GPG patients, preoperative thin slice CT scans of the knee were obtained using the Siemens 64 row spiral CT (Siemens, Munich, Germany). The scanning voltage was 130 kV, the scanning thickness was 1 mm, and the matrix was 512 × 512. Digital Imaging and Communications in Medicine format CT data were imported into Simpleware 7.2 (Simpleware Ltd., Exeter, UK) to reconstruct the 3D stereolithography format knee model. Calibrated weight-bearing full-length dual plane knee X-ray images were obtained [Figures 1 and 2]. 3D knee models were synthesized from calibrated dual plane images with SSM of the knee and registration.[4567] The automatic registration of the 3D knee model from the CT scan and from the SSM technique can be obtained digitally [Figure 3].
Photo of calibrated leg. (a) Frontal view. (b) Lateral view.
X-ray images of calibrated weight-bearing full-length lower limb. (a) Anteroposterior image. (b) Lateral image.
Registration between the three-dimensional model synthesized from calibrated X-ray and three-dimensional model from computed tomography. (a) The three dimensional diagrammatic sketch of registration. (b) The anteroposterior film after registration. (c) The lateral film after registration.
The hip center (center of the synthesized sphere) and the ankle center (center of the talar articular surface) were determined. Through the position relationship between the centers, points on the calibration device and the X-ray source, the relative spatial relationship between the two central points and the 3D model of the knee joint was determined. The central point of the distal femur was defined as the midpoint of the Whiteside line.[8] The central point of the proximal tibia was defined as the center of the tibial intercondylar eminence. The femoral anatomic axis was defined as the connection between the central point of the distal femur and the medullary cavity center 10 cm above the knee joint space. The femoral mechanical axis was defined as the connection between the central point of the distal femur and the central point of the femoral head. The mechanical axis of the tibia was defined as the connection between the central point of the proximal tibia and the central point of the ankle joint.
Prosthetic models were obtained by 3D scanning the NexGen LPS (Zimmer Biomet, Warsaw, Indiana, USA) with the Laser-RE (Serein Precision Machinery Company, Shenzhen, China).
The femoral distal osteotomy valgus angle was the angle measured between the femoral anatomy and the mechanical axis, while the osteotomy thickness was 9 mm from the lowest point of the medial femoral condyle.
Through the simulated operation, we were able to choose the suitable size of the prosthesis. The osteotomy line of the anterior and posterior condyles was parallel to the surgical transepicondylar axis (STEA) [Figure 4].
Anterior and posterior condylar osteotomy line is parallel to surgical transepicondylar axis.
The proximal tibial osteotomy line was perpendicular to the mechanical axis on the coronal plane, and was 7° posterior slope on the sagittal plane resulting from the design of the prosthesis. The thickness of the proximal tibial osteotomy was 10 mm from the highest point of the lateral tibial condyle.
According to the design above and the operative simulation, the hole position of the nonheaded screw and the size of the prosthesis were determined. The base of the guide plate was reverse engineered in accordance with the shape of the distal femur and proximal tibia. The screw hole and the base were then connected. The digital design was created using Geomagic (3D Systems, Valencia, CA, USA). In the reverse design stage of the base, the more shape characteristic bone surface was selected to be the attachment area for the guide plate to obtain a better match for the installation. The guide plate was 3D-printed with the polylactic acid material using a 3D Printer (3D Systems Company, Rock Hill, South Carolina, USA). After the operative exposure, the guide plates were attached to the bony surface where the articular cartilage was removed to determine the position of the nonheaded screw for the distal femoral, anterior and posterior condylar, and proximal tibial osteotomy devices. All surgeries were performed by a single surgeon [Figure 5]. Postoperative radiographs were obtained to verify the alignment of the reconstruction [Figure 6].
The nonhead screws’ positions were determined according the guide plates in order to locate the osteotomy devices. (a) The guide plate determined the positions of the nonhead screws in the proximal tibia. (b) The nonhead screws determined the positions of the proximal tibial osteotomy divices. (c) The guide plate determined the positions of the nonhead screws in the distal femur. (d)The nonhead screws determined the positions of the anterior and posterior condylar osteotomy divices.
Postoperative coronal femoral/tibial angles and the angle between posterior condylar osteotomy surface and the surgical transepicondylar axis. (a) The postoperative coronal femoral/tibial angles were 90°. (b) The postoperative angle between posterior condylar osteotomy surface and the surgical transepicondylar axis was 0°.
In the TSG, the angle between the femoral anatomic and mechanical axis was measured preoperatively using a weight-bearing full-length lower-extremity X-ray. The valgus angle for the distal femoral osteotomy was chosen using the closest available angle on the surgical instruments. The intramedullary alignment guide was applied to the femur while the extramedullary alignment guide was applied to the tibia. The rotational angle was chosen according to the experience of the surgeon.
Obtained data were statistically analyzed using SPSS 16.0 (IBM, Armonk, NY, USA). A t-test was applied to assess for differences between groups, with P < 0.05 considered significant.
RESULTS:
The operation time was 49.0 ± 10.5 min for GPG, and 62.0 ± 9.7 min for TSG. The coronal femoral angle, coronal tibial angle, posterior tibial slope, and the angle between the posterior condylar osteotomy surface and the STEA were 89.2 ± 1.7°, 89.0 ± 1.1°, 6.6 ± 1.4°, and 0.9 ± 0.3° in GPG, and 86.7 ± 2.9°, 87.6 ± 2.1°, 8.9 ± 2.8°, and 1.7 ± 0.8° in TSG, respectively. The Hospital for Special Surgery knee scores 3 months after surgery were 83.7 ± 18.4 after GPG and 71.5 ± 15.2 after TSG, respectively. Statistically significant differences were found between all previously noted comparisons [Table 1].
Comparing the outcomes of the GPG and the TSG
HSS: Hospital for Special Surgery; GPG: Guide plate group; TSG: Traditional surgery group; STEA: Surgical transepicondylar axis.
DISCUSSION:
TKA is an effective treatment for severe knee diseases. An accurate osteotomy and the reconstruction of the normal alignment of the lower limbs are key to obtaining a good curative effect.[12] There is significant anatomic variation of the knee joint, especially in the southwest part of China. The knee joint prosthesis and the matching operative instruments are designed with the anatomic characteristics of the European and American knee joint in mind. There may therefore be a large error when using conventional osteotomy instruments in East Asia, resulting in poor alignment of the lower limb, early component loosening, pain, activity limitation, and other complications. The traditional surgical osteotomy is based on the measurement of the weight-bearing full-length lower-extremity X-ray and the intra/extramedullary alignment guides. There is significant subjectivity when using these measurements. This increases the potential risk of infection, bleeding, and fat embolism in the traditional intramedullary alignment guide-based surgery.[910]
Modern computer technology has developed rapidly, providing a platform for preoperative planning. The orthopedic surgeon can perform computer-assisted precise operative measurements.[111213] Hafez et al.[14] digitally designed and 3D printed the osteotomy guide plate for a TKA for the first time, which can effectively avoid the fat embolisms that often result from the placement of the intramedullary alignment guide. Cai et al.[15] uses digital technology to control the axial alignment of total knee arthroplasties, but they needed a CT of the full-length lower limb. Zhang et al.[16] synthesized a 3D joint model from hip, knee, and ankle CTs and a weight-bearing full-length X-ray. 3D alignment reconstruction and the determination of the femoral prosthetic valgus angle can be made using the preoperative design based on the 3D model of the lower limb. In this study, the measurement and reconstruction of the alignment was conducted under physiologic load to be more consistent with normal biomechanical characteristics. However, it required a thin-cut CT scan of the three joints, which significantly increased the examination cost and the patient's radiation exposure.
In this study, the use of advanced 3D synthesis and 2D/3D registration technology allowed for the reconstruction of a weight-bearing 3D model of the lower limb, which was used to achieve an accurate 3D measurement of the coronal, sagittal and transversal plane, and to minimize medical expense and X-ray exposure. Our outcomes suggest that the customized guide plate can increase the accuracy of the operation, shorten operative times, and decrease the complications that can result from intramedullary alignment guide placement.
In digital medical studies, the 2D/3D registration of image data is accurate and important.[17181920] With the increased use of low-dose radiation imaging equipment in recent years, 2D/3D registration of X-ray and CT highlights the advantages.[2122] The construction of a 3D bone shape model plays an important role in both surgical navigation and image-based studies of the kinematics of the knee joint in vivo.[232425] In our study, the calibrated dual plane X-ray images were used to synthesize the SSM to conduct the feature-based registration of a 3D model from the CT. Although 2D/3D registration could be obtained by using a single plane X-ray, the accuracy of a single plane X-ray is far inferior to that of a dual plane X-ray.[2627] Li et al.[27] used a dual fluoroscopic imaging system in a kinematic study of the knee joint in vivo. Van de Velde et al.[28] also took the attitude that the 3D model could be registered with a single plane X-ray, but overall accuracy of this approach is much lower than that obtained with the dual plane X-ray. Zhu and Li[29] held the view that in the 2D/3D registration, the accuracy gap in the synthesized distal femur model between single plane and dual plane X-rays is significant, while there is almost no gap between dual plane X-rays and mutual plane X-rays. A number of studies have demonstrated that the irregularities in the 3D model created using a dual plane X-ray can be accepted in the medical field. Zhu et al.[29] introduced a technique to predict the 3D model of the distal femur. The deviation between the predicted 3D model and the model reconstructed from 3D medical images was <0.2 mm. Wang et al.[30] also applied the dual fluoroscopic image matching method in their study of in vivo spinal kinematics. The average positional deviation was 0.2 mm, and the average angular deviation was 0.4°. The repeatability was high. Baka et al.[31] indicated that the deviation between the distal femur model synthesized from dual plane X-rays and the 3D model reconstructed from the CT was <1.68 mm. Zheng et al.[32] confirmed the feasibility of this 2D/3D registration method. They also created the surface shape model from calibrated X-rays to conduct feature-based registration.
This study is a preliminary evaluation of a digitally customized osteotomy guide plate for TKA. The limitation of an insufficient sample size in this study may result in unnecessary variation. Future work will also add a cartilage model to the bony knee 3D model to decrease variability and reduce the surgical task of removing the articular cartilage. However, our findings suggest that the indications for a guide plate should be expanded, especially in cases of severe intra-or extra-articular deformity, given its customized and minimally invasive nature.
Financial support and sponsorship This work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).
This work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).
Conflicts of interest There are no conflicts of interest.
There are no conflicts of interest.
Financial support and sponsorship:
This work was supported by grants from Special Fund for Culture of Top Young Scientific Talents in Guizhou Province (No. 2011-30), Projects of Science and Technology of Guizhou Province (No. SY(2015) 3044), Fund of Youth of Guizhou Provincial People's Hospital (No. GZSYQN(2015) 05) and Medical Scientific Research Foundation of Guizhou Province (No. gzwjkj2015-1-018).
Conflicts of interest:
There are no conflicts of interest.
| Background:
The conventional method cannot guarantee the precise osteotomies required for a perfect realignment and a better prognosis after total knee arthroplasty (TKA). This study investigated a customized guide plate for osteotomy placement in TKAs with the aid of the statistical shape model technique using weight-bearing lower-extremity X-rays and computed tomography (CT) images of the knee.
Methods:
From October 2014 to June 2015, 42 patients who underwent a TKA in Guizhou Provincial People's Hospital were divided into a guide plate group (GPG, 21 cases) and a traditional surgery group (TSG, 21 cases) using a random number table method. In the GPG group, a guide plate was designed and printed using preoperative three-dimensional measurements to plan and digitally simulate the operation. TSG cases were treated with the conventional method. Outcomes were obtained from the postoperative image examination and short-term follow-up.
Results:
Operative time was 49.0 ± 10.5 min for GPG, and 62.0 ± 9.7 min in TSG. The coronal femoral angle, coronal tibial angle, posterior tibial slope, and the angle between the posterior condylar osteotomy surface and the surgical transepicondylar axis were 89.2 ± 1.7°, 89.0 ± 1.1°, 6.6 ± 1.4°, and 0.9 ± 0.3° in GPG, and 86.7 ± 2.9°, 87.6 ± 2.1°, 8.9 ± 2.8°, and 1.7 ± 0.8° in TSG, respectively. The Hospital for Special Surgery scores 3 months after surgery were 83.7 ± 18.4 in GPG and 71.5 ± 15.2 in TSG. Statistically significant differences were found between GPG and TSG in all measurements.
Conclusions:
A customized guide plate to create an accurate osteotomy in TKAs may be created using lower-extremity X-ray and knee CT images. This allows for shorter operative times and better postoperative alignment than the traditional surgery. Application of the digital guide plate may also result in better short-term outcomes. | null | null | 3,141 | 372 | [
77,
7
] | 6 | [
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"model",
"osteotomy",
"plane",
"ray",
"guide",
"angle",
"guizhou",
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] | [
"studies kinematics knee",
"use 3d knee",
"knee joint determined",
"accurate osteotomy reconstruction",
"osteotomy location angle"
] | null | null | [CONTENT] Digital | Guide Plate for Osteotomy | Lower-extremity X-ray | Statistical Shape Model | Total Knee Arthroplasty [SUMMARY] | [CONTENT] Digital | Guide Plate for Osteotomy | Lower-extremity X-ray | Statistical Shape Model | Total Knee Arthroplasty [SUMMARY] | [CONTENT] Digital | Guide Plate for Osteotomy | Lower-extremity X-ray | Statistical Shape Model | Total Knee Arthroplasty [SUMMARY] | null | [CONTENT] Digital | Guide Plate for Osteotomy | Lower-extremity X-ray | Statistical Shape Model | Total Knee Arthroplasty [SUMMARY] | null | [CONTENT] Aged | Arthroplasty, Replacement, Knee | Female | Humans | Knee | Male | Middle Aged | Osteotomy | Surgery, Computer-Assisted | Tomography, X-Ray Computed [SUMMARY] | [CONTENT] Aged | Arthroplasty, Replacement, Knee | Female | Humans | Knee | Male | Middle Aged | Osteotomy | Surgery, Computer-Assisted | Tomography, X-Ray Computed [SUMMARY] | [CONTENT] Aged | Arthroplasty, Replacement, Knee | Female | Humans | Knee | Male | Middle Aged | Osteotomy | Surgery, Computer-Assisted | Tomography, X-Ray Computed [SUMMARY] | null | [CONTENT] Aged | Arthroplasty, Replacement, Knee | Female | Humans | Knee | Male | Middle Aged | Osteotomy | Surgery, Computer-Assisted | Tomography, X-Ray Computed [SUMMARY] | null | [CONTENT] studies kinematics knee | use 3d knee | knee joint determined | accurate osteotomy reconstruction | osteotomy location angle [SUMMARY] | [CONTENT] studies kinematics knee | use 3d knee | knee joint determined | accurate osteotomy reconstruction | osteotomy location angle [SUMMARY] | [CONTENT] studies kinematics knee | use 3d knee | knee joint determined | accurate osteotomy reconstruction | osteotomy location angle [SUMMARY] | null | [CONTENT] studies kinematics knee | use 3d knee | knee joint determined | accurate osteotomy reconstruction | osteotomy location angle [SUMMARY] | null | [CONTENT] 3d | knee | model | osteotomy | plane | ray | guide | angle | guizhou | registration [SUMMARY] | [CONTENT] 3d | knee | model | osteotomy | plane | ray | guide | angle | guizhou | registration [SUMMARY] | [CONTENT] 3d | knee | model | osteotomy | plane | ray | guide | angle | guizhou | registration [SUMMARY] | null | [CONTENT] 3d | knee | model | osteotomy | plane | ray | guide | angle | guizhou | registration [SUMMARY] | null | [CONTENT] model | 3d | knee | osteotomy | joint | lower | ray | weight bearing | ct | bearing [SUMMARY] | [CONTENT] point | axis | central | proximal | osteotomy | femoral | central point | tibial | determined | distal [SUMMARY] | [CONTENT] gpg | tsg | surgery | min | tsg respectively | 89 | hospital special surgery | respectively | hospital special | special surgery [SUMMARY] | null | [CONTENT] conflicts | conflicts interest | interest | 3d | model | knee | guizhou | osteotomy | guizhou province | province [SUMMARY] | null | [CONTENT] TKA ||| [SUMMARY] | [CONTENT] October 2014 to June 2015 | 42 | TKA | Guizhou Provincial People's Hospital | GPG | 21 | TSG | 21 ||| GPG | three ||| TSG ||| [SUMMARY] | [CONTENT] 49.0 | 10.5 | GPG | 62.0 | 9.7 | TSG ||| 89.2 | 1.7 | 89.0 | 1.1 | 6.6 | 1.4 | 0.9 | 0.3 | GPG | 86.7 | 2.9 | 87.6 | 2.1 | 8.9 | 2.8 | 1.7 | 0.8 | TSG ||| The Hospital for Special Surgery | 3 months | 83.7 | 18.4 | GPG | 71.5 | 15.2 | TSG ||| GPG | TSG [SUMMARY] | null | [CONTENT] TKA ||| ||| October 2014 to June 2015 | 42 | TKA | Guizhou Provincial People's Hospital | GPG | 21 | TSG | 21 ||| GPG | three ||| TSG ||| ||| ||| 49.0 | 10.5 | GPG | 62.0 | 9.7 | TSG ||| 89.2 | 1.7 | 89.0 | 1.1 | 6.6 | 1.4 | 0.9 | 0.3 | GPG | 86.7 | 2.9 | 87.6 | 2.1 | 8.9 | 2.8 | 1.7 | 0.8 | TSG ||| The Hospital for Special Surgery | 3 months | 83.7 | 18.4 | GPG | 71.5 | 15.2 | TSG ||| GPG | TSG ||| ||| ||| [SUMMARY] | null |
||||
Integration of sustained low-efficiency dialysis into extracorporeal membrane oxygenation circuit in critically ill COVID-19 patients: A feasibility study. | 35490349 | Severe COVID-19 can necessitate multiple organ support including veno-venous extracorporeal membrane oxygenation (vvECMO) and renal replacement therapy. The therapy can be complicated by venous thromboembolism due to COVID-19-related hypercoagulability, thus restricting vascular access beyond the vvECMO cannula. Although continuous renal replacement therapy can be performed via a vvECMO circuit, studies addressing sustained low-efficiency dialysis (SLED) integration into vvECMO circuits are scarce. Here we address the lack of evidence by evaluating feasibility of SLED integration into vvECMO circuits. | BACKGROUND | Retrospective cohort study on nine critically ill COVID-19 patients, treated with integrated ECMO-SLED on a single intensive care unit at a tertiary healthcare facility between December 2020 and November 2021. The SLED circuits were established between the accessory arterial oxygenator outlets of a double-oxygenator vvECMO setup. Data on filter survival, quality of dialysis, and volume management were collected and compared with an internal control group receiving single SLED. | METHODS | This study demonstrates general feasibility of SLED integration into existing vvECMO circuits. Filter lifespans of ECMO-SLED compared with single SLED are significantly prolonged (median 18.3 h vs. 10.3 h, p < 0.01). ECMO-SLED treatment is furthermore able to sufficiently normalize creatinine, blood urea nitrogen, and serum sodium, and allows for adequate ultrafiltration rates. | RESULTS | We can show that ECMO-SLED is practical, safe, results in adequate dialysis quality and enables sufficient electrolyte and volume management. Our data indicate that SLED devices can serve as potential alternative to continuous-veno-venous-hemodialysis for integration in vvECMO circuits. | CONCLUSIONS | [
"Acute Kidney Injury",
"COVID-19",
"Critical Illness",
"Extracorporeal Membrane Oxygenation",
"Feasibility Studies",
"Humans",
"Hybrid Renal Replacement Therapy",
"Retrospective Studies"
] | 9347788 | INTRODUCTION | COVID‐19 is caused by an infection with the SARS‐CoV‐2 virus. Although the majority of infections lead to mild or no symptoms, some patients develop pneumonia.
1
Severe pneumonia can result in acute respiratory distress syndrome (ARDS).
2
Despite the predominance of respiratory manifestations, hospitalized COVID‐19 patients are under high risk for concomitant acute kidney injury (AKI).
3
This risk increases in critically ill COVID‐19 patients treated on intensive care units and especially in patients requiring extracorporeal organ support (ECOS).
4
A combination of severe pneumonia and AKI can necessitate initiation of ECOS consisting of extracorporeal‐membrane oxygenation (ECMO) and renal replacement therapy (RRT).
Multi‐organ support is standard procedure for tertiary care centers with respective experience. However, the establishment and maintenance of simultaneous ECOS in critically ill COVID‐19 patients can be challenging. Hypercoagulability is known to be associated with severe COVID‐19, increasing the risk for thromboembolic complications and reducing RRT filter lifespans.
5
,
6
Artificial surfaces of venous catheters can additionally trigger procoagulatory factors. In case of venous thromboembolic events, insertion sites for central venous catheters necessary for simultaneous ECOS can be limited. In worst case, establishment of ECMO and RRT via independent vascular accesses is not feasible. In these patients, the integration of an RRT device into the ECMO circuit is mandatory.
Studies have shown at least non‐inferiority concerning filter lifespans and effectiveness of solute removal and ultrafiltration compared with independent ECMO and RRT circuits.
7
,
8
However, past studies have focused on the integration of continuous RRT (continuous veno‐venous hemodialysis, CVVHD) into ECMO circuits.
9
In contrast, evidence for the integration of sustained low‐efficient dialysis (SLED) into ECMO circuits is scarce. This is surprising because SLEDs are widely used for RRT and non‐inferior compared with continuous RRT procedures.
10
,
11
Although the concept of an integrated ECMO‐SLED is mentioned in literature, evidence lacks beyond anecdotal notion.
12
,
13
,
14
Evidence concerning feasibility of an integrated ECMO‐SLED is especially pressing because outbreaks of COVID‐19 can lead to regional shortages in RRT devices and staff.
15
,
16
Consequently, ECOS experienced centers involved in the treatment of severe COVID‐19 will profit from a study addressing the integration of SLED into an ECMO circuit. In this report, we show that in a subset of critically ill COVID‐19 patients with restricted vascular access and need for ECMO‐RRT, SLED integration into ECMO circuits is practical, safe and results in adequate solute and fluid removal. | METHODS | General information In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).
In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).
ECMO and hemodialysis treatments Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.
SLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.
Schematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets
Anticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.
17
Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.
SLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.
Schematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets
Anticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.
17
Laboratory procedures Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.
18
Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.
18
Statistical analysis Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.
Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA. | RESULTS | In this retrospective single‐center study, we share results from nine patients, treated with integrated vvECMO‐SLED due to intermittent shortages of CVVHD devices and limited vascular access options. The vvECMO‐SLED setup is described in the methods section and illustrated in Figure 1. Four of the nine patients required continuation of SLED treatments after vvECMO weaning and serve as an internal control group. The primary aim of this study was to investigate the feasibility of ECMO‐SLED in critically ill COVID‐19 patients.
The patients had a mean age of 49 years (SD ± 9.2 years) with an equal sex distribution (female: 56%) (Table 1). The mean body mass index was elevated in the obese range (31.1 kg/m2, SD ± 5.3 kg/m2). A history of cardiac disease or diabetes mellitus type 2 was present in six patients (67%), respectively. Mean treatment time with vvECMO was 13.4 days (SD ± 4.5 days). Mean treatment time of simultaneous ECMO‐SLED was 7.9 days (SD ± 5.8 days). The mean blood flow rate (QB) of integrated SLED was 123.1 ml/min (SD ± 23.7 ml/min). Due to the single pump SLED system, dialysate flow rates (QD) were half as high as blood flow rates (QB:QD = 2:1). The mean ultrafiltration rate (UF) was 141.8 ml/h (SD ± 74.33 ml/min).
Baseline characteristics of cohort treated with ECMO‐SLED
Abbreviations: QB, SLED blood flow rate; UF, SLED ultrafiltration rate.
The patients did not suffer from advanced chronic kidney disease (baseline creatinine 1.0 mg/dl, SD ± 0.5 mg/dl). All patients developed COVID‐19‐associated AKI with a mean creatinine level of 4.7 mg/dl (SD ± 2.3 mg/dl) prior to SLED initiation. We observed pulmonary embolism in three patients (33%). We did not observe any major intra‐ or extracorporeal bleeding events or intracerebral complications. Six patients died (67%).
Filter lifespan is a major factor of RRT feasibility. Insufficient anticoagulation and low flow significantly reduce this lifespan. Median filter lifespan in our cohort was 18.3 h (IQR: 10.7–21.5 h), observed in 75 SLED treatments with SLED integrated into the vvECMO circuit (ECMO‐SLED group). Median filter lifespan of 84 SLED treatments of a subcohort of four patients after vvECMO explantation (SLED‐group) was significantly lower with 10.3 h (IQR: 5.4–18.5 h, p < 0.01) (Figure 2A,B). The same difference in filter lifespans can be observed, limiting the comparison with the four patients receiving ECMO‐SLED and SLED after vvECMO explantation (Figure S1). The characteristics of the SLED‐subgroup are shown in Table S1.
Extracorporeal‐membrane oxygenation–sustained low‐efficiency dialysis (ECMO‐SLED) leads to non‐inferior filter lifespans despite low anti‐Xa‐levels. (A) Kaplan–Meier estimators of filter survival in hours. Blue line () depicts isolated SLED circuits. Red line () depicts SLED circuits integrated into a veno‐venous extracorporeal membrane oxygenation setup. p‐value calculated using MantelCox test. (B) Median filter runtimes in the SLED () and ECMO‐SLED () group. Whiskers depict IQR, p‐value calculated using Wilcoxon–Mann–Whitney test. (C) Comparison of mean anti‐Xa levels. Whiskers depicting SD, p‐value calculated using Wilcoxon–Mann–Whitney test
Despite longer filter survival, mean anti‐Xa‐levels in the ECMO‐SLED group were significantly lower than in the SLED group (0.28 IU/ml vs. 0.74 IU/ml, p < 0.01) (Figure 2C). Comparison of aPTT showed mean values slightly above the normal range in both groups. ECMO‐SLED patients showed a 3.3 s longer aPTT (Figure S2).
Elevated creatinine and BUN levels were sufficiently reduced. Creatinine was lowered by 1.4 mg/dl (2.7 mg/dl vs. 1.3 mg/dl, p < 0.01), BUN by 58.5 mg/dl (118.3 mg/dl vs. 59.8 mg/dl, p < 0.01) by SLED operated in the vvECMO circuit (Figure 3A–D). All patients treated with ECMO‐SLED developed oliguria or anuria prior to and during ECMO‐SLED treatment (Figure S3).
Extracorporeal‐membrane oxygenation–sustained low‐efficiency dialysis (ECMO‐SLED) allows for sufficient dialysis. Time courses of creatinine (A), blood urea nitrogen (BUN) (C), potassium (E) and sodium (G) levels prior to and after SLED integration into the veno‐venous extracorporeal membrane oxygenation circuit. Numbers indicate patients with available data sets at respective time point. Adjacent graphs compare the means of creatinine (B), BUN (D), potassium (F) and sodium (H) levels before and after SLED initiation. Bars and whiskers depict mean and SEM. p‐values were calculated using Wilcoxon–Mann–Whitney test
In addition, ECMO‐SLED was able to control potassium levels within normal range (Figure 3E,F). Hypernatremia—often associated with severe COVID‐19—was normalized after SLED integration into the vvECMO circuit (148.1 mmol/L vs. 142.5 mmol/L, p < 0.01) (Figure 3G,H).
ECMO‐SLED allowed adequate and non‐inferior ultrafiltration rates compared with the SLED‐group. Balanced volume management was achieved (Figure S4). | null | null | [
"INTRODUCTION",
"General information",
"\nECMO and hemodialysis treatments",
"Laboratory procedures",
"Statistical analysis",
"AUTHOR CONTRIBUTIONS",
"ETHICS APPROVAL"
] | [
"COVID‐19 is caused by an infection with the SARS‐CoV‐2 virus. Although the majority of infections lead to mild or no symptoms, some patients develop pneumonia.\n1\n Severe pneumonia can result in acute respiratory distress syndrome (ARDS).\n2\n Despite the predominance of respiratory manifestations, hospitalized COVID‐19 patients are under high risk for concomitant acute kidney injury (AKI).\n3\n This risk increases in critically ill COVID‐19 patients treated on intensive care units and especially in patients requiring extracorporeal organ support (ECOS).\n4\n A combination of severe pneumonia and AKI can necessitate initiation of ECOS consisting of extracorporeal‐membrane oxygenation (ECMO) and renal replacement therapy (RRT).\nMulti‐organ support is standard procedure for tertiary care centers with respective experience. However, the establishment and maintenance of simultaneous ECOS in critically ill COVID‐19 patients can be challenging. Hypercoagulability is known to be associated with severe COVID‐19, increasing the risk for thromboembolic complications and reducing RRT filter lifespans.\n5\n, \n6\n Artificial surfaces of venous catheters can additionally trigger procoagulatory factors. In case of venous thromboembolic events, insertion sites for central venous catheters necessary for simultaneous ECOS can be limited. In worst case, establishment of ECMO and RRT via independent vascular accesses is not feasible. In these patients, the integration of an RRT device into the ECMO circuit is mandatory.\nStudies have shown at least non‐inferiority concerning filter lifespans and effectiveness of solute removal and ultrafiltration compared with independent ECMO and RRT circuits.\n7\n, \n8\n However, past studies have focused on the integration of continuous RRT (continuous veno‐venous hemodialysis, CVVHD) into ECMO circuits.\n9\n In contrast, evidence for the integration of sustained low‐efficient dialysis (SLED) into ECMO circuits is scarce. This is surprising because SLEDs are widely used for RRT and non‐inferior compared with continuous RRT procedures.\n10\n, \n11\n Although the concept of an integrated ECMO‐SLED is mentioned in literature, evidence lacks beyond anecdotal notion.\n12\n, \n13\n, \n14\n\n\nEvidence concerning feasibility of an integrated ECMO‐SLED is especially pressing because outbreaks of COVID‐19 can lead to regional shortages in RRT devices and staff.\n15\n, \n16\n Consequently, ECOS experienced centers involved in the treatment of severe COVID‐19 will profit from a study addressing the integration of SLED into an ECMO circuit. In this report, we show that in a subset of critically ill COVID‐19 patients with restricted vascular access and need for ECMO‐RRT, SLED integration into ECMO circuits is practical, safe and results in adequate solute and fluid removal.",
"In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).",
"Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.\nSLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.\nSchematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets\nAnticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.\n17\n\n",
"Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.\n18\n\n",
"Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.",
"Frederic Arnold, Johannes Kalbhenn, and Lukas Westermann conceived the study and its design, had full access to the patient records, and take responsibility for the accuracy and integrity of the data. Frederic Arnold and Lukas Westermann screened the electronic patient records, organized the data and performed statistical analysis. Johannes Kalbhenn and Rika Wobser critically contributed to data analysis. Frederic Arnold, Rika Wobser, Johannes Kalbhenn, and Lukas Westermann drafted the manuscript. Frederic Arnold and Lukas Westermann generated the figures. Illustrations were generated by Frederic Arnold. Rika Wobser and Johannes Kalbhenn were involved in clinical management of the patients and contributed to data interpretation. Frederic Arnold, Rika Wobser, Johannes Kalbhenn, Lukas Westermann critically revised the drafted manuscript and approve of the submission.",
"Analysis and publication of the data were approved by the ethics committee (405/20) of the University of Freiburg Medical Center, University of Freiburg, Faculty of Medicine, Germany."
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"General information",
"\nECMO and hemodialysis treatments",
"Laboratory procedures",
"Statistical analysis",
"RESULTS",
"DISCUSSION",
"CONFLICT OF INTEREST",
"AUTHOR CONTRIBUTIONS",
"ETHICS APPROVAL",
"Supporting information"
] | [
"COVID‐19 is caused by an infection with the SARS‐CoV‐2 virus. Although the majority of infections lead to mild or no symptoms, some patients develop pneumonia.\n1\n Severe pneumonia can result in acute respiratory distress syndrome (ARDS).\n2\n Despite the predominance of respiratory manifestations, hospitalized COVID‐19 patients are under high risk for concomitant acute kidney injury (AKI).\n3\n This risk increases in critically ill COVID‐19 patients treated on intensive care units and especially in patients requiring extracorporeal organ support (ECOS).\n4\n A combination of severe pneumonia and AKI can necessitate initiation of ECOS consisting of extracorporeal‐membrane oxygenation (ECMO) and renal replacement therapy (RRT).\nMulti‐organ support is standard procedure for tertiary care centers with respective experience. However, the establishment and maintenance of simultaneous ECOS in critically ill COVID‐19 patients can be challenging. Hypercoagulability is known to be associated with severe COVID‐19, increasing the risk for thromboembolic complications and reducing RRT filter lifespans.\n5\n, \n6\n Artificial surfaces of venous catheters can additionally trigger procoagulatory factors. In case of venous thromboembolic events, insertion sites for central venous catheters necessary for simultaneous ECOS can be limited. In worst case, establishment of ECMO and RRT via independent vascular accesses is not feasible. In these patients, the integration of an RRT device into the ECMO circuit is mandatory.\nStudies have shown at least non‐inferiority concerning filter lifespans and effectiveness of solute removal and ultrafiltration compared with independent ECMO and RRT circuits.\n7\n, \n8\n However, past studies have focused on the integration of continuous RRT (continuous veno‐venous hemodialysis, CVVHD) into ECMO circuits.\n9\n In contrast, evidence for the integration of sustained low‐efficient dialysis (SLED) into ECMO circuits is scarce. This is surprising because SLEDs are widely used for RRT and non‐inferior compared with continuous RRT procedures.\n10\n, \n11\n Although the concept of an integrated ECMO‐SLED is mentioned in literature, evidence lacks beyond anecdotal notion.\n12\n, \n13\n, \n14\n\n\nEvidence concerning feasibility of an integrated ECMO‐SLED is especially pressing because outbreaks of COVID‐19 can lead to regional shortages in RRT devices and staff.\n15\n, \n16\n Consequently, ECOS experienced centers involved in the treatment of severe COVID‐19 will profit from a study addressing the integration of SLED into an ECMO circuit. In this report, we show that in a subset of critically ill COVID‐19 patients with restricted vascular access and need for ECMO‐RRT, SLED integration into ECMO circuits is practical, safe and results in adequate solute and fluid removal.",
"General information In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).\nIn this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).\n\nECMO and hemodialysis treatments Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.\nSLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.\nSchematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets\nAnticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.\n17\n\n\nVeno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.\nSLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.\nSchematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets\nAnticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.\n17\n\n\nLaboratory procedures Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.\n18\n\n\nLaboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.\n18\n\n\nStatistical analysis Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.\nCategorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.",
"In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).",
"Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.\nSLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.\nSchematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets\nAnticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.\n17\n\n",
"Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.\n18\n\n",
"Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.",
"In this retrospective single‐center study, we share results from nine patients, treated with integrated vvECMO‐SLED due to intermittent shortages of CVVHD devices and limited vascular access options. The vvECMO‐SLED setup is described in the methods section and illustrated in Figure 1. Four of the nine patients required continuation of SLED treatments after vvECMO weaning and serve as an internal control group. The primary aim of this study was to investigate the feasibility of ECMO‐SLED in critically ill COVID‐19 patients.\nThe patients had a mean age of 49 years (SD ± 9.2 years) with an equal sex distribution (female: 56%) (Table 1). The mean body mass index was elevated in the obese range (31.1 kg/m2, SD ± 5.3 kg/m2). A history of cardiac disease or diabetes mellitus type 2 was present in six patients (67%), respectively. Mean treatment time with vvECMO was 13.4 days (SD ± 4.5 days). Mean treatment time of simultaneous ECMO‐SLED was 7.9 days (SD ± 5.8 days). The mean blood flow rate (QB) of integrated SLED was 123.1 ml/min (SD ± 23.7 ml/min). Due to the single pump SLED system, dialysate flow rates (QD) were half as high as blood flow rates (QB:QD = 2:1). The mean ultrafiltration rate (UF) was 141.8 ml/h (SD ± 74.33 ml/min).\nBaseline characteristics of cohort treated with ECMO‐SLED\nAbbreviations: QB, SLED blood flow rate; UF, SLED ultrafiltration rate.\nThe patients did not suffer from advanced chronic kidney disease (baseline creatinine 1.0 mg/dl, SD ± 0.5 mg/dl). All patients developed COVID‐19‐associated AKI with a mean creatinine level of 4.7 mg/dl (SD ± 2.3 mg/dl) prior to SLED initiation. We observed pulmonary embolism in three patients (33%). We did not observe any major intra‐ or extracorporeal bleeding events or intracerebral complications. Six patients died (67%).\nFilter lifespan is a major factor of RRT feasibility. Insufficient anticoagulation and low flow significantly reduce this lifespan. Median filter lifespan in our cohort was 18.3 h (IQR: 10.7–21.5 h), observed in 75 SLED treatments with SLED integrated into the vvECMO circuit (ECMO‐SLED group). Median filter lifespan of 84 SLED treatments of a subcohort of four patients after vvECMO explantation (SLED‐group) was significantly lower with 10.3 h (IQR: 5.4–18.5 h, p < 0.01) (Figure 2A,B). The same difference in filter lifespans can be observed, limiting the comparison with the four patients receiving ECMO‐SLED and SLED after vvECMO explantation (Figure S1). The characteristics of the SLED‐subgroup are shown in Table S1.\nExtracorporeal‐membrane oxygenation–sustained low‐efficiency dialysis (ECMO‐SLED) leads to non‐inferior filter lifespans despite low anti‐Xa‐levels. (A) Kaplan–Meier estimators of filter survival in hours. Blue line () depicts isolated SLED circuits. Red line () depicts SLED circuits integrated into a veno‐venous extracorporeal membrane oxygenation setup. p‐value calculated using MantelCox test. (B) Median filter runtimes in the SLED () and ECMO‐SLED () group. Whiskers depict IQR, p‐value calculated using Wilcoxon–Mann–Whitney test. (C) Comparison of mean anti‐Xa levels. Whiskers depicting SD, p‐value calculated using Wilcoxon–Mann–Whitney test\nDespite longer filter survival, mean anti‐Xa‐levels in the ECMO‐SLED group were significantly lower than in the SLED group (0.28 IU/ml vs. 0.74 IU/ml, p < 0.01) (Figure 2C). Comparison of aPTT showed mean values slightly above the normal range in both groups. ECMO‐SLED patients showed a 3.3 s longer aPTT (Figure S2).\nElevated creatinine and BUN levels were sufficiently reduced. Creatinine was lowered by 1.4 mg/dl (2.7 mg/dl vs. 1.3 mg/dl, p < 0.01), BUN by 58.5 mg/dl (118.3 mg/dl vs. 59.8 mg/dl, p < 0.01) by SLED operated in the vvECMO circuit (Figure 3A–D). All patients treated with ECMO‐SLED developed oliguria or anuria prior to and during ECMO‐SLED treatment (Figure S3).\nExtracorporeal‐membrane oxygenation–sustained low‐efficiency dialysis (ECMO‐SLED) allows for sufficient dialysis. Time courses of creatinine (A), blood urea nitrogen (BUN) (C), potassium (E) and sodium (G) levels prior to and after SLED integration into the veno‐venous extracorporeal membrane oxygenation circuit. Numbers indicate patients with available data sets at respective time point. Adjacent graphs compare the means of creatinine (B), BUN (D), potassium (F) and sodium (H) levels before and after SLED initiation. Bars and whiskers depict mean and SEM. p‐values were calculated using Wilcoxon–Mann–Whitney test\nIn addition, ECMO‐SLED was able to control potassium levels within normal range (Figure 3E,F). Hypernatremia—often associated with severe COVID‐19—was normalized after SLED integration into the vvECMO circuit (148.1 mmol/L vs. 142.5 mmol/L, p < 0.01) (Figure 3G,H).\nECMO‐SLED allowed adequate and non‐inferior ultrafiltration rates compared with the SLED‐group. Balanced volume management was achieved (Figure S4).",
"Severe COVID‐19 ARDS poses a significant risk for AKI.\n19\n Volume overload often additionally aggravates ARDS and must be addressed to allow for pulmonary recompensation and vvECMO weaning. Because many medical centers provide RRT devices for prolonged intermittent RRT such as SLED, these devices should be regarded as an alternative treatment option for integrated ECMO‐RRT. SLED devices offer the advantage of broad availability, rapid and easy installation, low maintenance time, plannable filter downtimes, and are more cost‐effective.\n11\n For these reasons, they are an attractive alternative for the treatment of patients requiring combined extracorporeal organ support.\nIn this single‐center study of nine critically ill COVID‐19 patients, we show that SLED integration into the vvECMO circuit is (i) technical feasible, (ii) provides adequate dialysis quality, (iii) is able to control electrolyte disturbances, and (iv) allows for sufficient fluid balance.\nThe integration of SLED into vvECMO circuits has several advantages. Firstly, RRT can be performed in absence of an alternative vascular insertion site besides the double lumen cannula used for vvECMO. Therefore, integration of SLED into the existing vvECMO circuit can be a life‐saving treatment option in case of limited vascular access. Because our center favors the use of two oxygenators, we were able to connect the SLED circuit quite easily at both oxygenators in the high‐pressure part of the ECMO. This arrangement decreases the risk for air embolism. Secondly, each central line poses a risk for complications due to the insertion procedure (e.g., local trauma, pneumothorax, bleeding, air embolism due to ECMO‐associated negative central venous pressure as well as central line–associated infection or thrombosis).\n20\n Often inguinal insertion sites have to be chosen, because jugular veins are already used for vvECMO, further aggravating the risk for infectious complications. Thirdly, handling of the patient (e.g., prone positioning, mobilization, nursing) is most likely facilitated with a single extracorporeal circuit. Fourthly, an integrated ECMO‐SLED circuit allows for higher and stable blood flow rates within the SLED circuit. The risk for central line dysfunction of a separate RRT catheter is, therefore, not present after SLED integration into the vvECMO circuit. Constant blood flow‐rates can reduce the risk of clotting events and premature termination of SLED. We therefore compared filter lifespans between patients with an integrated ECMO‐SLED circuit to a subgroup of patients receiving isolated SLED after vvECMO weaning. ECMO‐SLED filter lifespans were significantly higher compared with isolated SLED. The improved filter lifespan observed in the ECMO‐SLED cohort is most likely attributable to a reduction of low‐flow phases within the SLED circuit due to constant positive pressure in the vvECMO circuit. Our finding is in line with a previous study investigating filter lifespans in ECMO‐CVVHD. Integrated ECMO‐CVVHD also allowed for longer filter lifespans, compared with isolated circuits.\n7\n Of note, the increased filter lifespans were observed under less effective anticoagulation, as demonstrated by lower factor anti‐Xa levels in the ECMO‐SLED cohort. Although we found aPTT to be 3.3 s longer in the ECMO‐SLED cohort compared with patients treated with isolated SLED, all measured aPTTs were just slightly above the physiological range and the small difference is most likely without biological impact. Furthermore aPTT levels are not suitable for monitoring anticoagulation with LMWHs. In our study anticoagulation was performed exclusively with the LMWH enoxaparin. Anticoagulation with LMWH has been shown to be superior in CVVHD and SLED alike in cohorts of critically ill COVID‐19 patients.\n17\n, \n21\n We believe that constant blood flow through a combined ECMO‐SLED circuit might require an even less rigorous anticoagulation regimen. Less anticoagulation may reduce the risk for common bleeding complications during vvECMO treatment.\nThere are also disadvantages of an integrated ECMO‐SLED approach, favoring separate extracorporeal circuits: A separate SLED circuit may reduce the risk for vvECMO complications due to turbulences or clotting events in the SLED circuit. Maintenance of the SLED circuit does not have to be performed by ECMO trained staff. Furthermore, integrated SLED functionality may be limited by high circuit pressures if vvECMO blood flow rates need to be elevated (Table 2).\nAdvantages of SLED integration into vvECMO circuit\nfeasible in case of limited vascular access options (e.g., due to thrombosis, bleeding risk)\nreduced risk of complications associated with central catheter insertion (e.g., pneumothorax, local trauma, bleeding, risk for air embolism)\nreduced risk for central line associated complications (e.g., infection, thrombosis)\nlower risk for vvECMO circuit complications\nno ECMO trained personnel required for SLED circuit service\nbetter filter lifetime, less clotting events, reduced therapy downtime\nless anticoagulation required\nless pressure alarms\nlower risk for hemolysis due to flow turbulences\nBesides general feasibility, we investigated dialysis quality in this study. Dialysis parameters (blood and dialysate flow) were within the standard range of SLED therapy. Our data show adequate reduction of both creatinine and BUN over time. Thus, ECMO‐SLED allows for adequate hemodialysis quality. In addition, ECMO‐SLED was able to control electrolyte disorders. Hypernatremia is associated with higher mortality in critically ill COVID‐19 patients and was also present in our cohort prior to ECMO‐SLED initiation.\n22\n SLED treatment in the vvECMO circuit was efficient to normalize hypernatremia.\nLastly, we investigated ultrafiltration rates of ECMO‐SLED. Because oliguria or anuria persisted during the critical illness phase, ultrafiltration was required in all patients. Our results show that ECMO‐SLED allows for a wide range of desired daily ultrafiltration rates and, thus, is able to provide adequate volume control, which is especially necessary for successful pulmonary recovery and vvECMO weaning.\nThis study has several limitations. Major limitations are the small sample size, its retrospective design, short observational period and the lack of a matched control group being treated with ECMO and SLED through individual vascular accesses. Data of a cohort of nine patients does not allow general conclusions for a broader population. However, the baseline characteristics, incidence of pulmonary embolism and the observed fatality rate of our cohort was in line with other reports on critically ill COVID‐19 patients.\n23\n, \n24\n Furthermore, the primary aim of this study was to demonstrate feasibility of an integrated ECMO‐SLED approach.\nTo allow more precise conclusions, prospective follow‐up studies with a higher sample size should be conducted to further investigate safeness, filter lifespans, requirement for anticoagulation, quality of dialysis, and long‐term renal outcome of the ECMO‐SLED approach. Furthermore we exclusively report data from multiorgan failure due to severe COVID‐19. However, we believe that the concept of an integrated ECMO‐SLED is transferable to other situations of multi organ failure requiring parallel ECMO and RRT as well. Finally, data reported from our center was generated using two parallel oxygenators in the vvECMO circuit. This setup is well established and standard operating procedure at our intensive care department.\n25\n Two oxygenators provide several advantages such as the possibility of exchanging an oxygenator without stopping the ECMO pump, an increased blood flow of up to 7 L/min and an easy integration of SLED into the vvECMO circuit as illustrated in Figure 1. Many other centers routinely use single oxygenators in ECMO circuits. However, the concept of ECMO‐SLED is also feasible in single oxygenator vvECMO circuits by integrating two luer‐lock‐connection sites within the post‐pump‐part of the circuit.\nIn summary, this report shows that an integrated ECMO‐SLED approach is technically feasible, safe and allows for adequate dialysis quality and fluid control. Constant blood flow and sufficient pressure in the SLED circuit could improve filter lifespans and might allow reduction of anticoagulation. This feasibility study can be regarded as foundation for future research investigating ECMO‐SLED as potential alternative to more established protocols like ECMO‐CVVHD. High availability, cost efficiency, and low maintenance requirements of SLED devices compared with CVVHD devices are appealing arguments to further investigate our strategy.",
"Nothing do declare.",
"Frederic Arnold, Johannes Kalbhenn, and Lukas Westermann conceived the study and its design, had full access to the patient records, and take responsibility for the accuracy and integrity of the data. Frederic Arnold and Lukas Westermann screened the electronic patient records, organized the data and performed statistical analysis. Johannes Kalbhenn and Rika Wobser critically contributed to data analysis. Frederic Arnold, Rika Wobser, Johannes Kalbhenn, and Lukas Westermann drafted the manuscript. Frederic Arnold and Lukas Westermann generated the figures. Illustrations were generated by Frederic Arnold. Rika Wobser and Johannes Kalbhenn were involved in clinical management of the patients and contributed to data interpretation. Frederic Arnold, Rika Wobser, Johannes Kalbhenn, Lukas Westermann critically revised the drafted manuscript and approve of the submission.",
"Analysis and publication of the data were approved by the ethics committee (405/20) of the University of Freiburg Medical Center, University of Freiburg, Faculty of Medicine, Germany.",
"\nAppendix S1 Supplementary Information\nClick here for additional data file."
] | [
null,
"methods",
null,
null,
null,
null,
"results",
"discussion",
"COI-statement",
null,
null,
"supplementary-material"
] | [
"COVID‐19",
"ECMO",
"ECOS",
"MOF",
"RRT",
"SLED"
] | INTRODUCTION:
COVID‐19 is caused by an infection with the SARS‐CoV‐2 virus. Although the majority of infections lead to mild or no symptoms, some patients develop pneumonia.
1
Severe pneumonia can result in acute respiratory distress syndrome (ARDS).
2
Despite the predominance of respiratory manifestations, hospitalized COVID‐19 patients are under high risk for concomitant acute kidney injury (AKI).
3
This risk increases in critically ill COVID‐19 patients treated on intensive care units and especially in patients requiring extracorporeal organ support (ECOS).
4
A combination of severe pneumonia and AKI can necessitate initiation of ECOS consisting of extracorporeal‐membrane oxygenation (ECMO) and renal replacement therapy (RRT).
Multi‐organ support is standard procedure for tertiary care centers with respective experience. However, the establishment and maintenance of simultaneous ECOS in critically ill COVID‐19 patients can be challenging. Hypercoagulability is known to be associated with severe COVID‐19, increasing the risk for thromboembolic complications and reducing RRT filter lifespans.
5
,
6
Artificial surfaces of venous catheters can additionally trigger procoagulatory factors. In case of venous thromboembolic events, insertion sites for central venous catheters necessary for simultaneous ECOS can be limited. In worst case, establishment of ECMO and RRT via independent vascular accesses is not feasible. In these patients, the integration of an RRT device into the ECMO circuit is mandatory.
Studies have shown at least non‐inferiority concerning filter lifespans and effectiveness of solute removal and ultrafiltration compared with independent ECMO and RRT circuits.
7
,
8
However, past studies have focused on the integration of continuous RRT (continuous veno‐venous hemodialysis, CVVHD) into ECMO circuits.
9
In contrast, evidence for the integration of sustained low‐efficient dialysis (SLED) into ECMO circuits is scarce. This is surprising because SLEDs are widely used for RRT and non‐inferior compared with continuous RRT procedures.
10
,
11
Although the concept of an integrated ECMO‐SLED is mentioned in literature, evidence lacks beyond anecdotal notion.
12
,
13
,
14
Evidence concerning feasibility of an integrated ECMO‐SLED is especially pressing because outbreaks of COVID‐19 can lead to regional shortages in RRT devices and staff.
15
,
16
Consequently, ECOS experienced centers involved in the treatment of severe COVID‐19 will profit from a study addressing the integration of SLED into an ECMO circuit. In this report, we show that in a subset of critically ill COVID‐19 patients with restricted vascular access and need for ECMO‐RRT, SLED integration into ECMO circuits is practical, safe and results in adequate solute and fluid removal.
METHODS:
General information In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).
In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).
ECMO and hemodialysis treatments Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.
SLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.
Schematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets
Anticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.
17
Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.
SLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.
Schematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets
Anticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.
17
Laboratory procedures Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.
18
Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.
18
Statistical analysis Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.
Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.
General information:
In this single‐center retrospective feasibility study, we report data of nine patients from the University of Freiburg Medical Center, division of Anesthesiology and Intensive Care, Freiburg, Germany. Patients were adults (aged 18 years or older) with a laboratory‐confirmed SARS‐CoV‐2 infection. All patients required vvECMO and RRT therapy due to multiorgan failure during severe COVID‐19. All patients were treated on the same intensive care unit following identical standard operating procedures between December 2020 and November 2021. Demographic data, past medical history, clinical findings, laboratory values, treatment details, and outcome data of patients were extracted from electronic patient records by the investigators of the study (FA, JK, and LW). All data were reviewed and verified by two physicians (FA and LW). Any uncertain records were not included in the final data analysis. This study is in conformity with the ethical principles for medical research involving human subjects as laid down in the Helsinki Declaration (1964) and its amendments. Analysis and publication of the data were approved by the local ethics committee (405/20 to JK).
ECMO and hemodialysis treatments:
Veno‐venous ECMO (vvECMO) was applied in COVID‐19‐associated ARDS. Decision to initiate vvECMO was individually made after multidisciplinary discussion. Vascular access for vvECMO was established using a transjugular two‐stage Avalon Elite® catheter (Maquet Holding GmbH & Co. KG, Rastatt, Germany). The vvECMO circuit was composed by a Revolution® Centrifugal Blood Pump operated by the Stöckert Centrifugal Pump Console (SCPC), two EOS ECMO™ oxygenators (all LivaNova Deutschland, Munich, Germany) and a Sechrist 3500CP‐G gas blender (Sechrist Industries, Inc., Anaheim, CA, U.S.A.). A vvECMO setup with two oxygenators connected in parallel after the centrifugal pump is standard of care for severe ARDS at our center. The system setup allows blood flow up to 7 L/min. Usually vvECMO was adjusted to a blood flow of 3–5 L/min. Daily inspection of the pump and oxygenators, with special regard to thrombotic scaling, was performed by perfusionists. Extracorporeal components were changed exclusively when the function was impaired. No scheduled changes were applied.
SLED treatments were performed with the GENIUS®90 single‐pass batch system (Fresenius Medical Care GmbH, Bad Homburg, Germany) using suitable tubing kits and dialyzers. Dialysate solutions were individually prepared at site. The GENIUS®90 SLED system uses a single double‐sided roller pump that simultaneously generates blood (QB) and dialysate flow (QD) in a fixed ratio. To achieve treatment times up to 24 h, tubing kits allowing QB:QD ratios of 2:1 were used. Blood flow rates between 50 and 300 ml/min, corresponding dialysate flow rates of 25–150 ml/min and ultrafiltration rates between 100 and 1000 ml/h were chosen per treatment at the discretion of the treating physician. The SLED circuit was integrated between the sampling outlets of the oxygenators. SLED systems were used until dialysate was saturated or visual inspection of the dialysis filter indicated clotting events and prompted treatment termination. Figure 1 gives an overview of the ECMO‐SLED setup. In case of SLED maintenance after vvECMO explantation, a standard 11F double lumen dialysis catheter was implanted in the jugular vein.
Schematic of sustained low‐efficiency dialysis (SLED) integration within the veno‐venous extracorporeal membrane oxygenation (vvECMO) circuit. Vascular access is provided by a single double lumen central venous catheter. The vvECMO circuit consists of a centrifugal pump operated by an ECMO console regulating blood flow by adjusting the pump speed. Blood is pumped through two ECMO oxygenators connected in parallel to the pump outlet. Oxygenation and decarboxylation are regulated via a gas blender controlling gas flow and oxygen concentration. The integrated SLED circuit is established between the accessory arterial oxygenator outlets
Anticoagulation was performed with the low‐molecular‐weight heparin (LMWH) enoxaparin. Anticoagulation with LMWH was maintained by subcutaneous injection of enoxaparin in a bodyweight‐adapted individual dose once or twice a day. Sufficient anticoagulation was supposed with an anti‐factor‐Xa activity of 0.25–0.5 IU/ml determined 4 h after enoxaparin injection. Before connection of SLED to the vvECMO, an initial bolus of 1000 I.U. enoxaparin was added to the priming fluid of the SLED system. This protocol has been published recently and was associated with superior RRT circuit patency in severe COVID‐19.
17
Laboratory procedures:
Laboratory confirmation of SARS‐CoV‐2 infection was performed with real‐time RT‐PCR methods from throat swab samples. Concentrations of creatinine and blood urea nitrogen (BUN) were assessed in serum samples. Anti‐factor‐Xa activity and activated partial thromboplastin time (aPTT) were measured in citrate blood samples during hospitalization. Potassium‐ and sodium levels were assessed by point‐of‐care blood gas analyses. AKI was diagnosed according to the respective KDIGO clinical practice guidelines.
18
Statistical analysis:
Categorical variables were presented as n (%). Continuous variables were presented as median (IQR) or mean (SD), if not indicated otherwise. Wilcoxon–Mann–Whitney tests were performed to calculate p‐values. Comparison of dialysis filter survival were presented as Kaplan–Meier estimators and compared applying the Mantel–Cox test. A two‐sided α of less than 0.05 was considered statistically significant. All statistical analyses were performed using Prism (Version 9.1.3), GraphPad Software, San Diego, California, USA.
RESULTS:
In this retrospective single‐center study, we share results from nine patients, treated with integrated vvECMO‐SLED due to intermittent shortages of CVVHD devices and limited vascular access options. The vvECMO‐SLED setup is described in the methods section and illustrated in Figure 1. Four of the nine patients required continuation of SLED treatments after vvECMO weaning and serve as an internal control group. The primary aim of this study was to investigate the feasibility of ECMO‐SLED in critically ill COVID‐19 patients.
The patients had a mean age of 49 years (SD ± 9.2 years) with an equal sex distribution (female: 56%) (Table 1). The mean body mass index was elevated in the obese range (31.1 kg/m2, SD ± 5.3 kg/m2). A history of cardiac disease or diabetes mellitus type 2 was present in six patients (67%), respectively. Mean treatment time with vvECMO was 13.4 days (SD ± 4.5 days). Mean treatment time of simultaneous ECMO‐SLED was 7.9 days (SD ± 5.8 days). The mean blood flow rate (QB) of integrated SLED was 123.1 ml/min (SD ± 23.7 ml/min). Due to the single pump SLED system, dialysate flow rates (QD) were half as high as blood flow rates (QB:QD = 2:1). The mean ultrafiltration rate (UF) was 141.8 ml/h (SD ± 74.33 ml/min).
Baseline characteristics of cohort treated with ECMO‐SLED
Abbreviations: QB, SLED blood flow rate; UF, SLED ultrafiltration rate.
The patients did not suffer from advanced chronic kidney disease (baseline creatinine 1.0 mg/dl, SD ± 0.5 mg/dl). All patients developed COVID‐19‐associated AKI with a mean creatinine level of 4.7 mg/dl (SD ± 2.3 mg/dl) prior to SLED initiation. We observed pulmonary embolism in three patients (33%). We did not observe any major intra‐ or extracorporeal bleeding events or intracerebral complications. Six patients died (67%).
Filter lifespan is a major factor of RRT feasibility. Insufficient anticoagulation and low flow significantly reduce this lifespan. Median filter lifespan in our cohort was 18.3 h (IQR: 10.7–21.5 h), observed in 75 SLED treatments with SLED integrated into the vvECMO circuit (ECMO‐SLED group). Median filter lifespan of 84 SLED treatments of a subcohort of four patients after vvECMO explantation (SLED‐group) was significantly lower with 10.3 h (IQR: 5.4–18.5 h, p < 0.01) (Figure 2A,B). The same difference in filter lifespans can be observed, limiting the comparison with the four patients receiving ECMO‐SLED and SLED after vvECMO explantation (Figure S1). The characteristics of the SLED‐subgroup are shown in Table S1.
Extracorporeal‐membrane oxygenation–sustained low‐efficiency dialysis (ECMO‐SLED) leads to non‐inferior filter lifespans despite low anti‐Xa‐levels. (A) Kaplan–Meier estimators of filter survival in hours. Blue line () depicts isolated SLED circuits. Red line () depicts SLED circuits integrated into a veno‐venous extracorporeal membrane oxygenation setup. p‐value calculated using MantelCox test. (B) Median filter runtimes in the SLED () and ECMO‐SLED () group. Whiskers depict IQR, p‐value calculated using Wilcoxon–Mann–Whitney test. (C) Comparison of mean anti‐Xa levels. Whiskers depicting SD, p‐value calculated using Wilcoxon–Mann–Whitney test
Despite longer filter survival, mean anti‐Xa‐levels in the ECMO‐SLED group were significantly lower than in the SLED group (0.28 IU/ml vs. 0.74 IU/ml, p < 0.01) (Figure 2C). Comparison of aPTT showed mean values slightly above the normal range in both groups. ECMO‐SLED patients showed a 3.3 s longer aPTT (Figure S2).
Elevated creatinine and BUN levels were sufficiently reduced. Creatinine was lowered by 1.4 mg/dl (2.7 mg/dl vs. 1.3 mg/dl, p < 0.01), BUN by 58.5 mg/dl (118.3 mg/dl vs. 59.8 mg/dl, p < 0.01) by SLED operated in the vvECMO circuit (Figure 3A–D). All patients treated with ECMO‐SLED developed oliguria or anuria prior to and during ECMO‐SLED treatment (Figure S3).
Extracorporeal‐membrane oxygenation–sustained low‐efficiency dialysis (ECMO‐SLED) allows for sufficient dialysis. Time courses of creatinine (A), blood urea nitrogen (BUN) (C), potassium (E) and sodium (G) levels prior to and after SLED integration into the veno‐venous extracorporeal membrane oxygenation circuit. Numbers indicate patients with available data sets at respective time point. Adjacent graphs compare the means of creatinine (B), BUN (D), potassium (F) and sodium (H) levels before and after SLED initiation. Bars and whiskers depict mean and SEM. p‐values were calculated using Wilcoxon–Mann–Whitney test
In addition, ECMO‐SLED was able to control potassium levels within normal range (Figure 3E,F). Hypernatremia—often associated with severe COVID‐19—was normalized after SLED integration into the vvECMO circuit (148.1 mmol/L vs. 142.5 mmol/L, p < 0.01) (Figure 3G,H).
ECMO‐SLED allowed adequate and non‐inferior ultrafiltration rates compared with the SLED‐group. Balanced volume management was achieved (Figure S4).
DISCUSSION:
Severe COVID‐19 ARDS poses a significant risk for AKI.
19
Volume overload often additionally aggravates ARDS and must be addressed to allow for pulmonary recompensation and vvECMO weaning. Because many medical centers provide RRT devices for prolonged intermittent RRT such as SLED, these devices should be regarded as an alternative treatment option for integrated ECMO‐RRT. SLED devices offer the advantage of broad availability, rapid and easy installation, low maintenance time, plannable filter downtimes, and are more cost‐effective.
11
For these reasons, they are an attractive alternative for the treatment of patients requiring combined extracorporeal organ support.
In this single‐center study of nine critically ill COVID‐19 patients, we show that SLED integration into the vvECMO circuit is (i) technical feasible, (ii) provides adequate dialysis quality, (iii) is able to control electrolyte disturbances, and (iv) allows for sufficient fluid balance.
The integration of SLED into vvECMO circuits has several advantages. Firstly, RRT can be performed in absence of an alternative vascular insertion site besides the double lumen cannula used for vvECMO. Therefore, integration of SLED into the existing vvECMO circuit can be a life‐saving treatment option in case of limited vascular access. Because our center favors the use of two oxygenators, we were able to connect the SLED circuit quite easily at both oxygenators in the high‐pressure part of the ECMO. This arrangement decreases the risk for air embolism. Secondly, each central line poses a risk for complications due to the insertion procedure (e.g., local trauma, pneumothorax, bleeding, air embolism due to ECMO‐associated negative central venous pressure as well as central line–associated infection or thrombosis).
20
Often inguinal insertion sites have to be chosen, because jugular veins are already used for vvECMO, further aggravating the risk for infectious complications. Thirdly, handling of the patient (e.g., prone positioning, mobilization, nursing) is most likely facilitated with a single extracorporeal circuit. Fourthly, an integrated ECMO‐SLED circuit allows for higher and stable blood flow rates within the SLED circuit. The risk for central line dysfunction of a separate RRT catheter is, therefore, not present after SLED integration into the vvECMO circuit. Constant blood flow‐rates can reduce the risk of clotting events and premature termination of SLED. We therefore compared filter lifespans between patients with an integrated ECMO‐SLED circuit to a subgroup of patients receiving isolated SLED after vvECMO weaning. ECMO‐SLED filter lifespans were significantly higher compared with isolated SLED. The improved filter lifespan observed in the ECMO‐SLED cohort is most likely attributable to a reduction of low‐flow phases within the SLED circuit due to constant positive pressure in the vvECMO circuit. Our finding is in line with a previous study investigating filter lifespans in ECMO‐CVVHD. Integrated ECMO‐CVVHD also allowed for longer filter lifespans, compared with isolated circuits.
7
Of note, the increased filter lifespans were observed under less effective anticoagulation, as demonstrated by lower factor anti‐Xa levels in the ECMO‐SLED cohort. Although we found aPTT to be 3.3 s longer in the ECMO‐SLED cohort compared with patients treated with isolated SLED, all measured aPTTs were just slightly above the physiological range and the small difference is most likely without biological impact. Furthermore aPTT levels are not suitable for monitoring anticoagulation with LMWHs. In our study anticoagulation was performed exclusively with the LMWH enoxaparin. Anticoagulation with LMWH has been shown to be superior in CVVHD and SLED alike in cohorts of critically ill COVID‐19 patients.
17
,
21
We believe that constant blood flow through a combined ECMO‐SLED circuit might require an even less rigorous anticoagulation regimen. Less anticoagulation may reduce the risk for common bleeding complications during vvECMO treatment.
There are also disadvantages of an integrated ECMO‐SLED approach, favoring separate extracorporeal circuits: A separate SLED circuit may reduce the risk for vvECMO complications due to turbulences or clotting events in the SLED circuit. Maintenance of the SLED circuit does not have to be performed by ECMO trained staff. Furthermore, integrated SLED functionality may be limited by high circuit pressures if vvECMO blood flow rates need to be elevated (Table 2).
Advantages of SLED integration into vvECMO circuit
feasible in case of limited vascular access options (e.g., due to thrombosis, bleeding risk)
reduced risk of complications associated with central catheter insertion (e.g., pneumothorax, local trauma, bleeding, risk for air embolism)
reduced risk for central line associated complications (e.g., infection, thrombosis)
lower risk for vvECMO circuit complications
no ECMO trained personnel required for SLED circuit service
better filter lifetime, less clotting events, reduced therapy downtime
less anticoagulation required
less pressure alarms
lower risk for hemolysis due to flow turbulences
Besides general feasibility, we investigated dialysis quality in this study. Dialysis parameters (blood and dialysate flow) were within the standard range of SLED therapy. Our data show adequate reduction of both creatinine and BUN over time. Thus, ECMO‐SLED allows for adequate hemodialysis quality. In addition, ECMO‐SLED was able to control electrolyte disorders. Hypernatremia is associated with higher mortality in critically ill COVID‐19 patients and was also present in our cohort prior to ECMO‐SLED initiation.
22
SLED treatment in the vvECMO circuit was efficient to normalize hypernatremia.
Lastly, we investigated ultrafiltration rates of ECMO‐SLED. Because oliguria or anuria persisted during the critical illness phase, ultrafiltration was required in all patients. Our results show that ECMO‐SLED allows for a wide range of desired daily ultrafiltration rates and, thus, is able to provide adequate volume control, which is especially necessary for successful pulmonary recovery and vvECMO weaning.
This study has several limitations. Major limitations are the small sample size, its retrospective design, short observational period and the lack of a matched control group being treated with ECMO and SLED through individual vascular accesses. Data of a cohort of nine patients does not allow general conclusions for a broader population. However, the baseline characteristics, incidence of pulmonary embolism and the observed fatality rate of our cohort was in line with other reports on critically ill COVID‐19 patients.
23
,
24
Furthermore, the primary aim of this study was to demonstrate feasibility of an integrated ECMO‐SLED approach.
To allow more precise conclusions, prospective follow‐up studies with a higher sample size should be conducted to further investigate safeness, filter lifespans, requirement for anticoagulation, quality of dialysis, and long‐term renal outcome of the ECMO‐SLED approach. Furthermore we exclusively report data from multiorgan failure due to severe COVID‐19. However, we believe that the concept of an integrated ECMO‐SLED is transferable to other situations of multi organ failure requiring parallel ECMO and RRT as well. Finally, data reported from our center was generated using two parallel oxygenators in the vvECMO circuit. This setup is well established and standard operating procedure at our intensive care department.
25
Two oxygenators provide several advantages such as the possibility of exchanging an oxygenator without stopping the ECMO pump, an increased blood flow of up to 7 L/min and an easy integration of SLED into the vvECMO circuit as illustrated in Figure 1. Many other centers routinely use single oxygenators in ECMO circuits. However, the concept of ECMO‐SLED is also feasible in single oxygenator vvECMO circuits by integrating two luer‐lock‐connection sites within the post‐pump‐part of the circuit.
In summary, this report shows that an integrated ECMO‐SLED approach is technically feasible, safe and allows for adequate dialysis quality and fluid control. Constant blood flow and sufficient pressure in the SLED circuit could improve filter lifespans and might allow reduction of anticoagulation. This feasibility study can be regarded as foundation for future research investigating ECMO‐SLED as potential alternative to more established protocols like ECMO‐CVVHD. High availability, cost efficiency, and low maintenance requirements of SLED devices compared with CVVHD devices are appealing arguments to further investigate our strategy.
CONFLICT OF INTEREST:
Nothing do declare.
AUTHOR CONTRIBUTIONS:
Frederic Arnold, Johannes Kalbhenn, and Lukas Westermann conceived the study and its design, had full access to the patient records, and take responsibility for the accuracy and integrity of the data. Frederic Arnold and Lukas Westermann screened the electronic patient records, organized the data and performed statistical analysis. Johannes Kalbhenn and Rika Wobser critically contributed to data analysis. Frederic Arnold, Rika Wobser, Johannes Kalbhenn, and Lukas Westermann drafted the manuscript. Frederic Arnold and Lukas Westermann generated the figures. Illustrations were generated by Frederic Arnold. Rika Wobser and Johannes Kalbhenn were involved in clinical management of the patients and contributed to data interpretation. Frederic Arnold, Rika Wobser, Johannes Kalbhenn, Lukas Westermann critically revised the drafted manuscript and approve of the submission.
ETHICS APPROVAL:
Analysis and publication of the data were approved by the ethics committee (405/20) of the University of Freiburg Medical Center, University of Freiburg, Faculty of Medicine, Germany.
Supporting information:
Appendix S1 Supplementary Information
Click here for additional data file.
| Background:
Severe COVID-19 can necessitate multiple organ support including veno-venous extracorporeal membrane oxygenation (vvECMO) and renal replacement therapy. The therapy can be complicated by venous thromboembolism due to COVID-19-related hypercoagulability, thus restricting vascular access beyond the vvECMO cannula. Although continuous renal replacement therapy can be performed via a vvECMO circuit, studies addressing sustained low-efficiency dialysis (SLED) integration into vvECMO circuits are scarce. Here we address the lack of evidence by evaluating feasibility of SLED integration into vvECMO circuits.
Methods:
Retrospective cohort study on nine critically ill COVID-19 patients, treated with integrated ECMO-SLED on a single intensive care unit at a tertiary healthcare facility between December 2020 and November 2021. The SLED circuits were established between the accessory arterial oxygenator outlets of a double-oxygenator vvECMO setup. Data on filter survival, quality of dialysis, and volume management were collected and compared with an internal control group receiving single SLED.
Results:
This study demonstrates general feasibility of SLED integration into existing vvECMO circuits. Filter lifespans of ECMO-SLED compared with single SLED are significantly prolonged (median 18.3 h vs. 10.3 h, p < 0.01). ECMO-SLED treatment is furthermore able to sufficiently normalize creatinine, blood urea nitrogen, and serum sodium, and allows for adequate ultrafiltration rates.
Conclusions:
We can show that ECMO-SLED is practical, safe, results in adequate dialysis quality and enables sufficient electrolyte and volume management. Our data indicate that SLED devices can serve as potential alternative to continuous-veno-venous-hemodialysis for integration in vvECMO circuits. | null | null | 6,209 | 306 | [
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] | null | null | [CONTENT] COVID‐19 | ECMO | ECOS | MOF | RRT | SLED [SUMMARY] | [CONTENT] COVID‐19 | ECMO | ECOS | MOF | RRT | SLED [SUMMARY] | [CONTENT] COVID‐19 | ECMO | ECOS | MOF | RRT | SLED [SUMMARY] | null | [CONTENT] COVID‐19 | ECMO | ECOS | MOF | RRT | SLED [SUMMARY] | null | [CONTENT] Acute Kidney Injury | COVID-19 | Critical Illness | Extracorporeal Membrane Oxygenation | Feasibility Studies | Humans | Hybrid Renal Replacement Therapy | Retrospective Studies [SUMMARY] | [CONTENT] Acute Kidney Injury | COVID-19 | Critical Illness | Extracorporeal Membrane Oxygenation | Feasibility Studies | Humans | Hybrid Renal Replacement Therapy | Retrospective Studies [SUMMARY] | [CONTENT] Acute Kidney Injury | COVID-19 | Critical Illness | Extracorporeal Membrane Oxygenation | Feasibility Studies | Humans | Hybrid Renal Replacement Therapy | Retrospective Studies [SUMMARY] | null | [CONTENT] Acute Kidney Injury | COVID-19 | Critical Illness | Extracorporeal Membrane Oxygenation | Feasibility Studies | Humans | Hybrid Renal Replacement Therapy | Retrospective Studies [SUMMARY] | null | [CONTENT] result acute respiratory | extracorporeal organ support | hospitalized covid 19 | increases critically ill | combination severe pneumonia [SUMMARY] | [CONTENT] result acute respiratory | extracorporeal organ support | hospitalized covid 19 | increases critically ill | combination severe pneumonia [SUMMARY] | [CONTENT] result acute respiratory | extracorporeal organ support | hospitalized covid 19 | increases critically ill | combination severe pneumonia [SUMMARY] | null | [CONTENT] result acute respiratory | extracorporeal organ support | hospitalized covid 19 | increases critically ill | combination severe pneumonia [SUMMARY] | null | [CONTENT] sled | ecmo | vvecmo | patients | circuit | blood | ecmo sled | flow | data | pump [SUMMARY] | [CONTENT] sled | ecmo | vvecmo | patients | circuit | blood | ecmo sled | flow | data | pump [SUMMARY] | [CONTENT] sled | ecmo | vvecmo | patients | circuit | blood | ecmo sled | flow | data | pump [SUMMARY] | null | [CONTENT] sled | ecmo | vvecmo | patients | circuit | blood | ecmo sled | flow | data | pump [SUMMARY] | null | [CONTENT] ecmo | rrt | ecos | 19 | covid | covid 19 | patients | integration | sled | circuits [SUMMARY] | [CONTENT] vvecmo | blood | sled | pump | flow | oxygenators | circuit | centrifugal | ecmo | performed [SUMMARY] | [CONTENT] sled | dl | mg | mg dl | ecmo sled | ecmo | mean | patients | sd | figure [SUMMARY] | null | [CONTENT] sled | declare | ecmo | vvecmo | patients | data | blood | circuit | flow | ecmo sled [SUMMARY] | null | [CONTENT] COVID-19 ||| COVID-19 ||| ||| [SUMMARY] | [CONTENT] nine | COVID-19 | tertiary | between December 2020 and November 2021 ||| ||| [SUMMARY] | [CONTENT] ||| ECMO | 18.3 | 10.3 | 0.01 ||| urea nitrogen [SUMMARY] | null | [CONTENT] COVID-19 ||| COVID-19 ||| ||| ||| nine | COVID-19 | tertiary | between December 2020 and November 2021 ||| ||| ||| ||| ||| ECMO | 18.3 | 10.3 | 0.01 ||| urea nitrogen ||| ||| [SUMMARY] | null |
||||
Preparation and characterization of Tamoxifen citrate loaded nanoparticles for breast cancer therapy. | 25028549 | Four formulations of Tamoxifen citrate loaded polylactide-co-glycolide (PLGA) based nanoparticles (TNPs) were developed and characterized. Their internalization by Michigan Cancer Foundation-7 (MCF-7) breast cancer cells was also investigated. | BACKGROUND | Nanoparticles were prepared by a multiple emulsion solvent evaporation method. Then the following studies were carried out: drug-excipients interaction using Fourier transform infrared spectroscopy (FTIR), surface morphology by field emission scanning electron microscopy (FESEM), zeta potential and size distribution using a Zetasizer Nano ZS90 and particle size analyzer, and in vitro drug release. In vitro cellular uptake of nanoparticles was assessed by confocal microscopy and their cell viability (%) was studied. | METHODS | No chemical interaction was observed between the drug and the selected excipients. TNPs had a smooth surface, and a nanosize range (250-380 nm) with a negative surface charge. Drug loadings of the prepared particles were 1.5%±0.02% weight/weight (w/w), 2.68%±0.5% w/w, 4.09%±0.2% w/w, 27.16%±2.08% w/w for NP1-NP4, respectively. A sustained drug release pattern from the nanoparticles was observed for the entire period of study, ie, up to 60 days. Further, nanoparticles were internalized well by the MCF-7 breast cancer cells on a concentration dependent manner and were present in the cytoplasm. The nucleus was free from nanoparticle entry. Drug loaded nanoparticles were found to be more cytotoxic than the free drug. | RESULTS | TNPs (NP4) showed the highest drug loading, released the drug in a sustained manner for a prolonged period of time and were taken up well by the MCF-7 breast cancer cell line in vitro. Thus the formulation may be suitable for breast cancer treatment due to the good permeation of the formulation into the breast cancer cells. | CONCLUSION | [
"Antineoplastic Agents",
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"Female",
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"Lactic Acid",
"Microscopy, Electron",
"Nanoparticles",
"Particle Size",
"Polyglycolic Acid",
"Polylactic Acid-Polyglycolic Acid Copolymer",
"Spectroscopy, Fourier Transform Infrared",
"Surface Properties",
"Tamoxifen"
] | 4077606 | Introduction | About one-fifth of cancer patients suffer from breast cancer worldwide.1 Various chemotherapeutic agents are used to treat the breast cancer. The existing anticancer agents do not greatly differentiate between the cancerous and normal cells, leading to systemic toxicity and adverse effects. This greatly limits the maximum permissible dose of the drug. Drug permeation into the cancer cells from the conventional formulation is very poor due to less distribution and quick elimination. The extensive distribution and rapid elimination from targeted organs result in a greater requirement of the drug by the tissue, which causes undesirable toxicity as well as being economically unsound.2 Polymeric nanoparticles play an important role in delivering such kinds of chemotherapeutic agents in a controlled manner. Delivering the drugs through the nanoparticles makes it possible to achieve the desired concentration of drug in the specific site, thus minimizing the side effects and reducing the toxicity, dose dumping, etc. Polylactide-co-glycolide (PLGA) is one of the polymers approved by the US Food and Drug Administration and European Medicine Agency for various kinds of drug delivery system in humans due to its biodegradability and biocompatibility.3,4 This is a copolymer of lactic acid and glycolic acid and these two monomers are endogenous compounds and easily metabolized by the body via the Kreb’s cycle.5 Depending on the molecular weight and copolymer ratio, the degradation time of PLGA can vary from several months to years.3,6 Different anticancer drugs, including Tamoxifen citrate, were loaded in the PLGA nanoparticles by many researchers.7–10 Tamoxifen citrate, an antiestrogenic compound, is the first choice for hormonal treatment of breast cancer in both post and premenopausal women for the last few decades. It is often used as an adjuvant therapy following primary treatment of early stage breast cancer. Depending upon the dose and tissue, Tamoxifen citrate can act as an antiestrogenic or as an estrogenic agent. For breast cancer it shows an antiestrogenic effect, and on the uterus it shows an estrogenic effect. Depending upon the dose and the concentration it has several side effects, such as endometrial carcinoma for postmenopausal women. Other side effects include liver cancer, venous thrombosis, pulmonary emboli, and an ocular effect includes retinopathy and corneal opacities.11–14
To overcome such severe side effects, in our research work we have mainly concentrated on the parenteral sustained release delivery of Tamoxifen citrate in nanoparticles so that they can penetrate in the tumor tissue and can be taken up well by endocytosis into the affected cells. | Cellular uptake study | To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells. | Results | FTIR study The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.
The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.
Drug loading Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.
Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.
Determination of particle morphology of different formulations using FESEM FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.
FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.
Determination of particle size and zeta potential Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.
Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.
Transmission electron microscopy The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.
EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.
EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate). | Conclusion | The outcome of the present investigation proposes a novel formulation of TNPs, prepared by a multiple emulsion solvent evaporation technique. The polymeric particles in a nanosize range with a desired drug polymer ratio can be produced. The size, drug loading and the drug release kinetics can be optimally controlled. Further, TNPs were internalized well in breast cancer cells in vitro, suggesting their suitability in breast cancer treatment. Preferential uptake of nanoparticles rather than the free drug by MCF-7 cells causes the cells to be more viable to the free drug. However, in vivo studies are warranted with the nanoparticles. | [
"Preparation of nanoparticles",
"Drug-excipients interactions by Fourier transform infrared spectroscopy",
"Physicochemical characterization of nanoparticles",
"Determination of drug loading",
"Surface morphology and particle size measurement using field emission scanning electron microscopy",
"Transmission electron microscopy",
"Drug release study",
"Energy dispersive X-ray analysis",
"MTT assay for in vitro cell viability studies",
"FTIR study",
"Drug loading",
"Determination of particle morphology of different formulations using FESEM",
"Determination of particle size and zeta potential",
"Transmission electron microscopy",
"EDX study",
"Drug release study",
"Cellular uptake study",
"In vitro cytotoxic assay",
"Conclusion"
] | [
"Tamoxifen citrate nanoparticles (TNPs) were prepared by a multiple-emulsion solvent evaporation method, with some modifications of the reported methods.15–17 The compositions of the different formulations are given in Table 1. Tamoxifen citrate and subsequently PLGA (85:15) (~200 mg) were dissolved in 0.5 mL of methanol and 1.5 mL of dichloromethane mixture (phase 1). PVA was dissolved in water (2.5% weight per volume [w/v]) (phase 2), and 0.5 mL of phase 2 was added drop-wise into phase 1 with homogenization at an optimized speed (22,500 rpm) using a high-speed homogenizer (IKA Laboratory Equipment, Model T10B Ultras-Turrax, Staufen, Germany). The prepared primary emulsion was then added slowly into 75 mL of 1.5% (w/v) PVA solution (phase 3) with a continuous homogenization (22,500 rpm), which produced a secondary emulsion. The secondary emulsion was then placed on a magnetic stirrer and stirred overnight for evaporation to remove organic solvent and solidification of the particles. The nanoparticles were then first separated by centrifugation at 5,000 rpm for 5 minutes to separate larger particles and then the supernatant was collected and recentrifuged at 15,000 rpm for 45 minutes. The solid particles, thus separated, were resuspended in Milli-Q water (Millipore Corp., Billerica, MA, USA), and centrifuged to wash the particles to remove the excess PVA attached on the surface of the nanoparticles and to remove the free drug.15 The washing was repeated three times. The separated nanoparticles were frozen at −40°C and lyophilized (Laboratory Freeze Dryer, Instrumentation India Ltd., Kolkata, India) for 7–8 hours to obtain a solid product. The product obtained after lyophilization was kept overnight in a desiccator for the removal of the remaining moisture, then the lyophilized samples were stored in an airtight container at 4°C.\nFITC was used as a fluorescent marker to visualize TNPs. A stock solution of FITC (0.4% w/v) in ethanol: chloroform (1:3) was prepared, and 100 μL of this stock solution was added into phase 1 during the emulsification process, when necessary.",
"A Fourier transform infrared (FTIR) spectroscope (Magna-IR 750, Series II, Nicolet Instruments Inc., Madison, Wisconsin, USA) was used to obtain FTIR spectra of Tamoxifen citrate, PLGA, PVA, their physical mixture (1:1), and the prepared lyophilized formulation with and without the drug. The sample for analysis was mixed with potassium bromide (1:100 ratio) and compressed into a pellet by using a hydraulic press. The pellet was scanned under the FTIR spectroscope.",
" Determination of drug loading For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:\nActual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)\nFor the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:\nActual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)",
"For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:\nActual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)",
"Particle size and external morphology of the nanoparticles were studied by field emission scanning electron microscopy (FESEM) (Model-JSM-6700F; JEOL, Tokyo, Japan). The samples were coated with platinum before observation at an acceleration voltage of 5 kV.\nAverage particle sizes, polydispersity indices, and zeta potentials of different formulations were determined by a dynamic light scattering method using Data Transfer Assistance (DTA) software by Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) at 25°C. Samples were diluted with Milli-Q (Millipore Corp.) water before measurement.",
"The morphology and drug distribution in the experimental nanoparticles were examined by the transmission electron microscope (TEM) (FEI type FP5018/40 Tecnai G2 Spirit Bio TWIN). The nanoparticle suspension in Milli-Q water was dropped on standard carbon coated copper grid (mesh) and air dried for 5 hours. TEM images were taken and analyzed.",
"Drug release from the nanoparticles was studied in 1% HPβCD in phosphate buffered saline pH 7.4. Five milligrams of each formulation were taken in separate tubes, and 1.5 mL of the required medium was added separately in each tube. The samples were kept at 37°C in an incubator and were shaken slowly at 120 rotation/minute. After the scheduled time intervals, the tubes were centrifuged and 0.5 mL of supernatant was collected. The same volume of the respective fresh medium was replaced in the sample tube and incubated under the same condition as mentioned above. The collected samples were suitably diluted, if required, and analyzed spectrophotometrically at 234 nm.17",
"Encapsulation of the drug within the nanoparticles was confirmed by energy dispersive X-ray (EDX) analysis. It is a technique by which the elemental composition of the sample can be identified. The EDX analysis system works as an integrated feature of a scanning electron microscope (JSM 60, JEOL, Tokyo, Japan). Briefly, lyophilized formulations were spread on the metal stub and platinum coating was carried out. The samples were then examined under a scanning electron microscope. From the samples or from different areas of the sample, the elemental compositions were determined.",
"MCF-7 cells were cultured in DMEM without phenol red and supplemented with 10% fetal bovine serum. The cell culture medium was maintained at 37°C in a humidified incubator containing 5% CO2 atmosphere. Trypsinized confluent cell monolayers were grown (75%–80%) and the cells in the exponentially growing phase were used for cytotoxicity experiments.\nThe cytotoxicity study of TNPs (NP4) was investigated in MCF-7 cells using the MTT assay method.19 The cytotoxicity of the nanoparticles was determined after 48 hours incubation with MCF-7 cells. To determine the cell cytotoxicity/viability, the cells were plated at a density of 5×103 cells/well (optimal seeding density) in 96 well plates and the plate was kept at 37°C in 5% CO2 atmosphere in a CO2 incubator (Model MCO-15AC; Sanyo Electric Biomedical Co. Ltd., Osaka, Japan). After 12 hours of incubation, the medium in the wells was replaced by a fresh medium containing nanoparticles (added to the medium just before its incorporation in the well) with varying concentrations. After 48 hours, MTT dye solution was added to each well. The incubation was continued for a further 4 hours at 37°C and 5% CO2 for exponentially growing cells. Then the medium in each well containing unbound MTT and death cells was removed by suction. The formazan crystals were solubilized with 100 μL dimethyl-sulfoxide, and the solution was vigorously mixed to dissolve the reacted dye. All the experiments were performed in triplicate. The absorbance of each well was read on a microplate reader (multimode plate reader, SpectraMax M5; Molecular Devices, CA, USA) at 540 nm. Two different experimental control media, one containing nanoparticles without a drug and the other containing a free drug, were used. The cell viability (%) of the drug containing nanoparticles related to the control wells containing nanoparticles without a drug and a free drug respectively, was calculated by absorbance of test sample/absorbance of the control sample ×100.",
"The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.",
"Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.",
"FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.",
"Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.",
"The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.\n EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\nEDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\n Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\nA drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\n Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\nTo investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\n In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).\nThe proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).",
"EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.",
"A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.",
"To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.",
"The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).",
"The outcome of the present investigation proposes a novel formulation of TNPs, prepared by a multiple emulsion solvent evaporation technique. The polymeric particles in a nanosize range with a desired drug polymer ratio can be produced. The size, drug loading and the drug release kinetics can be optimally controlled. Further, TNPs were internalized well in breast cancer cells in vitro, suggesting their suitability in breast cancer treatment. Preferential uptake of nanoparticles rather than the free drug by MCF-7 cells causes the cells to be more viable to the free drug. However, in vivo studies are warranted with the nanoparticles."
] | [
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"methods",
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] | [
"Introduction",
"Materials and methods",
"Preparation of nanoparticles",
"Drug-excipients interactions by Fourier transform infrared spectroscopy",
"Physicochemical characterization of nanoparticles",
"Determination of drug loading",
"Surface morphology and particle size measurement using field emission scanning electron microscopy",
"Transmission electron microscopy",
"Drug release study",
"Energy dispersive X-ray analysis",
"Cellular uptake study",
"MTT assay for in vitro cell viability studies",
"Results",
"FTIR study",
"Drug loading",
"Determination of particle morphology of different formulations using FESEM",
"Determination of particle size and zeta potential",
"Transmission electron microscopy",
"EDX study",
"Drug release study",
"Cellular uptake study",
"In vitro cytotoxic assay",
"Discussion",
"Conclusion"
] | [
"About one-fifth of cancer patients suffer from breast cancer worldwide.1 Various chemotherapeutic agents are used to treat the breast cancer. The existing anticancer agents do not greatly differentiate between the cancerous and normal cells, leading to systemic toxicity and adverse effects. This greatly limits the maximum permissible dose of the drug. Drug permeation into the cancer cells from the conventional formulation is very poor due to less distribution and quick elimination. The extensive distribution and rapid elimination from targeted organs result in a greater requirement of the drug by the tissue, which causes undesirable toxicity as well as being economically unsound.2 Polymeric nanoparticles play an important role in delivering such kinds of chemotherapeutic agents in a controlled manner. Delivering the drugs through the nanoparticles makes it possible to achieve the desired concentration of drug in the specific site, thus minimizing the side effects and reducing the toxicity, dose dumping, etc. Polylactide-co-glycolide (PLGA) is one of the polymers approved by the US Food and Drug Administration and European Medicine Agency for various kinds of drug delivery system in humans due to its biodegradability and biocompatibility.3,4 This is a copolymer of lactic acid and glycolic acid and these two monomers are endogenous compounds and easily metabolized by the body via the Kreb’s cycle.5 Depending on the molecular weight and copolymer ratio, the degradation time of PLGA can vary from several months to years.3,6 Different anticancer drugs, including Tamoxifen citrate, were loaded in the PLGA nanoparticles by many researchers.7–10 Tamoxifen citrate, an antiestrogenic compound, is the first choice for hormonal treatment of breast cancer in both post and premenopausal women for the last few decades. It is often used as an adjuvant therapy following primary treatment of early stage breast cancer. Depending upon the dose and tissue, Tamoxifen citrate can act as an antiestrogenic or as an estrogenic agent. For breast cancer it shows an antiestrogenic effect, and on the uterus it shows an estrogenic effect. Depending upon the dose and the concentration it has several side effects, such as endometrial carcinoma for postmenopausal women. Other side effects include liver cancer, venous thrombosis, pulmonary emboli, and an ocular effect includes retinopathy and corneal opacities.11–14\nTo overcome such severe side effects, in our research work we have mainly concentrated on the parenteral sustained release delivery of Tamoxifen citrate in nanoparticles so that they can penetrate in the tumor tissue and can be taken up well by endocytosis into the affected cells.",
"Tamoxifen citrate (Sigma-Aldrich Co., St Louis, MO, USA), polyvinyl alcohol (PVA, MW 125,000; S.D. Fine Chem. Pvt. Ltd., Mumbai, India), PLGA (MW 50,000–75,000; lactide-co-glycolide ratio 85:15; Sigma-Aldrich Co.), hydroxypropyl-β-cyclodextrin (HPβCD; HiMedia Laboratories, Mumbai, India) and fluorescein isothiocyanate 98% (FITC) (HiMedia Laboratories) were used in the study. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum and tetrazolium dye 3-(4,5-dimers dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Co. All other chemicals used were of analytical reagent grade.",
"Tamoxifen citrate nanoparticles (TNPs) were prepared by a multiple-emulsion solvent evaporation method, with some modifications of the reported methods.15–17 The compositions of the different formulations are given in Table 1. Tamoxifen citrate and subsequently PLGA (85:15) (~200 mg) were dissolved in 0.5 mL of methanol and 1.5 mL of dichloromethane mixture (phase 1). PVA was dissolved in water (2.5% weight per volume [w/v]) (phase 2), and 0.5 mL of phase 2 was added drop-wise into phase 1 with homogenization at an optimized speed (22,500 rpm) using a high-speed homogenizer (IKA Laboratory Equipment, Model T10B Ultras-Turrax, Staufen, Germany). The prepared primary emulsion was then added slowly into 75 mL of 1.5% (w/v) PVA solution (phase 3) with a continuous homogenization (22,500 rpm), which produced a secondary emulsion. The secondary emulsion was then placed on a magnetic stirrer and stirred overnight for evaporation to remove organic solvent and solidification of the particles. The nanoparticles were then first separated by centrifugation at 5,000 rpm for 5 minutes to separate larger particles and then the supernatant was collected and recentrifuged at 15,000 rpm for 45 minutes. The solid particles, thus separated, were resuspended in Milli-Q water (Millipore Corp., Billerica, MA, USA), and centrifuged to wash the particles to remove the excess PVA attached on the surface of the nanoparticles and to remove the free drug.15 The washing was repeated three times. The separated nanoparticles were frozen at −40°C and lyophilized (Laboratory Freeze Dryer, Instrumentation India Ltd., Kolkata, India) for 7–8 hours to obtain a solid product. The product obtained after lyophilization was kept overnight in a desiccator for the removal of the remaining moisture, then the lyophilized samples were stored in an airtight container at 4°C.\nFITC was used as a fluorescent marker to visualize TNPs. A stock solution of FITC (0.4% w/v) in ethanol: chloroform (1:3) was prepared, and 100 μL of this stock solution was added into phase 1 during the emulsification process, when necessary.",
"A Fourier transform infrared (FTIR) spectroscope (Magna-IR 750, Series II, Nicolet Instruments Inc., Madison, Wisconsin, USA) was used to obtain FTIR spectra of Tamoxifen citrate, PLGA, PVA, their physical mixture (1:1), and the prepared lyophilized formulation with and without the drug. The sample for analysis was mixed with potassium bromide (1:100 ratio) and compressed into a pellet by using a hydraulic press. The pellet was scanned under the FTIR spectroscope.",
" Determination of drug loading For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:\nActual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)\nFor the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:\nActual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)",
"For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:\nActual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)",
"Particle size and external morphology of the nanoparticles were studied by field emission scanning electron microscopy (FESEM) (Model-JSM-6700F; JEOL, Tokyo, Japan). The samples were coated with platinum before observation at an acceleration voltage of 5 kV.\nAverage particle sizes, polydispersity indices, and zeta potentials of different formulations were determined by a dynamic light scattering method using Data Transfer Assistance (DTA) software by Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) at 25°C. Samples were diluted with Milli-Q (Millipore Corp.) water before measurement.",
"The morphology and drug distribution in the experimental nanoparticles were examined by the transmission electron microscope (TEM) (FEI type FP5018/40 Tecnai G2 Spirit Bio TWIN). The nanoparticle suspension in Milli-Q water was dropped on standard carbon coated copper grid (mesh) and air dried for 5 hours. TEM images were taken and analyzed.",
"Drug release from the nanoparticles was studied in 1% HPβCD in phosphate buffered saline pH 7.4. Five milligrams of each formulation were taken in separate tubes, and 1.5 mL of the required medium was added separately in each tube. The samples were kept at 37°C in an incubator and were shaken slowly at 120 rotation/minute. After the scheduled time intervals, the tubes were centrifuged and 0.5 mL of supernatant was collected. The same volume of the respective fresh medium was replaced in the sample tube and incubated under the same condition as mentioned above. The collected samples were suitably diluted, if required, and analyzed spectrophotometrically at 234 nm.17",
"Encapsulation of the drug within the nanoparticles was confirmed by energy dispersive X-ray (EDX) analysis. It is a technique by which the elemental composition of the sample can be identified. The EDX analysis system works as an integrated feature of a scanning electron microscope (JSM 60, JEOL, Tokyo, Japan). Briefly, lyophilized formulations were spread on the metal stub and platinum coating was carried out. The samples were then examined under a scanning electron microscope. From the samples or from different areas of the sample, the elemental compositions were determined.",
"Confocal laser scanning microscopy was used to visualize the uptake of the polymeric nanoparticles within the cancer cells. For fluorescence imaging of cellular uptake, MCF-7 cells (at 104 cells/mL) were cultivated for 24 hours on cover slips in six well culture plates (3 mL/well). TNP (NP4) suspensions, 50 μl/mL and 100 μl/mL, were then added to the cell culture medium at a concentration of 300 μg/mL. The cells were washed three times after incubation for 3 hours and then fixed using 4% paraformaldehyde aqueous solution. After fixing for 15 minutes, they were rinsed with phosphate buffered saline (pH 7.4) solution. After that the cover slips were taken out carefully and placed on the slide and air-dried. Finally, they were observed using a confocal laser scanning microscopy system (Andor Spinning Disc Confocal Microscope; Andor Technology Ltd., Belfast, Northern Ireland, UK).18",
"MCF-7 cells were cultured in DMEM without phenol red and supplemented with 10% fetal bovine serum. The cell culture medium was maintained at 37°C in a humidified incubator containing 5% CO2 atmosphere. Trypsinized confluent cell monolayers were grown (75%–80%) and the cells in the exponentially growing phase were used for cytotoxicity experiments.\nThe cytotoxicity study of TNPs (NP4) was investigated in MCF-7 cells using the MTT assay method.19 The cytotoxicity of the nanoparticles was determined after 48 hours incubation with MCF-7 cells. To determine the cell cytotoxicity/viability, the cells were plated at a density of 5×103 cells/well (optimal seeding density) in 96 well plates and the plate was kept at 37°C in 5% CO2 atmosphere in a CO2 incubator (Model MCO-15AC; Sanyo Electric Biomedical Co. Ltd., Osaka, Japan). After 12 hours of incubation, the medium in the wells was replaced by a fresh medium containing nanoparticles (added to the medium just before its incorporation in the well) with varying concentrations. After 48 hours, MTT dye solution was added to each well. The incubation was continued for a further 4 hours at 37°C and 5% CO2 for exponentially growing cells. Then the medium in each well containing unbound MTT and death cells was removed by suction. The formazan crystals were solubilized with 100 μL dimethyl-sulfoxide, and the solution was vigorously mixed to dissolve the reacted dye. All the experiments were performed in triplicate. The absorbance of each well was read on a microplate reader (multimode plate reader, SpectraMax M5; Molecular Devices, CA, USA) at 540 nm. Two different experimental control media, one containing nanoparticles without a drug and the other containing a free drug, were used. The cell viability (%) of the drug containing nanoparticles related to the control wells containing nanoparticles without a drug and a free drug respectively, was calculated by absorbance of test sample/absorbance of the control sample ×100.",
" FTIR study The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.\nThe interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.\n Drug loading Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.\nDrug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.\n Determination of particle morphology of different formulations using FESEM FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.\nFESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.\n Determination of particle size and zeta potential Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.\nParticle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.\n Transmission electron microscopy The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.\n EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\nEDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\n Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\nA drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\n Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\nTo investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\n In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).\nThe proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).\nThe morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.\n EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\nEDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\n Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\nA drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\n Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\nTo investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\n In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).\nThe proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).",
"The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.",
"Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.",
"FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.",
"Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.",
"The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.\n EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\nEDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.\n Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\nA drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.\n Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\nTo investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.\n In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).\nThe proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).",
"EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.",
"A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.",
"To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.",
"The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).",
"When FTIR spectra of the physical mixture of Tamoxifen citrate and the excipients were compared, no shifting of predominant peaks of the drug and the excipients was found, suggesting no chemical interaction had taken place. However, some physical interactions between the drug and the mixture of the excipients were observed. This might be due to the formation of weak bonds such as weak hydrogen bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc. The nonreactive nature of PLGA with the Tamoxifen citrate has been reported earlier also.14,21,22 Absence of the peaks of the drug in the FTIR spectrum for the formulations indicates that no free drug was available on the nanoparticle surface. This could be due to molecular distribution of the drug in the polymeric molecular scaffold in the nanoparticles.\nIn the drug loading study, increasing the amount of the drug in the formulations was found to increase drug loading. This may be because of the ability of the polymer matrix to accommodate a large amount of Tamoxifen citrate molecules in the polymeric network until it reached its saturation point. However, the saturation point (that is the maximum amount of drug molecules which can be accommodated in the polymer matrix of a definite quantity) has not been determined in the present study.\nFESEM images of the prepared nanoparticles show that all the particles prepared were spherical in shape, and had a smooth surface. The particles were found to be in submicron size with a relatively narrow range of distribution as supported by the PDI value. The particle size obtained from FESEM was further supported by the particle size obtained from the dynamic light scattering method. The average particle sizes of the formulations varied from 242 nm to 382 nm.\nThe zeta potential of the different batches varies from −10.5 mV to −16.5 mV. The zeta potential describes the nature of the electrostatic potential at the surface of a particle. An absolute zeta potential value of −30 mV to +30 mV suggests that the particles would remain in a suspended state for a longer period and are not susceptible to quick agglomeration in the liquid state.23,24 Thus, zeta potential values of the experimental TNPs suggest that the prepared nanoparticles should be stored in a lyophilized state and should be reconstituted only before injecting.\nTEM photographs of the nanoparticles confirmed their morphology, which reveals that homogeneous and molecular drug distribution was observed in the nanoparticles and the drug was not distributed in particulate forms.\nIn EDX analysis, the difference in values of weight % and atomic % of elements was the proportional increase of the elements due to the presence of Tamoxifen citrate in the nanoparticles. The study suggests that Tamoxifen citrate was encapsulated in the nanoparticles.\nTamoxifen citrate release was in a sustained manner over a period of 60 days from all the experimental formulations. In the NP1 case, maximum drug release was seen 83.76%±0.43% over the 60 days and in the NP4 case, the drug release was minimal during the said period. Much slower drug diffusion from the formulations might be responsible for that. Drug release kinetics of the different formulations were best fitted for the Korsmeyer–Peppas equation. When the release exponent n value is ≤0.43, it is Fickian release. The n-value between 0.43 and 0.85 is defined as non-Fickian release. When n is ≥0.85, it is case-II transport.25 In this study n-values of all the four formulations (ie, NP1, NP2, NP3 and NP4), were greater than 0.85. Interestingly, particles with less average diameter in the nanosize range were found to release the drug in a more sustained manner. This suggests that all the experimental formulations had greater affinity to obey the Korsmeyer–Peppas kinetic model which involves a case II transport (relaxation-controlled release) mechanism. More linearity (as assessed by R2 values) of drug release data toward Korsmeyer–Peppas kinetics implies anomalous diffusion controlled drug release by more than one process.26 Due to long-term slow degradation of PLGA, release of drug molecules follows a coupling of diffusion and erosion mechanism for PLGA degradation by slow hydrolysis of ester linkage by three phases.27 The three predominant sequences by which PLGA degrades in vivo are: random polymeric chain scission causing a decrease in molecular weight without much loss of molecular weight along with the formation of soluble monomers; a decrease in polymeric molecular weight with a rapid loss of mass due to the formation of oligomers and soluble monomers; and the conversion of soluble oligomers into soluble monomers causing complete solubilization. This ultimately causes anomalous drug diffusion.\nThe cellular uptake of nanoparticles by MCF-7 breast cancer cells is influenced by nanoparticle shape, size, surface properties, and concentration of nanoparticles in the medium, incubation time, and temperature, etc.28 In the present study, nanoparticles uptake by MCF-7 cells was good. Localization of TNPs was in the cytoplasm but not in the nucleus. Further, the uptake was found to be dependent on the nanoparticle concentration in the study MCF-7 cellular media of the experiment of endocytosis in vitro.\nThe cytotoxicity of TNPs was more than that of Tamoxifen citrate alone. The possible mechanism underlying the enhanced efficacy of TNPs against MCF-7 may include the enhanced intracellular drug accumulation by nanoparticle uptake.29–31 However, the advantage of TNPs over Tamoxifen citrate free drug is that a single dose of TNPs will provide a much longer drug action (sustained) as compared to a single dose of free drug and may provide passive targeting due to the enhanced permeability and retention effect as reported earlier.32\nSome literature has shown that TNPs have a greater potential than the free drug, however the present study is not just a similar mimicking document. The present study is predominantly different from those available reports.14,22,33 Mirzajani et al14 and Cirpanli et al33 have used a completely different variety of PLGA polymer in which the lactide-glycolide ratio was 50:50. In the present study, we used PLGA of lactide-glycolide ratio 85:15. Cirpanli et al33 used a completely different technique (nanoprecipitation method) than the multiemulsion solvent evaporation method used here to prepare the nanoparticles. Further, Cirpanli et al showed PLGA (50:50) Tamoxifen nanoparticles with positive zeta potentials, which have been reported to be cleared from the blood very quickly as compared to the nanoparticle with negative zeta value. In our study, the prepared nanoparticles had negative zeta potentials which generally allow them to be in the blood circulation for a longer time.34 Although Mirzajani et al used a similar technique to the present study, they used different homogenization speeds (13,500 rpm and 24,000 rpm) and stabilizer concentrations 2% and 3%. Earlier work from our laboratory22 has presented the fixed speeds of homogenization (16,000 rpm) and variable (different) speeds of centrifugation (5,000 rpm and 14,000 rpm). In the present study drug polymer ratios were different. Similarly the speed of homogenization (22,500 rpm) and separation by centrifugation were also varied. Last but the not least, none of the above mentioned studies have performed cellular uptake of the Tamoxifen citrate PLGA nanoparticle along with MTT assay, as investigated here, and the results of the study show concentration dependent cellular uptake of nanoparticle in vitro by MCF-7 breast cancer cells. Drug release also varied predominantly among the studies. Thus the present study has its own uniqueness.",
"The outcome of the present investigation proposes a novel formulation of TNPs, prepared by a multiple emulsion solvent evaporation technique. The polymeric particles in a nanosize range with a desired drug polymer ratio can be produced. The size, drug loading and the drug release kinetics can be optimally controlled. Further, TNPs were internalized well in breast cancer cells in vitro, suggesting their suitability in breast cancer treatment. Preferential uptake of nanoparticles rather than the free drug by MCF-7 cells causes the cells to be more viable to the free drug. However, in vivo studies are warranted with the nanoparticles."
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"polylactide-co-glycolide nanoparticle",
"PLGA",
"breast cancer",
"multiple emulsion"
] | Introduction:
About one-fifth of cancer patients suffer from breast cancer worldwide.1 Various chemotherapeutic agents are used to treat the breast cancer. The existing anticancer agents do not greatly differentiate between the cancerous and normal cells, leading to systemic toxicity and adverse effects. This greatly limits the maximum permissible dose of the drug. Drug permeation into the cancer cells from the conventional formulation is very poor due to less distribution and quick elimination. The extensive distribution and rapid elimination from targeted organs result in a greater requirement of the drug by the tissue, which causes undesirable toxicity as well as being economically unsound.2 Polymeric nanoparticles play an important role in delivering such kinds of chemotherapeutic agents in a controlled manner. Delivering the drugs through the nanoparticles makes it possible to achieve the desired concentration of drug in the specific site, thus minimizing the side effects and reducing the toxicity, dose dumping, etc. Polylactide-co-glycolide (PLGA) is one of the polymers approved by the US Food and Drug Administration and European Medicine Agency for various kinds of drug delivery system in humans due to its biodegradability and biocompatibility.3,4 This is a copolymer of lactic acid and glycolic acid and these two monomers are endogenous compounds and easily metabolized by the body via the Kreb’s cycle.5 Depending on the molecular weight and copolymer ratio, the degradation time of PLGA can vary from several months to years.3,6 Different anticancer drugs, including Tamoxifen citrate, were loaded in the PLGA nanoparticles by many researchers.7–10 Tamoxifen citrate, an antiestrogenic compound, is the first choice for hormonal treatment of breast cancer in both post and premenopausal women for the last few decades. It is often used as an adjuvant therapy following primary treatment of early stage breast cancer. Depending upon the dose and tissue, Tamoxifen citrate can act as an antiestrogenic or as an estrogenic agent. For breast cancer it shows an antiestrogenic effect, and on the uterus it shows an estrogenic effect. Depending upon the dose and the concentration it has several side effects, such as endometrial carcinoma for postmenopausal women. Other side effects include liver cancer, venous thrombosis, pulmonary emboli, and an ocular effect includes retinopathy and corneal opacities.11–14
To overcome such severe side effects, in our research work we have mainly concentrated on the parenteral sustained release delivery of Tamoxifen citrate in nanoparticles so that they can penetrate in the tumor tissue and can be taken up well by endocytosis into the affected cells.
Materials and methods:
Tamoxifen citrate (Sigma-Aldrich Co., St Louis, MO, USA), polyvinyl alcohol (PVA, MW 125,000; S.D. Fine Chem. Pvt. Ltd., Mumbai, India), PLGA (MW 50,000–75,000; lactide-co-glycolide ratio 85:15; Sigma-Aldrich Co.), hydroxypropyl-β-cyclodextrin (HPβCD; HiMedia Laboratories, Mumbai, India) and fluorescein isothiocyanate 98% (FITC) (HiMedia Laboratories) were used in the study. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum and tetrazolium dye 3-(4,5-dimers dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Co. All other chemicals used were of analytical reagent grade.
Preparation of nanoparticles:
Tamoxifen citrate nanoparticles (TNPs) were prepared by a multiple-emulsion solvent evaporation method, with some modifications of the reported methods.15–17 The compositions of the different formulations are given in Table 1. Tamoxifen citrate and subsequently PLGA (85:15) (~200 mg) were dissolved in 0.5 mL of methanol and 1.5 mL of dichloromethane mixture (phase 1). PVA was dissolved in water (2.5% weight per volume [w/v]) (phase 2), and 0.5 mL of phase 2 was added drop-wise into phase 1 with homogenization at an optimized speed (22,500 rpm) using a high-speed homogenizer (IKA Laboratory Equipment, Model T10B Ultras-Turrax, Staufen, Germany). The prepared primary emulsion was then added slowly into 75 mL of 1.5% (w/v) PVA solution (phase 3) with a continuous homogenization (22,500 rpm), which produced a secondary emulsion. The secondary emulsion was then placed on a magnetic stirrer and stirred overnight for evaporation to remove organic solvent and solidification of the particles. The nanoparticles were then first separated by centrifugation at 5,000 rpm for 5 minutes to separate larger particles and then the supernatant was collected and recentrifuged at 15,000 rpm for 45 minutes. The solid particles, thus separated, were resuspended in Milli-Q water (Millipore Corp., Billerica, MA, USA), and centrifuged to wash the particles to remove the excess PVA attached on the surface of the nanoparticles and to remove the free drug.15 The washing was repeated three times. The separated nanoparticles were frozen at −40°C and lyophilized (Laboratory Freeze Dryer, Instrumentation India Ltd., Kolkata, India) for 7–8 hours to obtain a solid product. The product obtained after lyophilization was kept overnight in a desiccator for the removal of the remaining moisture, then the lyophilized samples were stored in an airtight container at 4°C.
FITC was used as a fluorescent marker to visualize TNPs. A stock solution of FITC (0.4% w/v) in ethanol: chloroform (1:3) was prepared, and 100 μL of this stock solution was added into phase 1 during the emulsification process, when necessary.
Drug-excipients interactions by Fourier transform infrared spectroscopy:
A Fourier transform infrared (FTIR) spectroscope (Magna-IR 750, Series II, Nicolet Instruments Inc., Madison, Wisconsin, USA) was used to obtain FTIR spectra of Tamoxifen citrate, PLGA, PVA, their physical mixture (1:1), and the prepared lyophilized formulation with and without the drug. The sample for analysis was mixed with potassium bromide (1:100 ratio) and compressed into a pellet by using a hydraulic press. The pellet was scanned under the FTIR spectroscope.
Physicochemical characterization of nanoparticles:
Determination of drug loading For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:
Actual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)
For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:
Actual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)
Determination of drug loading:
For the determination of drug loading, an accurately weighed amount of TNPs (2 mg) was placed into a centrifuge tube, and 2 mL of 5% SLS-NaOH solution was added. It was continuously shaken for 3–4 hours at 37°C in an incubator shaker. The dispersed phase was separated from the continuous phase by centrifugation. Then the supernatant was collected and the released drug was assayed spectrophotometrically at 278 nm. The percentage of drug loading was calculated using Equation 1:
Actual drug loading=Amount of drug present in nanoparticlesWeight of nanoparticles sample analyzed×100.(1)
Surface morphology and particle size measurement using field emission scanning electron microscopy:
Particle size and external morphology of the nanoparticles were studied by field emission scanning electron microscopy (FESEM) (Model-JSM-6700F; JEOL, Tokyo, Japan). The samples were coated with platinum before observation at an acceleration voltage of 5 kV.
Average particle sizes, polydispersity indices, and zeta potentials of different formulations were determined by a dynamic light scattering method using Data Transfer Assistance (DTA) software by Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) at 25°C. Samples were diluted with Milli-Q (Millipore Corp.) water before measurement.
Transmission electron microscopy:
The morphology and drug distribution in the experimental nanoparticles were examined by the transmission electron microscope (TEM) (FEI type FP5018/40 Tecnai G2 Spirit Bio TWIN). The nanoparticle suspension in Milli-Q water was dropped on standard carbon coated copper grid (mesh) and air dried for 5 hours. TEM images were taken and analyzed.
Drug release study:
Drug release from the nanoparticles was studied in 1% HPβCD in phosphate buffered saline pH 7.4. Five milligrams of each formulation were taken in separate tubes, and 1.5 mL of the required medium was added separately in each tube. The samples were kept at 37°C in an incubator and were shaken slowly at 120 rotation/minute. After the scheduled time intervals, the tubes were centrifuged and 0.5 mL of supernatant was collected. The same volume of the respective fresh medium was replaced in the sample tube and incubated under the same condition as mentioned above. The collected samples were suitably diluted, if required, and analyzed spectrophotometrically at 234 nm.17
Energy dispersive X-ray analysis:
Encapsulation of the drug within the nanoparticles was confirmed by energy dispersive X-ray (EDX) analysis. It is a technique by which the elemental composition of the sample can be identified. The EDX analysis system works as an integrated feature of a scanning electron microscope (JSM 60, JEOL, Tokyo, Japan). Briefly, lyophilized formulations were spread on the metal stub and platinum coating was carried out. The samples were then examined under a scanning electron microscope. From the samples or from different areas of the sample, the elemental compositions were determined.
Cellular uptake study:
Confocal laser scanning microscopy was used to visualize the uptake of the polymeric nanoparticles within the cancer cells. For fluorescence imaging of cellular uptake, MCF-7 cells (at 104 cells/mL) were cultivated for 24 hours on cover slips in six well culture plates (3 mL/well). TNP (NP4) suspensions, 50 μl/mL and 100 μl/mL, were then added to the cell culture medium at a concentration of 300 μg/mL. The cells were washed three times after incubation for 3 hours and then fixed using 4% paraformaldehyde aqueous solution. After fixing for 15 minutes, they were rinsed with phosphate buffered saline (pH 7.4) solution. After that the cover slips were taken out carefully and placed on the slide and air-dried. Finally, they were observed using a confocal laser scanning microscopy system (Andor Spinning Disc Confocal Microscope; Andor Technology Ltd., Belfast, Northern Ireland, UK).18
MTT assay for in vitro cell viability studies:
MCF-7 cells were cultured in DMEM without phenol red and supplemented with 10% fetal bovine serum. The cell culture medium was maintained at 37°C in a humidified incubator containing 5% CO2 atmosphere. Trypsinized confluent cell monolayers were grown (75%–80%) and the cells in the exponentially growing phase were used for cytotoxicity experiments.
The cytotoxicity study of TNPs (NP4) was investigated in MCF-7 cells using the MTT assay method.19 The cytotoxicity of the nanoparticles was determined after 48 hours incubation with MCF-7 cells. To determine the cell cytotoxicity/viability, the cells were plated at a density of 5×103 cells/well (optimal seeding density) in 96 well plates and the plate was kept at 37°C in 5% CO2 atmosphere in a CO2 incubator (Model MCO-15AC; Sanyo Electric Biomedical Co. Ltd., Osaka, Japan). After 12 hours of incubation, the medium in the wells was replaced by a fresh medium containing nanoparticles (added to the medium just before its incorporation in the well) with varying concentrations. After 48 hours, MTT dye solution was added to each well. The incubation was continued for a further 4 hours at 37°C and 5% CO2 for exponentially growing cells. Then the medium in each well containing unbound MTT and death cells was removed by suction. The formazan crystals were solubilized with 100 μL dimethyl-sulfoxide, and the solution was vigorously mixed to dissolve the reacted dye. All the experiments were performed in triplicate. The absorbance of each well was read on a microplate reader (multimode plate reader, SpectraMax M5; Molecular Devices, CA, USA) at 540 nm. Two different experimental control media, one containing nanoparticles without a drug and the other containing a free drug, were used. The cell viability (%) of the drug containing nanoparticles related to the control wells containing nanoparticles without a drug and a free drug respectively, was calculated by absorbance of test sample/absorbance of the control sample ×100.
Results:
FTIR study The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.
The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.
Drug loading Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.
Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.
Determination of particle morphology of different formulations using FESEM FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.
FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.
Determination of particle size and zeta potential Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.
Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.
Transmission electron microscopy The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.
EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.
EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
FTIR study:
The interactions, if any, between the drug and the polymers (PLGA and PVA), were investigated by FTIR spectroscopy. All the important peaks (Figure 1, A–G) of Tamoxifen citrate were found to exist namely, ketonic group at 1,741 cm−1, amine (N-H bend) at 1,588 cm−1, phenyl ring substitution at 768 cm−1 to 707 cm−1, methyl group at 1,306 cm−1, amine (C-N stretch) at 1,044 cm−1, at 844 cm−1 for aromatic ring out of plane bend in the physical mixture of the drug and the excipients. However, few minor shiftings of peaks of the excipients were detected in the wave numbers from 940 cm−1 to 920 cm−1 responsible for alkane, from 3,406 cm−1 to 3,402 cm−1 responsible for benzene ring or substituted benzene, and at 3,435 cm−1 responsible for the OH group. The shifting of such peaks might have taken place due to the weak physical interactions such as formation of weak H-bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc, which could lead to the formation of a spherical structure of the nanoparticles. The blank formulation and the formulation with the drug had similar peaks, indicating that there was no free drug at the nanoparticle surface.
Drug loading:
Drug loading of TNPs varied from 1.5%±0.02%−27.16%±2.08% (Table 1). It was found to be maximum for NP4 and minimum for NP1. Theoretical drug loading of different formulations, NP1 to NP4, varied from 11%, to 50%, respectively. Drug loading was found to be directly proportional to the amount of the drug added during the preparation of the different formulations up to an approximate drug: PLGA ratio of 1:3. However, at a 1:1 (drug:PLGA) ratio, drug loading was found to be enhanced seven times more, than drug loading at a 1:3 ratio.
Determination of particle morphology of different formulations using FESEM:
FESEM photographs of different formulations are shown in Figure 2. Prepared nanoparticles were submicron in size, spherical in shape, and with a smooth surface.
Determination of particle size and zeta potential:
Particle sizes and zeta potentials of different formulations are shown in Figure 3 and Figure 4 respectively. The average particle sizes of different formulations were (Table 2) 242. 5 nm, 331.8 nm, 375.6 nm, 382 nm, with the polydispersity indices of 1.00, 0.143, 0.016, 0.611 for the formulations from NP1 to NP4 respectively. The zeta potential of different formulations had values from −10.5 mV to −16.5 mV, indicating the value was decreasing with the increasing amount of drug incorporation in the formulation.
Transmission electron microscopy:
The morphology and size of nanoparticles obtained were examined by TEM. TEM photographs of the prepared nanoparticles (Figure 5) show that there was homogeneous molecular distribution of the drug in the polymer based nanoparticles and the drug was not distributed in the particulate form. There was no diffraction of transmission of electrons through the particles and that is why uniform dark particles were seen without any spots. Spotted particles support the presence of a drug in particulate form rather than its distribution in molecular form.
EDX study EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
Drug release study A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
Cellular uptake study To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
In vitro cytotoxic assay The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
EDX study:
EDX analysis showed weight % and atomic % of various elements (C, O, and N) in various nanoparticle formulations (Figure 6 and Table 3). The weight % of C, O, and N in TNPs was 41.09%, 26% and 32.9% respectively; and the values for blank nanoparticles were 26.23%, 48.84%, and 24.92% respectively. The atomic % of C, O, and N in TNPs was 46.26%, 21.98%, and 31.76% respectively; in blank nanoparticles it was 31.13%, 43.51%, and 25.36% respectively. The differences in values of weight % and atomic % of elements were due to the presence of Tamoxifen citrate in the TNPs.
Drug release study:
A drug release study was carried out to understand in vitro drug release pattern from the formulations. A sustained drug release pattern was observed. After 60 days of in vitro drug release, variable amounts (in terms of percentages) (mean ± SD; n=3)namely, 83.76%±0.43%, 55.21%±0.77%, 40.69%±0.11%, and 9.47%±0.1% of Tamoxifen citrate were released from NP1, NP2, NP3, and NP4, respectively (Figure 7). To evaluate the drug-release kinetic patterns, drug release data were assessed using zero order, first order, Korsmeyer–Peppas, and Higuchi kinetic models.20 Calculated R2 values for the kinetics were tabulated (Table 4). The corresponding plot (log cumulative percent drug release versus log time) (data not shown) for the Korsmeyer–Pappas equation indicates a good linearity (R2=0.9976, 0.9652, 0.9721, 0.9736) for NP1 to NP4, respectively. The release exponents (n-value) for the formulations, (NP1 to NP4) were 1.09, 0.914, 0.901, and 0.852, respectively.
Cellular uptake study:
To investigate the cellular uptake of the nanoparticles in MCF-7 cells, a short-term in vitro particle endocytosis test was carried out using FITC-TNPs. Figure 8 shows that TNPs penetrated the cell membrane and were distributed in the cytoplasm, but not in the nuclei. Moreover, the green fluorescent dots in the samples increased with the increasing concentrations of the nanoparticles. These images have demonstrated concentration dependent endocytosis of nanoparticles in MCF-7 cells.
In vitro cytotoxic assay:
The proliferation/viability of MCF-7 cells was assessed by MTT assay after 48 hours of incubation with the free drug and nanoparticles with or without the drug, respectively (Figure 9). The cytotoxic effect of nanoparticles increased with an increase in Tamoxifen citrate concentration. The toxicity of Tamoxifen citrate increased as the drug concentration increased from 12.5 μM to 200 μM. The toxic effect of Tamoxifen citrate markedly decreased the cell, viability from 70.13% to 21.81%. In this cytotoxicity test, TNPs caused more death of viable cells than Tamoxifen citrate alone (free drug). Again, nanoparticles without the drug failed to produce any toxicity to viable cells and exhibited viability of 99.11%. This increased toxicity may be due to the preferential uptake of nanoparticles than that of the free drug (Tamoxifen citrate).
Discussion:
When FTIR spectra of the physical mixture of Tamoxifen citrate and the excipients were compared, no shifting of predominant peaks of the drug and the excipients was found, suggesting no chemical interaction had taken place. However, some physical interactions between the drug and the mixture of the excipients were observed. This might be due to the formation of weak bonds such as weak hydrogen bonding, van der Waals’ force of attraction, dipole–dipole interaction, etc. The nonreactive nature of PLGA with the Tamoxifen citrate has been reported earlier also.14,21,22 Absence of the peaks of the drug in the FTIR spectrum for the formulations indicates that no free drug was available on the nanoparticle surface. This could be due to molecular distribution of the drug in the polymeric molecular scaffold in the nanoparticles.
In the drug loading study, increasing the amount of the drug in the formulations was found to increase drug loading. This may be because of the ability of the polymer matrix to accommodate a large amount of Tamoxifen citrate molecules in the polymeric network until it reached its saturation point. However, the saturation point (that is the maximum amount of drug molecules which can be accommodated in the polymer matrix of a definite quantity) has not been determined in the present study.
FESEM images of the prepared nanoparticles show that all the particles prepared were spherical in shape, and had a smooth surface. The particles were found to be in submicron size with a relatively narrow range of distribution as supported by the PDI value. The particle size obtained from FESEM was further supported by the particle size obtained from the dynamic light scattering method. The average particle sizes of the formulations varied from 242 nm to 382 nm.
The zeta potential of the different batches varies from −10.5 mV to −16.5 mV. The zeta potential describes the nature of the electrostatic potential at the surface of a particle. An absolute zeta potential value of −30 mV to +30 mV suggests that the particles would remain in a suspended state for a longer period and are not susceptible to quick agglomeration in the liquid state.23,24 Thus, zeta potential values of the experimental TNPs suggest that the prepared nanoparticles should be stored in a lyophilized state and should be reconstituted only before injecting.
TEM photographs of the nanoparticles confirmed their morphology, which reveals that homogeneous and molecular drug distribution was observed in the nanoparticles and the drug was not distributed in particulate forms.
In EDX analysis, the difference in values of weight % and atomic % of elements was the proportional increase of the elements due to the presence of Tamoxifen citrate in the nanoparticles. The study suggests that Tamoxifen citrate was encapsulated in the nanoparticles.
Tamoxifen citrate release was in a sustained manner over a period of 60 days from all the experimental formulations. In the NP1 case, maximum drug release was seen 83.76%±0.43% over the 60 days and in the NP4 case, the drug release was minimal during the said period. Much slower drug diffusion from the formulations might be responsible for that. Drug release kinetics of the different formulations were best fitted for the Korsmeyer–Peppas equation. When the release exponent n value is ≤0.43, it is Fickian release. The n-value between 0.43 and 0.85 is defined as non-Fickian release. When n is ≥0.85, it is case-II transport.25 In this study n-values of all the four formulations (ie, NP1, NP2, NP3 and NP4), were greater than 0.85. Interestingly, particles with less average diameter in the nanosize range were found to release the drug in a more sustained manner. This suggests that all the experimental formulations had greater affinity to obey the Korsmeyer–Peppas kinetic model which involves a case II transport (relaxation-controlled release) mechanism. More linearity (as assessed by R2 values) of drug release data toward Korsmeyer–Peppas kinetics implies anomalous diffusion controlled drug release by more than one process.26 Due to long-term slow degradation of PLGA, release of drug molecules follows a coupling of diffusion and erosion mechanism for PLGA degradation by slow hydrolysis of ester linkage by three phases.27 The three predominant sequences by which PLGA degrades in vivo are: random polymeric chain scission causing a decrease in molecular weight without much loss of molecular weight along with the formation of soluble monomers; a decrease in polymeric molecular weight with a rapid loss of mass due to the formation of oligomers and soluble monomers; and the conversion of soluble oligomers into soluble monomers causing complete solubilization. This ultimately causes anomalous drug diffusion.
The cellular uptake of nanoparticles by MCF-7 breast cancer cells is influenced by nanoparticle shape, size, surface properties, and concentration of nanoparticles in the medium, incubation time, and temperature, etc.28 In the present study, nanoparticles uptake by MCF-7 cells was good. Localization of TNPs was in the cytoplasm but not in the nucleus. Further, the uptake was found to be dependent on the nanoparticle concentration in the study MCF-7 cellular media of the experiment of endocytosis in vitro.
The cytotoxicity of TNPs was more than that of Tamoxifen citrate alone. The possible mechanism underlying the enhanced efficacy of TNPs against MCF-7 may include the enhanced intracellular drug accumulation by nanoparticle uptake.29–31 However, the advantage of TNPs over Tamoxifen citrate free drug is that a single dose of TNPs will provide a much longer drug action (sustained) as compared to a single dose of free drug and may provide passive targeting due to the enhanced permeability and retention effect as reported earlier.32
Some literature has shown that TNPs have a greater potential than the free drug, however the present study is not just a similar mimicking document. The present study is predominantly different from those available reports.14,22,33 Mirzajani et al14 and Cirpanli et al33 have used a completely different variety of PLGA polymer in which the lactide-glycolide ratio was 50:50. In the present study, we used PLGA of lactide-glycolide ratio 85:15. Cirpanli et al33 used a completely different technique (nanoprecipitation method) than the multiemulsion solvent evaporation method used here to prepare the nanoparticles. Further, Cirpanli et al showed PLGA (50:50) Tamoxifen nanoparticles with positive zeta potentials, which have been reported to be cleared from the blood very quickly as compared to the nanoparticle with negative zeta value. In our study, the prepared nanoparticles had negative zeta potentials which generally allow them to be in the blood circulation for a longer time.34 Although Mirzajani et al used a similar technique to the present study, they used different homogenization speeds (13,500 rpm and 24,000 rpm) and stabilizer concentrations 2% and 3%. Earlier work from our laboratory22 has presented the fixed speeds of homogenization (16,000 rpm) and variable (different) speeds of centrifugation (5,000 rpm and 14,000 rpm). In the present study drug polymer ratios were different. Similarly the speed of homogenization (22,500 rpm) and separation by centrifugation were also varied. Last but the not least, none of the above mentioned studies have performed cellular uptake of the Tamoxifen citrate PLGA nanoparticle along with MTT assay, as investigated here, and the results of the study show concentration dependent cellular uptake of nanoparticle in vitro by MCF-7 breast cancer cells. Drug release also varied predominantly among the studies. Thus the present study has its own uniqueness.
Conclusion:
The outcome of the present investigation proposes a novel formulation of TNPs, prepared by a multiple emulsion solvent evaporation technique. The polymeric particles in a nanosize range with a desired drug polymer ratio can be produced. The size, drug loading and the drug release kinetics can be optimally controlled. Further, TNPs were internalized well in breast cancer cells in vitro, suggesting their suitability in breast cancer treatment. Preferential uptake of nanoparticles rather than the free drug by MCF-7 cells causes the cells to be more viable to the free drug. However, in vivo studies are warranted with the nanoparticles.
| Background:
Four formulations of Tamoxifen citrate loaded polylactide-co-glycolide (PLGA) based nanoparticles (TNPs) were developed and characterized. Their internalization by Michigan Cancer Foundation-7 (MCF-7) breast cancer cells was also investigated.
Methods:
Nanoparticles were prepared by a multiple emulsion solvent evaporation method. Then the following studies were carried out: drug-excipients interaction using Fourier transform infrared spectroscopy (FTIR), surface morphology by field emission scanning electron microscopy (FESEM), zeta potential and size distribution using a Zetasizer Nano ZS90 and particle size analyzer, and in vitro drug release. In vitro cellular uptake of nanoparticles was assessed by confocal microscopy and their cell viability (%) was studied.
Results:
No chemical interaction was observed between the drug and the selected excipients. TNPs had a smooth surface, and a nanosize range (250-380 nm) with a negative surface charge. Drug loadings of the prepared particles were 1.5%±0.02% weight/weight (w/w), 2.68%±0.5% w/w, 4.09%±0.2% w/w, 27.16%±2.08% w/w for NP1-NP4, respectively. A sustained drug release pattern from the nanoparticles was observed for the entire period of study, ie, up to 60 days. Further, nanoparticles were internalized well by the MCF-7 breast cancer cells on a concentration dependent manner and were present in the cytoplasm. The nucleus was free from nanoparticle entry. Drug loaded nanoparticles were found to be more cytotoxic than the free drug.
Conclusions:
TNPs (NP4) showed the highest drug loading, released the drug in a sustained manner for a prolonged period of time and were taken up well by the MCF-7 breast cancer cell line in vitro. Thus the formulation may be suitable for breast cancer treatment due to the good permeation of the formulation into the breast cancer cells. | Introduction:
About one-fifth of cancer patients suffer from breast cancer worldwide.1 Various chemotherapeutic agents are used to treat the breast cancer. The existing anticancer agents do not greatly differentiate between the cancerous and normal cells, leading to systemic toxicity and adverse effects. This greatly limits the maximum permissible dose of the drug. Drug permeation into the cancer cells from the conventional formulation is very poor due to less distribution and quick elimination. The extensive distribution and rapid elimination from targeted organs result in a greater requirement of the drug by the tissue, which causes undesirable toxicity as well as being economically unsound.2 Polymeric nanoparticles play an important role in delivering such kinds of chemotherapeutic agents in a controlled manner. Delivering the drugs through the nanoparticles makes it possible to achieve the desired concentration of drug in the specific site, thus minimizing the side effects and reducing the toxicity, dose dumping, etc. Polylactide-co-glycolide (PLGA) is one of the polymers approved by the US Food and Drug Administration and European Medicine Agency for various kinds of drug delivery system in humans due to its biodegradability and biocompatibility.3,4 This is a copolymer of lactic acid and glycolic acid and these two monomers are endogenous compounds and easily metabolized by the body via the Kreb’s cycle.5 Depending on the molecular weight and copolymer ratio, the degradation time of PLGA can vary from several months to years.3,6 Different anticancer drugs, including Tamoxifen citrate, were loaded in the PLGA nanoparticles by many researchers.7–10 Tamoxifen citrate, an antiestrogenic compound, is the first choice for hormonal treatment of breast cancer in both post and premenopausal women for the last few decades. It is often used as an adjuvant therapy following primary treatment of early stage breast cancer. Depending upon the dose and tissue, Tamoxifen citrate can act as an antiestrogenic or as an estrogenic agent. For breast cancer it shows an antiestrogenic effect, and on the uterus it shows an estrogenic effect. Depending upon the dose and the concentration it has several side effects, such as endometrial carcinoma for postmenopausal women. Other side effects include liver cancer, venous thrombosis, pulmonary emboli, and an ocular effect includes retinopathy and corneal opacities.11–14
To overcome such severe side effects, in our research work we have mainly concentrated on the parenteral sustained release delivery of Tamoxifen citrate in nanoparticles so that they can penetrate in the tumor tissue and can be taken up well by endocytosis into the affected cells.
Conclusion:
The outcome of the present investigation proposes a novel formulation of TNPs, prepared by a multiple emulsion solvent evaporation technique. The polymeric particles in a nanosize range with a desired drug polymer ratio can be produced. The size, drug loading and the drug release kinetics can be optimally controlled. Further, TNPs were internalized well in breast cancer cells in vitro, suggesting their suitability in breast cancer treatment. Preferential uptake of nanoparticles rather than the free drug by MCF-7 cells causes the cells to be more viable to the free drug. However, in vivo studies are warranted with the nanoparticles. | Background:
Four formulations of Tamoxifen citrate loaded polylactide-co-glycolide (PLGA) based nanoparticles (TNPs) were developed and characterized. Their internalization by Michigan Cancer Foundation-7 (MCF-7) breast cancer cells was also investigated.
Methods:
Nanoparticles were prepared by a multiple emulsion solvent evaporation method. Then the following studies were carried out: drug-excipients interaction using Fourier transform infrared spectroscopy (FTIR), surface morphology by field emission scanning electron microscopy (FESEM), zeta potential and size distribution using a Zetasizer Nano ZS90 and particle size analyzer, and in vitro drug release. In vitro cellular uptake of nanoparticles was assessed by confocal microscopy and their cell viability (%) was studied.
Results:
No chemical interaction was observed between the drug and the selected excipients. TNPs had a smooth surface, and a nanosize range (250-380 nm) with a negative surface charge. Drug loadings of the prepared particles were 1.5%±0.02% weight/weight (w/w), 2.68%±0.5% w/w, 4.09%±0.2% w/w, 27.16%±2.08% w/w for NP1-NP4, respectively. A sustained drug release pattern from the nanoparticles was observed for the entire period of study, ie, up to 60 days. Further, nanoparticles were internalized well by the MCF-7 breast cancer cells on a concentration dependent manner and were present in the cytoplasm. The nucleus was free from nanoparticle entry. Drug loaded nanoparticles were found to be more cytotoxic than the free drug.
Conclusions:
TNPs (NP4) showed the highest drug loading, released the drug in a sustained manner for a prolonged period of time and were taken up well by the MCF-7 breast cancer cell line in vitro. Thus the formulation may be suitable for breast cancer treatment due to the good permeation of the formulation into the breast cancer cells. | 9,788 | 350 | [
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] | [CONTENT] polylactide-co-glycolide nanoparticle | PLGA | breast cancer | multiple emulsion [SUMMARY] | [CONTENT] polylactide-co-glycolide nanoparticle | PLGA | breast cancer | multiple emulsion [SUMMARY] | [CONTENT] polylactide-co-glycolide nanoparticle | PLGA | breast cancer | multiple emulsion [SUMMARY] | [CONTENT] polylactide-co-glycolide nanoparticle | PLGA | breast cancer | multiple emulsion [SUMMARY] | [CONTENT] polylactide-co-glycolide nanoparticle | PLGA | breast cancer | multiple emulsion [SUMMARY] | [CONTENT] polylactide-co-glycolide nanoparticle | PLGA | breast cancer | multiple emulsion [SUMMARY] | [CONTENT] Antineoplastic Agents | Breast Neoplasms | Cell Line, Tumor | Cell Survival | Emulsions | Female | Humans | Lactic Acid | Microscopy, Electron | Nanoparticles | Particle Size | Polyglycolic Acid | Polylactic Acid-Polyglycolic Acid Copolymer | Spectroscopy, Fourier Transform Infrared | Surface Properties | Tamoxifen [SUMMARY] | [CONTENT] Antineoplastic Agents | Breast Neoplasms | Cell Line, Tumor | Cell Survival | Emulsions | Female | Humans | Lactic Acid | Microscopy, Electron | Nanoparticles | Particle Size | Polyglycolic Acid | Polylactic Acid-Polyglycolic Acid Copolymer | Spectroscopy, Fourier Transform Infrared | Surface Properties | Tamoxifen [SUMMARY] | [CONTENT] Antineoplastic Agents | Breast Neoplasms | Cell Line, Tumor | Cell Survival | Emulsions | Female | Humans | Lactic Acid | Microscopy, Electron | Nanoparticles | Particle Size | Polyglycolic Acid | Polylactic Acid-Polyglycolic Acid Copolymer | Spectroscopy, Fourier Transform Infrared | Surface Properties | Tamoxifen [SUMMARY] | [CONTENT] Antineoplastic Agents | Breast Neoplasms | Cell Line, Tumor | Cell Survival | Emulsions | Female | Humans | Lactic Acid | Microscopy, Electron | Nanoparticles | Particle Size | Polyglycolic Acid | Polylactic Acid-Polyglycolic Acid Copolymer | Spectroscopy, Fourier Transform Infrared | Surface Properties | Tamoxifen [SUMMARY] | [CONTENT] Antineoplastic Agents | Breast Neoplasms | Cell Line, Tumor | Cell Survival | Emulsions | Female | Humans | Lactic Acid | Microscopy, Electron | Nanoparticles | Particle Size | Polyglycolic Acid | Polylactic Acid-Polyglycolic Acid Copolymer | Spectroscopy, Fourier Transform Infrared | Surface Properties | Tamoxifen [SUMMARY] | [CONTENT] Antineoplastic Agents | Breast Neoplasms | Cell Line, Tumor | Cell Survival | Emulsions | Female | Humans | Lactic Acid | Microscopy, Electron | Nanoparticles | Particle Size | Polyglycolic Acid | Polylactic Acid-Polyglycolic Acid Copolymer | Spectroscopy, Fourier Transform Infrared | Surface Properties | Tamoxifen [SUMMARY] | [CONTENT] nanoparticles penetrate tumor | nanoparticles drug respectively | polylactide co glycolide | glycolide plga polymers | drug polymers plga [SUMMARY] | [CONTENT] nanoparticles penetrate tumor | nanoparticles drug respectively | polylactide co glycolide | glycolide plga polymers | drug polymers plga [SUMMARY] | [CONTENT] nanoparticles penetrate tumor | nanoparticles drug respectively | polylactide co glycolide | glycolide plga polymers | drug polymers plga [SUMMARY] | [CONTENT] nanoparticles penetrate tumor | nanoparticles drug respectively | polylactide co glycolide | glycolide plga polymers | drug polymers plga [SUMMARY] | [CONTENT] nanoparticles penetrate tumor | nanoparticles drug respectively | polylactide co glycolide | glycolide plga polymers | drug polymers plga [SUMMARY] | [CONTENT] nanoparticles penetrate tumor | nanoparticles drug respectively | polylactide co glycolide | glycolide plga polymers | drug polymers plga [SUMMARY] | [CONTENT] drug | nanoparticles | release | tamoxifen | citrate | tamoxifen citrate | respectively | drug release | tnps | cells [SUMMARY] | [CONTENT] drug | nanoparticles | release | tamoxifen | citrate | tamoxifen citrate | respectively | drug release | tnps | cells [SUMMARY] | [CONTENT] drug | nanoparticles | release | tamoxifen | citrate | tamoxifen citrate | 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uptake nanoparticles | warranted nanoparticles | drug loading drug release [SUMMARY] | [CONTENT] drug | nanoparticles | cells | release | respectively | tamoxifen | formulations | citrate | tamoxifen citrate | tnps [SUMMARY] | [CONTENT] drug | nanoparticles | cells | release | respectively | tamoxifen | formulations | citrate | tamoxifen citrate | tnps [SUMMARY] | [CONTENT] Four | Tamoxifen | PLGA ||| Michigan Cancer | MCF-7 [SUMMARY] | [CONTENT] ||| Fourier | Zetasizer Nano ||| [SUMMARY] | [CONTENT] ||| 250-380 ||| 1.5%±0.02% | 2.68%±0.5% | 4.09%±0.2% | w/w | 27.16%±2.08% ||| up to 60 days ||| MCF-7 ||| ||| [SUMMARY] | [CONTENT] MCF-7 ||| [SUMMARY] | [CONTENT] Four | Tamoxifen | PLGA ||| Michigan Cancer | MCF-7 ||| ||| Fourier | Zetasizer Nano ||| ||| ||| ||| 250-380 ||| 1.5%±0.02% | 2.68%±0.5% | 4.09%±0.2% | w/w | 27.16%±2.08% ||| up to 60 days ||| MCF-7 ||| ||| ||| MCF-7 ||| [SUMMARY] | [CONTENT] Four | Tamoxifen | PLGA ||| Michigan Cancer | MCF-7 ||| ||| Fourier | Zetasizer Nano ||| ||| ||| ||| 250-380 ||| 1.5%±0.02% | 2.68%±0.5% | 4.09%±0.2% | w/w | 27.16%±2.08% ||| up to 60 days ||| MCF-7 ||| ||| ||| MCF-7 ||| [SUMMARY] |
||||||
Effect of Endobronchial Coils on Exercise Tolerance and Lung Functions in Patients with Severe Emphysema - A Retrospective Cohort Study of 48 Patients. | 34675505 | Lung volume reduction with endobronchial coils treatment (ECT), for patients with severe emphysema, has shown modest improvement in exercise capacity and lung functions in clinical trials, yet the benefit of this procedure is still unclear. | BACKGROUND | We conducted a multicenter retrospective cohort study including all patients who underwent ECT in Israel and a propensity score matched control group of patients with chronic obstructive pulmonary disease (COPD) that were treated with usual care. The primary outcome was six-minute walk test distance (6MWTD), secondary outcomes were lung function tests and patient survival. | METHODS | Overall, 46 patients were included in the ECT group. Their mean 6MWTD at baseline and at 6 and at 24 months post procedure was 331.0±101.4, 372.9±76.8 and 338.8±104.8, respectively (overall P=0.04, pairwise comparison: baseline to 6 months (P=0.1), baseline to 24 months (P=1.0)). Mean FEV1 values at baseline and at 6 and at 24 months post procedure were 0.86±0.38, 0.92±0.37 and 0.82±0.36 liters, respectively (overall P=0.003, pairwise comparison: baseline to 6 months (P=0.04), baseline to 24 months (P=0.75)). The median 6MWTD for the ECT and control groups at 24 months were 333.0 (262.5-390) and 280 (210-405), respectively (P=0.16). There was no difference in overall survival (P=0.84). Heterogenous emphysema was a significant predictor of treatment success in univariate analysis (p=0.004). | RESULTS | Lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit-harm ratio in a highly selected patient population. | CONCLUSION | [
"Bronchoscopy",
"Emphysema",
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"Humans",
"Lung",
"Pneumonectomy",
"Pulmonary Disease, Chronic Obstructive",
"Pulmonary Emphysema",
"Retrospective Studies",
"Treatment Outcome"
] | 8517418 | Introduction | Endoscopic lung volume reduction (ELVR) with endobronchial nitinol coils is a viable treatment option for patients with advanced chronic obstructive pulmonary disease (COPD).1 The RESET trial, published in 2013, was the first randomized controlled trial (RCT) to evaluate the benefit of endobronchial coils treatment (ECT). The study included 23 patients in the ECT groups and 24 patients in the control group and reported a significant improvement in St George’s Respiratory Questionnaire (SGRQ), 90 days after the last coils treatment.2 Another RCT, with a similar sample size, by Zoumot et al reported a significant improvement in SGRQ, FEV1 and six-minute walk test distance (6MWTD) one year after ECT.3 The REVOLENS trial which included 100 patients, 50 patients in each treatment group, reported a modest improvement in 6MWTD, lung functions and SGRQ at 6 and 12 months.4 The RENEW trial, which is currently, the largest RCT conducted to evaluate ECT, included 315 patients, 158 in the ECT group and 157 in the usual care group. The results showed a statistically significant improvement in 6MWTD at 12 months. Yet, this change was modest and with questionable clinical significance. Moreover, the rate of adverse events was higher in the ECT group.5 The benefit of ECT was also reported in two retrospective trials that showed an improvement in arterial blood gas and a decrease in anxiety and depression symptoms.6,7 To improve clinical outcomes, a post hoc analyses of the RENEW trial was conducted to identify baseline predictors of procedure success. The analysis showed that significant hyperinflation and quantitative CT scan analysis are important for patient selection.8,9 To prospectively validate these results, the ELEVATE study was designed to include patients with significant hyperinflation (residual volume (RV) > 200%). The trial, that was stopped prematurely by the sponsor, included 120 patients, 80 in the ECT group and 40 in the usual care group, reported a clinically significant improvement in lung function and quality of life at six months. Nevertheless, with a higher probability of serious adverse events.10 Since clinical trials published to date, reported a modest improvement in lung functions with a higher risk of adverse events and long-term data is sparse, the benefit of ECT is yet undetermined. In this trial, we present the Israeli long-term experience with ECT in an effort to contribute to the growing body of evidence regarding the benefit of this procedure. | null | null | null | null | Discussion | In this study, we have presented the Israeli experience with endoscopic lung volume reduction using endobronchial coils. The results showed that ECT generate a short-term improvement in lung functions and exercise capacity but does not create a change in the course of the disease. Slight improvement is seen after 6 months which then slowly dwindles until the patient is back to the baseline values at 12 to 24 months. When compared to the control group, the graphs that first diverged at 6 months start to converge and at 24 months both groups are again at a similar clinical state. Nevertheless, if not treated, these patients would most probably have a progression of their emphysema during the 2 observed years, which we could obviously postpone. Survival was similar between groups. These results are in agreement with the RCTs and single-arm studies conducted to evaluate ECT, which showed an improvement in lung functions within 1 year after the procedure.2–5,10–12 The results are also in agreement with the two-year follow-up of the REVOLENS study which showed that by 2 years there was no difference in 6MWTD and FEV1 between groups.13 In comparison to other methods of endoscopic lung volume reduction, a study that evaluated long-term outcomes with endobronchial valve therapy reported superior results with a higher proportion of patient maintaining the initial improvement in FEV1, RV and 6MWTD.14
The general positive effect seen in the results is not the effect seen at the individual patient level, while some patients had a significant clinical improvement after ECT, others had no clinical improvement or even deterioration. To further evaluate these results, we conducted a post hoc analysis for predictors of procedure success. Only 25% (12/46) of the patients fulfilled the requirements for treatment success. Although the number of outcomes was too small for multivariate analysis, univariate analysis showed that heterogenous emphysema and lack of supplemental oxygen dependence were predictors of procedure success (Table 4). The baseline RV was not different between the success and no success groups however, the baseline RV was quite high in the entire cohort (median 245%). Similar results were shown in a post hoc analysis of the RENEW trial that reported significant hyperinflation (residual volume ≥ 200% predicted) and Quantitative CT analysis as critical factors for patient selection.9
The overall survival was similar between groups. The median BODE score in the intervention group was 6 (IQR 5–7) and the 4-year survival was 69.6% (32/46) which is slightly higher than the historical BODE cohort that reported a survival of 57% at 4 years and in agreement with the extended follow-up of the RESET trial which reported a survival of 64.4% at 4 years.15,16
This study has several limitations, first its retrospective design. Second, lack of quality-of-life evaluation and third, incomplete individual patient data in various time periods which reduced the number of patients included in the repeated measures analysis. The control group was matched according to age, gender, baseline FEV1 and 6MWTD. However, differences were seen in RV and BODE score. These differences are probably because the ECT group was chosen according to parameters for intervention, eg, hyperinflation and severe disease, while the control group was matched from a cohort of ambulatory clinic patients.
In conclusion, lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit–harm ratio in a highly selected patient population. | [
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"Statistical Methods",
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"Discussion"
] | [
"We conducted a multicenter retrospective cohort study, inclusion criteria were all patients who underwent unilateral or bilateral endoscopic lung volume reduction procedure with endobronchial coils at Rabin and Shaare Zedek Medical Centers in Israel (RMC and SZMC), there were no exclusion criteria. We have also included a propensity score matched control group of 49 COPD patients from Rabin Medical Center ambulatory clinic. The inclusion criteria for the control group were diagnosis of COPD and data regarding pulmonary function tests. The exclusion criteria were thoracic surgical procedure (eg, lung volume reduction surgery, wedge resection, lobectomy or pneumonectomy) or endoscopic treatment for lung volume reduction. The control group was matched for age, gender, baseline FEV1 and 6MWT distance. The primary outcome was 6-minute walk test distance at 24 months post procedure in comparison to the distance at baseline and at 6 months. Secondary outcomes were change from baseline in FEV1, carbon monoxide diffusing capacity (DLCO) and residual volume (RV) within the ECT group and the change in FEV1, RV, 6MWT distance and survival in comparison to the control group. The 6MWD at baseline was calculated as the mean of the last two tests before the first procedure. The distance at 6 months was calculated as the mean of all values registered between 1 and 6 months follow-up. The value at 24 months was calculated as the mean of all values registered between 18 and 30 months. FEV1, RV and DLCO were collected as described for the primary outcome. Pulmonary function tests were measured with the Pulmonary function testing system ZAN 300 nSpire health. Lung volumes including total lung capacity (TLC) and residual volume were measured with a Pressure (Closed-Type) Plethysmograph. Diffusion capacity was measured using the single breath methods with 0.3% carbon monoxide. Survival was analyzed as time to death or lung transplant. A post hoc analysis for predictors of procedure success was added after data analysis. Treatment success was defined by two objective parameters in the 6MWT, first the 6MWTD had to improve after the procedure by 30 meters or more and second the 6MWTD at 24 months had to be longer than the baseline value. Both parameters had to be fulfilled for the procedure to be considered successful. The study was approved by the RMC institutional ethical review board (IRB number: 0785–16-RMC).",
"Dispersion variables were presented as average with standard deviation (SD) or median and interquartile range (IQR) as appropriate. The demographic and baseline variables were compared with the chi-square test, or the Mann–Whitney U-test, as appropriate. The primary outcome was analysed with general linear model and results were reported in estimated marginal means. Pairwise comparison was adjusted for multiple comparisons (Bonferroni). Only patients with data in all time points were included in the repeated measures analysis. The control group was matched to the intervention group by propensity score matching with a caliper of 0.2 for age, gender, baseline FEV1 and baseline 6MWT distance. Comparison of outcomes between the intervention and control groups was analysed with the chi-square test and the Mann–Whitney U-test. Survival analysis was analysed with the Kaplan–Meier curve and the Log rank test. A P-value of 0.05 was considered as significant. Statistical analysis was conducted with the SPSS version 27 software.",
"All procedures were completed in the bronchoscopy suite under moderate sedation using midazolam (1 mg), fentanyl (50–100 mcg) and propofol (dosing according to procedure length). First, the treated lobe and subsegments were identified. The bronchoscope was placed at the ostium of the treated subsegment. A catheter was inserted to measure the distance from the pleura in order to determine the appropriate coil length (100, 150 or 200 mm). The coil was loaded to the loading system and then deployed under fluoroscopic guidance into the desired lung segment. Between 8 and 12 coils were deployed into each lobe. At first, patients were hospitalized after the procedure for observation. However, due to the low complication rate, the procedure protocol was changed, and patients were discharged on the same day, if the chest x-ray was with no sign of pneumothorax and the patient was feeling well.",
"Overall, 39 patients from Rabin Medical Center and 7 patients from Shaare Zedek medical center were included. This cohort includes all endobronchial coils procedures performed in Israel. Thirty patients underwent bilateral coils placement, and 16 patients underwent unilateral coils placement (Table S1).\nThe mean age (SD) of the cohort was 64.1 (6.9), 29 patients were males (63.0%). The majority of patients were in GOLD stage 3 and 4 (95.6%) and the median (IQR) BODE score (BMI, obstruction, dyspnea, and exercise capacity) were 6 (5–7) (Table 1). Mean 6MWTD at baseline and at 6 and 24 months post procedure was 331.0±101.4, 372.9±76.8 and 338.8±104.8, respectively (overall P value = 0.04), Pairwise comparison of baseline to 6 and baseline to 24 months yielded P values of 0.1 and 1.0, respectively. Mean FEV1 values at baseline and at 6 and 24 months post procedure were 0.86±0.38, 0.92±0.37 and 0.82±0.36 liters (L), respectively (P=0.003). Pairwise comparison between FEV1 values at baseline to values at 6 and 24 months yielded P values of 0.04 and 0.75, respectively. Mean RV values at baseline, 6 and 24 months were 5.28±1.59, 4.91±1.43 and 5.07±1.30 L, respectively (P=0.45). Pairwise comparison between RV values at baseline to values at 6 and 24 months yielded P values of 0.6 and 1.0, respectively (Table 2 and Figure 1). Overall complication rate was low. Out of 76 procedures (30 bilateral and 16 unilateral), four patients suffered from post procedural pneumothorax and three from post procedural hemoptysis. During the post procedural follow-up period, three patients were hospitalized due to pneumonia (Table S1). There were no procedural-related deaths. The demographic and baseline clinical characteristics of the intervention and control groups were similar except for a higher baseline RV and TLC for the intervention group (Tables 1 and 3). The median (IQR) 6MWTD at 24 months was 333.0 (262.5–390) and 280 (210–405) for the ECT and control groups, respectively (P=0.16). The median FEV1 at 24 months was 0.77 (0.57–0.92) and 0.73 (0.56–1.05) for the ECT and control groups, respectively (P=0.78) and median RV at 24 months was 4.66 (3.89–6.21) and 4.22 (3.28–5.49) for the ECT and control groups, respectively (P=0.15). Median follow-up time was 42.89 months (IQR 29.9–59.66) for the ECT group and 34.9 (IQR 21.59–56.96) for the control group (P=0.19). During this time 9 patients died in the ECT group and 11 in the control group (P=0.77). Additionally, six patients had undergone lung transplantation in the ECT group and five in the control group (P=0.63) (Table 3). There was no difference in overall survival between groups (P=0.84) (Figure 2). Treatment success was seen in 25% (12/46) of patients, Heterogenous emphysema was seen in 83.3% of patients with a successful procedure in comparison to 35.5% in patients with unsuccessful procedure (p=0.004) (Table 4).Table 1Baseline Clinical and Demographic CharacteristicsMedical CenterCoils TreatmentControlP valueRMCSZMCRMCNumber of Patients39749Age (mean ± SD)64.1 ± 6.965.0±10.30.62Male gender (%)29 (63.0)31 (63.3)0.98Baseline FEV1 %pred a29.0 (23.0–38.2)31.0 (21.5–42.0)0.56Baseline DLCO %pred a39.0 (30.0–44.75)39.0 (29.0–49.0)0.58Baseline TLC %pred a127 (122–141)111.5 (105.75–126.75)0.01Residual volume mL a5.42 (4.32–5.96)3.97 (3.22–5.01)0.001GOLD stage423180.2132024225100BODE score a6 (5–7)5 (4–7)0.11Supplemental oxygen (%)8 (17.4)6 (12.8)0.53Pack years a50 (40–75)50 (40–70)0.80DM (%)7 (15.6)14 (29.8)0.10HTN (%)17 (37.8)13 (27.7)0.30IHD (%)10 (22.7)8 (17.0)0.49Note:\naData presented in median and IQR.Abbreviations: RMC, Rabin Medical Center; SZMC, Shaare Zedek Medical Center; FEV1 %pred, forced expiratory volume in the first second percent predicted; DLCO %pred, carbon monoxide diffusing capacity percent predicted; Baseline TLC %pred, total lung capacity percent predicted; BODE score, BMI, obstruction, dyspnea, and exercise capacity; DM, diabetes mellitus; HTN, hypertension; IHD, ischemic heart disease; IQR, interquartile range.\nTable 2Within Group Comparison. Repeated Measures Analysis Showing Estimated Marginal Means of Six-Minute Walk Test Distance, FEV1 and Residual Volume at Baseline, 6 Months and 24 Months in Patients After Lung Volume Reduction with Endobronchial CoilsPulmonary Function TestN*Baseline6 Months24 MonthsP valueOverallBaseline vs 6 MonthsBaseline vs 24 Months6MWTD23331.0±101.4372.9±76.8338.8±104.80.040.101.0FEV1320.86±0.380.92±0.370.82±0.360.0030.040.75RV255.28±1.594.91±1.435.07±1.300.450.601.0Note: *The analysis included only patients with data in all time points.Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume.\nTable 3Comparison of Six-Minute Walk Test Distance, FEV1, Residual Volume, Lung Transplant Recipients and Mortality Between the Endobronchial Coils and Control GroupEndobronchial Coils N=46Control N=49P value6MWTDaBaseline325.0 (196.25–390.0)342.0 (240–405)0.5824 months333.0 (262.5–390)280 (210–405)0.16FEV1aBaseline0.76 (0.61–0.97)0.82 (0.63–1.06)0.4724 months0.77 (0.57–0.92)0.73 (0.56–1.05)0.78RVaBaseline5.42 (4.32–5.96)3.97 (3.22–5.01)0.00124 months4.66 (3.89–6.21)4.22 (3.28–5.49)0.15TLCaBaseline7.83 (6.70–8.65)6.87 (5.66–7.46)0.0324 months7.01 (6.14–8.36)6.64(5.28–7.39)0.10Median follow-up time (months)a42.89 (29.90–59.66)34.9 (21.59–56.96)0.19Mortality (%)9 (20.0)11 (22.4)0.77Median survival for deceased patientsa20.27 (8.86–42.90)18.16 (16.09–22.04)0.87Lung transplant (%)6 (13.3)5 (10.2)0.63Note:\naData presented as median and IQR.Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume; TLC, total lung capacity; IQR, interquartile range.\nTable 4Post Hoc Analysis for Predictors of Procedure SuccessDemographic/Clinical PredictorSuccessP value+–n1234Age63.17±5.4964.4±7.480.48Male gender9 (75.0)20(58.8)0.26Heterogeneous emphysema10 (83.3)12 (35.3)0.004Baseline residual volume (%predicted)235.5 (206.5–269.25)249.0 (218.25–277.5)0.39Baseline FEV1 (%predicted)35 (24.75–38.75)26.5 (22.75–36.75)0.23Supplemental oxygen08 (23.5)0.07Abbreviation: FEV1, forced expiratory volume in the first second.\nFigure 1Six-minute walk test distance (6MWTD), FEV1 and residual volume (RV) values for the intervention and control groups at baseline and at 6 and 24 months post procedure (intervention) or since follow-up commencement (control). Values for the control group are presented in Table S2.Figure 2Kaplan–Meier Curve presenting overall survival (death or lung transplant) for the intervention and control groups. Log Rank value is presented at the bottom of the graph.\nBaseline Clinical and Demographic Characteristics\nNote:\naData presented in median and IQR.\nAbbreviations: RMC, Rabin Medical Center; SZMC, Shaare Zedek Medical Center; FEV1 %pred, forced expiratory volume in the first second percent predicted; DLCO %pred, carbon monoxide diffusing capacity percent predicted; Baseline TLC %pred, total lung capacity percent predicted; BODE score, BMI, obstruction, dyspnea, and exercise capacity; DM, diabetes mellitus; HTN, hypertension; IHD, ischemic heart disease; IQR, interquartile range.\nWithin Group Comparison. Repeated Measures Analysis Showing Estimated Marginal Means of Six-Minute Walk Test Distance, FEV1 and Residual Volume at Baseline, 6 Months and 24 Months in Patients After Lung Volume Reduction with Endobronchial Coils\nNote: *The analysis included only patients with data in all time points.\nAbbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume.\nComparison of Six-Minute Walk Test Distance, FEV1, Residual Volume, Lung Transplant Recipients and Mortality Between the Endobronchial Coils and Control Group\nNote:\naData presented as median and IQR.\nAbbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume; TLC, total lung capacity; IQR, interquartile range.\nPost Hoc Analysis for Predictors of Procedure Success\nAbbreviation: FEV1, forced expiratory volume in the first second.\nSix-minute walk test distance (6MWTD), FEV1 and residual volume (RV) values for the intervention and control groups at baseline and at 6 and 24 months post procedure (intervention) or since follow-up commencement (control). Values for the control group are presented in Table S2.\nKaplan–Meier Curve presenting overall survival (death or lung transplant) for the intervention and control groups. Log Rank value is presented at the bottom of the graph.",
"In this study, we have presented the Israeli experience with endoscopic lung volume reduction using endobronchial coils. The results showed that ECT generate a short-term improvement in lung functions and exercise capacity but does not create a change in the course of the disease. Slight improvement is seen after 6 months which then slowly dwindles until the patient is back to the baseline values at 12 to 24 months. When compared to the control group, the graphs that first diverged at 6 months start to converge and at 24 months both groups are again at a similar clinical state. Nevertheless, if not treated, these patients would most probably have a progression of their emphysema during the 2 observed years, which we could obviously postpone. Survival was similar between groups. These results are in agreement with the RCTs and single-arm studies conducted to evaluate ECT, which showed an improvement in lung functions within 1 year after the procedure.2–5,10–12 The results are also in agreement with the two-year follow-up of the REVOLENS study which showed that by 2 years there was no difference in 6MWTD and FEV1 between groups.13 In comparison to other methods of endoscopic lung volume reduction, a study that evaluated long-term outcomes with endobronchial valve therapy reported superior results with a higher proportion of patient maintaining the initial improvement in FEV1, RV and 6MWTD.14\nThe general positive effect seen in the results is not the effect seen at the individual patient level, while some patients had a significant clinical improvement after ECT, others had no clinical improvement or even deterioration. To further evaluate these results, we conducted a post hoc analysis for predictors of procedure success. Only 25% (12/46) of the patients fulfilled the requirements for treatment success. Although the number of outcomes was too small for multivariate analysis, univariate analysis showed that heterogenous emphysema and lack of supplemental oxygen dependence were predictors of procedure success (Table 4). The baseline RV was not different between the success and no success groups however, the baseline RV was quite high in the entire cohort (median 245%). Similar results were shown in a post hoc analysis of the RENEW trial that reported significant hyperinflation (residual volume ≥ 200% predicted) and Quantitative CT analysis as critical factors for patient selection.9\nThe overall survival was similar between groups. The median BODE score in the intervention group was 6 (IQR 5–7) and the 4-year survival was 69.6% (32/46) which is slightly higher than the historical BODE cohort that reported a survival of 57% at 4 years and in agreement with the extended follow-up of the RESET trial which reported a survival of 64.4% at 4 years.15,16\nThis study has several limitations, first its retrospective design. Second, lack of quality-of-life evaluation and third, incomplete individual patient data in various time periods which reduced the number of patients included in the repeated measures analysis. The control group was matched according to age, gender, baseline FEV1 and 6MWTD. However, differences were seen in RV and BODE score. These differences are probably because the ECT group was chosen according to parameters for intervention, eg, hyperinflation and severe disease, while the control group was matched from a cohort of ambulatory clinic patients.\nIn conclusion, lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit–harm ratio in a highly selected patient population."
] | [
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Statistical Methods",
"Procedure Flow",
"Results",
"Discussion"
] | [
"Endoscopic lung volume reduction (ELVR) with endobronchial nitinol coils is a viable treatment option for patients with advanced chronic obstructive pulmonary disease (COPD).1 The RESET trial, published in 2013, was the first randomized controlled trial (RCT) to evaluate the benefit of endobronchial coils treatment (ECT). The study included 23 patients in the ECT groups and 24 patients in the control group and reported a significant improvement in St George’s Respiratory Questionnaire (SGRQ), 90 days after the last coils treatment.2 Another RCT, with a similar sample size, by Zoumot et al reported a significant improvement in SGRQ, FEV1 and six-minute walk test distance (6MWTD) one year after ECT.3 The REVOLENS trial which included 100 patients, 50 patients in each treatment group, reported a modest improvement in 6MWTD, lung functions and SGRQ at 6 and 12 months.4 The RENEW trial, which is currently, the largest RCT conducted to evaluate ECT, included 315 patients, 158 in the ECT group and 157 in the usual care group. The results showed a statistically significant improvement in 6MWTD at 12 months. Yet, this change was modest and with questionable clinical significance. Moreover, the rate of adverse events was higher in the ECT group.5 The benefit of ECT was also reported in two retrospective trials that showed an improvement in arterial blood gas and a decrease in anxiety and depression symptoms.6,7 To improve clinical outcomes, a post hoc analyses of the RENEW trial was conducted to identify baseline predictors of procedure success. The analysis showed that significant hyperinflation and quantitative CT scan analysis are important for patient selection.8,9 To prospectively validate these results, the ELEVATE study was designed to include patients with significant hyperinflation (residual volume (RV) > 200%). The trial, that was stopped prematurely by the sponsor, included 120 patients, 80 in the ECT group and 40 in the usual care group, reported a clinically significant improvement in lung function and quality of life at six months. Nevertheless, with a higher probability of serious adverse events.10 Since clinical trials published to date, reported a modest improvement in lung functions with a higher risk of adverse events and long-term data is sparse, the benefit of ECT is yet undetermined. In this trial, we present the Israeli long-term experience with ECT in an effort to contribute to the growing body of evidence regarding the benefit of this procedure.",
"We conducted a multicenter retrospective cohort study, inclusion criteria were all patients who underwent unilateral or bilateral endoscopic lung volume reduction procedure with endobronchial coils at Rabin and Shaare Zedek Medical Centers in Israel (RMC and SZMC), there were no exclusion criteria. We have also included a propensity score matched control group of 49 COPD patients from Rabin Medical Center ambulatory clinic. The inclusion criteria for the control group were diagnosis of COPD and data regarding pulmonary function tests. The exclusion criteria were thoracic surgical procedure (eg, lung volume reduction surgery, wedge resection, lobectomy or pneumonectomy) or endoscopic treatment for lung volume reduction. The control group was matched for age, gender, baseline FEV1 and 6MWT distance. The primary outcome was 6-minute walk test distance at 24 months post procedure in comparison to the distance at baseline and at 6 months. Secondary outcomes were change from baseline in FEV1, carbon monoxide diffusing capacity (DLCO) and residual volume (RV) within the ECT group and the change in FEV1, RV, 6MWT distance and survival in comparison to the control group. The 6MWD at baseline was calculated as the mean of the last two tests before the first procedure. The distance at 6 months was calculated as the mean of all values registered between 1 and 6 months follow-up. The value at 24 months was calculated as the mean of all values registered between 18 and 30 months. FEV1, RV and DLCO were collected as described for the primary outcome. Pulmonary function tests were measured with the Pulmonary function testing system ZAN 300 nSpire health. Lung volumes including total lung capacity (TLC) and residual volume were measured with a Pressure (Closed-Type) Plethysmograph. Diffusion capacity was measured using the single breath methods with 0.3% carbon monoxide. Survival was analyzed as time to death or lung transplant. A post hoc analysis for predictors of procedure success was added after data analysis. Treatment success was defined by two objective parameters in the 6MWT, first the 6MWTD had to improve after the procedure by 30 meters or more and second the 6MWTD at 24 months had to be longer than the baseline value. Both parameters had to be fulfilled for the procedure to be considered successful. The study was approved by the RMC institutional ethical review board (IRB number: 0785–16-RMC).",
"Dispersion variables were presented as average with standard deviation (SD) or median and interquartile range (IQR) as appropriate. The demographic and baseline variables were compared with the chi-square test, or the Mann–Whitney U-test, as appropriate. The primary outcome was analysed with general linear model and results were reported in estimated marginal means. Pairwise comparison was adjusted for multiple comparisons (Bonferroni). Only patients with data in all time points were included in the repeated measures analysis. The control group was matched to the intervention group by propensity score matching with a caliper of 0.2 for age, gender, baseline FEV1 and baseline 6MWT distance. Comparison of outcomes between the intervention and control groups was analysed with the chi-square test and the Mann–Whitney U-test. Survival analysis was analysed with the Kaplan–Meier curve and the Log rank test. A P-value of 0.05 was considered as significant. Statistical analysis was conducted with the SPSS version 27 software.",
"All procedures were completed in the bronchoscopy suite under moderate sedation using midazolam (1 mg), fentanyl (50–100 mcg) and propofol (dosing according to procedure length). First, the treated lobe and subsegments were identified. The bronchoscope was placed at the ostium of the treated subsegment. A catheter was inserted to measure the distance from the pleura in order to determine the appropriate coil length (100, 150 or 200 mm). The coil was loaded to the loading system and then deployed under fluoroscopic guidance into the desired lung segment. Between 8 and 12 coils were deployed into each lobe. At first, patients were hospitalized after the procedure for observation. However, due to the low complication rate, the procedure protocol was changed, and patients were discharged on the same day, if the chest x-ray was with no sign of pneumothorax and the patient was feeling well.",
"Overall, 39 patients from Rabin Medical Center and 7 patients from Shaare Zedek medical center were included. This cohort includes all endobronchial coils procedures performed in Israel. Thirty patients underwent bilateral coils placement, and 16 patients underwent unilateral coils placement (Table S1).\nThe mean age (SD) of the cohort was 64.1 (6.9), 29 patients were males (63.0%). The majority of patients were in GOLD stage 3 and 4 (95.6%) and the median (IQR) BODE score (BMI, obstruction, dyspnea, and exercise capacity) were 6 (5–7) (Table 1). Mean 6MWTD at baseline and at 6 and 24 months post procedure was 331.0±101.4, 372.9±76.8 and 338.8±104.8, respectively (overall P value = 0.04), Pairwise comparison of baseline to 6 and baseline to 24 months yielded P values of 0.1 and 1.0, respectively. Mean FEV1 values at baseline and at 6 and 24 months post procedure were 0.86±0.38, 0.92±0.37 and 0.82±0.36 liters (L), respectively (P=0.003). Pairwise comparison between FEV1 values at baseline to values at 6 and 24 months yielded P values of 0.04 and 0.75, respectively. Mean RV values at baseline, 6 and 24 months were 5.28±1.59, 4.91±1.43 and 5.07±1.30 L, respectively (P=0.45). Pairwise comparison between RV values at baseline to values at 6 and 24 months yielded P values of 0.6 and 1.0, respectively (Table 2 and Figure 1). Overall complication rate was low. Out of 76 procedures (30 bilateral and 16 unilateral), four patients suffered from post procedural pneumothorax and three from post procedural hemoptysis. During the post procedural follow-up period, three patients were hospitalized due to pneumonia (Table S1). There were no procedural-related deaths. The demographic and baseline clinical characteristics of the intervention and control groups were similar except for a higher baseline RV and TLC for the intervention group (Tables 1 and 3). The median (IQR) 6MWTD at 24 months was 333.0 (262.5–390) and 280 (210–405) for the ECT and control groups, respectively (P=0.16). The median FEV1 at 24 months was 0.77 (0.57–0.92) and 0.73 (0.56–1.05) for the ECT and control groups, respectively (P=0.78) and median RV at 24 months was 4.66 (3.89–6.21) and 4.22 (3.28–5.49) for the ECT and control groups, respectively (P=0.15). Median follow-up time was 42.89 months (IQR 29.9–59.66) for the ECT group and 34.9 (IQR 21.59–56.96) for the control group (P=0.19). During this time 9 patients died in the ECT group and 11 in the control group (P=0.77). Additionally, six patients had undergone lung transplantation in the ECT group and five in the control group (P=0.63) (Table 3). There was no difference in overall survival between groups (P=0.84) (Figure 2). Treatment success was seen in 25% (12/46) of patients, Heterogenous emphysema was seen in 83.3% of patients with a successful procedure in comparison to 35.5% in patients with unsuccessful procedure (p=0.004) (Table 4).Table 1Baseline Clinical and Demographic CharacteristicsMedical CenterCoils TreatmentControlP valueRMCSZMCRMCNumber of Patients39749Age (mean ± SD)64.1 ± 6.965.0±10.30.62Male gender (%)29 (63.0)31 (63.3)0.98Baseline FEV1 %pred a29.0 (23.0–38.2)31.0 (21.5–42.0)0.56Baseline DLCO %pred a39.0 (30.0–44.75)39.0 (29.0–49.0)0.58Baseline TLC %pred a127 (122–141)111.5 (105.75–126.75)0.01Residual volume mL a5.42 (4.32–5.96)3.97 (3.22–5.01)0.001GOLD stage423180.2132024225100BODE score a6 (5–7)5 (4–7)0.11Supplemental oxygen (%)8 (17.4)6 (12.8)0.53Pack years a50 (40–75)50 (40–70)0.80DM (%)7 (15.6)14 (29.8)0.10HTN (%)17 (37.8)13 (27.7)0.30IHD (%)10 (22.7)8 (17.0)0.49Note:\naData presented in median and IQR.Abbreviations: RMC, Rabin Medical Center; SZMC, Shaare Zedek Medical Center; FEV1 %pred, forced expiratory volume in the first second percent predicted; DLCO %pred, carbon monoxide diffusing capacity percent predicted; Baseline TLC %pred, total lung capacity percent predicted; BODE score, BMI, obstruction, dyspnea, and exercise capacity; DM, diabetes mellitus; HTN, hypertension; IHD, ischemic heart disease; IQR, interquartile range.\nTable 2Within Group Comparison. Repeated Measures Analysis Showing Estimated Marginal Means of Six-Minute Walk Test Distance, FEV1 and Residual Volume at Baseline, 6 Months and 24 Months in Patients After Lung Volume Reduction with Endobronchial CoilsPulmonary Function TestN*Baseline6 Months24 MonthsP valueOverallBaseline vs 6 MonthsBaseline vs 24 Months6MWTD23331.0±101.4372.9±76.8338.8±104.80.040.101.0FEV1320.86±0.380.92±0.370.82±0.360.0030.040.75RV255.28±1.594.91±1.435.07±1.300.450.601.0Note: *The analysis included only patients with data in all time points.Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume.\nTable 3Comparison of Six-Minute Walk Test Distance, FEV1, Residual Volume, Lung Transplant Recipients and Mortality Between the Endobronchial Coils and Control GroupEndobronchial Coils N=46Control N=49P value6MWTDaBaseline325.0 (196.25–390.0)342.0 (240–405)0.5824 months333.0 (262.5–390)280 (210–405)0.16FEV1aBaseline0.76 (0.61–0.97)0.82 (0.63–1.06)0.4724 months0.77 (0.57–0.92)0.73 (0.56–1.05)0.78RVaBaseline5.42 (4.32–5.96)3.97 (3.22–5.01)0.00124 months4.66 (3.89–6.21)4.22 (3.28–5.49)0.15TLCaBaseline7.83 (6.70–8.65)6.87 (5.66–7.46)0.0324 months7.01 (6.14–8.36)6.64(5.28–7.39)0.10Median follow-up time (months)a42.89 (29.90–59.66)34.9 (21.59–56.96)0.19Mortality (%)9 (20.0)11 (22.4)0.77Median survival for deceased patientsa20.27 (8.86–42.90)18.16 (16.09–22.04)0.87Lung transplant (%)6 (13.3)5 (10.2)0.63Note:\naData presented as median and IQR.Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume; TLC, total lung capacity; IQR, interquartile range.\nTable 4Post Hoc Analysis for Predictors of Procedure SuccessDemographic/Clinical PredictorSuccessP value+–n1234Age63.17±5.4964.4±7.480.48Male gender9 (75.0)20(58.8)0.26Heterogeneous emphysema10 (83.3)12 (35.3)0.004Baseline residual volume (%predicted)235.5 (206.5–269.25)249.0 (218.25–277.5)0.39Baseline FEV1 (%predicted)35 (24.75–38.75)26.5 (22.75–36.75)0.23Supplemental oxygen08 (23.5)0.07Abbreviation: FEV1, forced expiratory volume in the first second.\nFigure 1Six-minute walk test distance (6MWTD), FEV1 and residual volume (RV) values for the intervention and control groups at baseline and at 6 and 24 months post procedure (intervention) or since follow-up commencement (control). Values for the control group are presented in Table S2.Figure 2Kaplan–Meier Curve presenting overall survival (death or lung transplant) for the intervention and control groups. Log Rank value is presented at the bottom of the graph.\nBaseline Clinical and Demographic Characteristics\nNote:\naData presented in median and IQR.\nAbbreviations: RMC, Rabin Medical Center; SZMC, Shaare Zedek Medical Center; FEV1 %pred, forced expiratory volume in the first second percent predicted; DLCO %pred, carbon monoxide diffusing capacity percent predicted; Baseline TLC %pred, total lung capacity percent predicted; BODE score, BMI, obstruction, dyspnea, and exercise capacity; DM, diabetes mellitus; HTN, hypertension; IHD, ischemic heart disease; IQR, interquartile range.\nWithin Group Comparison. Repeated Measures Analysis Showing Estimated Marginal Means of Six-Minute Walk Test Distance, FEV1 and Residual Volume at Baseline, 6 Months and 24 Months in Patients After Lung Volume Reduction with Endobronchial Coils\nNote: *The analysis included only patients with data in all time points.\nAbbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume.\nComparison of Six-Minute Walk Test Distance, FEV1, Residual Volume, Lung Transplant Recipients and Mortality Between the Endobronchial Coils and Control Group\nNote:\naData presented as median and IQR.\nAbbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume; TLC, total lung capacity; IQR, interquartile range.\nPost Hoc Analysis for Predictors of Procedure Success\nAbbreviation: FEV1, forced expiratory volume in the first second.\nSix-minute walk test distance (6MWTD), FEV1 and residual volume (RV) values for the intervention and control groups at baseline and at 6 and 24 months post procedure (intervention) or since follow-up commencement (control). Values for the control group are presented in Table S2.\nKaplan–Meier Curve presenting overall survival (death or lung transplant) for the intervention and control groups. Log Rank value is presented at the bottom of the graph.",
"In this study, we have presented the Israeli experience with endoscopic lung volume reduction using endobronchial coils. The results showed that ECT generate a short-term improvement in lung functions and exercise capacity but does not create a change in the course of the disease. Slight improvement is seen after 6 months which then slowly dwindles until the patient is back to the baseline values at 12 to 24 months. When compared to the control group, the graphs that first diverged at 6 months start to converge and at 24 months both groups are again at a similar clinical state. Nevertheless, if not treated, these patients would most probably have a progression of their emphysema during the 2 observed years, which we could obviously postpone. Survival was similar between groups. These results are in agreement with the RCTs and single-arm studies conducted to evaluate ECT, which showed an improvement in lung functions within 1 year after the procedure.2–5,10–12 The results are also in agreement with the two-year follow-up of the REVOLENS study which showed that by 2 years there was no difference in 6MWTD and FEV1 between groups.13 In comparison to other methods of endoscopic lung volume reduction, a study that evaluated long-term outcomes with endobronchial valve therapy reported superior results with a higher proportion of patient maintaining the initial improvement in FEV1, RV and 6MWTD.14\nThe general positive effect seen in the results is not the effect seen at the individual patient level, while some patients had a significant clinical improvement after ECT, others had no clinical improvement or even deterioration. To further evaluate these results, we conducted a post hoc analysis for predictors of procedure success. Only 25% (12/46) of the patients fulfilled the requirements for treatment success. Although the number of outcomes was too small for multivariate analysis, univariate analysis showed that heterogenous emphysema and lack of supplemental oxygen dependence were predictors of procedure success (Table 4). The baseline RV was not different between the success and no success groups however, the baseline RV was quite high in the entire cohort (median 245%). Similar results were shown in a post hoc analysis of the RENEW trial that reported significant hyperinflation (residual volume ≥ 200% predicted) and Quantitative CT analysis as critical factors for patient selection.9\nThe overall survival was similar between groups. The median BODE score in the intervention group was 6 (IQR 5–7) and the 4-year survival was 69.6% (32/46) which is slightly higher than the historical BODE cohort that reported a survival of 57% at 4 years and in agreement with the extended follow-up of the RESET trial which reported a survival of 64.4% at 4 years.15,16\nThis study has several limitations, first its retrospective design. Second, lack of quality-of-life evaluation and third, incomplete individual patient data in various time periods which reduced the number of patients included in the repeated measures analysis. The control group was matched according to age, gender, baseline FEV1 and 6MWTD. However, differences were seen in RV and BODE score. These differences are probably because the ECT group was chosen according to parameters for intervention, eg, hyperinflation and severe disease, while the control group was matched from a cohort of ambulatory clinic patients.\nIn conclusion, lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit–harm ratio in a highly selected patient population."
] | [
"intro",
null,
null,
null,
null,
null
] | [
"emphysema",
"endobronchial coils",
"lung volume reduction",
"six-minute walk",
"residual volume",
"FEV1",
"survival"
] | Introduction:
Endoscopic lung volume reduction (ELVR) with endobronchial nitinol coils is a viable treatment option for patients with advanced chronic obstructive pulmonary disease (COPD).1 The RESET trial, published in 2013, was the first randomized controlled trial (RCT) to evaluate the benefit of endobronchial coils treatment (ECT). The study included 23 patients in the ECT groups and 24 patients in the control group and reported a significant improvement in St George’s Respiratory Questionnaire (SGRQ), 90 days after the last coils treatment.2 Another RCT, with a similar sample size, by Zoumot et al reported a significant improvement in SGRQ, FEV1 and six-minute walk test distance (6MWTD) one year after ECT.3 The REVOLENS trial which included 100 patients, 50 patients in each treatment group, reported a modest improvement in 6MWTD, lung functions and SGRQ at 6 and 12 months.4 The RENEW trial, which is currently, the largest RCT conducted to evaluate ECT, included 315 patients, 158 in the ECT group and 157 in the usual care group. The results showed a statistically significant improvement in 6MWTD at 12 months. Yet, this change was modest and with questionable clinical significance. Moreover, the rate of adverse events was higher in the ECT group.5 The benefit of ECT was also reported in two retrospective trials that showed an improvement in arterial blood gas and a decrease in anxiety and depression symptoms.6,7 To improve clinical outcomes, a post hoc analyses of the RENEW trial was conducted to identify baseline predictors of procedure success. The analysis showed that significant hyperinflation and quantitative CT scan analysis are important for patient selection.8,9 To prospectively validate these results, the ELEVATE study was designed to include patients with significant hyperinflation (residual volume (RV) > 200%). The trial, that was stopped prematurely by the sponsor, included 120 patients, 80 in the ECT group and 40 in the usual care group, reported a clinically significant improvement in lung function and quality of life at six months. Nevertheless, with a higher probability of serious adverse events.10 Since clinical trials published to date, reported a modest improvement in lung functions with a higher risk of adverse events and long-term data is sparse, the benefit of ECT is yet undetermined. In this trial, we present the Israeli long-term experience with ECT in an effort to contribute to the growing body of evidence regarding the benefit of this procedure.
Methods:
We conducted a multicenter retrospective cohort study, inclusion criteria were all patients who underwent unilateral or bilateral endoscopic lung volume reduction procedure with endobronchial coils at Rabin and Shaare Zedek Medical Centers in Israel (RMC and SZMC), there were no exclusion criteria. We have also included a propensity score matched control group of 49 COPD patients from Rabin Medical Center ambulatory clinic. The inclusion criteria for the control group were diagnosis of COPD and data regarding pulmonary function tests. The exclusion criteria were thoracic surgical procedure (eg, lung volume reduction surgery, wedge resection, lobectomy or pneumonectomy) or endoscopic treatment for lung volume reduction. The control group was matched for age, gender, baseline FEV1 and 6MWT distance. The primary outcome was 6-minute walk test distance at 24 months post procedure in comparison to the distance at baseline and at 6 months. Secondary outcomes were change from baseline in FEV1, carbon monoxide diffusing capacity (DLCO) and residual volume (RV) within the ECT group and the change in FEV1, RV, 6MWT distance and survival in comparison to the control group. The 6MWD at baseline was calculated as the mean of the last two tests before the first procedure. The distance at 6 months was calculated as the mean of all values registered between 1 and 6 months follow-up. The value at 24 months was calculated as the mean of all values registered between 18 and 30 months. FEV1, RV and DLCO were collected as described for the primary outcome. Pulmonary function tests were measured with the Pulmonary function testing system ZAN 300 nSpire health. Lung volumes including total lung capacity (TLC) and residual volume were measured with a Pressure (Closed-Type) Plethysmograph. Diffusion capacity was measured using the single breath methods with 0.3% carbon monoxide. Survival was analyzed as time to death or lung transplant. A post hoc analysis for predictors of procedure success was added after data analysis. Treatment success was defined by two objective parameters in the 6MWT, first the 6MWTD had to improve after the procedure by 30 meters or more and second the 6MWTD at 24 months had to be longer than the baseline value. Both parameters had to be fulfilled for the procedure to be considered successful. The study was approved by the RMC institutional ethical review board (IRB number: 0785–16-RMC).
Statistical Methods:
Dispersion variables were presented as average with standard deviation (SD) or median and interquartile range (IQR) as appropriate. The demographic and baseline variables were compared with the chi-square test, or the Mann–Whitney U-test, as appropriate. The primary outcome was analysed with general linear model and results were reported in estimated marginal means. Pairwise comparison was adjusted for multiple comparisons (Bonferroni). Only patients with data in all time points were included in the repeated measures analysis. The control group was matched to the intervention group by propensity score matching with a caliper of 0.2 for age, gender, baseline FEV1 and baseline 6MWT distance. Comparison of outcomes between the intervention and control groups was analysed with the chi-square test and the Mann–Whitney U-test. Survival analysis was analysed with the Kaplan–Meier curve and the Log rank test. A P-value of 0.05 was considered as significant. Statistical analysis was conducted with the SPSS version 27 software.
Procedure Flow:
All procedures were completed in the bronchoscopy suite under moderate sedation using midazolam (1 mg), fentanyl (50–100 mcg) and propofol (dosing according to procedure length). First, the treated lobe and subsegments were identified. The bronchoscope was placed at the ostium of the treated subsegment. A catheter was inserted to measure the distance from the pleura in order to determine the appropriate coil length (100, 150 or 200 mm). The coil was loaded to the loading system and then deployed under fluoroscopic guidance into the desired lung segment. Between 8 and 12 coils were deployed into each lobe. At first, patients were hospitalized after the procedure for observation. However, due to the low complication rate, the procedure protocol was changed, and patients were discharged on the same day, if the chest x-ray was with no sign of pneumothorax and the patient was feeling well.
Results:
Overall, 39 patients from Rabin Medical Center and 7 patients from Shaare Zedek medical center were included. This cohort includes all endobronchial coils procedures performed in Israel. Thirty patients underwent bilateral coils placement, and 16 patients underwent unilateral coils placement (Table S1).
The mean age (SD) of the cohort was 64.1 (6.9), 29 patients were males (63.0%). The majority of patients were in GOLD stage 3 and 4 (95.6%) and the median (IQR) BODE score (BMI, obstruction, dyspnea, and exercise capacity) were 6 (5–7) (Table 1). Mean 6MWTD at baseline and at 6 and 24 months post procedure was 331.0±101.4, 372.9±76.8 and 338.8±104.8, respectively (overall P value = 0.04), Pairwise comparison of baseline to 6 and baseline to 24 months yielded P values of 0.1 and 1.0, respectively. Mean FEV1 values at baseline and at 6 and 24 months post procedure were 0.86±0.38, 0.92±0.37 and 0.82±0.36 liters (L), respectively (P=0.003). Pairwise comparison between FEV1 values at baseline to values at 6 and 24 months yielded P values of 0.04 and 0.75, respectively. Mean RV values at baseline, 6 and 24 months were 5.28±1.59, 4.91±1.43 and 5.07±1.30 L, respectively (P=0.45). Pairwise comparison between RV values at baseline to values at 6 and 24 months yielded P values of 0.6 and 1.0, respectively (Table 2 and Figure 1). Overall complication rate was low. Out of 76 procedures (30 bilateral and 16 unilateral), four patients suffered from post procedural pneumothorax and three from post procedural hemoptysis. During the post procedural follow-up period, three patients were hospitalized due to pneumonia (Table S1). There were no procedural-related deaths. The demographic and baseline clinical characteristics of the intervention and control groups were similar except for a higher baseline RV and TLC for the intervention group (Tables 1 and 3). The median (IQR) 6MWTD at 24 months was 333.0 (262.5–390) and 280 (210–405) for the ECT and control groups, respectively (P=0.16). The median FEV1 at 24 months was 0.77 (0.57–0.92) and 0.73 (0.56–1.05) for the ECT and control groups, respectively (P=0.78) and median RV at 24 months was 4.66 (3.89–6.21) and 4.22 (3.28–5.49) for the ECT and control groups, respectively (P=0.15). Median follow-up time was 42.89 months (IQR 29.9–59.66) for the ECT group and 34.9 (IQR 21.59–56.96) for the control group (P=0.19). During this time 9 patients died in the ECT group and 11 in the control group (P=0.77). Additionally, six patients had undergone lung transplantation in the ECT group and five in the control group (P=0.63) (Table 3). There was no difference in overall survival between groups (P=0.84) (Figure 2). Treatment success was seen in 25% (12/46) of patients, Heterogenous emphysema was seen in 83.3% of patients with a successful procedure in comparison to 35.5% in patients with unsuccessful procedure (p=0.004) (Table 4).Table 1Baseline Clinical and Demographic CharacteristicsMedical CenterCoils TreatmentControlP valueRMCSZMCRMCNumber of Patients39749Age (mean ± SD)64.1 ± 6.965.0±10.30.62Male gender (%)29 (63.0)31 (63.3)0.98Baseline FEV1 %pred a29.0 (23.0–38.2)31.0 (21.5–42.0)0.56Baseline DLCO %pred a39.0 (30.0–44.75)39.0 (29.0–49.0)0.58Baseline TLC %pred a127 (122–141)111.5 (105.75–126.75)0.01Residual volume mL a5.42 (4.32–5.96)3.97 (3.22–5.01)0.001GOLD stage423180.2132024225100BODE score a6 (5–7)5 (4–7)0.11Supplemental oxygen (%)8 (17.4)6 (12.8)0.53Pack years a50 (40–75)50 (40–70)0.80DM (%)7 (15.6)14 (29.8)0.10HTN (%)17 (37.8)13 (27.7)0.30IHD (%)10 (22.7)8 (17.0)0.49Note:
aData presented in median and IQR.Abbreviations: RMC, Rabin Medical Center; SZMC, Shaare Zedek Medical Center; FEV1 %pred, forced expiratory volume in the first second percent predicted; DLCO %pred, carbon monoxide diffusing capacity percent predicted; Baseline TLC %pred, total lung capacity percent predicted; BODE score, BMI, obstruction, dyspnea, and exercise capacity; DM, diabetes mellitus; HTN, hypertension; IHD, ischemic heart disease; IQR, interquartile range.
Table 2Within Group Comparison. Repeated Measures Analysis Showing Estimated Marginal Means of Six-Minute Walk Test Distance, FEV1 and Residual Volume at Baseline, 6 Months and 24 Months in Patients After Lung Volume Reduction with Endobronchial CoilsPulmonary Function TestN*Baseline6 Months24 MonthsP valueOverallBaseline vs 6 MonthsBaseline vs 24 Months6MWTD23331.0±101.4372.9±76.8338.8±104.80.040.101.0FEV1320.86±0.380.92±0.370.82±0.360.0030.040.75RV255.28±1.594.91±1.435.07±1.300.450.601.0Note: *The analysis included only patients with data in all time points.Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume.
Table 3Comparison of Six-Minute Walk Test Distance, FEV1, Residual Volume, Lung Transplant Recipients and Mortality Between the Endobronchial Coils and Control GroupEndobronchial Coils N=46Control N=49P value6MWTDaBaseline325.0 (196.25–390.0)342.0 (240–405)0.5824 months333.0 (262.5–390)280 (210–405)0.16FEV1aBaseline0.76 (0.61–0.97)0.82 (0.63–1.06)0.4724 months0.77 (0.57–0.92)0.73 (0.56–1.05)0.78RVaBaseline5.42 (4.32–5.96)3.97 (3.22–5.01)0.00124 months4.66 (3.89–6.21)4.22 (3.28–5.49)0.15TLCaBaseline7.83 (6.70–8.65)6.87 (5.66–7.46)0.0324 months7.01 (6.14–8.36)6.64(5.28–7.39)0.10Median follow-up time (months)a42.89 (29.90–59.66)34.9 (21.59–56.96)0.19Mortality (%)9 (20.0)11 (22.4)0.77Median survival for deceased patientsa20.27 (8.86–42.90)18.16 (16.09–22.04)0.87Lung transplant (%)6 (13.3)5 (10.2)0.63Note:
aData presented as median and IQR.Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume; TLC, total lung capacity; IQR, interquartile range.
Table 4Post Hoc Analysis for Predictors of Procedure SuccessDemographic/Clinical PredictorSuccessP value+–n1234Age63.17±5.4964.4±7.480.48Male gender9 (75.0)20(58.8)0.26Heterogeneous emphysema10 (83.3)12 (35.3)0.004Baseline residual volume (%predicted)235.5 (206.5–269.25)249.0 (218.25–277.5)0.39Baseline FEV1 (%predicted)35 (24.75–38.75)26.5 (22.75–36.75)0.23Supplemental oxygen08 (23.5)0.07Abbreviation: FEV1, forced expiratory volume in the first second.
Figure 1Six-minute walk test distance (6MWTD), FEV1 and residual volume (RV) values for the intervention and control groups at baseline and at 6 and 24 months post procedure (intervention) or since follow-up commencement (control). Values for the control group are presented in Table S2.Figure 2Kaplan–Meier Curve presenting overall survival (death or lung transplant) for the intervention and control groups. Log Rank value is presented at the bottom of the graph.
Baseline Clinical and Demographic Characteristics
Note:
aData presented in median and IQR.
Abbreviations: RMC, Rabin Medical Center; SZMC, Shaare Zedek Medical Center; FEV1 %pred, forced expiratory volume in the first second percent predicted; DLCO %pred, carbon monoxide diffusing capacity percent predicted; Baseline TLC %pred, total lung capacity percent predicted; BODE score, BMI, obstruction, dyspnea, and exercise capacity; DM, diabetes mellitus; HTN, hypertension; IHD, ischemic heart disease; IQR, interquartile range.
Within Group Comparison. Repeated Measures Analysis Showing Estimated Marginal Means of Six-Minute Walk Test Distance, FEV1 and Residual Volume at Baseline, 6 Months and 24 Months in Patients After Lung Volume Reduction with Endobronchial Coils
Note: *The analysis included only patients with data in all time points.
Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume.
Comparison of Six-Minute Walk Test Distance, FEV1, Residual Volume, Lung Transplant Recipients and Mortality Between the Endobronchial Coils and Control Group
Note:
aData presented as median and IQR.
Abbreviations: 6MWTD, six-minute walk test distance; FEV1, forced expiratory volume in the first second; RV, residual volume; TLC, total lung capacity; IQR, interquartile range.
Post Hoc Analysis for Predictors of Procedure Success
Abbreviation: FEV1, forced expiratory volume in the first second.
Six-minute walk test distance (6MWTD), FEV1 and residual volume (RV) values for the intervention and control groups at baseline and at 6 and 24 months post procedure (intervention) or since follow-up commencement (control). Values for the control group are presented in Table S2.
Kaplan–Meier Curve presenting overall survival (death or lung transplant) for the intervention and control groups. Log Rank value is presented at the bottom of the graph.
Discussion:
In this study, we have presented the Israeli experience with endoscopic lung volume reduction using endobronchial coils. The results showed that ECT generate a short-term improvement in lung functions and exercise capacity but does not create a change in the course of the disease. Slight improvement is seen after 6 months which then slowly dwindles until the patient is back to the baseline values at 12 to 24 months. When compared to the control group, the graphs that first diverged at 6 months start to converge and at 24 months both groups are again at a similar clinical state. Nevertheless, if not treated, these patients would most probably have a progression of their emphysema during the 2 observed years, which we could obviously postpone. Survival was similar between groups. These results are in agreement with the RCTs and single-arm studies conducted to evaluate ECT, which showed an improvement in lung functions within 1 year after the procedure.2–5,10–12 The results are also in agreement with the two-year follow-up of the REVOLENS study which showed that by 2 years there was no difference in 6MWTD and FEV1 between groups.13 In comparison to other methods of endoscopic lung volume reduction, a study that evaluated long-term outcomes with endobronchial valve therapy reported superior results with a higher proportion of patient maintaining the initial improvement in FEV1, RV and 6MWTD.14
The general positive effect seen in the results is not the effect seen at the individual patient level, while some patients had a significant clinical improvement after ECT, others had no clinical improvement or even deterioration. To further evaluate these results, we conducted a post hoc analysis for predictors of procedure success. Only 25% (12/46) of the patients fulfilled the requirements for treatment success. Although the number of outcomes was too small for multivariate analysis, univariate analysis showed that heterogenous emphysema and lack of supplemental oxygen dependence were predictors of procedure success (Table 4). The baseline RV was not different between the success and no success groups however, the baseline RV was quite high in the entire cohort (median 245%). Similar results were shown in a post hoc analysis of the RENEW trial that reported significant hyperinflation (residual volume ≥ 200% predicted) and Quantitative CT analysis as critical factors for patient selection.9
The overall survival was similar between groups. The median BODE score in the intervention group was 6 (IQR 5–7) and the 4-year survival was 69.6% (32/46) which is slightly higher than the historical BODE cohort that reported a survival of 57% at 4 years and in agreement with the extended follow-up of the RESET trial which reported a survival of 64.4% at 4 years.15,16
This study has several limitations, first its retrospective design. Second, lack of quality-of-life evaluation and third, incomplete individual patient data in various time periods which reduced the number of patients included in the repeated measures analysis. The control group was matched according to age, gender, baseline FEV1 and 6MWTD. However, differences were seen in RV and BODE score. These differences are probably because the ECT group was chosen according to parameters for intervention, eg, hyperinflation and severe disease, while the control group was matched from a cohort of ambulatory clinic patients.
In conclusion, lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit–harm ratio in a highly selected patient population.
| Background:
Lung volume reduction with endobronchial coils treatment (ECT), for patients with severe emphysema, has shown modest improvement in exercise capacity and lung functions in clinical trials, yet the benefit of this procedure is still unclear.
Methods:
We conducted a multicenter retrospective cohort study including all patients who underwent ECT in Israel and a propensity score matched control group of patients with chronic obstructive pulmonary disease (COPD) that were treated with usual care. The primary outcome was six-minute walk test distance (6MWTD), secondary outcomes were lung function tests and patient survival.
Results:
Overall, 46 patients were included in the ECT group. Their mean 6MWTD at baseline and at 6 and at 24 months post procedure was 331.0±101.4, 372.9±76.8 and 338.8±104.8, respectively (overall P=0.04, pairwise comparison: baseline to 6 months (P=0.1), baseline to 24 months (P=1.0)). Mean FEV1 values at baseline and at 6 and at 24 months post procedure were 0.86±0.38, 0.92±0.37 and 0.82±0.36 liters, respectively (overall P=0.003, pairwise comparison: baseline to 6 months (P=0.04), baseline to 24 months (P=0.75)). The median 6MWTD for the ECT and control groups at 24 months were 333.0 (262.5-390) and 280 (210-405), respectively (P=0.16). There was no difference in overall survival (P=0.84). Heterogenous emphysema was a significant predictor of treatment success in univariate analysis (p=0.004).
Conclusions:
Lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit-harm ratio in a highly selected patient population. | Introduction:
Endoscopic lung volume reduction (ELVR) with endobronchial nitinol coils is a viable treatment option for patients with advanced chronic obstructive pulmonary disease (COPD).1 The RESET trial, published in 2013, was the first randomized controlled trial (RCT) to evaluate the benefit of endobronchial coils treatment (ECT). The study included 23 patients in the ECT groups and 24 patients in the control group and reported a significant improvement in St George’s Respiratory Questionnaire (SGRQ), 90 days after the last coils treatment.2 Another RCT, with a similar sample size, by Zoumot et al reported a significant improvement in SGRQ, FEV1 and six-minute walk test distance (6MWTD) one year after ECT.3 The REVOLENS trial which included 100 patients, 50 patients in each treatment group, reported a modest improvement in 6MWTD, lung functions and SGRQ at 6 and 12 months.4 The RENEW trial, which is currently, the largest RCT conducted to evaluate ECT, included 315 patients, 158 in the ECT group and 157 in the usual care group. The results showed a statistically significant improvement in 6MWTD at 12 months. Yet, this change was modest and with questionable clinical significance. Moreover, the rate of adverse events was higher in the ECT group.5 The benefit of ECT was also reported in two retrospective trials that showed an improvement in arterial blood gas and a decrease in anxiety and depression symptoms.6,7 To improve clinical outcomes, a post hoc analyses of the RENEW trial was conducted to identify baseline predictors of procedure success. The analysis showed that significant hyperinflation and quantitative CT scan analysis are important for patient selection.8,9 To prospectively validate these results, the ELEVATE study was designed to include patients with significant hyperinflation (residual volume (RV) > 200%). The trial, that was stopped prematurely by the sponsor, included 120 patients, 80 in the ECT group and 40 in the usual care group, reported a clinically significant improvement in lung function and quality of life at six months. Nevertheless, with a higher probability of serious adverse events.10 Since clinical trials published to date, reported a modest improvement in lung functions with a higher risk of adverse events and long-term data is sparse, the benefit of ECT is yet undetermined. In this trial, we present the Israeli long-term experience with ECT in an effort to contribute to the growing body of evidence regarding the benefit of this procedure.
Discussion:
In this study, we have presented the Israeli experience with endoscopic lung volume reduction using endobronchial coils. The results showed that ECT generate a short-term improvement in lung functions and exercise capacity but does not create a change in the course of the disease. Slight improvement is seen after 6 months which then slowly dwindles until the patient is back to the baseline values at 12 to 24 months. When compared to the control group, the graphs that first diverged at 6 months start to converge and at 24 months both groups are again at a similar clinical state. Nevertheless, if not treated, these patients would most probably have a progression of their emphysema during the 2 observed years, which we could obviously postpone. Survival was similar between groups. These results are in agreement with the RCTs and single-arm studies conducted to evaluate ECT, which showed an improvement in lung functions within 1 year after the procedure.2–5,10–12 The results are also in agreement with the two-year follow-up of the REVOLENS study which showed that by 2 years there was no difference in 6MWTD and FEV1 between groups.13 In comparison to other methods of endoscopic lung volume reduction, a study that evaluated long-term outcomes with endobronchial valve therapy reported superior results with a higher proportion of patient maintaining the initial improvement in FEV1, RV and 6MWTD.14
The general positive effect seen in the results is not the effect seen at the individual patient level, while some patients had a significant clinical improvement after ECT, others had no clinical improvement or even deterioration. To further evaluate these results, we conducted a post hoc analysis for predictors of procedure success. Only 25% (12/46) of the patients fulfilled the requirements for treatment success. Although the number of outcomes was too small for multivariate analysis, univariate analysis showed that heterogenous emphysema and lack of supplemental oxygen dependence were predictors of procedure success (Table 4). The baseline RV was not different between the success and no success groups however, the baseline RV was quite high in the entire cohort (median 245%). Similar results were shown in a post hoc analysis of the RENEW trial that reported significant hyperinflation (residual volume ≥ 200% predicted) and Quantitative CT analysis as critical factors for patient selection.9
The overall survival was similar between groups. The median BODE score in the intervention group was 6 (IQR 5–7) and the 4-year survival was 69.6% (32/46) which is slightly higher than the historical BODE cohort that reported a survival of 57% at 4 years and in agreement with the extended follow-up of the RESET trial which reported a survival of 64.4% at 4 years.15,16
This study has several limitations, first its retrospective design. Second, lack of quality-of-life evaluation and third, incomplete individual patient data in various time periods which reduced the number of patients included in the repeated measures analysis. The control group was matched according to age, gender, baseline FEV1 and 6MWTD. However, differences were seen in RV and BODE score. These differences are probably because the ECT group was chosen according to parameters for intervention, eg, hyperinflation and severe disease, while the control group was matched from a cohort of ambulatory clinic patients.
In conclusion, lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit–harm ratio in a highly selected patient population. | Background:
Lung volume reduction with endobronchial coils treatment (ECT), for patients with severe emphysema, has shown modest improvement in exercise capacity and lung functions in clinical trials, yet the benefit of this procedure is still unclear.
Methods:
We conducted a multicenter retrospective cohort study including all patients who underwent ECT in Israel and a propensity score matched control group of patients with chronic obstructive pulmonary disease (COPD) that were treated with usual care. The primary outcome was six-minute walk test distance (6MWTD), secondary outcomes were lung function tests and patient survival.
Results:
Overall, 46 patients were included in the ECT group. Their mean 6MWTD at baseline and at 6 and at 24 months post procedure was 331.0±101.4, 372.9±76.8 and 338.8±104.8, respectively (overall P=0.04, pairwise comparison: baseline to 6 months (P=0.1), baseline to 24 months (P=1.0)). Mean FEV1 values at baseline and at 6 and at 24 months post procedure were 0.86±0.38, 0.92±0.37 and 0.82±0.36 liters, respectively (overall P=0.003, pairwise comparison: baseline to 6 months (P=0.04), baseline to 24 months (P=0.75)). The median 6MWTD for the ECT and control groups at 24 months were 333.0 (262.5-390) and 280 (210-405), respectively (P=0.16). There was no difference in overall survival (P=0.84). Heterogenous emphysema was a significant predictor of treatment success in univariate analysis (p=0.004).
Conclusions:
Lung volume reduction with endobronchial coils may improve the exercise capacity and FEV1 of COPD patients. However, the majority of the effect was diminished after 24 months. The current state of evidence does not support regulatory approval of ECT and warrant its use only after consideration of the benefit-harm ratio in a highly selected patient population. | 3,483 | 344 | [
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||||
α1-Antitrypsin promotes SPLUNC1-mediated lung defense against Pseudomonas aeruginosa infection in mice. | 24209388 | Pseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection. | BACKGROUND | SPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2 hrs after the PA infection, and mice were sacrificed 24 hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot. | METHOD | In WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production. | RESULTS | A1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation. | CONCLUSION | [
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] | 3829673 | Background | Bacterial infection is involved in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [1]. Persistent lung Pseudomonas aeruginosa (PA) infection is one the most common causes associated with the exacerbations of CF and COPD [1-3]. One of the major challenges in the treatment of PA infection is the rising antibiotic resistance [4]. A previous study has demonstrated the therapeutic effect of α1-antitrypsin (A1AT) against PA infection in CF patients [5]. Animal studies also suggested the anti-PA effects of A1AT [5,6], but how A1AT improves host defense against PA infection remains unknown.
PA infection in the lung elicits a robust inflammatory response such as recruitment and activation of neutrophils. Neutrophil elastase (NE) is released during lung inflammation, which in turn may cause detrimental effects such as tissue destruction seen in emphysema. We have demonstrated that human NE (HNE) impairs airway epithelial defense functions against bacterial infections by degrading short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein [7]. SPLUNC1 is a 25-kDa secretory protein from large airway epithelial cells. It has been shown to exert host defense function against several strains of bacteria including PA, non-typeable Haemophilus influenzae (NTHi) and Mycoplasma pneumoniae[7-9]. However, whether NE in vivo degrades SPLUNC1 and subsequently impair lung defense against PA infection has not been investigated.
A1AT is a 52-kDa serine protease inhibitor mainly produced by the liver and in the lung by macrophages and epithelial cells. In the lung, A1AT is a potent inhibitor of neutrophil elastase protecting the lung tissue from proteolytic degradation [10]. In individuals with a genetic deficiency of A1AT there is an imbalance between neutrophil elastase and A1AT in the lung increasing the risk of developing emphysema [11]. Purified human A1AT has been used to treat A1AT deficiency by augmenting A1AT levels and inhibiting NE activity [12,13]. In the current study, we hypothesized that A1AT prevents NE-mediated SPLUNC1 degradation and subsequently enhances lung defense against PA infection. To test our hypothesis, we utilized the PA infection model in wild-type and SPLUNC1 knockout (KO) mice with or without A1AT treatment. First, we confirmed that PA infection reduces SPLUNC1 expression in mouse bronchoalveolar lavage (BAL) fluid. Second, we demonstrated that A1AT increased SPLUNC1 levels in PA-infected wild-type mice, and decreased lung PA load. Third, we found that SLPUNC1 is required to improve lung defense against PA. | Methods | Animals SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.
SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.
Pseudomonas aeruginosa culture Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).
Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).
Mouse model of PA infection with α1-antitrypsin treatment PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.
PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.
BAL and lung tissue processing Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.
Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.
Western blot analysis of mouse SPLUNC1 protein Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.
Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.
ELISA of mouse KC KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.
KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.
Neutrophil elastase (NE) activity assay in mouse BAL fluid NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.
NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.
Statistical analysis Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.
Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant. | Results | Reduced SPLUNC1, but increased NE activity in BAL fluid of PA-infected wild-type mice After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).
Pseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.
After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).
Pseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.
Therapeutic effect of A1AT on lung SPLUNC1, NE activity, bacterial load and inflammation in wild-type mice To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).
A1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.
Notably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).
A1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.
A1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).
A1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.
Notably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).
A1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.
A1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
No therapeutic effect of A1AT on lung bacterial load and inflammation in SPLUNC1 KO mice To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).
A1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).
A1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM. | Conclusion | Our current study has provided a novel mechanism underlying A1AT-mediated host defense against bacterial infection. Our research findings indicate that A1AT exerts host defense functions against PA infection in part by modulating the host defense protein SPLUNC1. | [
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"Neutrophil elastase (NE) activity assay in mouse BAL fluid",
"Statistical analysis",
"Reduced SPLUNC1, but increased NE activity in BAL fluid of PA-infected wild-type mice",
"Therapeutic effect of A1AT on lung SPLUNC1, NE activity, bacterial load and inflammation in wild-type mice",
"No therapeutic effect of A1AT on lung bacterial load and inflammation in SPLUNC1 KO mice",
"Abbreviations",
"Competing interest",
"Authors’ contributions"
] | [
"Bacterial infection is involved in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [1]. Persistent lung Pseudomonas aeruginosa (PA) infection is one the most common causes associated with the exacerbations of CF and COPD [1-3]. One of the major challenges in the treatment of PA infection is the rising antibiotic resistance [4]. A previous study has demonstrated the therapeutic effect of α1-antitrypsin (A1AT) against PA infection in CF patients [5]. Animal studies also suggested the anti-PA effects of A1AT [5,6], but how A1AT improves host defense against PA infection remains unknown.\nPA infection in the lung elicits a robust inflammatory response such as recruitment and activation of neutrophils. Neutrophil elastase (NE) is released during lung inflammation, which in turn may cause detrimental effects such as tissue destruction seen in emphysema. We have demonstrated that human NE (HNE) impairs airway epithelial defense functions against bacterial infections by degrading short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein [7]. SPLUNC1 is a 25-kDa secretory protein from large airway epithelial cells. It has been shown to exert host defense function against several strains of bacteria including PA, non-typeable Haemophilus influenzae (NTHi) and Mycoplasma pneumoniae[7-9]. However, whether NE in vivo degrades SPLUNC1 and subsequently impair lung defense against PA infection has not been investigated.\nA1AT is a 52-kDa serine protease inhibitor mainly produced by the liver and in the lung by macrophages and epithelial cells. In the lung, A1AT is a potent inhibitor of neutrophil elastase protecting the lung tissue from proteolytic degradation [10]. In individuals with a genetic deficiency of A1AT there is an imbalance between neutrophil elastase and A1AT in the lung increasing the risk of developing emphysema [11]. Purified human A1AT has been used to treat A1AT deficiency by augmenting A1AT levels and inhibiting NE activity [12,13]. In the current study, we hypothesized that A1AT prevents NE-mediated SPLUNC1 degradation and subsequently enhances lung defense against PA infection. To test our hypothesis, we utilized the PA infection model in wild-type and SPLUNC1 knockout (KO) mice with or without A1AT treatment. First, we confirmed that PA infection reduces SPLUNC1 expression in mouse bronchoalveolar lavage (BAL) fluid. Second, we demonstrated that A1AT increased SPLUNC1 levels in PA-infected wild-type mice, and decreased lung PA load. Third, we found that SLPUNC1 is required to improve lung defense against PA.",
"SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.",
"Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).",
"PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.",
"Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.",
"Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.",
"KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.",
"NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.",
"Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.",
"After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).\nPseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.",
"To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).\nA1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.\nNotably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).\nA1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.\nA1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.",
"To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).\nA1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.",
"A1AT: α1-antitrypsin; BAL: Bronchoalveolar lavage; CF: Cystic fibrosis; COPD: Chronic obstructive pulmonary disease; KO: Knockout; NE: Neutrophil elastase; NTHi: Non-typeable Haemophilus influenzae; PA: Pseudomonas aeruginosa; SPLUNC1: Short palate, lung, and nasal epithelium clone 1; WT: Wild-type.",
"All authors declare not having competing interests that might have influenced the performance or presentation of our work in this manuscript.",
"DJ contributed to the experimental designs, collected the data, carried out data analysis and drafted the manuscript. RP provided the A1AT and revised the manuscript. QW contributed to the data collection, analysis and revised the manuscript. AG contributed to the data collection and revised the manuscript. HWC contributed to the experimental designs, data analysis and critically reviewed the manuscript. All authors read and approved the final manuscript."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
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null,
null
] | [
"Background",
"Methods",
"Animals",
"Pseudomonas aeruginosa culture",
"Mouse model of PA infection with α1-antitrypsin treatment",
"BAL and lung tissue processing",
"Western blot analysis of mouse SPLUNC1 protein",
"ELISA of mouse KC",
"Neutrophil elastase (NE) activity assay in mouse BAL fluid",
"Statistical analysis",
"Results",
"Reduced SPLUNC1, but increased NE activity in BAL fluid of PA-infected wild-type mice",
"Therapeutic effect of A1AT on lung SPLUNC1, NE activity, bacterial load and inflammation in wild-type mice",
"No therapeutic effect of A1AT on lung bacterial load and inflammation in SPLUNC1 KO mice",
"Discussion",
"Conclusion",
"Abbreviations",
"Competing interest",
"Authors’ contributions"
] | [
"Bacterial infection is involved in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [1]. Persistent lung Pseudomonas aeruginosa (PA) infection is one the most common causes associated with the exacerbations of CF and COPD [1-3]. One of the major challenges in the treatment of PA infection is the rising antibiotic resistance [4]. A previous study has demonstrated the therapeutic effect of α1-antitrypsin (A1AT) against PA infection in CF patients [5]. Animal studies also suggested the anti-PA effects of A1AT [5,6], but how A1AT improves host defense against PA infection remains unknown.\nPA infection in the lung elicits a robust inflammatory response such as recruitment and activation of neutrophils. Neutrophil elastase (NE) is released during lung inflammation, which in turn may cause detrimental effects such as tissue destruction seen in emphysema. We have demonstrated that human NE (HNE) impairs airway epithelial defense functions against bacterial infections by degrading short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein [7]. SPLUNC1 is a 25-kDa secretory protein from large airway epithelial cells. It has been shown to exert host defense function against several strains of bacteria including PA, non-typeable Haemophilus influenzae (NTHi) and Mycoplasma pneumoniae[7-9]. However, whether NE in vivo degrades SPLUNC1 and subsequently impair lung defense against PA infection has not been investigated.\nA1AT is a 52-kDa serine protease inhibitor mainly produced by the liver and in the lung by macrophages and epithelial cells. In the lung, A1AT is a potent inhibitor of neutrophil elastase protecting the lung tissue from proteolytic degradation [10]. In individuals with a genetic deficiency of A1AT there is an imbalance between neutrophil elastase and A1AT in the lung increasing the risk of developing emphysema [11]. Purified human A1AT has been used to treat A1AT deficiency by augmenting A1AT levels and inhibiting NE activity [12,13]. In the current study, we hypothesized that A1AT prevents NE-mediated SPLUNC1 degradation and subsequently enhances lung defense against PA infection. To test our hypothesis, we utilized the PA infection model in wild-type and SPLUNC1 knockout (KO) mice with or without A1AT treatment. First, we confirmed that PA infection reduces SPLUNC1 expression in mouse bronchoalveolar lavage (BAL) fluid. Second, we demonstrated that A1AT increased SPLUNC1 levels in PA-infected wild-type mice, and decreased lung PA load. Third, we found that SLPUNC1 is required to improve lung defense against PA.",
" Animals SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.\nSPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.\n Pseudomonas aeruginosa culture Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).\nPseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).\n Mouse model of PA infection with α1-antitrypsin treatment PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.\nPA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.\n BAL and lung tissue processing Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.\nMouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.\n Western blot analysis of mouse SPLUNC1 protein Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.\nWestern blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.\n ELISA of mouse KC KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.\nKC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.\n Neutrophil elastase (NE) activity assay in mouse BAL fluid NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.\nNE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.\n Statistical analysis Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.\nData are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.",
"SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.",
"Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).",
"PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.",
"Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.",
"Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.",
"KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.",
"NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.",
"Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.",
" Reduced SPLUNC1, but increased NE activity in BAL fluid of PA-infected wild-type mice After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).\nPseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.\nAfter 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).\nPseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.\n Therapeutic effect of A1AT on lung SPLUNC1, NE activity, bacterial load and inflammation in wild-type mice To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).\nA1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.\nNotably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).\nA1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.\nA1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.\nTo determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).\nA1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.\nNotably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).\nA1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.\nA1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.\n No therapeutic effect of A1AT on lung bacterial load and inflammation in SPLUNC1 KO mice To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).\nA1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.\nTo determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).\nA1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.",
"After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).\nPseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.",
"To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).\nA1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.\nNotably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).\nA1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.\nA1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.",
"To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).\nA1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.",
"The current study provides a novel mechanism by which A1AT exerts host defense function against lung PA infection. By using a SPLUNC1 KO mouse model, we were able to reveal a critical role of SPLUNC1 in A1AT-mediated host defense function. First, PA infection significantly reduced SPLUNC1 expression in wild-type mice, which was accompanied by increased NE activity. Second, A1AT treatment significantly reduced lung PA load and inflammation in wild-type mice, but not in SPLUNC1 KO mice.\nPersistent lung PA infection is a significant challenge in the management of CF and COPD patients, particularly during disease acute exacerbations. Activation of airway neutrophils during bacterial infection results in the release of various mediators such as NE that degrades proteins (e.g., elastin, fibronectin), leading to disease progression [7,16]. Although previous studies suggest a therapeutic role of A1AT, an inhibitor of NE, in host defense against bacterial (e.g., PA) infection in vivo[6], the underlying mechanisms remain poorly understood. We and others have previously demonstrated that SPLUNC1 exerts host defense function against bacterial infection [7-9], and HNE can degrade secreted SPLUNC1 from airway epithelial cells [7]. We clearly showed that A1AT is effective in reducing PA load in wild-type mice, but not in SPLUNC1 KO mice. Our results suggest that SPLUNC1 is critical to A1AT-mediated host defense function against PA infection.\nThe impact of an in vivo PA infection on SPLUNC1 expression is unclear. Bingle and co-workers have found increased SPLUNC1 protein expression by airway epithelial cells of CF patients [17]. However, this study did not measure the levels of released SPLUNC1 in the airway lumen, and the association of SPLUNC1 levels with NE levels. To clarify the role of an in vivo PA infection on SPLUNC1 levels, we examined SPLUNC1 protein in BAL fluid of PA-infected mice. We, for the first time, demonstrated that PA infection reduces SPLUNC1 protein in mouse BAL fluid. The reduced SPLUNC1 may be in part explained by increased NE activity following PA infection as we have shown SPLUNC1 degradation by NE [7]. Additionally, PA-derived elastase may contribute to SPLUNC1 reduction in BAL fluid because it has been shown to degrade some innate immunity components (e.g., surfactant protein A) [18]. We speculate that during PA infection, airway epithelial cells increase intracellular SPLUNC1 expression to compensate the loss of secreted SPLUNC1 due to its degradation by proteases including NE. Although we have shown SPLUNC1 up-regulation by an atypical bacterium Mycoplasma pneumoniae[19], the direct effect of PA infection on mouse or human airway epithelial SPLUNC1 expression warrants future study.\nOne of our major research questions is about whether A1AT can rescue PA infection-induced SPLUNC1 reduction and subsequently enhance host defense against PA infection. To address this question, A1AT was aerosolized to mice 2 hrs after PA infection. This therapeutic approach clearly demonstrated that A1AT treatment restored SPLUNC1 levels in BAL fluid of wild-type mice, which was accompanied by reduced NE activity. Our results have suggested an in vivo mechanism of A1AT function. To further demonstrate the role of SPLUNC1 in A1AT therapeutic effect, we treated PA-infected SPLUNC1 KO mice with A1AT. Unlike the wild-type mice, SPLUNC1 KO mice did not show reduced lung PA load. These results emphasize that A1AT therapeutic effect during PA infection depends on SPLUNC1. Given the fact that A1AT can enter into the cells, it would be interesting to determine whether it has a direct effect on intracellular SPLUNC1 mRNA or protein expression. In our primary normal human airway epithelial cell culture experiments, we found that A1AT has no direct effects on intracellular SPLUNC1 expression. Together, A1AT functions primarily through preventing extracellular SPLUNC1 protein from degradation by NE and perhaps other proteases.\nWe found that lung pro-inflammatory cytokine KC was significantly reduced by A1AT in PA-infected wild-type mouse lungs. However, leukocytes including neutrophils were not as significantly reduced as KC. This may be explained by several factors. First, we only gave one dose of A1AT treatment in PA-infected mice. As a previous study demonstrated a significant inhibition of lung neutrophils following repeated A1AT treatments in cigarette smoke-exposed mice [20], we may need to deliver A1AT multiple times (e.g., before and after PA infection) to markedly suppress lung neutrophil recruitment. Second, A1AT has diverse NE-dependent and NE-independent functions [21]. Lastly, leukocyte numbers may not reflect cell activity. Future studies will explore the direct effects of A1AT on inflammatory cell functions.\nWe realize that SPLUNC1 KO mice demonstrated greater variability of PA load in the lung than the wild-type mice although the sample size of SPLUNC1 KO and wild-type mice is similar. Thus, it appears that bacterial load variability in our current study is related to the genetic background. We confirmed such greater variability of lung bacterial load in SPLUNC1 KO mice that were pre-treated with A1AT and then infected with PA. We found that A1AT aerosolized 2 hrs prior to PA infection decreased lung PA load in wild-type mice, but not in SPLUNC1 KO mice (Figure 6).\nA1AT pre-treatment reduces lung PA load in wild-type (WT) mice, but not in SPLUNC1 knockout (KO) mice. WT and SPLUNC1 KO mice were treated with aerosolized A1AT (0.5 mg/ml). Two hours later, mice were intranasally infected with PA (1.5 × 107 CFUs/mouse). After 24 hrs of PA infection, lung tissues were processed to quantify PA load. While A1AT significantly reduced lung PA load in WT mice (A), it did not alter the PA load in SPLUNC1 KO mice (B). N = 4 – 5 mice per group. The horizontal solid red lines indicate medians of CFUs.\nThere are several limitations to our current study. First, only one strain of bacteria (i.e., PA) was used in our study. Future studies using other strains of bacteria (e.g., NTHi) are warranted to examine the broad-spectrum effect of A1AT against lung infection. Second, an acute (e.g., 24 hr) PA infection model was carried out to address our research questions. Although we were able to demonstrate a critical role of SPLUNC1 in A1AT-mediated host defense against PA infection with an acute model, a chronic infection model will certainly be beneficial to study the role of SPLUNC1 in disease progression. Lastly, to increase the relevance of our animal research findings to patients, we plan to measure SPLUNC1 and PA in sputum and BAL samples from CF and COPD patients treated with A1AT or placebo.",
"Our current study has provided a novel mechanism underlying A1AT-mediated host defense against bacterial infection. Our research findings indicate that A1AT exerts host defense functions against PA infection in part by modulating the host defense protein SPLUNC1.",
"A1AT: α1-antitrypsin; BAL: Bronchoalveolar lavage; CF: Cystic fibrosis; COPD: Chronic obstructive pulmonary disease; KO: Knockout; NE: Neutrophil elastase; NTHi: Non-typeable Haemophilus influenzae; PA: Pseudomonas aeruginosa; SPLUNC1: Short palate, lung, and nasal epithelium clone 1; WT: Wild-type.",
"All authors declare not having competing interests that might have influenced the performance or presentation of our work in this manuscript.",
"DJ contributed to the experimental designs, collected the data, carried out data analysis and drafted the manuscript. RP provided the A1AT and revised the manuscript. QW contributed to the data collection, analysis and revised the manuscript. AG contributed to the data collection and revised the manuscript. HWC contributed to the experimental designs, data analysis and critically reviewed the manuscript. All authors read and approved the final manuscript."
] | [
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"methods",
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null,
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null,
null,
null,
null,
"results",
null,
null,
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"discussion",
"conclusions",
null,
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null
] | [
"SPLUNC1",
"α1-antitrypsin",
"Pseudomonas aeruginosa infection",
"Human neutrophil elastase"
] | Background:
Bacterial infection is involved in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [1]. Persistent lung Pseudomonas aeruginosa (PA) infection is one the most common causes associated with the exacerbations of CF and COPD [1-3]. One of the major challenges in the treatment of PA infection is the rising antibiotic resistance [4]. A previous study has demonstrated the therapeutic effect of α1-antitrypsin (A1AT) against PA infection in CF patients [5]. Animal studies also suggested the anti-PA effects of A1AT [5,6], but how A1AT improves host defense against PA infection remains unknown.
PA infection in the lung elicits a robust inflammatory response such as recruitment and activation of neutrophils. Neutrophil elastase (NE) is released during lung inflammation, which in turn may cause detrimental effects such as tissue destruction seen in emphysema. We have demonstrated that human NE (HNE) impairs airway epithelial defense functions against bacterial infections by degrading short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein [7]. SPLUNC1 is a 25-kDa secretory protein from large airway epithelial cells. It has been shown to exert host defense function against several strains of bacteria including PA, non-typeable Haemophilus influenzae (NTHi) and Mycoplasma pneumoniae[7-9]. However, whether NE in vivo degrades SPLUNC1 and subsequently impair lung defense against PA infection has not been investigated.
A1AT is a 52-kDa serine protease inhibitor mainly produced by the liver and in the lung by macrophages and epithelial cells. In the lung, A1AT is a potent inhibitor of neutrophil elastase protecting the lung tissue from proteolytic degradation [10]. In individuals with a genetic deficiency of A1AT there is an imbalance between neutrophil elastase and A1AT in the lung increasing the risk of developing emphysema [11]. Purified human A1AT has been used to treat A1AT deficiency by augmenting A1AT levels and inhibiting NE activity [12,13]. In the current study, we hypothesized that A1AT prevents NE-mediated SPLUNC1 degradation and subsequently enhances lung defense against PA infection. To test our hypothesis, we utilized the PA infection model in wild-type and SPLUNC1 knockout (KO) mice with or without A1AT treatment. First, we confirmed that PA infection reduces SPLUNC1 expression in mouse bronchoalveolar lavage (BAL) fluid. Second, we demonstrated that A1AT increased SPLUNC1 levels in PA-infected wild-type mice, and decreased lung PA load. Third, we found that SLPUNC1 is required to improve lung defense against PA.
Methods:
Animals SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.
SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.
Pseudomonas aeruginosa culture Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).
Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).
Mouse model of PA infection with α1-antitrypsin treatment PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.
PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.
BAL and lung tissue processing Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.
Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.
Western blot analysis of mouse SPLUNC1 protein Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.
Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.
ELISA of mouse KC KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.
KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.
Neutrophil elastase (NE) activity assay in mouse BAL fluid NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.
NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.
Statistical analysis Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.
Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.
Animals:
SPLUNC1 knockout (KO) and littermate control wild-type (WT) mice on the C57BL/6 background were used for the current study. SPLUNC1 KO mice were generated as we previously reported [14]. All mice were bred and housed in our biological resource center under pathogen-free conditions, and tested to establish that they were virus and M. pulmonis free. All the animal procedures were approved by the IACUC at National Jewish Health.
Pseudomonas aeruginosa culture:
Pseudomonas aeruginosa (PA) strain PAO1 was kindly provided by Dr. Michael Schurr at the University of Colorado Denver, and was stored at −80°C. For each experiment, bacteria were first streaked onto a Tryptic-Soy agar plate and cultured for 18–22 hrs at 37°C. An individual colony was then inoculated into Tryptic-Soy medium and shaked at 37°C to grow the bacteria until 1 × 108 CFUs/ml were achieved as determined by spectrophotometry (optical density at 600 nm = 0.5).
Mouse model of PA infection with α1-antitrypsin treatment:
PA (1.5 × 107 CFUs/mouse in 30 μl saline) or saline (control) was intranasally inoculated to WT and SPLUNC1 KO mice. After 2 hrs of PA infection, purified human alpha-1 proteinase inhibitor (A1AT or Prolastin-C, Grifols Inc., Research Triangle Park, NC) or bovine serum albumin (BSA, Sigma-Aldrich, as the A1AT control) were aerosolized to mice. To deliver A1AT or BSA, WT and SPLUNC1 KO mice were placed in a Plexiglas chamber and treated with aerosolized A1AT or BSA (0.5 mg/ml, total volume = 10 ml) for 30 minutes by using an ultrasonic nebulizer (De Vilbiss) at an airflow rate of 8 L/min. After 24 hrs of PA infection, lung lavage was performed to collect bronchoalveolar lavage (BAL) fluid for cell count, and measurement of KC and NE activity.
BAL and lung tissue processing:
Mouse lungs were lavaged with 1 ml of sterile saline. Cell-free BAL fluid were stored at −80°C for cytokine analysis and Western blot. Cytospins of BAL cells were stained with a Diff-Quick Kit (IMEB INC., San Marcos, CA), and leukocyte differentials were determined as percentage of 500 counted leukocytes. The left lung lobe from infected mice was homogenized in PBS, the homogenates were then cultured on Tryptic-Soy agar plates to quantify PA levels.
Western blot analysis of mouse SPLUNC1 protein:
Western blot analysis was carried out to quantify SPLUNC1 protein. In brief, 30 μl BAL fluid was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked with the Western blocking buffer, and incubated with a sheep anti-mouse SPLUNC1 antibody (R&D Systems, Inc., Minneapolis, MN) overnight at 4°C. After washes in PBS with 0.1% Tween 20, the membranes were incubated with an anti-sheep IgG conjugated to horseradish peroxidase. Membranes were stained with Ponceau S solution to normalize total protein load by comparing the 68-kDa protein (corresponding to albumin) levels. Densitometry was performed using the NIH Image-J software. The ratio of SPLUNC1/albumin was used to normalize SPLUNC1 protein expression in BAL fluid.
ELISA of mouse KC:
KC, a homolog of human IL-8, in mouse BAL fluid was determined by using a mouse KC DuoSet ELISA Development kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction.
Neutrophil elastase (NE) activity assay in mouse BAL fluid:
NE activity in mouse BAL fluid samples was measured using the substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich) [15]. The samples were mixed with an equal volume of 0.1 M HEPES buffer containing 0.5 mmol/L NaCl (pH 7.5), and then transferred to a 96-well plate. Saline solution mixed with NaCl/HEPES buffer was used as a negative control. The substrate, 2 mmol/L N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide, was added to each well. After 5 minutes of incubation, liberation of p-nitroaniline was measured at 405 nm using a spectrophotometer. The OD readings were used to reflect NE activity.
Statistical analysis:
Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons, and a Tukey’s post hoc test was applied to illustrate the significant differences between two groups. Student’s t test was used when only two groups were compared. A p value < 0.05 was considered significant.
Results:
Reduced SPLUNC1, but increased NE activity in BAL fluid of PA-infected wild-type mice After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).
Pseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.
After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).
Pseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.
Therapeutic effect of A1AT on lung SPLUNC1, NE activity, bacterial load and inflammation in wild-type mice To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).
A1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.
Notably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).
A1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.
A1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).
A1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.
Notably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).
A1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.
A1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
No therapeutic effect of A1AT on lung bacterial load and inflammation in SPLUNC1 KO mice To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).
A1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).
A1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
Reduced SPLUNC1, but increased NE activity in BAL fluid of PA-infected wild-type mice:
After 24 hrs of PA infection in wild-type mice, SPLUNC1 protein in BAL fluid was examined by Western blot analysis. As shown in Figure 1A and 1B, PA infection reduced SPLUNC1 protein expression in BAL fluid. In contrast to the SPLUNC1 data, NE activity was significantly increased after the PA infection (Figure 1C).
Pseudomonas aeruginosa (PA) infection reduces SPLUNC1 and increases neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. BAL fluid from WT mice was processed for SPLUNC1 protein Western blot. (A) – Quantitative analysis of BAL fluid SPLUNC1 protein expression normalized by albumin. (B) – Representative Western blot image of SPLUNC1 and albumin. The vertical dotted red line separates the saline group from the PA infection group. The two lanes under saline or PA treatment represent SPLUNC1 data from two different mice. (C) – NE activity was examined by an NE activity assay as described in Materials and methods. N = 4 – 5 mice per group. Data are expressed as means ± SEM.
Therapeutic effect of A1AT on lung SPLUNC1, NE activity, bacterial load and inflammation in wild-type mice:
To determine whether A1AT restores SPLUNC1 levels in PA-infected mouse lungs, A1AT was aerosolized to mice 2 hrs after an intranasal inoculation of PA. After 24 hrs of PA infection, A1AT treatment significantly increased SPLUNC1 protein levels in BAL fluid as compared to BSA treatment (Figure 2A and 2B). In line with the SPLUNC1 data, NE activity was reduced following A1AT treatment (Figure 2C).
A1AT treatment enhances SPLUNC1 and reduces NE activity in bronchoalveolar lavage (BAL) fluid of PA-infected WT mice. PA-infected mice were treated with A1AT for 22 hrs and sacrificed after 24 hrs of infection as described in Materials and Methods. Quantitative analysis (A) and representative image (B) of BAL fluid SPLUNC1 Western blot were shown to demonstrate the therapeutic effect of A1AT treatment. The two lanes of Western blot image under saline or PA treatment represent SPLUNC1 data from two different mice. NE activity (C) was examined by an NE activity assay. N = 4 – 7 mice per group. The vertical dotted red line in Figure 2B separates the saline group from the PA infection group. NS indicates no significant differences. Data are expressed as means ± SEM.
Notably, the PA load was reduced by A1AT treatment (Figure 3). Likewise, A1AT significantly reduced PA infection-induced KC (Figure 4A) and trended to reduce total leukocytes (Figure 4B) in BAL fluid. However, the total number of neutrophils in BAL fluid was not significantly reduced by A1AT (Figure 4C).
A1AT treatment reduces lung PA load in wild-type (WT) mice. Left lungs from PA-infected WT mice were homogenized, and PA load was quantified by culture. The horizontal solid red lines indicate medians of CFUs.
A1AT treatment reduces PA-induced KC level and trend to reduce total leukocytes in BAL fluid of wild-type (WT) mice. (A) – KC; (B) – total leukocytes; and (C) – BAL neutrophils; N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
No therapeutic effect of A1AT on lung bacterial load and inflammation in SPLUNC1 KO mice:
To determine if the therapeutic effect of A1AT is dependent on SPLUNC1, PA-infected SPLUNC1 KO mice were treated with A1AT or BSA. Unlike the WT mice, SPLUNC1 KO mice, following the A1AT treatment, failed to decrease lung PA load and inflammation (Figure 5A and 5B).
A1AT treatment does not reduce lung PA load and inflammation in SPLUNC1 knockout (KO) mice. (A) – PA load was quantified in homogenized left lungs. The horizontal solid red lines indicate medians of CFUs. (B) – KC levels were quantified by using an ELISA. N = 4 – 7 mice per group. NS indicates no significant differences. Data are expressed as means ± SEM.
Discussion:
The current study provides a novel mechanism by which A1AT exerts host defense function against lung PA infection. By using a SPLUNC1 KO mouse model, we were able to reveal a critical role of SPLUNC1 in A1AT-mediated host defense function. First, PA infection significantly reduced SPLUNC1 expression in wild-type mice, which was accompanied by increased NE activity. Second, A1AT treatment significantly reduced lung PA load and inflammation in wild-type mice, but not in SPLUNC1 KO mice.
Persistent lung PA infection is a significant challenge in the management of CF and COPD patients, particularly during disease acute exacerbations. Activation of airway neutrophils during bacterial infection results in the release of various mediators such as NE that degrades proteins (e.g., elastin, fibronectin), leading to disease progression [7,16]. Although previous studies suggest a therapeutic role of A1AT, an inhibitor of NE, in host defense against bacterial (e.g., PA) infection in vivo[6], the underlying mechanisms remain poorly understood. We and others have previously demonstrated that SPLUNC1 exerts host defense function against bacterial infection [7-9], and HNE can degrade secreted SPLUNC1 from airway epithelial cells [7]. We clearly showed that A1AT is effective in reducing PA load in wild-type mice, but not in SPLUNC1 KO mice. Our results suggest that SPLUNC1 is critical to A1AT-mediated host defense function against PA infection.
The impact of an in vivo PA infection on SPLUNC1 expression is unclear. Bingle and co-workers have found increased SPLUNC1 protein expression by airway epithelial cells of CF patients [17]. However, this study did not measure the levels of released SPLUNC1 in the airway lumen, and the association of SPLUNC1 levels with NE levels. To clarify the role of an in vivo PA infection on SPLUNC1 levels, we examined SPLUNC1 protein in BAL fluid of PA-infected mice. We, for the first time, demonstrated that PA infection reduces SPLUNC1 protein in mouse BAL fluid. The reduced SPLUNC1 may be in part explained by increased NE activity following PA infection as we have shown SPLUNC1 degradation by NE [7]. Additionally, PA-derived elastase may contribute to SPLUNC1 reduction in BAL fluid because it has been shown to degrade some innate immunity components (e.g., surfactant protein A) [18]. We speculate that during PA infection, airway epithelial cells increase intracellular SPLUNC1 expression to compensate the loss of secreted SPLUNC1 due to its degradation by proteases including NE. Although we have shown SPLUNC1 up-regulation by an atypical bacterium Mycoplasma pneumoniae[19], the direct effect of PA infection on mouse or human airway epithelial SPLUNC1 expression warrants future study.
One of our major research questions is about whether A1AT can rescue PA infection-induced SPLUNC1 reduction and subsequently enhance host defense against PA infection. To address this question, A1AT was aerosolized to mice 2 hrs after PA infection. This therapeutic approach clearly demonstrated that A1AT treatment restored SPLUNC1 levels in BAL fluid of wild-type mice, which was accompanied by reduced NE activity. Our results have suggested an in vivo mechanism of A1AT function. To further demonstrate the role of SPLUNC1 in A1AT therapeutic effect, we treated PA-infected SPLUNC1 KO mice with A1AT. Unlike the wild-type mice, SPLUNC1 KO mice did not show reduced lung PA load. These results emphasize that A1AT therapeutic effect during PA infection depends on SPLUNC1. Given the fact that A1AT can enter into the cells, it would be interesting to determine whether it has a direct effect on intracellular SPLUNC1 mRNA or protein expression. In our primary normal human airway epithelial cell culture experiments, we found that A1AT has no direct effects on intracellular SPLUNC1 expression. Together, A1AT functions primarily through preventing extracellular SPLUNC1 protein from degradation by NE and perhaps other proteases.
We found that lung pro-inflammatory cytokine KC was significantly reduced by A1AT in PA-infected wild-type mouse lungs. However, leukocytes including neutrophils were not as significantly reduced as KC. This may be explained by several factors. First, we only gave one dose of A1AT treatment in PA-infected mice. As a previous study demonstrated a significant inhibition of lung neutrophils following repeated A1AT treatments in cigarette smoke-exposed mice [20], we may need to deliver A1AT multiple times (e.g., before and after PA infection) to markedly suppress lung neutrophil recruitment. Second, A1AT has diverse NE-dependent and NE-independent functions [21]. Lastly, leukocyte numbers may not reflect cell activity. Future studies will explore the direct effects of A1AT on inflammatory cell functions.
We realize that SPLUNC1 KO mice demonstrated greater variability of PA load in the lung than the wild-type mice although the sample size of SPLUNC1 KO and wild-type mice is similar. Thus, it appears that bacterial load variability in our current study is related to the genetic background. We confirmed such greater variability of lung bacterial load in SPLUNC1 KO mice that were pre-treated with A1AT and then infected with PA. We found that A1AT aerosolized 2 hrs prior to PA infection decreased lung PA load in wild-type mice, but not in SPLUNC1 KO mice (Figure 6).
A1AT pre-treatment reduces lung PA load in wild-type (WT) mice, but not in SPLUNC1 knockout (KO) mice. WT and SPLUNC1 KO mice were treated with aerosolized A1AT (0.5 mg/ml). Two hours later, mice were intranasally infected with PA (1.5 × 107 CFUs/mouse). After 24 hrs of PA infection, lung tissues were processed to quantify PA load. While A1AT significantly reduced lung PA load in WT mice (A), it did not alter the PA load in SPLUNC1 KO mice (B). N = 4 – 5 mice per group. The horizontal solid red lines indicate medians of CFUs.
There are several limitations to our current study. First, only one strain of bacteria (i.e., PA) was used in our study. Future studies using other strains of bacteria (e.g., NTHi) are warranted to examine the broad-spectrum effect of A1AT against lung infection. Second, an acute (e.g., 24 hr) PA infection model was carried out to address our research questions. Although we were able to demonstrate a critical role of SPLUNC1 in A1AT-mediated host defense against PA infection with an acute model, a chronic infection model will certainly be beneficial to study the role of SPLUNC1 in disease progression. Lastly, to increase the relevance of our animal research findings to patients, we plan to measure SPLUNC1 and PA in sputum and BAL samples from CF and COPD patients treated with A1AT or placebo.
Conclusion:
Our current study has provided a novel mechanism underlying A1AT-mediated host defense against bacterial infection. Our research findings indicate that A1AT exerts host defense functions against PA infection in part by modulating the host defense protein SPLUNC1.
Abbreviations:
A1AT: α1-antitrypsin; BAL: Bronchoalveolar lavage; CF: Cystic fibrosis; COPD: Chronic obstructive pulmonary disease; KO: Knockout; NE: Neutrophil elastase; NTHi: Non-typeable Haemophilus influenzae; PA: Pseudomonas aeruginosa; SPLUNC1: Short palate, lung, and nasal epithelium clone 1; WT: Wild-type.
Competing interest:
All authors declare not having competing interests that might have influenced the performance or presentation of our work in this manuscript.
Authors’ contributions:
DJ contributed to the experimental designs, collected the data, carried out data analysis and drafted the manuscript. RP provided the A1AT and revised the manuscript. QW contributed to the data collection, analysis and revised the manuscript. AG contributed to the data collection and revised the manuscript. HWC contributed to the experimental designs, data analysis and critically reviewed the manuscript. All authors read and approved the final manuscript.
| Background:
Pseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection.
Methods:
SPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2 hrs after the PA infection, and mice were sacrificed 24 hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot.
Results:
In WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production.
Conclusions:
A1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation. | Background:
Bacterial infection is involved in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [1]. Persistent lung Pseudomonas aeruginosa (PA) infection is one the most common causes associated with the exacerbations of CF and COPD [1-3]. One of the major challenges in the treatment of PA infection is the rising antibiotic resistance [4]. A previous study has demonstrated the therapeutic effect of α1-antitrypsin (A1AT) against PA infection in CF patients [5]. Animal studies also suggested the anti-PA effects of A1AT [5,6], but how A1AT improves host defense against PA infection remains unknown.
PA infection in the lung elicits a robust inflammatory response such as recruitment and activation of neutrophils. Neutrophil elastase (NE) is released during lung inflammation, which in turn may cause detrimental effects such as tissue destruction seen in emphysema. We have demonstrated that human NE (HNE) impairs airway epithelial defense functions against bacterial infections by degrading short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein [7]. SPLUNC1 is a 25-kDa secretory protein from large airway epithelial cells. It has been shown to exert host defense function against several strains of bacteria including PA, non-typeable Haemophilus influenzae (NTHi) and Mycoplasma pneumoniae[7-9]. However, whether NE in vivo degrades SPLUNC1 and subsequently impair lung defense against PA infection has not been investigated.
A1AT is a 52-kDa serine protease inhibitor mainly produced by the liver and in the lung by macrophages and epithelial cells. In the lung, A1AT is a potent inhibitor of neutrophil elastase protecting the lung tissue from proteolytic degradation [10]. In individuals with a genetic deficiency of A1AT there is an imbalance between neutrophil elastase and A1AT in the lung increasing the risk of developing emphysema [11]. Purified human A1AT has been used to treat A1AT deficiency by augmenting A1AT levels and inhibiting NE activity [12,13]. In the current study, we hypothesized that A1AT prevents NE-mediated SPLUNC1 degradation and subsequently enhances lung defense against PA infection. To test our hypothesis, we utilized the PA infection model in wild-type and SPLUNC1 knockout (KO) mice with or without A1AT treatment. First, we confirmed that PA infection reduces SPLUNC1 expression in mouse bronchoalveolar lavage (BAL) fluid. Second, we demonstrated that A1AT increased SPLUNC1 levels in PA-infected wild-type mice, and decreased lung PA load. Third, we found that SLPUNC1 is required to improve lung defense against PA.
Conclusion:
Our current study has provided a novel mechanism underlying A1AT-mediated host defense against bacterial infection. Our research findings indicate that A1AT exerts host defense functions against PA infection in part by modulating the host defense protein SPLUNC1. | Background:
Pseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection.
Methods:
SPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2 hrs after the PA infection, and mice were sacrificed 24 hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot.
Results:
In WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production.
Conclusions:
A1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation. | 7,136 | 338 | [
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Fluid | Cytokines | Disease Models, Animal | Glycoproteins | Leukocyte Elastase | Lung | Lung Diseases | Mice | Mice, Inbred C57BL | Mice, Knockout | Phosphoproteins | Pseudomonas Infections | Pseudomonas aeruginosa | alpha 1-Antitrypsin [SUMMARY] | [CONTENT] Animals | Bronchoalveolar Lavage Fluid | Cytokines | Disease Models, Animal | Glycoproteins | Leukocyte Elastase | Lung | Lung Diseases | Mice | Mice, Inbred C57BL | Mice, Knockout | Phosphoproteins | Pseudomonas Infections | Pseudomonas aeruginosa | alpha 1-Antitrypsin [SUMMARY] | [CONTENT] Animals | Bronchoalveolar Lavage Fluid | Cytokines | Disease Models, Animal | Glycoproteins | Leukocyte Elastase | Lung | Lung Diseases | Mice | Mice, Inbred C57BL | Mice, Knockout | Phosphoproteins | Pseudomonas Infections | Pseudomonas aeruginosa | alpha 1-Antitrypsin [SUMMARY] | [CONTENT] Animals | Bronchoalveolar Lavage Fluid | Cytokines | Disease Models, Animal | Glycoproteins | Leukocyte Elastase | Lung | Lung Diseases | Mice | Mice, Inbred C57BL | Mice, Knockout | Phosphoproteins | Pseudomonas Infections | Pseudomonas aeruginosa | alpha 1-Antitrypsin [SUMMARY] | [CONTENT] Animals | Bronchoalveolar Lavage Fluid | Cytokines | Disease Models, Animal | Glycoproteins | Leukocyte Elastase | Lung | Lung Diseases | Mice | Mice, Inbred C57BL | Mice, Knockout | Phosphoproteins | Pseudomonas Infections | Pseudomonas aeruginosa | alpha 1-Antitrypsin [SUMMARY] | [CONTENT] airway neutrophils bacterial | improve lung defense | lung pro inflammatory | infection lung elicits | inhibition lung neutrophils [SUMMARY] | [CONTENT] airway neutrophils bacterial | improve lung defense | lung pro inflammatory | infection lung elicits | inhibition lung neutrophils [SUMMARY] | [CONTENT] airway neutrophils bacterial | improve lung defense | lung pro inflammatory | infection lung elicits | inhibition lung neutrophils [SUMMARY] | [CONTENT] airway neutrophils bacterial | improve lung defense | lung pro inflammatory | infection lung elicits | 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||||||
Parent artery-initiated and stent-mediated neointima formation in a rat saccular side wall model. | 35110397 | Unlike clipping that forms an immediate barrier of blood flow into intracranial aneurysms, endovascular treatments rely on thrombus organization and neointima formation. Therefore, a continuous endothelial cell layer is crucial to prevent blood flow in the former aneurysm. This study investigates the origin of endothelial cells in the neointima of endovascular treated aneurysms, specifically whether cells from the parent artery play a role in neointima formation. | BACKGROUND | In male rats, decellularized and vital side wall aneurysms were treated by coil (n=16) or stent embolization (n=15). The cell tracer CM-Dil dye was injected into the clamped aorta before aneurysm suture to mark initial endothelial cells in the parent artery and enable tracking of their proliferation during follow-up. Aneurysms were analyzed for growth, thrombus formation, and recurrence. Histological evaluation followed with cell counts for specific regions-of-interest. | METHODS | During follow-up, none of the 31 aneurysms ruptured. Macroscopic residual perfusion was observed in 12/16 rats after coiling and in 1/15 after stenting. Amounts of CM-Dil +cells in coiled versus stented decellularized aneurysms significantly decreased in the thrombus on day 7 (p=0.01) and neointima on day 21 (p=0.04). For vital aneurysms, the number of CM-Dil +cells in the neointima on day 21 showed no significant difference. | RESULTS | Healing patterns were worse in coil-treated than stent-treated aneurysms. Cell migration forming a neointima seemed mainly dependent on the adjacent vessel in decellularized aneurysms, but appeared buoyed by recruitment from aneurysm wall cells in vital aneurysms. Therefore, a cell-rich parent artery might be crucial. | CONCLUSIONS | [
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"Treatment Outcome"
] | 9685721 | Introduction | During the past decades, endovascular treatment has gained increasing importance in the management of both ruptured and unruptured intracranial aneurysms. Nevertheless, recurrence rates after coil embolization remain notably high compared with clip ligation.1–4 Unlike direct mechanical occlusion of an aneurysm sac by clipping, an endovascularly treated patient awaits a biological healing response of thrombus organization and neointima formation as mediated by cell migration.
Smooth muscle cells (SMCs) play an important role in inducing aneurysm healing on a biological level. Preclinical and clinical studies have revealed that loss of mural cells predisposes aneurysm growth and rupture,5–7 and that healing after coil embolization is worse in aneurysms with highly degenerated walls than in those with SMC-rich walls.8 Cell-based therapies with intraluminal transplantation of SMCs were shown to promote complete aneurysm healing along with prevention of growth and rupture.9 In a comparison of endovascular devices, better aneurysm healing was demonstrated in stented compared with coiled aneurysms10; likewise neointima formation and thrombus organization are concurrent processes in aneurysm healing, that depend on cell migration from both the parent artery and aneurysm wall. Neointima formation relied more on cell migration from the aneurysm wall in coiled aneurysms, whereas the role of cells originating from the parent artery seemed to be higher in stent-treated aneurysms.11 These factors suggest that cell migration, allowing for a continuous endothelial lining along the parent artery’s lumen, substantially contributes to complete aneurysm healing after endovascular treatment.
Until now, the origin of cells (aneurysm wall, parent artery or progenitor cells) responsible for aneurysm healing has remained unclear.12 13 Whether the endothelial lining is triggered by progenitor cells, which have already been identified as attaching in stented animal aneurysm models,14 or the parent artery itself has not yet been adequately analyzed. Toward this aim, our experimental study in a rat saccular side wall aneurysm model examined the origin of neointima-forming cells. Using a cell tracer injection, we analyzed the role of endothelial cells originating in the parent artery in neointima formation. | null | null | Results | In decellularized aneurysms, the amounts of CM-Dil +cells in the neointima did not significantly differ between stent-treated or coil-treated animals at 7 days (p=0.77), but were significantly higher in stent-treated animals at 21 days (p=0.04) (figure 2A). For vital aneurysms, no significant differences were noted at either 7 days (p=1.0) or 21 days (p=0.66) (figure 2B). A pooled-analysis comparing decellularized coiled versus stented aneurysms showed no significant differences regarding neointima formation (p=0.38), cells in the aneurysm wall (p=0.81), or periadventitia (p=0.35). Significant differences of CM-Dil dye +cells were found for thrombus invasion in the stent group (p=0.017). A pooled-analysis comparing vital coiled and stented aneurysms showed no overall significant differences in neointima (p=0.87), thrombus (p=0.83), aneurysm wall (p=0.74), and parent artery (p=0.26).
Left panel shows cell count comparison between different regions of interest (ROI) in coil- and stent-treated decellularized aneurysms. Graph depicts the cumulative relative cell counts for CM-Dil +cells in decellularized aneurysms sutured on the abdominal part of the aorta, either coil- or stent-treated. Treatments were compared for coiled and stented groups in decellularized aneurysms on days 7 and 21. Significant differences in the proportion of CM-Dil +cells in the neointima for day 21 and thrombus for day 7 (p<0.05) were observed. Right panel shows cell count comparison between different ROIs in coil- and stent-treated vital aneurysms. Graph depicts the cumulative relative cell counts for CM-Dil +cells in vital aneurysms sutured on the abdominal part of the aorta, either coil- or stent-treated. Treatments were compared for coiled and stented groups in vital aneurysms on days 7 and 21. Significant differences in the proportion of CM-Dil +cells in the parent artery for day 7 (p<0.05) were observed.
Overall, four of 35 animals died, yielding an 11.4% mortality rate. The four deaths included two animals with irreparable vessel defect suffered intraoperatively during stent implantation, and two animals in the decellularized stenting group whose euthanasia was planned for day 21 (one for abdominal wound dehiscence, and one for dehydration due to inadequate food and water intake).
Aneurysm growth after treatment Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.
Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.
Microscopic healing status All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).
Periadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).
All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).
Periadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).
Fluorescence analysis for neointima, thrombus, aneurysm wall and parent artery In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).
Illustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.
For vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.
In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).
Illustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.
For vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.
Surgical characteristics Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).
Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).
Physiological parameters Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.
Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences. | Conclusion | Our study in a rat side wall model corroborates previous findings that biological healing of endovascularly treated aneurysms depends on cell migration from the adjacent vessel and is additionally supported by recruitment of cells from the aneurysm wall in vital (cell-rich) aneurysms. However, in decellularized aneurysms, the adjacent vessel segment is the most important source of cells to promote healing. Therefore, cell migration is facilitated by use of endovascular devices, such as stents, that connect adjacent cell-rich tissues to the aneurysm orifice. In consequence, scaffolds should be placed in cell-rich healthy vessel regions, particularly for treatment of highly degenerated aneurysms. Vice versa, the success of these treatment strategies may depend on the presence of a healthy (cell-rich) endothelium in the adjacent vessel. Coil treatment alone might be sufficient for aneurysms with relatively healthy vessel walls. | [
"Material and methods",
"Animals",
"Study design",
"Anesthesia protocol",
"Cell tracer injection",
"Patency evaluation, macroscopic inspection and soft tissue preparation",
"Measurement of the pre- and post-mortem aneurysm volume, histology",
"Exclusion criteria and statistical analysis",
"Aneurysm growth after treatment",
"Microscopic healing status",
"Fluorescence analysis for neointima, thrombus, aneurysm wall and parent artery",
"Surgical characteristics",
"Physiological parameters",
"Study limitations"
] | [
"Animals After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.\nAfter randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.\nStudy design The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5\n\nArterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.\nBefore surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.\nFlowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).\nPrimary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.\nThe study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5\n\nArterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.\nBefore surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.\nFlowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).\nPrimary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.\nAnesthesia protocol For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).\n\n\n\nFor initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).\n\n\n\nCell tracer injection The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.\nLikewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).\nThe fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.\nLikewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).\nPatency evaluation, macroscopic inspection and soft tissue preparation Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.\nFluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.\nMeasurement of the pre- and post-mortem aneurysm volume, histology The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).\nPost-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.\nFor fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.\nThe aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).\nPost-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.\nFor fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.\nExclusion criteria and statistical analysis Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.\nExclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.",
"After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.",
"The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5\n\nArterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.\nBefore surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.\nFlowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).\nPrimary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.",
"For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).\n\n\n",
"The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.\nLikewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).",
"Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.",
"The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).\nPost-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.\nFor fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.",
"Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.",
"Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.",
"All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).\nPeriadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).",
"In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).\nIllustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.\nFor vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.",
"Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).",
"Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.",
"Recent findings by Morel et al\n22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23\n"
] | [
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] | [
"Introduction",
"Material and methods",
"Animals",
"Study design",
"Anesthesia protocol",
"Cell tracer injection",
"Patency evaluation, macroscopic inspection and soft tissue preparation",
"Measurement of the pre- and post-mortem aneurysm volume, histology",
"Exclusion criteria and statistical analysis",
"Results",
"Aneurysm growth after treatment",
"Microscopic healing status",
"Fluorescence analysis for neointima, thrombus, aneurysm wall and parent artery",
"Surgical characteristics",
"Physiological parameters",
"Discussion",
"Study limitations",
"Conclusion"
] | [
"During the past decades, endovascular treatment has gained increasing importance in the management of both ruptured and unruptured intracranial aneurysms. Nevertheless, recurrence rates after coil embolization remain notably high compared with clip ligation.1–4 Unlike direct mechanical occlusion of an aneurysm sac by clipping, an endovascularly treated patient awaits a biological healing response of thrombus organization and neointima formation as mediated by cell migration.\nSmooth muscle cells (SMCs) play an important role in inducing aneurysm healing on a biological level. Preclinical and clinical studies have revealed that loss of mural cells predisposes aneurysm growth and rupture,5–7 and that healing after coil embolization is worse in aneurysms with highly degenerated walls than in those with SMC-rich walls.8 Cell-based therapies with intraluminal transplantation of SMCs were shown to promote complete aneurysm healing along with prevention of growth and rupture.9 In a comparison of endovascular devices, better aneurysm healing was demonstrated in stented compared with coiled aneurysms10; likewise neointima formation and thrombus organization are concurrent processes in aneurysm healing, that depend on cell migration from both the parent artery and aneurysm wall. Neointima formation relied more on cell migration from the aneurysm wall in coiled aneurysms, whereas the role of cells originating from the parent artery seemed to be higher in stent-treated aneurysms.11 These factors suggest that cell migration, allowing for a continuous endothelial lining along the parent artery’s lumen, substantially contributes to complete aneurysm healing after endovascular treatment.\nUntil now, the origin of cells (aneurysm wall, parent artery or progenitor cells) responsible for aneurysm healing has remained unclear.12 13 Whether the endothelial lining is triggered by progenitor cells, which have already been identified as attaching in stented animal aneurysm models,14 or the parent artery itself has not yet been adequately analyzed. Toward this aim, our experimental study in a rat saccular side wall aneurysm model examined the origin of neointima-forming cells. Using a cell tracer injection, we analyzed the role of endothelial cells originating in the parent artery in neointima formation.",
"Animals After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.\nAfter randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.\nStudy design The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5\n\nArterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.\nBefore surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.\nFlowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).\nPrimary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.\nThe study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5\n\nArterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.\nBefore surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.\nFlowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).\nPrimary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.\nAnesthesia protocol For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).\n\n\n\nFor initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).\n\n\n\nCell tracer injection The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.\nLikewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).\nThe fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.\nLikewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).\nPatency evaluation, macroscopic inspection and soft tissue preparation Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.\nFluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.\nMeasurement of the pre- and post-mortem aneurysm volume, histology The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).\nPost-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.\nFor fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.\nThe aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).\nPost-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.\nFor fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.\nExclusion criteria and statistical analysis Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.\nExclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.",
"After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.",
"The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5\n\nArterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.\nBefore surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.\nFlowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).\nPrimary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.",
"For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).\n\n\n",
"The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.\nLikewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).",
"Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.",
"The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).\nPost-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.\nFor fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.",
"Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.",
"In decellularized aneurysms, the amounts of CM-Dil +cells in the neointima did not significantly differ between stent-treated or coil-treated animals at 7 days (p=0.77), but were significantly higher in stent-treated animals at 21 days (p=0.04) (figure 2A). For vital aneurysms, no significant differences were noted at either 7 days (p=1.0) or 21 days (p=0.66) (figure 2B). A pooled-analysis comparing decellularized coiled versus stented aneurysms showed no significant differences regarding neointima formation (p=0.38), cells in the aneurysm wall (p=0.81), or periadventitia (p=0.35). Significant differences of CM-Dil dye +cells were found for thrombus invasion in the stent group (p=0.017). A pooled-analysis comparing vital coiled and stented aneurysms showed no overall significant differences in neointima (p=0.87), thrombus (p=0.83), aneurysm wall (p=0.74), and parent artery (p=0.26).\nLeft panel shows cell count comparison between different regions of interest (ROI) in coil- and stent-treated decellularized aneurysms. Graph depicts the cumulative relative cell counts for CM-Dil +cells in decellularized aneurysms sutured on the abdominal part of the aorta, either coil- or stent-treated. Treatments were compared for coiled and stented groups in decellularized aneurysms on days 7 and 21. Significant differences in the proportion of CM-Dil +cells in the neointima for day 21 and thrombus for day 7 (p<0.05) were observed. Right panel shows cell count comparison between different ROIs in coil- and stent-treated vital aneurysms. Graph depicts the cumulative relative cell counts for CM-Dil +cells in vital aneurysms sutured on the abdominal part of the aorta, either coil- or stent-treated. Treatments were compared for coiled and stented groups in vital aneurysms on days 7 and 21. Significant differences in the proportion of CM-Dil +cells in the parent artery for day 7 (p<0.05) were observed.\nOverall, four of 35 animals died, yielding an 11.4% mortality rate. The four deaths included two animals with irreparable vessel defect suffered intraoperatively during stent implantation, and two animals in the decellularized stenting group whose euthanasia was planned for day 21 (one for abdominal wound dehiscence, and one for dehydration due to inadequate food and water intake).\nAneurysm growth after treatment Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.\nBaseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.\nMicroscopic healing status All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).\nPeriadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).\nAll aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).\nPeriadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).\nFluorescence analysis for neointima, thrombus, aneurysm wall and parent artery In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).\nIllustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.\nFor vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.\nIn decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).\nIllustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.\nFor vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.\nSurgical characteristics Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).\nComparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).\nPhysiological parameters Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.\nVital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.",
"Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.",
"All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).\nPeriadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).",
"In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).\nIllustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.\nFor vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.",
"Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).",
"Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.",
"In comparing healing of decellularized and vital aneurysms after coil and stent treatments, we demonstrated that neointima formation was mediated by endothelial cells originating in the adjacent parent artery in decellularized aneurysms whereas this lining was supported by cells derived from the aneurysm wall in vital aneurysms. This finding was consistent in short-term and intermediate follow-up. In turn, previous studies have already shown that the amount of circulating progenitor cells contribute only a minor role in aneurysm healing.19 20\n\nA growing body of literature suggests that the various treatment options for intracranial aneurysms are not only efficacious but can be associated with the biological response of the wall itself.5 7 9 11 12 Specifically, ligation provides an immediate direct endothelial contact whereas endovascular therapies rely more on biological processes, particularly the newly developed minimally invasive techniques, such as stent-assisted coiling, flow-diversion, web-device management, bioactive endovascular devices, systemic drug therapies or cell-based therapies.9 10\n\nPrevious studies have shown that neointima formation and thrombus organization are concurrent processes in aneurysm healing after endovascular therapies: both rely on cell migration from the parent artery and aneurysm wall, and both are facilitated by the presence of endovascular devices. Thrombus-organizing cells typically originate from the parent artery for both types of endovascular treatment, whereas neointima formation in coiled aneurysms relies more on cell migration from the aneurysm wall, in stent-treated aneurysms mostly from the parent artery.11 In our experimental study, the parent artery was identified as an important source of migrating cells that form the neointima. This process happened in both forms of endovascular treatment and was observed within a few days from onset of aneurysm healing. During subsequent weeks, fading of the CM-Dil dye indicates the loss of signal intensity as these cells continue to divide at their new location, predominantly in the neointima and thrombus. The intensity of the lipophilic cell tracer decreases with each mitosis. These findings confirm the important role of endovascularly applied stents in a cell-rich region that serves as a principal structure for cell migration, which allows continuous endothelial lining, neointima formation, and progressive aneurysm healing.\nIn decellularized aneurysms, the number of CM-Dil +cells after stent versus coil treatments were higher on day 7 (72.25% vs 68%, p=0.77) and significantly higher on day 21 in the neointima (34.44% vs 7.75%, p=0.04). This finding suggests that cells involved in the aneurysm healing process migrated from the parent artery rather than the aneurysm wall. More importantly, we believe that the significantly greater number of CM-Dil +cells found in the thrombus after 7 days could be because the stent struts facilitated intraluminal thrombus formation in the aneurysm sac, which also seems explainable because the neointima is not sealed after 7 days. For vital aneurysms after coiling and stenting for days 7 (56.75% vs 58.25%, p=1) and 21 (11.5% vs 16.5%, p=0.66), no significant differences were found regarding CM-Dil +cells in the neointima; this finding supports the hypothesis that healing mechanisms in the coiled animals are stronger because of recruiting cells from the aneurysm wall (for endothelial staining via F8 please see online supplemental figure 9). In vital aneurysms, comparison of the number of CM-Dil +cells in coil versus stent treatments showed similar distributions in the aneurysm wall on day 7 (13.25% vs 13.5%, p=0.88) and day 21 (10.25% vs 10.75%, p=0.55); were similar for intraluminal thrombus formation on day 7 (62% vs 71.25%, p=1) and day 21 (26.25 % vs 23.75 %, p=0.55); and decreased in the parent artery on day 7 (60.0% vs 81.5%, p=0.02) and day 21 (24.25% vs 26.5%, p=0.46). The significantly higher amount of CM-Dil +cells in the parent artery of stented aneurysms might be because stent insertion causes more local reactions than coils and that neointima formation was initiated via stents overlapping the parent artery. Therefore, endovascular treatment via stent treatment might be crucial for highly degenerated aneurysm walls whereas coiling or stent-assisted coiling might be predominantly useful for aneurysms with relatively healthy vessel walls.\nInterestingly, the proportion of CM-Dil +cells in the parent artery was higher at both day 7 and day 21 in the decellularized stent-treated group (76.6% vs 35.66% day) than in the coil-treated group (75.5% vs 10.5%). P values for both time-points, comparing the amount of CM-Dil +cells in the parent artery in the decellularized coil versus stented group on day 7, were non-significantly altered (p=0.77), and also when compared with 21 days (p=0.07). At 21-day follow-up, variations in levels of CM-Dil +cells of <10% in five rats might be explained by the natural course of the fading dye or longer operating times that potentially degraded the light sensitive dye.\nOn days 7 and 21, respectively, more CM-Dil +cells were detected inside the thrombus of the stent-treated group (25.25% vs 8.33%) than the coiled group (7.5% vs 5.5%). This might be reasonable because after 21 days the neointima is completely sealed in nearly all of the cases. CM-Dil +cell migration into the thrombus was significantly enhanced for stent treatments on day 7 (p=0.01), possibly facilitated by the strut-like stents themselves; after 21 days, the p value was not significantly altered (p=0.1). CM-Dil +cells in the aneurysm wall showed no differences between coil or stent treatments on day 7 (12.25% vs 11.75%, p=0.55) and day 21 (8.5% vs 9%, p=1). Regarding endothelial thrombus invasion, Raymond et al elucidated the role of stents in lowering aneurysm recanalization rate and decreasing recurrences after embolization in a side-wall aneurysm model with venous pouches.21 In our experiments, the amounts and extent of invasion of endothelial cells into the thrombus was higher in the coil-treated than stent-treated groups; this finding corresponded with a higher aneurysm reperfusion rate in the coiled group.\nIn summary, cell migration, allowing for a continuous endothelial lining via stents along the cell-rich parent artery’s lumen, may be the substantial prerequisite for complete aneurysm healing after endovascular therapy, and may be especially critical in highly degenerated aneurysms. The implication is that coiling in ruptured cerebral aneurysms might suffice to promote healing in patients with relative healthy vessel walls, but is likely insufficient in aneurysms with degenerated walls. In terms of clinical translation and future perspectives, our results align with a preliminary report in a preclinical series by Nevzati et al.8 Coating endovascular devices, such as coils, with chemo-attractants might be a therapeutic strategy to stimulate adjacent cells in a healthy parent artery to divide and migrate into the aneurysm thrombus.\nStudy limitations Recent findings by Morel et al\n22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23\n\nRecent findings by Morel et al\n22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23\n",
"Recent findings by Morel et al\n22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23\n",
"Our study in a rat side wall model corroborates previous findings that biological healing of endovascularly treated aneurysms depends on cell migration from the adjacent vessel and is additionally supported by recruitment of cells from the aneurysm wall in vital (cell-rich) aneurysms. However, in decellularized aneurysms, the adjacent vessel segment is the most important source of cells to promote healing. Therefore, cell migration is facilitated by use of endovascular devices, such as stents, that connect adjacent cell-rich tissues to the aneurysm orifice. In consequence, scaffolds should be placed in cell-rich healthy vessel regions, particularly for treatment of highly degenerated aneurysms. Vice versa, the success of these treatment strategies may depend on the presence of a healthy (cell-rich) endothelium in the adjacent vessel. Coil treatment alone might be sufficient for aneurysms with relatively healthy vessel walls."
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] | [
"aneurysm",
"coil",
"stent",
"vessel wall",
"intervention"
] | Introduction:
During the past decades, endovascular treatment has gained increasing importance in the management of both ruptured and unruptured intracranial aneurysms. Nevertheless, recurrence rates after coil embolization remain notably high compared with clip ligation.1–4 Unlike direct mechanical occlusion of an aneurysm sac by clipping, an endovascularly treated patient awaits a biological healing response of thrombus organization and neointima formation as mediated by cell migration.
Smooth muscle cells (SMCs) play an important role in inducing aneurysm healing on a biological level. Preclinical and clinical studies have revealed that loss of mural cells predisposes aneurysm growth and rupture,5–7 and that healing after coil embolization is worse in aneurysms with highly degenerated walls than in those with SMC-rich walls.8 Cell-based therapies with intraluminal transplantation of SMCs were shown to promote complete aneurysm healing along with prevention of growth and rupture.9 In a comparison of endovascular devices, better aneurysm healing was demonstrated in stented compared with coiled aneurysms10; likewise neointima formation and thrombus organization are concurrent processes in aneurysm healing, that depend on cell migration from both the parent artery and aneurysm wall. Neointima formation relied more on cell migration from the aneurysm wall in coiled aneurysms, whereas the role of cells originating from the parent artery seemed to be higher in stent-treated aneurysms.11 These factors suggest that cell migration, allowing for a continuous endothelial lining along the parent artery’s lumen, substantially contributes to complete aneurysm healing after endovascular treatment.
Until now, the origin of cells (aneurysm wall, parent artery or progenitor cells) responsible for aneurysm healing has remained unclear.12 13 Whether the endothelial lining is triggered by progenitor cells, which have already been identified as attaching in stented animal aneurysm models,14 or the parent artery itself has not yet been adequately analyzed. Toward this aim, our experimental study in a rat saccular side wall aneurysm model examined the origin of neointima-forming cells. Using a cell tracer injection, we analyzed the role of endothelial cells originating in the parent artery in neointima formation.
Material and methods:
Animals After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.
After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.
Study design The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5
Arterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.
Before surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.
Flowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).
Primary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.
The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5
Arterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.
Before surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.
Flowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).
Primary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.
Anesthesia protocol For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).
For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).
Cell tracer injection The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.
Likewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).
The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.
Likewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).
Patency evaluation, macroscopic inspection and soft tissue preparation Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.
Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.
Measurement of the pre- and post-mortem aneurysm volume, histology The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).
Post-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.
For fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.
The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).
Post-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.
For fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.
Exclusion criteria and statistical analysis Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.
Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.
Animals:
After randomly assigning 12-week-old male Lewis rats (n=31; mean weight 498±8 g at baseline, 491±8 g at follow-up) to one of the four experimental groups, all underwent coiling or stenting treatment by using the Helsinki saccular side wall model within 6 weeks.6 The rats were housed in groups of four animals at a room temperature accompanied by a 12 hour light/dark cycle. Free access to pellet and water was provided and care was in accordance with the local institutional guidelines. Animal experiments were approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (BE 108/16; BE 60/19). For all experimental procedures the ARRIVE (Animals in Research: Reporting of In Vivo Experiments) guidelines were strictly followed.15 Animal caregivers were blinded to all therapeutic steps and outcomes.
Study design:
The study setting consisted of four groups that included (1) decellularized coiled, (2) vital coiled, (3) decellularized stented, and (4) vital stented aneurysms. Techniques have been described elsewhere for the coil device (2 cm of Target 360 TM Ultra, 2 mm diameter; Stryker, Kalamazoo, MI) and stent device (modified magmaris device, AMS with polymer coating, 6 mm length, 2 mm diameter, square-shaped struts 150 µm in thickness and 150 µm in width; nominal pressure of 8 bar; Biotronik, Bülach, Switzerland).8 10 16 Side wall aneurysms were created by end-to-side anastomosis of a previously ligated arterial vessel pouch from the thoracic part of a donor animal. Each saccular pouch was sutured onto the clamped abdominal aorta of the recipient directly followed by either coil or stent implantation.5
Arterial pouches for decellularization were incubated in 0.1% sodium dodecyl sulfate for 10 hours at 37°C as previously described.11 Vital aneurysm pouches were characterized as cell-rich aneurysm pouches. Coil and stent groups were designated as decellularized or vital at follow-up evaluation on days 7 and 21.
Before surgery, randomization to either the coil or stent treatment was performed using a web-based randomization system (www.sealedenvelope.com) (figure 1). Of the 38 rats operated on, 31 animals underwent coil (n=16) or stent (n=15) treatments and seven animals were excluded (three in the pilot study, four early dropouts). After euthanasia, the aneurysm and its parent artery were harvested for further histological processing.
Flowchart of the study design. Of a total of 38 rats, 31 were included for final analysis and seven were excluded (three in the pilot study and four early dropouts).
Primary endpoints were defined as the proportion (%) CM-Dil +cells tracked for one of the following regions of interest: (1) aneurysm wall, (2) intraluminal thrombus, (3) neointima, and (4) parent artery. Secondary endpoints analyzed were aneurysm healing (by macroscopic and fluorescence examination) and histological analysis as defined by neointima formation, aneurysm wall cellularity and inflammation, periadventitial inflammation, and fibrosis.
Anesthesia protocol:
For initial induction of anesthesia, all rats were placed in a clean box with oxygen (O2) provided until the animal lost consciousness (5–10 min). Next, rats were anesthetized with a subcutaneous injection of a mixture of fentanyl 0.005 mg/kg (Sintetica, S.A. Switzerland), medetomidine 0.15 mg/kg (Virbac, Switzerland), and midazolam 2 mg/kg (Roche, Switzerland). This protocol ensured a surgical plane of at least 45 min. In cases requiring prolonged anesthesia, isoflurane was started (1.0–2.0% titrated to effect in 100% O2 administered via face mask) to allow adequate surgical anesthetic depth and analgesic coverage throughout (detailed anesthesiological protocol given in online supplemental methods).
Cell tracer injection:
The fluorescent lipophilic CellTracker CM-Dil dye (Invitrogen, Molecular Probes, MW 1051.50; Eugene, OR) was stored light-protected at ≤ −20°C at all times (pretesting of the functionality is described in online supplemental figure 1). On the day of the experiment, CM-Dil dye (2 µL was dissolved in 1 mL phosphate-buffered saline and transferred to a 1 mL syringe with a 27-1/2 gauge (0.4×13 mm) cannula; both steps were taken under light protection. Lights in the operating room were turned off. After clamping the proximal and distal parts of the abdominal aorta, the corresponding vessel segment was flushed with heparinized 0.9% saline solution and the CM-Dil dye was carefully injected. Immediately, the microscope light was also turned off. After a 15 min incubation period, room lights and the microscope light were turned on and the longitudinal arteriotomy and suturing of the aneurysm proceeded.
Likewise, three pilots were created with cell tracer injection. Next, decellularized aneurysms were sutured end-to-side on the aorta. With euthanasia of the animals immediately after labeling, the dye appeared uniformly distributed and the proportions of CM-Dil dye +cells were 100% in the vessel wall (online supplemental figure 2).
Patency evaluation, macroscopic inspection and soft tissue preparation:
Fluorescence angiography was performed to assess dynamic perfusion status of the aneurysm after intravenous injection of 2 mL fluorescein (fluorescein 10% Faure, 0.5 g/5 mL), followed by illumination (light source 465–490 nm) that was filmed with a specific detection filter as previously described.17 18 After the animals were euthanized with an overdose of intracardial ketamine hydrochloride (Narketan, 120 mg/kg ketamine injection, Vetoquinol, Switzerland), the aneurysms were harvested and measured in three dimensions (length, width, height). The posterior aorta was opened to inspect the aneurysm orifice in coiled animals. Tissues were immediately fixed in formalin (4% weight/volume solution, J.T. Baker, Arnhem, The Netherlands) until paraffin embedding for histological analysis was performed.
Measurement of the pre- and post-mortem aneurysm volume, histology:
The aneurysm was documented before implantation and at harvesting using a digital camera (Sony NEX-5R, Sony, Tokyo, Japan) attached to a microscope (OPMI, Carl Zeiss AG, Oberkochen, Germany). Aneurysm volume was calculated with the cylindric formula: π × height × width/2 × length/2 (online supplemental table 1).
Post-mortem, under the microscope, coils were carefully retrieved with micro instruments. Further histological processing was proceeded with stents in situ. Paraffin-embedded aneurysms were cut along the axis of the corresponding parent artery, in 2 µm sections, and stained; stains included hematoxylin and eosin (H&E), smooth muscle actin (SMA), Masson-Goldner trichrome (MASA), and Von Willebrand factor (F8). After digitalization of slides followed by Omnyx (VL 120, GE), each slide was evaluated using the JVS viewer (JVS view 1.2 full version, http://jvsmicroscope.uta.fi/software/, University of Tampere, Finland). All light microscopy samples underwent blinded qualitative analysis by two independent observers (SW and BEG) and were rated with a previously used four-tier grading system.5 Periadventitial inflammation, aneurysm wall inflammation, number of neutrophils in the thrombus, and aneurysm wall cellularity were analyzed in H&E stained, periadventitial fibrosis, and neointima formation in MASA-stained slices. Furthermore, the number of endothelial cells in the thrombus was analyzed, specifically regarding the potential role of endothelial clefts as the main reason for aneurysm recurrence.
For fluorescence microscopy, slides were cut to a 4 µm thickness, deparaffined, and stained by DAPI (4',6-diamidino-2-phenylindole) application. CM-Dil dye staining had been completed intraoperatively in vivo. Slides were photographed digitally using a fluorescence microscope (Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8) with exposure times of 50 ms for DAPI and 90–130 ms for TXRED. The proportions of CM-Dil dye +cells on all cells were calculated using a semi-automated, cell count software (Image-J version 1.52 n, US National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij/) for four regions of interest: (1) aneurysm wall, (2) inside thrombus and (3) neointima, and (4) parent artery. For cell counting, a size from 7 to 100 (infinity) and a circularity for corresponding analyzed particles from 0.3 to 1.00 was applied.
Exclusion criteria and statistical analysis:
Exclusion from final analysis were premature death or euthanasia for any reason. Statistical analyses were performed using the non-parametric Wilcoxon-Mann-Whitney-U test. A probability value of p<0.05 (*) and p<0.01 (**) was considered significant. Data were analyzed by IBM SPSS (version 22, USA) and visualized by Graph Pad Prism 8 (Version 8.2.0.435, GraphPad software, San Diego, CA). Sample size per group was determined using a priori sample size calculation (BiAS.for. Windows Version 11, epsilon Verlag, Germany). To achieve α=0.05 at β=0.2 with a sigma (σ) of 0.2, the sample size calculation showed that n=4 animals per group were appropriate to achieve a delta (δ) between 0.3 and 0.5. All values given in the text are expressed as mean±SD or median and IQR.
Results:
In decellularized aneurysms, the amounts of CM-Dil +cells in the neointima did not significantly differ between stent-treated or coil-treated animals at 7 days (p=0.77), but were significantly higher in stent-treated animals at 21 days (p=0.04) (figure 2A). For vital aneurysms, no significant differences were noted at either 7 days (p=1.0) or 21 days (p=0.66) (figure 2B). A pooled-analysis comparing decellularized coiled versus stented aneurysms showed no significant differences regarding neointima formation (p=0.38), cells in the aneurysm wall (p=0.81), or periadventitia (p=0.35). Significant differences of CM-Dil dye +cells were found for thrombus invasion in the stent group (p=0.017). A pooled-analysis comparing vital coiled and stented aneurysms showed no overall significant differences in neointima (p=0.87), thrombus (p=0.83), aneurysm wall (p=0.74), and parent artery (p=0.26).
Left panel shows cell count comparison between different regions of interest (ROI) in coil- and stent-treated decellularized aneurysms. Graph depicts the cumulative relative cell counts for CM-Dil +cells in decellularized aneurysms sutured on the abdominal part of the aorta, either coil- or stent-treated. Treatments were compared for coiled and stented groups in decellularized aneurysms on days 7 and 21. Significant differences in the proportion of CM-Dil +cells in the neointima for day 21 and thrombus for day 7 (p<0.05) were observed. Right panel shows cell count comparison between different ROIs in coil- and stent-treated vital aneurysms. Graph depicts the cumulative relative cell counts for CM-Dil +cells in vital aneurysms sutured on the abdominal part of the aorta, either coil- or stent-treated. Treatments were compared for coiled and stented groups in vital aneurysms on days 7 and 21. Significant differences in the proportion of CM-Dil +cells in the parent artery for day 7 (p<0.05) were observed.
Overall, four of 35 animals died, yielding an 11.4% mortality rate. The four deaths included two animals with irreparable vessel defect suffered intraoperatively during stent implantation, and two animals in the decellularized stenting group whose euthanasia was planned for day 21 (one for abdominal wound dehiscence, and one for dehydration due to inadequate food and water intake).
Aneurysm growth after treatment Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.
Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.
Microscopic healing status All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).
Periadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).
All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).
Periadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).
Fluorescence analysis for neointima, thrombus, aneurysm wall and parent artery In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).
Illustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.
For vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.
In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).
Illustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.
For vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.
Surgical characteristics Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).
Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).
Physiological parameters Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.
Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.
Aneurysm growth after treatment:
Baseline aneurysm volumes did not statistically differ between coiling and stenting, respectively, in the decellularized group (17.15±6.40 mm3 vs. 14.60±3.50 mm3; p=0.86) or vital group (17.10±3.01 mm3 vs. 11.87±5.87 mm3; p=0.10) (online supplemental figure 3); aneurysm volumes depicted were pooled (ie, combined follow-up days 7 and 21) as a single value. In follow-up comparisons, decellularized aneurysms showed a non-significant tendency toward more pronounced aneurysm growth in the coiled group than stented group (38.15±23.47 mm3 vs. 22.42±7.92 mm3; p=0.28). At follow-up, this growth was significantly greater in the vital group for coiled versus stent-treated aneurysms (60.10±31.12 mm3 vs. 20.51±20.65 mm3; p=0.002) (online supplemental figure 3). At follow up, fluorescence angiography indicated reperfusion in 6/6 (100%) coiled animals and in 1/8 (12.5%) with stent treatment (online supplemental figure 4). Evolution of aneurysm healing is depicted by time in online supplemental figure 5.
Microscopic healing status:
All aneurysms demonstrated progressive healing patterns with increased neointima thickness and advanced thrombus organization (detailed histological analysis of all aneurysms in online supplemental figure 6; aneurysm volumes are depicted pooled).
Periadventitial inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.003); no effects were observed between vital-coiled and vital-stented aneurysms (p=0.36). Periadventitial fibrosis was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0006) and vital counterparts (p=0.014). Aneurysm wall inflammation was significantly reduced in the decellularized coil group compared with the decellularized stent group (p=0.0046). Neointima formation was significantly enhanced in the decellularized stent group compared with the decellularized coil group (p=0.036), and in the vital group in favor to stent treatment (p=0.0012).
Fluorescence analysis for neointima, thrombus, aneurysm wall and parent artery:
In decellularized aneurysms, the amount of CM-Dil +cells in the neointima did not differ between stent-treated and coil-treated animals at 7 days (p=0.77), but was significantly higher in stent-treated animals at 21 days (p=0.04) (figures 2 and 3). For vital aneurysms, no significant differences were noted between groups at 7 days (p=1.0) or 21 days (p=0.66) (figure 2). In decellularized aneurysms, after 7 days significantly more CM-Dil +cells were found in the thrombus of the stented-treated group (p=0.01).
Illustrative panel of CM-Dil +cell distribution in a decellularized aneurysm with coil- and stent-treatment postoperatively. Panel A, coil day 7/stent day 7. Left-side depicts a coil-treated aneurysm, immunostaining for neointima (A) and parent artery (B) performed. Right side shows same setting performed for stenting 7 days postoperatively. CM-Dil +cells are found in the neointima more pronounced for stent-treatment (scale bar 100 μm left, 200 μm right). Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells (twofold magnification) with stent-treatment 7 days postoperatively; $ shows fixation artifact. In panel B (coil day 21/stent day 21), immunostaining for neointima (A) and parent artery (B) demonstrates decreased CM-Dil +cells in coil-treated compared with stent-treated aneurysms (p<0.05). (A) Magnified area of the neointima. CM-Dil +cells are found in the neointima (scale bar 100 μm left, 100 μm right). (B) As well, CM-Dil uptake is registrated in the parent artery. Middle, image overview of monoclonal anti-α muscle actin (a-SMA) positive cells in an aneurysm with a twofold magnification after coil treatment 21 days postoperatively is shown. *Lumen of the parent artery; #coil artifact.
For vital aneurysms, a significant enhancement in CM-Dil +cells was observed in the parent artery of stented animals on day 7 (p=0.02). In a pooled analysis, decellularized aneurysms showed significance in CM-Dil +cells in the thrombus for the stent group (p=0.017), whereas no differences were observed in the vital group for either coil or stent treatment. Proportions of CM-Dil +cells for all aneurysms are depicted in online supplemental table 2.
Surgical characteristics:
Comparisons of surgical characteristics between coil- and stent-treated animals are depicted in online supplemental figure 7. Significant differences were found between coil and stent treatments, respectively, for operative time (119.06±21.35 min vs 154.13±30.20 min; p=0.001), the number of stitches to suture the aneurysm (15.62±2.87 vs 11.26±1.09; p=0.000002), and the total number of stitches (including further stitches performed when anastomosis was leaking) used for aneurysm suturing (15.93±2.86 vs 11.40±1.05; p=0.000002).
Physiological parameters:
Vital signs monitored for the duration of the operation are depicted in online supplemental figure 8. In addition to pulse distension, we also analyzed breath distension, heart rate, oxygen saturation, breath rate, and temperature. Although breath distension was significantly reduced in stent-treated (12.95±0.71) versus coil-treated (13.51±0.63) rats (p=0.027), the other physiological variables did not show any significant differences.
Discussion:
In comparing healing of decellularized and vital aneurysms after coil and stent treatments, we demonstrated that neointima formation was mediated by endothelial cells originating in the adjacent parent artery in decellularized aneurysms whereas this lining was supported by cells derived from the aneurysm wall in vital aneurysms. This finding was consistent in short-term and intermediate follow-up. In turn, previous studies have already shown that the amount of circulating progenitor cells contribute only a minor role in aneurysm healing.19 20
A growing body of literature suggests that the various treatment options for intracranial aneurysms are not only efficacious but can be associated with the biological response of the wall itself.5 7 9 11 12 Specifically, ligation provides an immediate direct endothelial contact whereas endovascular therapies rely more on biological processes, particularly the newly developed minimally invasive techniques, such as stent-assisted coiling, flow-diversion, web-device management, bioactive endovascular devices, systemic drug therapies or cell-based therapies.9 10
Previous studies have shown that neointima formation and thrombus organization are concurrent processes in aneurysm healing after endovascular therapies: both rely on cell migration from the parent artery and aneurysm wall, and both are facilitated by the presence of endovascular devices. Thrombus-organizing cells typically originate from the parent artery for both types of endovascular treatment, whereas neointima formation in coiled aneurysms relies more on cell migration from the aneurysm wall, in stent-treated aneurysms mostly from the parent artery.11 In our experimental study, the parent artery was identified as an important source of migrating cells that form the neointima. This process happened in both forms of endovascular treatment and was observed within a few days from onset of aneurysm healing. During subsequent weeks, fading of the CM-Dil dye indicates the loss of signal intensity as these cells continue to divide at their new location, predominantly in the neointima and thrombus. The intensity of the lipophilic cell tracer decreases with each mitosis. These findings confirm the important role of endovascularly applied stents in a cell-rich region that serves as a principal structure for cell migration, which allows continuous endothelial lining, neointima formation, and progressive aneurysm healing.
In decellularized aneurysms, the number of CM-Dil +cells after stent versus coil treatments were higher on day 7 (72.25% vs 68%, p=0.77) and significantly higher on day 21 in the neointima (34.44% vs 7.75%, p=0.04). This finding suggests that cells involved in the aneurysm healing process migrated from the parent artery rather than the aneurysm wall. More importantly, we believe that the significantly greater number of CM-Dil +cells found in the thrombus after 7 days could be because the stent struts facilitated intraluminal thrombus formation in the aneurysm sac, which also seems explainable because the neointima is not sealed after 7 days. For vital aneurysms after coiling and stenting for days 7 (56.75% vs 58.25%, p=1) and 21 (11.5% vs 16.5%, p=0.66), no significant differences were found regarding CM-Dil +cells in the neointima; this finding supports the hypothesis that healing mechanisms in the coiled animals are stronger because of recruiting cells from the aneurysm wall (for endothelial staining via F8 please see online supplemental figure 9). In vital aneurysms, comparison of the number of CM-Dil +cells in coil versus stent treatments showed similar distributions in the aneurysm wall on day 7 (13.25% vs 13.5%, p=0.88) and day 21 (10.25% vs 10.75%, p=0.55); were similar for intraluminal thrombus formation on day 7 (62% vs 71.25%, p=1) and day 21 (26.25 % vs 23.75 %, p=0.55); and decreased in the parent artery on day 7 (60.0% vs 81.5%, p=0.02) and day 21 (24.25% vs 26.5%, p=0.46). The significantly higher amount of CM-Dil +cells in the parent artery of stented aneurysms might be because stent insertion causes more local reactions than coils and that neointima formation was initiated via stents overlapping the parent artery. Therefore, endovascular treatment via stent treatment might be crucial for highly degenerated aneurysm walls whereas coiling or stent-assisted coiling might be predominantly useful for aneurysms with relatively healthy vessel walls.
Interestingly, the proportion of CM-Dil +cells in the parent artery was higher at both day 7 and day 21 in the decellularized stent-treated group (76.6% vs 35.66% day) than in the coil-treated group (75.5% vs 10.5%). P values for both time-points, comparing the amount of CM-Dil +cells in the parent artery in the decellularized coil versus stented group on day 7, were non-significantly altered (p=0.77), and also when compared with 21 days (p=0.07). At 21-day follow-up, variations in levels of CM-Dil +cells of <10% in five rats might be explained by the natural course of the fading dye or longer operating times that potentially degraded the light sensitive dye.
On days 7 and 21, respectively, more CM-Dil +cells were detected inside the thrombus of the stent-treated group (25.25% vs 8.33%) than the coiled group (7.5% vs 5.5%). This might be reasonable because after 21 days the neointima is completely sealed in nearly all of the cases. CM-Dil +cell migration into the thrombus was significantly enhanced for stent treatments on day 7 (p=0.01), possibly facilitated by the strut-like stents themselves; after 21 days, the p value was not significantly altered (p=0.1). CM-Dil +cells in the aneurysm wall showed no differences between coil or stent treatments on day 7 (12.25% vs 11.75%, p=0.55) and day 21 (8.5% vs 9%, p=1). Regarding endothelial thrombus invasion, Raymond et al elucidated the role of stents in lowering aneurysm recanalization rate and decreasing recurrences after embolization in a side-wall aneurysm model with venous pouches.21 In our experiments, the amounts and extent of invasion of endothelial cells into the thrombus was higher in the coil-treated than stent-treated groups; this finding corresponded with a higher aneurysm reperfusion rate in the coiled group.
In summary, cell migration, allowing for a continuous endothelial lining via stents along the cell-rich parent artery’s lumen, may be the substantial prerequisite for complete aneurysm healing after endovascular therapy, and may be especially critical in highly degenerated aneurysms. The implication is that coiling in ruptured cerebral aneurysms might suffice to promote healing in patients with relative healthy vessel walls, but is likely insufficient in aneurysms with degenerated walls. In terms of clinical translation and future perspectives, our results align with a preliminary report in a preclinical series by Nevzati et al.8 Coating endovascular devices, such as coils, with chemo-attractants might be a therapeutic strategy to stimulate adjacent cells in a healthy parent artery to divide and migrate into the aneurysm thrombus.
Study limitations Recent findings by Morel et al
22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23
Recent findings by Morel et al
22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23
Study limitations:
Recent findings by Morel et al
22 showed the influence of sex hormones on aneurysm growth, intraluminal thrombus resolution, and wall inflammation in the same aneurysm model used for our experiments. Therefore, to avoid the confounding effect of estrogen or androgen or both hormones, we included only male rats. Moreover, we used a specific cell tracer for labeling the parent artery in an effort to differentiate which cells were derived from circulating cells in the blood stream and which were derived from true migration of neighboring cells. Although previous studies found strong signal intensity of myofibroblasts at days 7 and 21, we cannot exclude a slightly diminished signal intensity in endothelial cells that was related to time and cell division.9 Lastly, our model used hemodynamic characteristics and subsequent biological processes, such as the rate of spontaneous thrombosis or aneurysm healing, which are relevantly influenced by the side wall constellation of the aneurysm.23
Conclusion:
Our study in a rat side wall model corroborates previous findings that biological healing of endovascularly treated aneurysms depends on cell migration from the adjacent vessel and is additionally supported by recruitment of cells from the aneurysm wall in vital (cell-rich) aneurysms. However, in decellularized aneurysms, the adjacent vessel segment is the most important source of cells to promote healing. Therefore, cell migration is facilitated by use of endovascular devices, such as stents, that connect adjacent cell-rich tissues to the aneurysm orifice. In consequence, scaffolds should be placed in cell-rich healthy vessel regions, particularly for treatment of highly degenerated aneurysms. Vice versa, the success of these treatment strategies may depend on the presence of a healthy (cell-rich) endothelium in the adjacent vessel. Coil treatment alone might be sufficient for aneurysms with relatively healthy vessel walls.
| Background:
Unlike clipping that forms an immediate barrier of blood flow into intracranial aneurysms, endovascular treatments rely on thrombus organization and neointima formation. Therefore, a continuous endothelial cell layer is crucial to prevent blood flow in the former aneurysm. This study investigates the origin of endothelial cells in the neointima of endovascular treated aneurysms, specifically whether cells from the parent artery play a role in neointima formation.
Methods:
In male rats, decellularized and vital side wall aneurysms were treated by coil (n=16) or stent embolization (n=15). The cell tracer CM-Dil dye was injected into the clamped aorta before aneurysm suture to mark initial endothelial cells in the parent artery and enable tracking of their proliferation during follow-up. Aneurysms were analyzed for growth, thrombus formation, and recurrence. Histological evaluation followed with cell counts for specific regions-of-interest.
Results:
During follow-up, none of the 31 aneurysms ruptured. Macroscopic residual perfusion was observed in 12/16 rats after coiling and in 1/15 after stenting. Amounts of CM-Dil +cells in coiled versus stented decellularized aneurysms significantly decreased in the thrombus on day 7 (p=0.01) and neointima on day 21 (p=0.04). For vital aneurysms, the number of CM-Dil +cells in the neointima on day 21 showed no significant difference.
Conclusions:
Healing patterns were worse in coil-treated than stent-treated aneurysms. Cell migration forming a neointima seemed mainly dependent on the adjacent vessel in decellularized aneurysms, but appeared buoyed by recruitment from aneurysm wall cells in vital aneurysms. Therefore, a cell-rich parent artery might be crucial. | Introduction:
During the past decades, endovascular treatment has gained increasing importance in the management of both ruptured and unruptured intracranial aneurysms. Nevertheless, recurrence rates after coil embolization remain notably high compared with clip ligation.1–4 Unlike direct mechanical occlusion of an aneurysm sac by clipping, an endovascularly treated patient awaits a biological healing response of thrombus organization and neointima formation as mediated by cell migration.
Smooth muscle cells (SMCs) play an important role in inducing aneurysm healing on a biological level. Preclinical and clinical studies have revealed that loss of mural cells predisposes aneurysm growth and rupture,5–7 and that healing after coil embolization is worse in aneurysms with highly degenerated walls than in those with SMC-rich walls.8 Cell-based therapies with intraluminal transplantation of SMCs were shown to promote complete aneurysm healing along with prevention of growth and rupture.9 In a comparison of endovascular devices, better aneurysm healing was demonstrated in stented compared with coiled aneurysms10; likewise neointima formation and thrombus organization are concurrent processes in aneurysm healing, that depend on cell migration from both the parent artery and aneurysm wall. Neointima formation relied more on cell migration from the aneurysm wall in coiled aneurysms, whereas the role of cells originating from the parent artery seemed to be higher in stent-treated aneurysms.11 These factors suggest that cell migration, allowing for a continuous endothelial lining along the parent artery’s lumen, substantially contributes to complete aneurysm healing after endovascular treatment.
Until now, the origin of cells (aneurysm wall, parent artery or progenitor cells) responsible for aneurysm healing has remained unclear.12 13 Whether the endothelial lining is triggered by progenitor cells, which have already been identified as attaching in stented animal aneurysm models,14 or the parent artery itself has not yet been adequately analyzed. Toward this aim, our experimental study in a rat saccular side wall aneurysm model examined the origin of neointima-forming cells. Using a cell tracer injection, we analyzed the role of endothelial cells originating in the parent artery in neointima formation.
Conclusion:
Our study in a rat side wall model corroborates previous findings that biological healing of endovascularly treated aneurysms depends on cell migration from the adjacent vessel and is additionally supported by recruitment of cells from the aneurysm wall in vital (cell-rich) aneurysms. However, in decellularized aneurysms, the adjacent vessel segment is the most important source of cells to promote healing. Therefore, cell migration is facilitated by use of endovascular devices, such as stents, that connect adjacent cell-rich tissues to the aneurysm orifice. In consequence, scaffolds should be placed in cell-rich healthy vessel regions, particularly for treatment of highly degenerated aneurysms. Vice versa, the success of these treatment strategies may depend on the presence of a healthy (cell-rich) endothelium in the adjacent vessel. Coil treatment alone might be sufficient for aneurysms with relatively healthy vessel walls. | Background:
Unlike clipping that forms an immediate barrier of blood flow into intracranial aneurysms, endovascular treatments rely on thrombus organization and neointima formation. Therefore, a continuous endothelial cell layer is crucial to prevent blood flow in the former aneurysm. This study investigates the origin of endothelial cells in the neointima of endovascular treated aneurysms, specifically whether cells from the parent artery play a role in neointima formation.
Methods:
In male rats, decellularized and vital side wall aneurysms were treated by coil (n=16) or stent embolization (n=15). The cell tracer CM-Dil dye was injected into the clamped aorta before aneurysm suture to mark initial endothelial cells in the parent artery and enable tracking of their proliferation during follow-up. Aneurysms were analyzed for growth, thrombus formation, and recurrence. Histological evaluation followed with cell counts for specific regions-of-interest.
Results:
During follow-up, none of the 31 aneurysms ruptured. Macroscopic residual perfusion was observed in 12/16 rats after coiling and in 1/15 after stenting. Amounts of CM-Dil +cells in coiled versus stented decellularized aneurysms significantly decreased in the thrombus on day 7 (p=0.01) and neointima on day 21 (p=0.04). For vital aneurysms, the number of CM-Dil +cells in the neointima on day 21 showed no significant difference.
Conclusions:
Healing patterns were worse in coil-treated than stent-treated aneurysms. Cell migration forming a neointima seemed mainly dependent on the adjacent vessel in decellularized aneurysms, but appeared buoyed by recruitment from aneurysm wall cells in vital aneurysms. Therefore, a cell-rich parent artery might be crucial. | 11,022 | 312 | [
3449,
152,
418,
136,
243,
140,
454,
159,
188,
153,
474,
88,
77,
167
] | 18 | [
"aneurysm",
"stent",
"cells",
"aneurysms",
"coil",
"cm",
"cm dil",
"dil",
"decellularized",
"group"
] | [
"better aneurysm healing",
"inducing aneurysm healing",
"progressive aneurysm healing",
"aneurysm healing macroscopic",
"aneurysm healing endovascular"
] | null | [CONTENT] aneurysm | coil | stent | vessel wall | intervention [SUMMARY] | null | [CONTENT] aneurysm | coil | stent | vessel wall | intervention [SUMMARY] | [CONTENT] aneurysm | coil | stent | vessel wall | intervention [SUMMARY] | [CONTENT] aneurysm | coil | stent | vessel wall | intervention [SUMMARY] | [CONTENT] aneurysm | coil | stent | vessel wall | intervention [SUMMARY] | [CONTENT] Male | Rats | Animals | Neointima | Endothelial Cells | Intracranial Aneurysm | Stents | Embolization, Therapeutic | Arteries | Thrombosis | Treatment Outcome [SUMMARY] | null | [CONTENT] Male | Rats | Animals | Neointima | Endothelial Cells | Intracranial Aneurysm | Stents | Embolization, Therapeutic | Arteries | Thrombosis | Treatment Outcome [SUMMARY] | [CONTENT] Male | Rats | Animals | Neointima | Endothelial Cells | Intracranial Aneurysm | Stents | Embolization, Therapeutic | Arteries | Thrombosis | Treatment Outcome [SUMMARY] | [CONTENT] Male | Rats | Animals | Neointima | Endothelial Cells | Intracranial Aneurysm | Stents | Embolization, Therapeutic | Arteries | Thrombosis | Treatment Outcome [SUMMARY] | [CONTENT] Male | Rats | Animals | Neointima | Endothelial Cells | Intracranial Aneurysm | Stents | Embolization, Therapeutic | Arteries | Thrombosis | Treatment Outcome [SUMMARY] | [CONTENT] better aneurysm healing | inducing aneurysm healing | progressive aneurysm healing | aneurysm healing macroscopic | aneurysm healing endovascular [SUMMARY] | null | [CONTENT] better aneurysm healing | inducing aneurysm healing | progressive aneurysm healing | aneurysm healing macroscopic | aneurysm healing endovascular [SUMMARY] | [CONTENT] better aneurysm healing | inducing aneurysm healing | progressive aneurysm healing | aneurysm healing macroscopic | aneurysm healing endovascular [SUMMARY] | [CONTENT] better aneurysm healing | inducing aneurysm healing | progressive aneurysm healing | aneurysm healing macroscopic | aneurysm healing endovascular [SUMMARY] | [CONTENT] better aneurysm healing | inducing aneurysm healing | progressive aneurysm healing | aneurysm healing macroscopic | aneurysm healing endovascular [SUMMARY] | [CONTENT] aneurysm | stent | cells | aneurysms | coil | cm | cm dil | dil | decellularized | group [SUMMARY] | null | [CONTENT] aneurysm | stent | cells | aneurysms | coil | cm | cm dil | dil | decellularized | group [SUMMARY] | [CONTENT] aneurysm | stent | cells | aneurysms | coil | cm | cm dil | dil | decellularized | group [SUMMARY] | [CONTENT] aneurysm | stent | cells | aneurysms | coil | cm | cm dil | dil | decellularized | group [SUMMARY] | [CONTENT] aneurysm | stent | cells | aneurysms | coil | cm | cm dil | dil | decellularized | group [SUMMARY] | [CONTENT] aneurysm | healing | cells | aneurysm healing | parent | artery | parent artery | cell migration | migration | cell [SUMMARY] | null | [CONTENT] stent | group | coil | decellularized | treated | dil | cm | cm dil | aneurysms | mm3 [SUMMARY] | [CONTENT] adjacent | vessel | adjacent vessel | cell rich | cell | rich | healthy | aneurysms | healthy vessel | cell migration [SUMMARY] | [CONTENT] aneurysm | cells | stent | group | coil | aneurysms | cm | dil | cm dil | decellularized [SUMMARY] | [CONTENT] aneurysm | cells | stent | group | coil | aneurysms | cm | dil | cm dil | decellularized [SUMMARY] | [CONTENT] ||| ||| [SUMMARY] | null | [CONTENT] 31 ||| 12/16 | 1/15 ||| day 7 | day 21 | p=0.04 ||| CM-Dil | day 21 [SUMMARY] | [CONTENT] ||| ||| [SUMMARY] | [CONTENT] ||| ||| ||| ||| CM-Dil ||| ||| ||| 31 ||| 12/16 | 1/15 ||| day 7 | day 21 | p=0.04 ||| CM-Dil | day 21 ||| ||| ||| [SUMMARY] | [CONTENT] ||| ||| ||| ||| CM-Dil ||| ||| ||| 31 ||| 12/16 | 1/15 ||| day 7 | day 21 | p=0.04 ||| CM-Dil | day 21 ||| ||| ||| [SUMMARY] |
|||||
[Contemporary use of instruments during clinical examination in German ENT departments and private practices]. | 34402928 | The classical forehead reflector as traditionally used by ear, nose, and throat (ENT) physicians for the ENT examination is now iconic for doctors in general. It is unknown which instruments are currently used in Germany to clinically examine ENT patients. Therefore, this study aims to present results of a survey about commonly used instruments. | BACKGROUND | An evaluation of 321 questionnaires from ENT doctors working in general and university hospitals (172) and in private practices (149) was performed. | MATERIALS AND METHODS | The ENT mirror examination is nowadays carried out with a self-illuminating headlamp with battery and/or light guide cable. Approximately 20% of respondents also use a forehead mirror. The microscope is used by 90% of doctors to examine the ears; a rigid endoscope was used in 53.3% to examine the larynx, epipharynx (41.1%), and the nose/sinuses (34.6%). Flexible endoscopes and otoscopes are used only rarely. | RESULTS | The self-illuminating headlamp, which is more often wireless in eastern Germany, has largely replaced the classical forehead reflector, with which doctors younger than 40 years were no longer trained. At least some organs are also examined very regularly with the microscope or rigid endoscope. The flexible endoscope and otoscope are used much less frequently overall, mainly by younger physicians and ENT doctors working in hospitals. The diagnostic potential of flexible endoscopy may be compromised by the outpatient remuneration structures in Germany. | CONCLUSION | [
"Endoscopes",
"Germany",
"Humans",
"Nose",
"Pharynx",
"Private Practice"
] | 8813715 | null | null | null | null | null | null | Fazit für die Praxis |
Die klinische HNO-Untersuchung in Deutschland wird mit einer Stirnlampe in den neuen Bundesländern häufiger ohne Kabel durchgeführt.Der Stirnreflektor spielt mit weiter rückläufiger Tendenz kaum noch eine Rolle.Die Diagnostik der Ohren erfolgt fast immer mit einem Mikroskop, die übrigen Organe werden regelmäßig auch mit einem starren Endoskop untersucht.Auf deutlich niedrigerem Niveau sind flexible Endoskope und Otoskope bei jüngeren, klinisch tätigen Kollegen beliebter.Eine konsequente Nutzung des diagnostischen Potenzials der flexiblen Endoskopie des Larynx (und Epi‑/Hypopharynx) wird durch die ambulanten Vergütungsstrukturen in Deutschland möglicherweise kompromittiert.
Die klinische HNO-Untersuchung in Deutschland wird mit einer Stirnlampe in den neuen Bundesländern häufiger ohne Kabel durchgeführt.
Der Stirnreflektor spielt mit weiter rückläufiger Tendenz kaum noch eine Rolle.
Die Diagnostik der Ohren erfolgt fast immer mit einem Mikroskop, die übrigen Organe werden regelmäßig auch mit einem starren Endoskop untersucht.
Auf deutlich niedrigerem Niveau sind flexible Endoskope und Otoskope bei jüngeren, klinisch tätigen Kollegen beliebter.
Eine konsequente Nutzung des diagnostischen Potenzials der flexiblen Endoskopie des Larynx (und Epi‑/Hypopharynx) wird durch die ambulanten Vergütungsstrukturen in Deutschland möglicherweise kompromittiert. | [
"Hintergrund und Fragestellung",
"Studiendesign und Untersuchungsmethoden",
"Ergebnisse",
"Diskussion"
] | [
"Die Organe des oberen Aerodigestivtrakts gehören exemplarisch zu den schwer zugänglichen Körperregionen, deren Beurteilung besondere Untersuchungsinstrumente erforderlich machen. Von großer Bedeutung für die Hals-Nasen-Ohren-Heilkunde war die erste Beschreibung eines „Hohlspiegels mit zentraler Durchlöcherung“ durch Friedrich Hofmann, Burgsteinfurt, im Jahr 1841, der um 1855 durch von Tröltsch zunächst für die Otoskopie etabliert wurde [5]. Der Hohlspiegel war ein ideales Instrument zur Beleuchtung und gleichzeitigen parallaxenfreien Betrachtung. Zunächst waren das Tageslicht oder eine Kerze die erforderlichen Lichtquellen, später wurde elektrisches Licht verwendet. Die über die Grenzen der Medizin hinausgehende Bedeutung des Stirnspiegels wird auch dadurch verdeutlicht, dass der verkehrt herum getragene, nach oben geklappte Spiegel in diversen Medien als Symbol des Arztes weit verbreitet ist (Abb. 1; [10]).\nAls Alternative zu dem an einem Stirnreif vor einem Auge fixierten Stirnspiegel wanderte später die Lichtquelle an eine zentrale Position in der Stirnmitte [11].\nAb den 1920er-Jahren stehen außerdem stereoskopische Mikroskope, ab den 1960er-Jahren nach der Entwicklung von Stablinsenoptiken starre Kaltlichtendoskope und später auch flexible, auch für die Passage durch die Nase geeignete Endoskope für die klinische Untersuchung von HNO-Patienten zur Verfügung [8].\nMit welchen Instrumenten heute angesichts der zahlreichen Optionen Patienten in der Praxis und im Krankenhaus untersucht werden, ist nicht bekannt. Es ist das Ziel der vorgelegten Untersuchung, dies auf dem Weg einer Befragung zu ermitteln. Außerdem sollen ggf. bestehende Unterschiede, z. B. zwischen den Generationen, zwischen Klinik und Praxis oder innerhalb Deutschlands detektiert werden. Von ausdrücklichem Interesse ist zuletzt auch die Frage, welche Rolle der Stirnreflektor in der aktuellen Praxis noch spielt.",
"Der von uns entwickelte und im Herbst 2019 versandte Fragebogen (Abb. 2, Abb. 3) umfasste 18 Fragen zur Person (u. a. Alter in Dekaden, Geschlecht, Ausbildungs- und derzeitiges Tätigkeitsumfeld) und zu Untersuchungsmethoden und eingesetzten Geräten (Stirnreflektor, Stirnlampe, Mikroskop, starres/flexibles Endoskop, Otoskop). Explizit gestellt wurde die Frage nach der Ausbildung mit dem Stirnreflektor; die Intensität der Nutzung der einzelnen Instrumente konnte differenziert werden (immer, situativ, nie).\nAdressaten der Befragung waren niedergelassene und klinisch tätige HNO-Ärzte. Zur Weiterleitung der Fragebögen an niedergelassene HNO-Ärzte nahmen wir Kontakt zum Deutschen HNO-Berufsverband (Organisationsgrad ≥ 90 % der niedergelassenen Ärzte) auf, zur Weiterleitung an die in 160 HNO-Hauptabteilungen und 35 Universitätsklinika klinisch tätigen HNO-Ärzte kontaktierten wir die Deutsche Gesellschaft für HNO-Heilkunde, Kopf- und Hals-Chirurgie.\nZur Untersuchung des Einflusses der Region Deutschlands, in der die Befragten ausgebildet wurden bzw. derzeit tätig sind, etablierten wir drei Untergruppen: Stadtstaaten, neue Bundesländer und alte Bundesländer. Relevante Daten der Bundesärztekammer und der Kassenärztlichen Bundesvereinigung wurden soweit verfügbar abgefragt.\nDie vorwiegend deskriptive statistische Auswertung der Daten wurde mit SPSS Statistics 23 (Statistical Package for the Social Sciences, IBM®, Armonk, NY, USA) vorgenommen. Neben dem Darstellen univariater Häufigkeiten wurden Assoziationen zwischen kategorialen Variablen mit dem Chi-Quadrat-Test analysiert.",
"Insgesamt 321 vollständig/auswertbar beantwortete Fragebögen erhielten wir von 172 in einer Klinik und 149 niedergelassenen oder in einem MVZ (Medizinisches Versorgungszentrum) tätigen Ärzten. Die Fragebögen wurden aus unterschiedlichen Gründen (z. B. Datenschutz) in lediglich 8/18 Landesverbänden an die Mitglieder des Berufsverbands versandt (39,5 % aller Mitglieder) [2]. Auch über die Deutsche HNO-Gesellschaft konnten die Fragebögen direkt lediglich an die Leiter der HNO-Hauptabteilungen und Universitätsklinika – mit der Bitte um Weiterleitung an die nachgeordneten Mitarbeiter – versandt werden. Wir schätzen, dass insgesamt ungefähr 1350 HNO-Ärzte die Einladung zur Befragung direkt erhalten haben. Die Response-Rate wird deshalb mit ca. 321/1350 (24 %) geschätzt.\nAn der Befragung nahmen 110 (34,4 %) Frauen und 209 (65,4 %) Männer teil. Andere und keine Angabe machten zwei (0,6 %). Die am stärksten vertretene Altersgruppe war die der 50- bis 60-Jährgen (Abb. 4). Von den 172 Klinik- und 149 niedergelassenen Ärzten waren 10,2 % in Stadtstaaten (Berlin, Hamburg), 45,4 % in den neuen Bundesländern (vor allem Brandenburg und Sachsen) und 44,4 % in den alten Bundesländern (vor allem Bayern und Nordrhein-Westfalen) tätig. Die Verteilung der Bundesländer, in denen die Befragten ausgebildet wurden, war mit 11,3 %, 45,8 % bzw. 42,4 % ähnlich. Darüber hinaus wurden 13 HNO-Ärzte (4 %) außerhalb Deutschlands, vornehmlich in Osteuropa, ausgebildet. Zum Zeitpunkt der Befragung waren 267 (83,2 %) als Fachärzte tätig, 54 als Ausbildungsassistenten. Unter den niedergelassenen Ärzten lag der Anteil der Fachärzte bei 98 %; unter den 172 Klinikärzten waren 59 Chefärzte und 62 Oberärzte.\n213 Befragte (66,4 %) gaben an, mit dem Stirnreflektor ausgebildet worden zu sein (Tab. 1). Von den 13 im Ausland Ausgebildeten (11 jünger als 40 Jahre) wurden 11 (85 %) mit dem Stirnreflektor ausgebildet. 66 Befragte (20,5 %) gaben an, den Reflektor auch aktuell zumindest manchmal einzusetzen, insbesondere zur Untersuchung von Mundhöhle/Oropharynx (65/66) und Nase (56/66). Aktuelle Nutzer eines Stirnreflektors sind vor allem > 60-jährige Fachärzte (47 % dieser Gruppe), etwas häufiger weiblich und doppelt so häufig in einer Praxis tätig wie in einer Klinik (30 % vs. 15 %).Immer Anzahl n (%)Situativ Anzahl n (%)Nie Anzahl n (%)MikroskopOhr289 (90,3 %)28 (8,8 %)3 (0,9 %)Nase/Nasenebenhöhlen43 (13,9 %)213 (69,7 %)54 (17,4 %)Mundhöhle/Oropharynx30 (9,7 %)214 (69,3 %)65 (21 %)Haut48 (15,1 %)239 (75,4 %)30 (9,5 %)Starres EndoskopNase/Nasennebenhöhlen111 (35,1 %)201 (63,6 %)4 (1,3 %)Kehlkopf/Hypopharynx171 (53,6 %)138 (43,3 %)10 (3,1 %)Epipharynx132 (41,5 %)170 (53,5 %)16 (5 %)Flexibles EndoskopNase/Nasennebenhöhlen28 (9,2 %)225 (74,3 %)50 (16,5 %)Kehlkopf/Hypopharynx36 (11,4 %)257 (81,1 %)24 (7,6 %)Epipharynx37 (11,7 %)251 (79,7 %)27 (8,6 %)Otoskop11 (3,4 %)121 (37,9 %)187 (58,6 %)\nZur Untersuchung ihrer Patienten setzten 217 Befragte eine Kopflampe mit Akku und 205 mit einem Lichtleitkabel ein, 101 benutzten beide Varianten; in den neuen Bundesländern war der Anteil derer, die (auch) eine kabellose Lampe benutzten, höher (74,4 % vs. 61,6 %).\nDas Mikroskop wird von 289 (90 %) HNO-Ärzten bei jedem Patienten zur Untersuchung der Ohren eingesetzt. Andere Organe werden in weniger als 15 % immer mit einem Mikroskop untersucht; situativ setzen die Befragten das Mikroskop zur Untersuchung von Nase/Nasennebenhöhlen und Mund‑/Rachenraum in 2/3 der Fälle und zur Untersuchung der Haut in ca. 75 % der Fälle ein.\nEin starres Endoskop mit unterschiedlichen Winkeloptiken verwenden 53,3 % der teilnehmenden HNO-Ärzte immer zur Untersuchung des Kehlkopfs, 41,1 % zur Untersuchung des Epipharynx und 34,6 % bei Nase/Nebenhöhlen. In allen übrigen Fällen wird das Gerät zumindest situativ verwandt.\nDas flexible Endoskop verwenden nur 10 % der Befragten bei jedem Patienten, zwischen 70 % (Nase/Nasennebenhöhlen) und 80 % (Kehlkopf) setzen das Gerät situativ ein. Während ca. 20 % der in der Praxis tätigen niemals ein flexibles Endoskop nutzen, tun dies 15 %, insbesondere jüngere Kollegen in der Klinik bei jedem Patienten.\nDas Otoskop wird von HNO-Ärzten in Klinik und Praxis lediglich von 3,4 % immer eingesetzt und von 58,3 % nie; die übrigen (38,3 %) nutzen es situativ. 70 % der niedergelassenen HNO-Ärzte verwenden das Otoskop nie; unter den klinisch tätigen Ärzten sind es vor allem die jüngsten, die das Otoskop situativ einsetzen (55,9 %), während ca. 65 % der über 50-Jährigen Otoskope nie verwenden.\nDer Umstand, dass der Stirnreflektor auch aktuell noch als Symbol für (HNO-)Ärzte verwandt wird, ist den meisten Befragten egal (45,8 %) oder stößt insbesondere bei den < 30-Jährigen sogar auf Ablehnung (24,6 %).",
"Wie HNO-Ärzte in deutschen Kliniken und Praxen ihre Patienten klinisch untersuchen, also unter Einsatz welcher Untersuchungsmethoden und Geräte heute eine „Spiegeluntersuchung“ stattfindet, ist nicht bekannt. Die durchgeführte Literaturrecherche machte außerdem deutlich, dass dies auch international kaum anders aussieht [11].\nIm Rahmen der von uns durchgeführten Untersuchung werteten wir 321 beantwortete Fragebögen aus den neuen und alten Bundesländern und Stadtstaaten aus. Die Altersverteilung der antwortenden Kollegen mit einem Maximum bei den 50- bis 60-Jährigen entsprach exakt der tatsächlichen Verteilung in Deutschland. Die Antwortenden waren überwiegend als Fachärzte (82 %) entweder in der Praxis oder in der Klinik tätig. Aus den Kliniken erhielten wir überrepräsentativ viele Antworten von leitenden Ärzten.\nDie klinische HNO-Untersuchung (Spiegeluntersuchung) wird in allen deutschen HNO-Einrichtungen mit einer fokussierbaren Stirnlampe durchgeführt; in den neuen Bundesländern häufiger mit einem System ohne Kabel. Lediglich ca. 20 % der Antwortenden untersuchen heute vor allem Nase und Mundhöhle mit dem Stirnreflektor. Wurden nach unseren Ergebnissen die > 40-Jährigen überwiegend noch mit dem Stirnreflektor ausgebildet, gilt dies für jüngere nicht mehr! Die Antworten von insbesondere in Osteuropa ausgebildeten Ärzten deuten an, dass es international Unterschiede geben könnte. Das Schicksal des Stirnspiegels für die klinische HNO-Untersuchung in Deutschland scheint also besiegelt.\nIm Unterschied dazu steht die aktuelle Präsenz des Stirnreflektors in primär nichtmedizinischen Medien als das verbreitetste Symbol des Arztes überhaupt [4]. Dieser Aspekt hat für die Befragten überwiegend keine Bedeutung oder stößt, häufiger bei jüngeren, auf Ablehnung.\nNahezu alle Patienten werden sowohl in Kliniken als auch in Praxen über die Spiegeluntersuchung hinaus auch mit einem Mikroskop und/oder Endoskop untersucht.\nDies gilt vor allem für die Untersuchung der Ohren, die nahezu bei jedem Patienten mit dem Mikroskop untersucht werden. Bei anderen Organen wird das Mikroskop nur selten regelmäßig angewendet, in einem nennenswerten Umfang allerdings fakultativ. In diesem Kontext nennen die meisten Befragten die Beurteilung von Hautveränderungen. Auch im Fachgebiet Dermatologie spielt das Mikroskop bekanntlich im Rahmen der Tumordiagnostik eine große Rolle. Insbesondere zur regelmäßigen Untersuchung von Nase/Nasennebenhöhlen wird das Mikroskop seltener verwendet als das Endoskop; der erhöhte Aufwand für die Reinigung des Endoskops scheint bei der Wahl nicht ins Gewicht zu fallen. Auch die Untersuchung des Kehlkopfs mit Mikroskop in Verbindung mit einem Spiegel ist möglich, wurde allerdings von uns nicht ausdrücklich abgefragt. Bei einer Auswertung der Abrechnungsdaten der US-amerikanischen Krankenversicherung „Medicare“ wurden mikroskopische Ohruntersuchungen in Verbindung mit einer Zerumenentfernung bei Weitem am häufigsten abgerechnet [6].\nDas in der Häufigkeit des Einsatzes nach dem Mikroskop folgende Instrument ist das starre Endoskop mit unterschiedlichen Winkeloptiken, das bei mehr als jedem zweiten Patienten zur Untersuchung des Kehlkopfs eingesetzt wird. Regelmäßig werden auch Epipharynx (> 40 %) und Nase/Nasennebenhöhlen (ca. 35 %) so untersucht. Darüber hinaus berichten alle Antwortenden, starre Endoskope zumindest situativ zu verwenden. Die Bedeutung der starren Lupenlaryngoskopie für die Beurteilung des Situs und im Rahmen einer Stroboskopie, gute Untersuchungsbedingungen vorausgesetzt, ist sicher evident. Die Bedeutung der endoskopischen Untersuchung im Vergleich zur klassischen anterioren Rhinoskopie konnte vor allem bei anatomischen Veränderungen wie z. B. einer Septumdeviation, aber auch bei einem Vergleich mit einer CT demonstriert werden [9]. Hur et al. [6] fanden eine Steigerung der durch niedergelassene US-amerikanische Ärzte durchgeführten Nasenendoskopien um 313 % zwischen 2000 und 2016. Ein Zusammenhang zwischen den erheblichen regionalen Schwankungen und der Dichte der HNO-Ärzte, der Zahl der Versicherten und der Höhe der Vergütung wurde vermutet. Im Unterschied dazu nutzten in einer 1989 publizierten Studie aus England nur ca. 10 % der befragten niedergelassenen HNO-Ärzte starre Winkeloptiken zur Untersuchung von Nasopharynx und Larynx; insbesondere die Laryngoskopie betreffend wird dieses Verhalten damit erklärt, dass die starre Endoskopie weder hinsichtlich des Würgereizes noch der Übersicht (überhängende Epiglottis) Vorteile gegenüber der Untersuchung mit dem Spiegel hat [1].\nLetztgenannte Aspekte betreffend stellt das flexible Endoskop eine wirkliche Alternative dar, wird aber nach unseren Ergebnissen nur sehr selten bei jedem Patienten eingesetzt; auch unter Berücksichtigung eines situativen Einsatzes wird deutlich, dass in der Praxis tätige Kollegen das flexible Endoskop seltener und jüngere Kollegen (in der Klinik) es häufiger nutzen. Im Unterschied hierzu ist die flexible Laryngoskopie die zweithäufigste Prozedur, die niedergelassenen HNO-Ärzte in den USA aktuell abrechnen. Zwischen den Jahren 2000 und 2016 wurde ein Anstieg der durchgeführten Prozeduren um 87 % festgestellt [7].\nUrsache für den seltenen Einsatz vor allem flexibler Endoskope in deutschen HNO-Praxen könnte ein Missverhältnis von Kosten und Erlösen sein. Mit der Novellierung der Abrechnung ambulanter Leistungen (EBM 2000Plus) wurde die Einzelleistungsvergütung weitgehend durch eine Pauschale ersetzt. Aktuell wird lediglich die Lupenlaryngoskopie mit 74 Punkten (8,23 €) vergütet; weitere mikroskopische und endoskopische Untersuchungen einschließlich der flexiblen Rhinolaryngoskopie sind in der Konsultationspauschale enthalten. Ein weiterer betriebswirtschaftlicher Aspekt ist die im EBM 2000Plus fehlende Vergütung für die Aufbereitung von Medizinprodukten, die seit der Einführung des Medizinproduktegesetzes, der Medizinprodukte-Betreiberverordnung etc. mit einem erheblichen Aufwand (und Kosten) verbunden ist.\nIn den USA wurde die flexible Rhinolaryngoskopie im Jahr 2016 als Einzelleistung mit 85 bis 104 US-$ vergütet. Bereits in den 1980er-Jahren wurde mehr als jeder zweite ambulante Patient in England (auch) mit einem flexiblen Rhinolaryngoskop untersucht. Als Begründung werden nicht nur die gute Übersicht über praktisch jeden Kehlkopf bei einem wachen Patienten benannt, sondern vor allem die eben auch kostenrelevante Vermeidung von ggf. stationären Untersuchungen in Narkose [1].\nDas Otoskop ist ein Instrument, das von ca. 60 % der HNO-Ärzte nie eingesetzt wird. Die offensichtlichen Nachteile des Otoskops, also Monokularität, kurze Brennweite und die Blockierung einer Hand und die offensichtlich ubiquitäre Verfügbarkeit binokularer Mikroskope in Praxen und Kliniken könnte dies erklären. Häufiger nehmen klinisch als ambulant tätige HNO-Ärzte die Nachteile des Otoskops zumindest situativ in Kauf, möglicherweise zur Durchführung von konsiliarischen Untersuchungen bei immobilen Patienten. Wir finden darüber hinaus einen Unterschied zwischen jüngeren HNO-Ärzten, die das Otoskop häufiger einsetzen, im Vergleich zu älteren HNO-Ärzten [3]. Bei Pädiatern und Allgemeinärzten ist dagegen die Ohruntersuchung mit dem Otoskop Standard [3].\nDer Umfang unserer Befragung mit 321 Teilnehmern war relativ gering verglichen mit der Gesamtzahl von etwa 5000 in Deutschland klinisch und ambulant tätigen HNO-Ärzten. Die für die eingeschränkte Weiterleitung der Fragebögen verantwortlichen Datenschutz-Probleme wurden dargelegt. Zumindest geografisch scheint eine Selektion unwahrscheinlich, da der Rücklauf aus den verschiedenen Bundesländern repräsentativ war und daher ein regionaler Vergleich möglich. Ob der hohe Anteil leitender Klinikärzte an der Studie die Repräsentativität der Angaben zu den angewandten Untersuchungstechniken erhöht, muss offen bleiben."
] | [
null,
null,
null,
null
] | [
"Hintergrund und Fragestellung",
"Studiendesign und Untersuchungsmethoden",
"Ergebnisse",
"Diskussion",
"Fazit für die Praxis"
] | [
"Die Organe des oberen Aerodigestivtrakts gehören exemplarisch zu den schwer zugänglichen Körperregionen, deren Beurteilung besondere Untersuchungsinstrumente erforderlich machen. Von großer Bedeutung für die Hals-Nasen-Ohren-Heilkunde war die erste Beschreibung eines „Hohlspiegels mit zentraler Durchlöcherung“ durch Friedrich Hofmann, Burgsteinfurt, im Jahr 1841, der um 1855 durch von Tröltsch zunächst für die Otoskopie etabliert wurde [5]. Der Hohlspiegel war ein ideales Instrument zur Beleuchtung und gleichzeitigen parallaxenfreien Betrachtung. Zunächst waren das Tageslicht oder eine Kerze die erforderlichen Lichtquellen, später wurde elektrisches Licht verwendet. Die über die Grenzen der Medizin hinausgehende Bedeutung des Stirnspiegels wird auch dadurch verdeutlicht, dass der verkehrt herum getragene, nach oben geklappte Spiegel in diversen Medien als Symbol des Arztes weit verbreitet ist (Abb. 1; [10]).\nAls Alternative zu dem an einem Stirnreif vor einem Auge fixierten Stirnspiegel wanderte später die Lichtquelle an eine zentrale Position in der Stirnmitte [11].\nAb den 1920er-Jahren stehen außerdem stereoskopische Mikroskope, ab den 1960er-Jahren nach der Entwicklung von Stablinsenoptiken starre Kaltlichtendoskope und später auch flexible, auch für die Passage durch die Nase geeignete Endoskope für die klinische Untersuchung von HNO-Patienten zur Verfügung [8].\nMit welchen Instrumenten heute angesichts der zahlreichen Optionen Patienten in der Praxis und im Krankenhaus untersucht werden, ist nicht bekannt. Es ist das Ziel der vorgelegten Untersuchung, dies auf dem Weg einer Befragung zu ermitteln. Außerdem sollen ggf. bestehende Unterschiede, z. B. zwischen den Generationen, zwischen Klinik und Praxis oder innerhalb Deutschlands detektiert werden. Von ausdrücklichem Interesse ist zuletzt auch die Frage, welche Rolle der Stirnreflektor in der aktuellen Praxis noch spielt.",
"Der von uns entwickelte und im Herbst 2019 versandte Fragebogen (Abb. 2, Abb. 3) umfasste 18 Fragen zur Person (u. a. Alter in Dekaden, Geschlecht, Ausbildungs- und derzeitiges Tätigkeitsumfeld) und zu Untersuchungsmethoden und eingesetzten Geräten (Stirnreflektor, Stirnlampe, Mikroskop, starres/flexibles Endoskop, Otoskop). Explizit gestellt wurde die Frage nach der Ausbildung mit dem Stirnreflektor; die Intensität der Nutzung der einzelnen Instrumente konnte differenziert werden (immer, situativ, nie).\nAdressaten der Befragung waren niedergelassene und klinisch tätige HNO-Ärzte. Zur Weiterleitung der Fragebögen an niedergelassene HNO-Ärzte nahmen wir Kontakt zum Deutschen HNO-Berufsverband (Organisationsgrad ≥ 90 % der niedergelassenen Ärzte) auf, zur Weiterleitung an die in 160 HNO-Hauptabteilungen und 35 Universitätsklinika klinisch tätigen HNO-Ärzte kontaktierten wir die Deutsche Gesellschaft für HNO-Heilkunde, Kopf- und Hals-Chirurgie.\nZur Untersuchung des Einflusses der Region Deutschlands, in der die Befragten ausgebildet wurden bzw. derzeit tätig sind, etablierten wir drei Untergruppen: Stadtstaaten, neue Bundesländer und alte Bundesländer. Relevante Daten der Bundesärztekammer und der Kassenärztlichen Bundesvereinigung wurden soweit verfügbar abgefragt.\nDie vorwiegend deskriptive statistische Auswertung der Daten wurde mit SPSS Statistics 23 (Statistical Package for the Social Sciences, IBM®, Armonk, NY, USA) vorgenommen. Neben dem Darstellen univariater Häufigkeiten wurden Assoziationen zwischen kategorialen Variablen mit dem Chi-Quadrat-Test analysiert.",
"Insgesamt 321 vollständig/auswertbar beantwortete Fragebögen erhielten wir von 172 in einer Klinik und 149 niedergelassenen oder in einem MVZ (Medizinisches Versorgungszentrum) tätigen Ärzten. Die Fragebögen wurden aus unterschiedlichen Gründen (z. B. Datenschutz) in lediglich 8/18 Landesverbänden an die Mitglieder des Berufsverbands versandt (39,5 % aller Mitglieder) [2]. Auch über die Deutsche HNO-Gesellschaft konnten die Fragebögen direkt lediglich an die Leiter der HNO-Hauptabteilungen und Universitätsklinika – mit der Bitte um Weiterleitung an die nachgeordneten Mitarbeiter – versandt werden. Wir schätzen, dass insgesamt ungefähr 1350 HNO-Ärzte die Einladung zur Befragung direkt erhalten haben. Die Response-Rate wird deshalb mit ca. 321/1350 (24 %) geschätzt.\nAn der Befragung nahmen 110 (34,4 %) Frauen und 209 (65,4 %) Männer teil. Andere und keine Angabe machten zwei (0,6 %). Die am stärksten vertretene Altersgruppe war die der 50- bis 60-Jährgen (Abb. 4). Von den 172 Klinik- und 149 niedergelassenen Ärzten waren 10,2 % in Stadtstaaten (Berlin, Hamburg), 45,4 % in den neuen Bundesländern (vor allem Brandenburg und Sachsen) und 44,4 % in den alten Bundesländern (vor allem Bayern und Nordrhein-Westfalen) tätig. Die Verteilung der Bundesländer, in denen die Befragten ausgebildet wurden, war mit 11,3 %, 45,8 % bzw. 42,4 % ähnlich. Darüber hinaus wurden 13 HNO-Ärzte (4 %) außerhalb Deutschlands, vornehmlich in Osteuropa, ausgebildet. Zum Zeitpunkt der Befragung waren 267 (83,2 %) als Fachärzte tätig, 54 als Ausbildungsassistenten. Unter den niedergelassenen Ärzten lag der Anteil der Fachärzte bei 98 %; unter den 172 Klinikärzten waren 59 Chefärzte und 62 Oberärzte.\n213 Befragte (66,4 %) gaben an, mit dem Stirnreflektor ausgebildet worden zu sein (Tab. 1). Von den 13 im Ausland Ausgebildeten (11 jünger als 40 Jahre) wurden 11 (85 %) mit dem Stirnreflektor ausgebildet. 66 Befragte (20,5 %) gaben an, den Reflektor auch aktuell zumindest manchmal einzusetzen, insbesondere zur Untersuchung von Mundhöhle/Oropharynx (65/66) und Nase (56/66). Aktuelle Nutzer eines Stirnreflektors sind vor allem > 60-jährige Fachärzte (47 % dieser Gruppe), etwas häufiger weiblich und doppelt so häufig in einer Praxis tätig wie in einer Klinik (30 % vs. 15 %).Immer Anzahl n (%)Situativ Anzahl n (%)Nie Anzahl n (%)MikroskopOhr289 (90,3 %)28 (8,8 %)3 (0,9 %)Nase/Nasenebenhöhlen43 (13,9 %)213 (69,7 %)54 (17,4 %)Mundhöhle/Oropharynx30 (9,7 %)214 (69,3 %)65 (21 %)Haut48 (15,1 %)239 (75,4 %)30 (9,5 %)Starres EndoskopNase/Nasennebenhöhlen111 (35,1 %)201 (63,6 %)4 (1,3 %)Kehlkopf/Hypopharynx171 (53,6 %)138 (43,3 %)10 (3,1 %)Epipharynx132 (41,5 %)170 (53,5 %)16 (5 %)Flexibles EndoskopNase/Nasennebenhöhlen28 (9,2 %)225 (74,3 %)50 (16,5 %)Kehlkopf/Hypopharynx36 (11,4 %)257 (81,1 %)24 (7,6 %)Epipharynx37 (11,7 %)251 (79,7 %)27 (8,6 %)Otoskop11 (3,4 %)121 (37,9 %)187 (58,6 %)\nZur Untersuchung ihrer Patienten setzten 217 Befragte eine Kopflampe mit Akku und 205 mit einem Lichtleitkabel ein, 101 benutzten beide Varianten; in den neuen Bundesländern war der Anteil derer, die (auch) eine kabellose Lampe benutzten, höher (74,4 % vs. 61,6 %).\nDas Mikroskop wird von 289 (90 %) HNO-Ärzten bei jedem Patienten zur Untersuchung der Ohren eingesetzt. Andere Organe werden in weniger als 15 % immer mit einem Mikroskop untersucht; situativ setzen die Befragten das Mikroskop zur Untersuchung von Nase/Nasennebenhöhlen und Mund‑/Rachenraum in 2/3 der Fälle und zur Untersuchung der Haut in ca. 75 % der Fälle ein.\nEin starres Endoskop mit unterschiedlichen Winkeloptiken verwenden 53,3 % der teilnehmenden HNO-Ärzte immer zur Untersuchung des Kehlkopfs, 41,1 % zur Untersuchung des Epipharynx und 34,6 % bei Nase/Nebenhöhlen. In allen übrigen Fällen wird das Gerät zumindest situativ verwandt.\nDas flexible Endoskop verwenden nur 10 % der Befragten bei jedem Patienten, zwischen 70 % (Nase/Nasennebenhöhlen) und 80 % (Kehlkopf) setzen das Gerät situativ ein. Während ca. 20 % der in der Praxis tätigen niemals ein flexibles Endoskop nutzen, tun dies 15 %, insbesondere jüngere Kollegen in der Klinik bei jedem Patienten.\nDas Otoskop wird von HNO-Ärzten in Klinik und Praxis lediglich von 3,4 % immer eingesetzt und von 58,3 % nie; die übrigen (38,3 %) nutzen es situativ. 70 % der niedergelassenen HNO-Ärzte verwenden das Otoskop nie; unter den klinisch tätigen Ärzten sind es vor allem die jüngsten, die das Otoskop situativ einsetzen (55,9 %), während ca. 65 % der über 50-Jährigen Otoskope nie verwenden.\nDer Umstand, dass der Stirnreflektor auch aktuell noch als Symbol für (HNO-)Ärzte verwandt wird, ist den meisten Befragten egal (45,8 %) oder stößt insbesondere bei den < 30-Jährigen sogar auf Ablehnung (24,6 %).",
"Wie HNO-Ärzte in deutschen Kliniken und Praxen ihre Patienten klinisch untersuchen, also unter Einsatz welcher Untersuchungsmethoden und Geräte heute eine „Spiegeluntersuchung“ stattfindet, ist nicht bekannt. Die durchgeführte Literaturrecherche machte außerdem deutlich, dass dies auch international kaum anders aussieht [11].\nIm Rahmen der von uns durchgeführten Untersuchung werteten wir 321 beantwortete Fragebögen aus den neuen und alten Bundesländern und Stadtstaaten aus. Die Altersverteilung der antwortenden Kollegen mit einem Maximum bei den 50- bis 60-Jährigen entsprach exakt der tatsächlichen Verteilung in Deutschland. Die Antwortenden waren überwiegend als Fachärzte (82 %) entweder in der Praxis oder in der Klinik tätig. Aus den Kliniken erhielten wir überrepräsentativ viele Antworten von leitenden Ärzten.\nDie klinische HNO-Untersuchung (Spiegeluntersuchung) wird in allen deutschen HNO-Einrichtungen mit einer fokussierbaren Stirnlampe durchgeführt; in den neuen Bundesländern häufiger mit einem System ohne Kabel. Lediglich ca. 20 % der Antwortenden untersuchen heute vor allem Nase und Mundhöhle mit dem Stirnreflektor. Wurden nach unseren Ergebnissen die > 40-Jährigen überwiegend noch mit dem Stirnreflektor ausgebildet, gilt dies für jüngere nicht mehr! Die Antworten von insbesondere in Osteuropa ausgebildeten Ärzten deuten an, dass es international Unterschiede geben könnte. Das Schicksal des Stirnspiegels für die klinische HNO-Untersuchung in Deutschland scheint also besiegelt.\nIm Unterschied dazu steht die aktuelle Präsenz des Stirnreflektors in primär nichtmedizinischen Medien als das verbreitetste Symbol des Arztes überhaupt [4]. Dieser Aspekt hat für die Befragten überwiegend keine Bedeutung oder stößt, häufiger bei jüngeren, auf Ablehnung.\nNahezu alle Patienten werden sowohl in Kliniken als auch in Praxen über die Spiegeluntersuchung hinaus auch mit einem Mikroskop und/oder Endoskop untersucht.\nDies gilt vor allem für die Untersuchung der Ohren, die nahezu bei jedem Patienten mit dem Mikroskop untersucht werden. Bei anderen Organen wird das Mikroskop nur selten regelmäßig angewendet, in einem nennenswerten Umfang allerdings fakultativ. In diesem Kontext nennen die meisten Befragten die Beurteilung von Hautveränderungen. Auch im Fachgebiet Dermatologie spielt das Mikroskop bekanntlich im Rahmen der Tumordiagnostik eine große Rolle. Insbesondere zur regelmäßigen Untersuchung von Nase/Nasennebenhöhlen wird das Mikroskop seltener verwendet als das Endoskop; der erhöhte Aufwand für die Reinigung des Endoskops scheint bei der Wahl nicht ins Gewicht zu fallen. Auch die Untersuchung des Kehlkopfs mit Mikroskop in Verbindung mit einem Spiegel ist möglich, wurde allerdings von uns nicht ausdrücklich abgefragt. Bei einer Auswertung der Abrechnungsdaten der US-amerikanischen Krankenversicherung „Medicare“ wurden mikroskopische Ohruntersuchungen in Verbindung mit einer Zerumenentfernung bei Weitem am häufigsten abgerechnet [6].\nDas in der Häufigkeit des Einsatzes nach dem Mikroskop folgende Instrument ist das starre Endoskop mit unterschiedlichen Winkeloptiken, das bei mehr als jedem zweiten Patienten zur Untersuchung des Kehlkopfs eingesetzt wird. Regelmäßig werden auch Epipharynx (> 40 %) und Nase/Nasennebenhöhlen (ca. 35 %) so untersucht. Darüber hinaus berichten alle Antwortenden, starre Endoskope zumindest situativ zu verwenden. Die Bedeutung der starren Lupenlaryngoskopie für die Beurteilung des Situs und im Rahmen einer Stroboskopie, gute Untersuchungsbedingungen vorausgesetzt, ist sicher evident. Die Bedeutung der endoskopischen Untersuchung im Vergleich zur klassischen anterioren Rhinoskopie konnte vor allem bei anatomischen Veränderungen wie z. B. einer Septumdeviation, aber auch bei einem Vergleich mit einer CT demonstriert werden [9]. Hur et al. [6] fanden eine Steigerung der durch niedergelassene US-amerikanische Ärzte durchgeführten Nasenendoskopien um 313 % zwischen 2000 und 2016. Ein Zusammenhang zwischen den erheblichen regionalen Schwankungen und der Dichte der HNO-Ärzte, der Zahl der Versicherten und der Höhe der Vergütung wurde vermutet. Im Unterschied dazu nutzten in einer 1989 publizierten Studie aus England nur ca. 10 % der befragten niedergelassenen HNO-Ärzte starre Winkeloptiken zur Untersuchung von Nasopharynx und Larynx; insbesondere die Laryngoskopie betreffend wird dieses Verhalten damit erklärt, dass die starre Endoskopie weder hinsichtlich des Würgereizes noch der Übersicht (überhängende Epiglottis) Vorteile gegenüber der Untersuchung mit dem Spiegel hat [1].\nLetztgenannte Aspekte betreffend stellt das flexible Endoskop eine wirkliche Alternative dar, wird aber nach unseren Ergebnissen nur sehr selten bei jedem Patienten eingesetzt; auch unter Berücksichtigung eines situativen Einsatzes wird deutlich, dass in der Praxis tätige Kollegen das flexible Endoskop seltener und jüngere Kollegen (in der Klinik) es häufiger nutzen. Im Unterschied hierzu ist die flexible Laryngoskopie die zweithäufigste Prozedur, die niedergelassenen HNO-Ärzte in den USA aktuell abrechnen. Zwischen den Jahren 2000 und 2016 wurde ein Anstieg der durchgeführten Prozeduren um 87 % festgestellt [7].\nUrsache für den seltenen Einsatz vor allem flexibler Endoskope in deutschen HNO-Praxen könnte ein Missverhältnis von Kosten und Erlösen sein. Mit der Novellierung der Abrechnung ambulanter Leistungen (EBM 2000Plus) wurde die Einzelleistungsvergütung weitgehend durch eine Pauschale ersetzt. Aktuell wird lediglich die Lupenlaryngoskopie mit 74 Punkten (8,23 €) vergütet; weitere mikroskopische und endoskopische Untersuchungen einschließlich der flexiblen Rhinolaryngoskopie sind in der Konsultationspauschale enthalten. Ein weiterer betriebswirtschaftlicher Aspekt ist die im EBM 2000Plus fehlende Vergütung für die Aufbereitung von Medizinprodukten, die seit der Einführung des Medizinproduktegesetzes, der Medizinprodukte-Betreiberverordnung etc. mit einem erheblichen Aufwand (und Kosten) verbunden ist.\nIn den USA wurde die flexible Rhinolaryngoskopie im Jahr 2016 als Einzelleistung mit 85 bis 104 US-$ vergütet. Bereits in den 1980er-Jahren wurde mehr als jeder zweite ambulante Patient in England (auch) mit einem flexiblen Rhinolaryngoskop untersucht. Als Begründung werden nicht nur die gute Übersicht über praktisch jeden Kehlkopf bei einem wachen Patienten benannt, sondern vor allem die eben auch kostenrelevante Vermeidung von ggf. stationären Untersuchungen in Narkose [1].\nDas Otoskop ist ein Instrument, das von ca. 60 % der HNO-Ärzte nie eingesetzt wird. Die offensichtlichen Nachteile des Otoskops, also Monokularität, kurze Brennweite und die Blockierung einer Hand und die offensichtlich ubiquitäre Verfügbarkeit binokularer Mikroskope in Praxen und Kliniken könnte dies erklären. Häufiger nehmen klinisch als ambulant tätige HNO-Ärzte die Nachteile des Otoskops zumindest situativ in Kauf, möglicherweise zur Durchführung von konsiliarischen Untersuchungen bei immobilen Patienten. Wir finden darüber hinaus einen Unterschied zwischen jüngeren HNO-Ärzten, die das Otoskop häufiger einsetzen, im Vergleich zu älteren HNO-Ärzten [3]. Bei Pädiatern und Allgemeinärzten ist dagegen die Ohruntersuchung mit dem Otoskop Standard [3].\nDer Umfang unserer Befragung mit 321 Teilnehmern war relativ gering verglichen mit der Gesamtzahl von etwa 5000 in Deutschland klinisch und ambulant tätigen HNO-Ärzten. Die für die eingeschränkte Weiterleitung der Fragebögen verantwortlichen Datenschutz-Probleme wurden dargelegt. Zumindest geografisch scheint eine Selektion unwahrscheinlich, da der Rücklauf aus den verschiedenen Bundesländern repräsentativ war und daher ein regionaler Vergleich möglich. Ob der hohe Anteil leitender Klinikärzte an der Studie die Repräsentativität der Angaben zu den angewandten Untersuchungstechniken erhöht, muss offen bleiben.",
"\nDie klinische HNO-Untersuchung in Deutschland wird mit einer Stirnlampe in den neuen Bundesländern häufiger ohne Kabel durchgeführt.Der Stirnreflektor spielt mit weiter rückläufiger Tendenz kaum noch eine Rolle.Die Diagnostik der Ohren erfolgt fast immer mit einem Mikroskop, die übrigen Organe werden regelmäßig auch mit einem starren Endoskop untersucht.Auf deutlich niedrigerem Niveau sind flexible Endoskope und Otoskope bei jüngeren, klinisch tätigen Kollegen beliebter.Eine konsequente Nutzung des diagnostischen Potenzials der flexiblen Endoskopie des Larynx (und Epi‑/Hypopharynx) wird durch die ambulanten Vergütungsstrukturen in Deutschland möglicherweise kompromittiert.\n\nDie klinische HNO-Untersuchung in Deutschland wird mit einer Stirnlampe in den neuen Bundesländern häufiger ohne Kabel durchgeführt.\nDer Stirnreflektor spielt mit weiter rückläufiger Tendenz kaum noch eine Rolle.\nDie Diagnostik der Ohren erfolgt fast immer mit einem Mikroskop, die übrigen Organe werden regelmäßig auch mit einem starren Endoskop untersucht.\nAuf deutlich niedrigerem Niveau sind flexible Endoskope und Otoskope bei jüngeren, klinisch tätigen Kollegen beliebter.\nEine konsequente Nutzung des diagnostischen Potenzials der flexiblen Endoskopie des Larynx (und Epi‑/Hypopharynx) wird durch die ambulanten Vergütungsstrukturen in Deutschland möglicherweise kompromittiert."
] | [
null,
null,
null,
null,
"conclusion"
] | [
"Hals-Nasen-Ohren-Heilkunde",
"Stirnspiegel",
"Mikroskop",
"Endoskop",
"Otoskop",
"Otorhinolaryngology",
"Forehead mirror",
"Microscope",
"Endoscope",
"Otoscope"
] | Hintergrund und Fragestellung:
Die Organe des oberen Aerodigestivtrakts gehören exemplarisch zu den schwer zugänglichen Körperregionen, deren Beurteilung besondere Untersuchungsinstrumente erforderlich machen. Von großer Bedeutung für die Hals-Nasen-Ohren-Heilkunde war die erste Beschreibung eines „Hohlspiegels mit zentraler Durchlöcherung“ durch Friedrich Hofmann, Burgsteinfurt, im Jahr 1841, der um 1855 durch von Tröltsch zunächst für die Otoskopie etabliert wurde [5]. Der Hohlspiegel war ein ideales Instrument zur Beleuchtung und gleichzeitigen parallaxenfreien Betrachtung. Zunächst waren das Tageslicht oder eine Kerze die erforderlichen Lichtquellen, später wurde elektrisches Licht verwendet. Die über die Grenzen der Medizin hinausgehende Bedeutung des Stirnspiegels wird auch dadurch verdeutlicht, dass der verkehrt herum getragene, nach oben geklappte Spiegel in diversen Medien als Symbol des Arztes weit verbreitet ist (Abb. 1; [10]).
Als Alternative zu dem an einem Stirnreif vor einem Auge fixierten Stirnspiegel wanderte später die Lichtquelle an eine zentrale Position in der Stirnmitte [11].
Ab den 1920er-Jahren stehen außerdem stereoskopische Mikroskope, ab den 1960er-Jahren nach der Entwicklung von Stablinsenoptiken starre Kaltlichtendoskope und später auch flexible, auch für die Passage durch die Nase geeignete Endoskope für die klinische Untersuchung von HNO-Patienten zur Verfügung [8].
Mit welchen Instrumenten heute angesichts der zahlreichen Optionen Patienten in der Praxis und im Krankenhaus untersucht werden, ist nicht bekannt. Es ist das Ziel der vorgelegten Untersuchung, dies auf dem Weg einer Befragung zu ermitteln. Außerdem sollen ggf. bestehende Unterschiede, z. B. zwischen den Generationen, zwischen Klinik und Praxis oder innerhalb Deutschlands detektiert werden. Von ausdrücklichem Interesse ist zuletzt auch die Frage, welche Rolle der Stirnreflektor in der aktuellen Praxis noch spielt.
Studiendesign und Untersuchungsmethoden:
Der von uns entwickelte und im Herbst 2019 versandte Fragebogen (Abb. 2, Abb. 3) umfasste 18 Fragen zur Person (u. a. Alter in Dekaden, Geschlecht, Ausbildungs- und derzeitiges Tätigkeitsumfeld) und zu Untersuchungsmethoden und eingesetzten Geräten (Stirnreflektor, Stirnlampe, Mikroskop, starres/flexibles Endoskop, Otoskop). Explizit gestellt wurde die Frage nach der Ausbildung mit dem Stirnreflektor; die Intensität der Nutzung der einzelnen Instrumente konnte differenziert werden (immer, situativ, nie).
Adressaten der Befragung waren niedergelassene und klinisch tätige HNO-Ärzte. Zur Weiterleitung der Fragebögen an niedergelassene HNO-Ärzte nahmen wir Kontakt zum Deutschen HNO-Berufsverband (Organisationsgrad ≥ 90 % der niedergelassenen Ärzte) auf, zur Weiterleitung an die in 160 HNO-Hauptabteilungen und 35 Universitätsklinika klinisch tätigen HNO-Ärzte kontaktierten wir die Deutsche Gesellschaft für HNO-Heilkunde, Kopf- und Hals-Chirurgie.
Zur Untersuchung des Einflusses der Region Deutschlands, in der die Befragten ausgebildet wurden bzw. derzeit tätig sind, etablierten wir drei Untergruppen: Stadtstaaten, neue Bundesländer und alte Bundesländer. Relevante Daten der Bundesärztekammer und der Kassenärztlichen Bundesvereinigung wurden soweit verfügbar abgefragt.
Die vorwiegend deskriptive statistische Auswertung der Daten wurde mit SPSS Statistics 23 (Statistical Package for the Social Sciences, IBM®, Armonk, NY, USA) vorgenommen. Neben dem Darstellen univariater Häufigkeiten wurden Assoziationen zwischen kategorialen Variablen mit dem Chi-Quadrat-Test analysiert.
Ergebnisse:
Insgesamt 321 vollständig/auswertbar beantwortete Fragebögen erhielten wir von 172 in einer Klinik und 149 niedergelassenen oder in einem MVZ (Medizinisches Versorgungszentrum) tätigen Ärzten. Die Fragebögen wurden aus unterschiedlichen Gründen (z. B. Datenschutz) in lediglich 8/18 Landesverbänden an die Mitglieder des Berufsverbands versandt (39,5 % aller Mitglieder) [2]. Auch über die Deutsche HNO-Gesellschaft konnten die Fragebögen direkt lediglich an die Leiter der HNO-Hauptabteilungen und Universitätsklinika – mit der Bitte um Weiterleitung an die nachgeordneten Mitarbeiter – versandt werden. Wir schätzen, dass insgesamt ungefähr 1350 HNO-Ärzte die Einladung zur Befragung direkt erhalten haben. Die Response-Rate wird deshalb mit ca. 321/1350 (24 %) geschätzt.
An der Befragung nahmen 110 (34,4 %) Frauen und 209 (65,4 %) Männer teil. Andere und keine Angabe machten zwei (0,6 %). Die am stärksten vertretene Altersgruppe war die der 50- bis 60-Jährgen (Abb. 4). Von den 172 Klinik- und 149 niedergelassenen Ärzten waren 10,2 % in Stadtstaaten (Berlin, Hamburg), 45,4 % in den neuen Bundesländern (vor allem Brandenburg und Sachsen) und 44,4 % in den alten Bundesländern (vor allem Bayern und Nordrhein-Westfalen) tätig. Die Verteilung der Bundesländer, in denen die Befragten ausgebildet wurden, war mit 11,3 %, 45,8 % bzw. 42,4 % ähnlich. Darüber hinaus wurden 13 HNO-Ärzte (4 %) außerhalb Deutschlands, vornehmlich in Osteuropa, ausgebildet. Zum Zeitpunkt der Befragung waren 267 (83,2 %) als Fachärzte tätig, 54 als Ausbildungsassistenten. Unter den niedergelassenen Ärzten lag der Anteil der Fachärzte bei 98 %; unter den 172 Klinikärzten waren 59 Chefärzte und 62 Oberärzte.
213 Befragte (66,4 %) gaben an, mit dem Stirnreflektor ausgebildet worden zu sein (Tab. 1). Von den 13 im Ausland Ausgebildeten (11 jünger als 40 Jahre) wurden 11 (85 %) mit dem Stirnreflektor ausgebildet. 66 Befragte (20,5 %) gaben an, den Reflektor auch aktuell zumindest manchmal einzusetzen, insbesondere zur Untersuchung von Mundhöhle/Oropharynx (65/66) und Nase (56/66). Aktuelle Nutzer eines Stirnreflektors sind vor allem > 60-jährige Fachärzte (47 % dieser Gruppe), etwas häufiger weiblich und doppelt so häufig in einer Praxis tätig wie in einer Klinik (30 % vs. 15 %).Immer Anzahl n (%)Situativ Anzahl n (%)Nie Anzahl n (%)MikroskopOhr289 (90,3 %)28 (8,8 %)3 (0,9 %)Nase/Nasenebenhöhlen43 (13,9 %)213 (69,7 %)54 (17,4 %)Mundhöhle/Oropharynx30 (9,7 %)214 (69,3 %)65 (21 %)Haut48 (15,1 %)239 (75,4 %)30 (9,5 %)Starres EndoskopNase/Nasennebenhöhlen111 (35,1 %)201 (63,6 %)4 (1,3 %)Kehlkopf/Hypopharynx171 (53,6 %)138 (43,3 %)10 (3,1 %)Epipharynx132 (41,5 %)170 (53,5 %)16 (5 %)Flexibles EndoskopNase/Nasennebenhöhlen28 (9,2 %)225 (74,3 %)50 (16,5 %)Kehlkopf/Hypopharynx36 (11,4 %)257 (81,1 %)24 (7,6 %)Epipharynx37 (11,7 %)251 (79,7 %)27 (8,6 %)Otoskop11 (3,4 %)121 (37,9 %)187 (58,6 %)
Zur Untersuchung ihrer Patienten setzten 217 Befragte eine Kopflampe mit Akku und 205 mit einem Lichtleitkabel ein, 101 benutzten beide Varianten; in den neuen Bundesländern war der Anteil derer, die (auch) eine kabellose Lampe benutzten, höher (74,4 % vs. 61,6 %).
Das Mikroskop wird von 289 (90 %) HNO-Ärzten bei jedem Patienten zur Untersuchung der Ohren eingesetzt. Andere Organe werden in weniger als 15 % immer mit einem Mikroskop untersucht; situativ setzen die Befragten das Mikroskop zur Untersuchung von Nase/Nasennebenhöhlen und Mund‑/Rachenraum in 2/3 der Fälle und zur Untersuchung der Haut in ca. 75 % der Fälle ein.
Ein starres Endoskop mit unterschiedlichen Winkeloptiken verwenden 53,3 % der teilnehmenden HNO-Ärzte immer zur Untersuchung des Kehlkopfs, 41,1 % zur Untersuchung des Epipharynx und 34,6 % bei Nase/Nebenhöhlen. In allen übrigen Fällen wird das Gerät zumindest situativ verwandt.
Das flexible Endoskop verwenden nur 10 % der Befragten bei jedem Patienten, zwischen 70 % (Nase/Nasennebenhöhlen) und 80 % (Kehlkopf) setzen das Gerät situativ ein. Während ca. 20 % der in der Praxis tätigen niemals ein flexibles Endoskop nutzen, tun dies 15 %, insbesondere jüngere Kollegen in der Klinik bei jedem Patienten.
Das Otoskop wird von HNO-Ärzten in Klinik und Praxis lediglich von 3,4 % immer eingesetzt und von 58,3 % nie; die übrigen (38,3 %) nutzen es situativ. 70 % der niedergelassenen HNO-Ärzte verwenden das Otoskop nie; unter den klinisch tätigen Ärzten sind es vor allem die jüngsten, die das Otoskop situativ einsetzen (55,9 %), während ca. 65 % der über 50-Jährigen Otoskope nie verwenden.
Der Umstand, dass der Stirnreflektor auch aktuell noch als Symbol für (HNO-)Ärzte verwandt wird, ist den meisten Befragten egal (45,8 %) oder stößt insbesondere bei den < 30-Jährigen sogar auf Ablehnung (24,6 %).
Diskussion:
Wie HNO-Ärzte in deutschen Kliniken und Praxen ihre Patienten klinisch untersuchen, also unter Einsatz welcher Untersuchungsmethoden und Geräte heute eine „Spiegeluntersuchung“ stattfindet, ist nicht bekannt. Die durchgeführte Literaturrecherche machte außerdem deutlich, dass dies auch international kaum anders aussieht [11].
Im Rahmen der von uns durchgeführten Untersuchung werteten wir 321 beantwortete Fragebögen aus den neuen und alten Bundesländern und Stadtstaaten aus. Die Altersverteilung der antwortenden Kollegen mit einem Maximum bei den 50- bis 60-Jährigen entsprach exakt der tatsächlichen Verteilung in Deutschland. Die Antwortenden waren überwiegend als Fachärzte (82 %) entweder in der Praxis oder in der Klinik tätig. Aus den Kliniken erhielten wir überrepräsentativ viele Antworten von leitenden Ärzten.
Die klinische HNO-Untersuchung (Spiegeluntersuchung) wird in allen deutschen HNO-Einrichtungen mit einer fokussierbaren Stirnlampe durchgeführt; in den neuen Bundesländern häufiger mit einem System ohne Kabel. Lediglich ca. 20 % der Antwortenden untersuchen heute vor allem Nase und Mundhöhle mit dem Stirnreflektor. Wurden nach unseren Ergebnissen die > 40-Jährigen überwiegend noch mit dem Stirnreflektor ausgebildet, gilt dies für jüngere nicht mehr! Die Antworten von insbesondere in Osteuropa ausgebildeten Ärzten deuten an, dass es international Unterschiede geben könnte. Das Schicksal des Stirnspiegels für die klinische HNO-Untersuchung in Deutschland scheint also besiegelt.
Im Unterschied dazu steht die aktuelle Präsenz des Stirnreflektors in primär nichtmedizinischen Medien als das verbreitetste Symbol des Arztes überhaupt [4]. Dieser Aspekt hat für die Befragten überwiegend keine Bedeutung oder stößt, häufiger bei jüngeren, auf Ablehnung.
Nahezu alle Patienten werden sowohl in Kliniken als auch in Praxen über die Spiegeluntersuchung hinaus auch mit einem Mikroskop und/oder Endoskop untersucht.
Dies gilt vor allem für die Untersuchung der Ohren, die nahezu bei jedem Patienten mit dem Mikroskop untersucht werden. Bei anderen Organen wird das Mikroskop nur selten regelmäßig angewendet, in einem nennenswerten Umfang allerdings fakultativ. In diesem Kontext nennen die meisten Befragten die Beurteilung von Hautveränderungen. Auch im Fachgebiet Dermatologie spielt das Mikroskop bekanntlich im Rahmen der Tumordiagnostik eine große Rolle. Insbesondere zur regelmäßigen Untersuchung von Nase/Nasennebenhöhlen wird das Mikroskop seltener verwendet als das Endoskop; der erhöhte Aufwand für die Reinigung des Endoskops scheint bei der Wahl nicht ins Gewicht zu fallen. Auch die Untersuchung des Kehlkopfs mit Mikroskop in Verbindung mit einem Spiegel ist möglich, wurde allerdings von uns nicht ausdrücklich abgefragt. Bei einer Auswertung der Abrechnungsdaten der US-amerikanischen Krankenversicherung „Medicare“ wurden mikroskopische Ohruntersuchungen in Verbindung mit einer Zerumenentfernung bei Weitem am häufigsten abgerechnet [6].
Das in der Häufigkeit des Einsatzes nach dem Mikroskop folgende Instrument ist das starre Endoskop mit unterschiedlichen Winkeloptiken, das bei mehr als jedem zweiten Patienten zur Untersuchung des Kehlkopfs eingesetzt wird. Regelmäßig werden auch Epipharynx (> 40 %) und Nase/Nasennebenhöhlen (ca. 35 %) so untersucht. Darüber hinaus berichten alle Antwortenden, starre Endoskope zumindest situativ zu verwenden. Die Bedeutung der starren Lupenlaryngoskopie für die Beurteilung des Situs und im Rahmen einer Stroboskopie, gute Untersuchungsbedingungen vorausgesetzt, ist sicher evident. Die Bedeutung der endoskopischen Untersuchung im Vergleich zur klassischen anterioren Rhinoskopie konnte vor allem bei anatomischen Veränderungen wie z. B. einer Septumdeviation, aber auch bei einem Vergleich mit einer CT demonstriert werden [9]. Hur et al. [6] fanden eine Steigerung der durch niedergelassene US-amerikanische Ärzte durchgeführten Nasenendoskopien um 313 % zwischen 2000 und 2016. Ein Zusammenhang zwischen den erheblichen regionalen Schwankungen und der Dichte der HNO-Ärzte, der Zahl der Versicherten und der Höhe der Vergütung wurde vermutet. Im Unterschied dazu nutzten in einer 1989 publizierten Studie aus England nur ca. 10 % der befragten niedergelassenen HNO-Ärzte starre Winkeloptiken zur Untersuchung von Nasopharynx und Larynx; insbesondere die Laryngoskopie betreffend wird dieses Verhalten damit erklärt, dass die starre Endoskopie weder hinsichtlich des Würgereizes noch der Übersicht (überhängende Epiglottis) Vorteile gegenüber der Untersuchung mit dem Spiegel hat [1].
Letztgenannte Aspekte betreffend stellt das flexible Endoskop eine wirkliche Alternative dar, wird aber nach unseren Ergebnissen nur sehr selten bei jedem Patienten eingesetzt; auch unter Berücksichtigung eines situativen Einsatzes wird deutlich, dass in der Praxis tätige Kollegen das flexible Endoskop seltener und jüngere Kollegen (in der Klinik) es häufiger nutzen. Im Unterschied hierzu ist die flexible Laryngoskopie die zweithäufigste Prozedur, die niedergelassenen HNO-Ärzte in den USA aktuell abrechnen. Zwischen den Jahren 2000 und 2016 wurde ein Anstieg der durchgeführten Prozeduren um 87 % festgestellt [7].
Ursache für den seltenen Einsatz vor allem flexibler Endoskope in deutschen HNO-Praxen könnte ein Missverhältnis von Kosten und Erlösen sein. Mit der Novellierung der Abrechnung ambulanter Leistungen (EBM 2000Plus) wurde die Einzelleistungsvergütung weitgehend durch eine Pauschale ersetzt. Aktuell wird lediglich die Lupenlaryngoskopie mit 74 Punkten (8,23 €) vergütet; weitere mikroskopische und endoskopische Untersuchungen einschließlich der flexiblen Rhinolaryngoskopie sind in der Konsultationspauschale enthalten. Ein weiterer betriebswirtschaftlicher Aspekt ist die im EBM 2000Plus fehlende Vergütung für die Aufbereitung von Medizinprodukten, die seit der Einführung des Medizinproduktegesetzes, der Medizinprodukte-Betreiberverordnung etc. mit einem erheblichen Aufwand (und Kosten) verbunden ist.
In den USA wurde die flexible Rhinolaryngoskopie im Jahr 2016 als Einzelleistung mit 85 bis 104 US-$ vergütet. Bereits in den 1980er-Jahren wurde mehr als jeder zweite ambulante Patient in England (auch) mit einem flexiblen Rhinolaryngoskop untersucht. Als Begründung werden nicht nur die gute Übersicht über praktisch jeden Kehlkopf bei einem wachen Patienten benannt, sondern vor allem die eben auch kostenrelevante Vermeidung von ggf. stationären Untersuchungen in Narkose [1].
Das Otoskop ist ein Instrument, das von ca. 60 % der HNO-Ärzte nie eingesetzt wird. Die offensichtlichen Nachteile des Otoskops, also Monokularität, kurze Brennweite und die Blockierung einer Hand und die offensichtlich ubiquitäre Verfügbarkeit binokularer Mikroskope in Praxen und Kliniken könnte dies erklären. Häufiger nehmen klinisch als ambulant tätige HNO-Ärzte die Nachteile des Otoskops zumindest situativ in Kauf, möglicherweise zur Durchführung von konsiliarischen Untersuchungen bei immobilen Patienten. Wir finden darüber hinaus einen Unterschied zwischen jüngeren HNO-Ärzten, die das Otoskop häufiger einsetzen, im Vergleich zu älteren HNO-Ärzten [3]. Bei Pädiatern und Allgemeinärzten ist dagegen die Ohruntersuchung mit dem Otoskop Standard [3].
Der Umfang unserer Befragung mit 321 Teilnehmern war relativ gering verglichen mit der Gesamtzahl von etwa 5000 in Deutschland klinisch und ambulant tätigen HNO-Ärzten. Die für die eingeschränkte Weiterleitung der Fragebögen verantwortlichen Datenschutz-Probleme wurden dargelegt. Zumindest geografisch scheint eine Selektion unwahrscheinlich, da der Rücklauf aus den verschiedenen Bundesländern repräsentativ war und daher ein regionaler Vergleich möglich. Ob der hohe Anteil leitender Klinikärzte an der Studie die Repräsentativität der Angaben zu den angewandten Untersuchungstechniken erhöht, muss offen bleiben.
Fazit für die Praxis:
Die klinische HNO-Untersuchung in Deutschland wird mit einer Stirnlampe in den neuen Bundesländern häufiger ohne Kabel durchgeführt.Der Stirnreflektor spielt mit weiter rückläufiger Tendenz kaum noch eine Rolle.Die Diagnostik der Ohren erfolgt fast immer mit einem Mikroskop, die übrigen Organe werden regelmäßig auch mit einem starren Endoskop untersucht.Auf deutlich niedrigerem Niveau sind flexible Endoskope und Otoskope bei jüngeren, klinisch tätigen Kollegen beliebter.Eine konsequente Nutzung des diagnostischen Potenzials der flexiblen Endoskopie des Larynx (und Epi‑/Hypopharynx) wird durch die ambulanten Vergütungsstrukturen in Deutschland möglicherweise kompromittiert.
Die klinische HNO-Untersuchung in Deutschland wird mit einer Stirnlampe in den neuen Bundesländern häufiger ohne Kabel durchgeführt.
Der Stirnreflektor spielt mit weiter rückläufiger Tendenz kaum noch eine Rolle.
Die Diagnostik der Ohren erfolgt fast immer mit einem Mikroskop, die übrigen Organe werden regelmäßig auch mit einem starren Endoskop untersucht.
Auf deutlich niedrigerem Niveau sind flexible Endoskope und Otoskope bei jüngeren, klinisch tätigen Kollegen beliebter.
Eine konsequente Nutzung des diagnostischen Potenzials der flexiblen Endoskopie des Larynx (und Epi‑/Hypopharynx) wird durch die ambulanten Vergütungsstrukturen in Deutschland möglicherweise kompromittiert.
| Background:
The classical forehead reflector as traditionally used by ear, nose, and throat (ENT) physicians for the ENT examination is now iconic for doctors in general. It is unknown which instruments are currently used in Germany to clinically examine ENT patients. Therefore, this study aims to present results of a survey about commonly used instruments.
Methods:
An evaluation of 321 questionnaires from ENT doctors working in general and university hospitals (172) and in private practices (149) was performed.
Results:
The ENT mirror examination is nowadays carried out with a self-illuminating headlamp with battery and/or light guide cable. Approximately 20% of respondents also use a forehead mirror. The microscope is used by 90% of doctors to examine the ears; a rigid endoscope was used in 53.3% to examine the larynx, epipharynx (41.1%), and the nose/sinuses (34.6%). Flexible endoscopes and otoscopes are used only rarely.
Conclusions:
The self-illuminating headlamp, which is more often wireless in eastern Germany, has largely replaced the classical forehead reflector, with which doctors younger than 40 years were no longer trained. At least some organs are also examined very regularly with the microscope or rigid endoscope. The flexible endoscope and otoscope are used much less frequently overall, mainly by younger physicians and ENT doctors working in hospitals. The diagnostic potential of flexible endoscopy may be compromised by the outpatient remuneration structures in Germany. | null | null | 3,143 | 284 | [
310,
271,
1086,
1250
] | 5 | [
"der",
"die",
"und",
"mit",
"hno",
"den",
"von",
"das",
"des",
"bei"
] | [
"und gleichzeitigen parallaxenfreien",
"lichtquelle eine zentrale",
"organe des oberen",
"aerodigestivtrakts",
"aerodigestivtrakts gehören"
] | null | null | null | null | null | null | null | [CONTENT] Hals-Nasen-Ohren-Heilkunde | Stirnspiegel | Mikroskop | Endoskop | Otoskop | Otorhinolaryngology | Forehead mirror | Microscope | Endoscope | Otoscope [SUMMARY] | [CONTENT] Hals-Nasen-Ohren-Heilkunde | Stirnspiegel | Mikroskop | Endoskop | Otoskop | Otorhinolaryngology | Forehead mirror | Microscope | Endoscope | Otoscope [SUMMARY] | null | null | null | null | [CONTENT] Endoscopes | Germany | Humans | Nose | Pharynx | Private Practice [SUMMARY] | [CONTENT] Endoscopes | Germany | Humans | Nose | Pharynx | Private Practice [SUMMARY] | null | null | null | null | [CONTENT] und gleichzeitigen parallaxenfreien | lichtquelle eine zentrale | organe des oberen | aerodigestivtrakts | aerodigestivtrakts gehören [SUMMARY] | [CONTENT] und gleichzeitigen parallaxenfreien | lichtquelle eine zentrale | organe des oberen | aerodigestivtrakts | aerodigestivtrakts gehören [SUMMARY] | null | null | null | null | [CONTENT] der | die | und | mit | hno | den | von | das | des | bei [SUMMARY] | [CONTENT] der | die | und | mit | hno | den | von | das | des | bei [SUMMARY] | null | null | null | null | [CONTENT] die | mit | deutschland | der | mit einem | wird | einem | eine | starren endoskop | ambulanten [SUMMARY] | [CONTENT] der | die | und | mit | hno | den | von | das | zur | bei [SUMMARY] | null | null | null | null | [CONTENT] eastern Germany | 40 years ||| ||| ENT ||| Germany [SUMMARY] | [CONTENT] ENT ||| Germany ||| ||| 321 | ENT | 172 | 149 ||| ENT ||| Approximately 20% ||| 90% | 53.3% | 41.1% | 34.6% ||| ||| eastern Germany | 40 years ||| ||| ENT ||| Germany [SUMMARY] | null |
||
Sex- and age-specific trends in antibiotic resistance patterns of Escherichia coli urinary isolates from outpatients. | 23433241 | Urinary tract infections (UTIs) are one of the most common infections treated in ambulatory care settings, however the epidemiology differs by age and sex. The incidence of UTI is far greater in females than males, and infection in pediatric patients is more often due to anatomical abnormalities. The purpose of this research was to describe age- and sex-specific trends in antibiotic susceptibility to common urinary anti-infectives among urinary isolates of Escherichia coli from ambulatory primary care patients in a regional health maintenance organization. | BACKGROUND | Clinical microbiology data were collected for all urine cultures from patients with visits to primary care clinics in a regional health maintenance organization between 2005 and 2010. The first positive culture for E. coli tested for antibiotic susceptibilities per patient per year was included in the analysis dataset. The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for male and female patients. The Cochrane-Mantel-Haenzel test was used to test for differences in age-stratified susceptibility to each antibiotic between males and females. | METHODS | A total of 43,493 E. coli isolates from 34,539 unique patients were identified for study inclusion. After stratifying by age, E. coli susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin differed significantly between males and females. However, the magnitude of the differences was less than 10% for all strata except amoxicillin-clavulanate susceptibility in E. coli isolated from males age 18-64 compared to females of the same age. | RESULTS | We did not observe clinically meaningful differences in antibiotic susceptibility to common urinary anti-infectives among E. coli isolated from males versus females. These data suggest that male sex alone should not be used as an indication for empiric use of second-line broad-spectrum antibiotic agents for the treatment of UTIs. | CONCLUSIONS | [
"Adolescent",
"Adult",
"Age Factors",
"Aged",
"Ambulatory Care",
"Amoxicillin-Potassium Clavulanate Combination",
"Ampicillin",
"Anti-Bacterial Agents",
"Ciprofloxacin",
"Cross-Sectional Studies",
"Drug Resistance, Bacterial",
"Escherichia coli",
"Female",
"Humans",
"Male",
"Microbial Sensitivity Tests",
"Middle Aged",
"Nitrofurantoin",
"Sex Factors",
"Trimethoprim, Sulfamethoxazole Drug Combination",
"Urinary Tract Infections",
"Young Adult"
] | 3610120 | Introduction | Urinary tract infections (UTIs) are one of the most commonly treated bacterial infections and account for over 10 million ambulatory care visits annually in the United States [1]. Antibiotic treatment is typically selected empirically, based on the patient clinical presentation, medical history and local patterns of antibiotic susceptibility [2]. Because the incidence of UTIs is significantly greater among women, much of the research has focused on women. Thus there exists a paucity of research on UTIs in men. As such, guidelines on the diagnosis, treatment, and management of UTIs focus largely on infection in women [2-5].
The incidence, presentation, and course of infection for UTIs in men and women differ in large part due to anatomical differences. For the treatment of acute uncomplicated cystitis in women, trimethoprim/sulfamethoxazole (TMP/SMX), nitrofurantoin, fosfomycin, and pivmecillinam are recommended first-line empiric therapies [2,6]. In men, because prostate involvement occurs in roughly 90% of cases, an empiric agent should be selected that achieves therapeutic concentrations in prostatic tissues (e.g., trimethoprim or ciprofloxacin) [7,8].
While historically it was believed that the causative organism in UTIs differed between men and women, more recent data has shown that for both sexes the primary causative pathogen is Escherichia coli, which accounts for 75-90% of UTIs [8,9]. With the increases in antibiotic resistance among E. coli and other Enterobacteriaceae over the past several decades, surveillance data have become critical for appropriate empiric selection of antibiotic therapy. U.S. guidelines specify that TMP/SMX should be avoided for empiric treatment of uncomplicated acute cystitis or pyelonephritis in populations where non-susceptibility to this agent exceeds 20% in uropathogens [2].
While some surveillance studies have identified significant differences in the frequency of susceptibility to common urinary anti-infectives between isolates collected from male and female patients, this has not been consistently observed [10,11]. The objective of this study was to describe age- and sex-specific antibiotic susceptibility patterns for common urinary anti-infectives among E. coli urine isolates using six years of data from a large ambulatory primary care patient population. | Methods | Study design and patient population We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].
We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].
Data collection Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.
Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.
Data analysis The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.
The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater. | Results | Description of study sample During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.
Patient characteristics based on first
E. coli
isolate from urine specimen in KPNW outpatients 2005-2010
a
aData are No. (%) unless otherwise indicated.
During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.
Patient characteristics based on first
E. coli
isolate from urine specimen in KPNW outpatients 2005-2010
a
aData are No. (%) unless otherwise indicated.
Antibiotic susceptibility of urinary Escherichia coli isolates by patient sex Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.
Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.
Trends in antibiotic susceptibility of urinary Escherichia coli isolates over time Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.
Susceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).
Susceptibility to urinary anti-infectives by sex and age for
E. coli
cultured from urine*
*Represents the first isolate for each patient and year.
CMH: Cochrane Maentel Haenszel correlation test.
Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.
Susceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).
Susceptibility to urinary anti-infectives by sex and age for
E. coli
cultured from urine*
*Represents the first isolate for each patient and year.
CMH: Cochrane Maentel Haenszel correlation test. | Conclusions | In the era of increasing antibiotic resistance, prudent use of antibiotics is critical to prolong the clinical effectiveness of existing agents. Excessive use of broad-spectrum agents, such as fluoroquinolones, increases the evolutionary selective pressures that drive the increasing prevalence of resistance. These data suggest that first-line urinary anti-infectives such as TMP/SMX may be effective agents for treating UTIs in men. While more data are needed, clinicians should use local surveillance data to guide the prudent, empiric selection of antibiotic therapy for UTIs. | [
"Introduction",
"Study design and patient population",
"Data collection",
"Data analysis",
"Description of study sample",
"Antibiotic susceptibility of urinary Escherichia coli isolates by patient sex",
"Trends in antibiotic susceptibility of urinary Escherichia coli isolates over time",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Urinary tract infections (UTIs) are one of the most commonly treated bacterial infections and account for over 10 million ambulatory care visits annually in the United States [1]. Antibiotic treatment is typically selected empirically, based on the patient clinical presentation, medical history and local patterns of antibiotic susceptibility [2]. Because the incidence of UTIs is significantly greater among women, much of the research has focused on women. Thus there exists a paucity of research on UTIs in men. As such, guidelines on the diagnosis, treatment, and management of UTIs focus largely on infection in women [2-5].\nThe incidence, presentation, and course of infection for UTIs in men and women differ in large part due to anatomical differences. For the treatment of acute uncomplicated cystitis in women, trimethoprim/sulfamethoxazole (TMP/SMX), nitrofurantoin, fosfomycin, and pivmecillinam are recommended first-line empiric therapies [2,6]. In men, because prostate involvement occurs in roughly 90% of cases, an empiric agent should be selected that achieves therapeutic concentrations in prostatic tissues (e.g., trimethoprim or ciprofloxacin) [7,8].\nWhile historically it was believed that the causative organism in UTIs differed between men and women, more recent data has shown that for both sexes the primary causative pathogen is Escherichia coli, which accounts for 75-90% of UTIs [8,9]. With the increases in antibiotic resistance among E. coli and other Enterobacteriaceae over the past several decades, surveillance data have become critical for appropriate empiric selection of antibiotic therapy. U.S. guidelines specify that TMP/SMX should be avoided for empiric treatment of uncomplicated acute cystitis or pyelonephritis in populations where non-susceptibility to this agent exceeds 20% in uropathogens [2].\nWhile some surveillance studies have identified significant differences in the frequency of susceptibility to common urinary anti-infectives between isolates collected from male and female patients, this has not been consistently observed [10,11]. The objective of this study was to describe age- and sex-specific antibiotic susceptibility patterns for common urinary anti-infectives among E. coli urine isolates using six years of data from a large ambulatory primary care patient population.",
"We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].",
"Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.",
"The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.",
"During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.\n\nPatient characteristics based on first \n\nE. coli \n\nisolate from urine specimen in KPNW outpatients 2005-2010\n\na\n\n\naData are No. (%) unless otherwise indicated.",
"Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.",
"Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.\nSusceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).\n\nSusceptibility to urinary anti-infectives by sex and age for \n\nE. coli \n\ncultured from urine*\n\n*Represents the first isolate for each patient and year.\nCMH: Cochrane Maentel Haenszel correlation test.",
"UTI: Urinary Tract Infection; KPNW: Kaiser Permanente Northwest; TMP/SMX: trimethoprim/sulfamethoxazole",
"The authors declare that they have no competing interests.",
"JCM participated in the conception and design of the study; the acquisition, statistical analysis, and interpretation of the data; and the drafting of the manuscript. JCM, MRE, DTB, and DHS assisted with the interpretation of the data and participated in the critical revision of the manuscript. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/14/25/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Study design and patient population",
"Data collection",
"Data analysis",
"Results",
"Description of study sample",
"Antibiotic susceptibility of urinary Escherichia coli isolates by patient sex",
"Trends in antibiotic susceptibility of urinary Escherichia coli isolates over time",
"Discussion",
"Conclusions",
"Abbreviations",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Urinary tract infections (UTIs) are one of the most commonly treated bacterial infections and account for over 10 million ambulatory care visits annually in the United States [1]. Antibiotic treatment is typically selected empirically, based on the patient clinical presentation, medical history and local patterns of antibiotic susceptibility [2]. Because the incidence of UTIs is significantly greater among women, much of the research has focused on women. Thus there exists a paucity of research on UTIs in men. As such, guidelines on the diagnosis, treatment, and management of UTIs focus largely on infection in women [2-5].\nThe incidence, presentation, and course of infection for UTIs in men and women differ in large part due to anatomical differences. For the treatment of acute uncomplicated cystitis in women, trimethoprim/sulfamethoxazole (TMP/SMX), nitrofurantoin, fosfomycin, and pivmecillinam are recommended first-line empiric therapies [2,6]. In men, because prostate involvement occurs in roughly 90% of cases, an empiric agent should be selected that achieves therapeutic concentrations in prostatic tissues (e.g., trimethoprim or ciprofloxacin) [7,8].\nWhile historically it was believed that the causative organism in UTIs differed between men and women, more recent data has shown that for both sexes the primary causative pathogen is Escherichia coli, which accounts for 75-90% of UTIs [8,9]. With the increases in antibiotic resistance among E. coli and other Enterobacteriaceae over the past several decades, surveillance data have become critical for appropriate empiric selection of antibiotic therapy. U.S. guidelines specify that TMP/SMX should be avoided for empiric treatment of uncomplicated acute cystitis or pyelonephritis in populations where non-susceptibility to this agent exceeds 20% in uropathogens [2].\nWhile some surveillance studies have identified significant differences in the frequency of susceptibility to common urinary anti-infectives between isolates collected from male and female patients, this has not been consistently observed [10,11]. The objective of this study was to describe age- and sex-specific antibiotic susceptibility patterns for common urinary anti-infectives among E. coli urine isolates using six years of data from a large ambulatory primary care patient population.",
" Study design and patient population We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].\nWe conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].\n Data collection Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.\nData were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.\n Data analysis The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.\nThe frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.",
"We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].",
"Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.",
"The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.",
" Description of study sample During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.\n\nPatient characteristics based on first \n\nE. coli \n\nisolate from urine specimen in KPNW outpatients 2005-2010\n\na\n\n\naData are No. (%) unless otherwise indicated.\nDuring the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.\n\nPatient characteristics based on first \n\nE. coli \n\nisolate from urine specimen in KPNW outpatients 2005-2010\n\na\n\n\naData are No. (%) unless otherwise indicated.\n Antibiotic susceptibility of urinary Escherichia coli isolates by patient sex Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.\nOverall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.\n Trends in antibiotic susceptibility of urinary Escherichia coli isolates over time Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.\nSusceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).\n\nSusceptibility to urinary anti-infectives by sex and age for \n\nE. coli \n\ncultured from urine*\n\n*Represents the first isolate for each patient and year.\nCMH: Cochrane Maentel Haenszel correlation test.\nFigure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.\nSusceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).\n\nSusceptibility to urinary anti-infectives by sex and age for \n\nE. coli \n\ncultured from urine*\n\n*Represents the first isolate for each patient and year.\nCMH: Cochrane Maentel Haenszel correlation test.",
"During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.\n\nPatient characteristics based on first \n\nE. coli \n\nisolate from urine specimen in KPNW outpatients 2005-2010\n\na\n\n\naData are No. (%) unless otherwise indicated.",
"Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.",
"Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.\nSusceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).\n\nSusceptibility to urinary anti-infectives by sex and age for \n\nE. coli \n\ncultured from urine*\n\n*Represents the first isolate for each patient and year.\nCMH: Cochrane Maentel Haenszel correlation test.",
"In this study, we observed statistically significant differences between males and females in the age-specific susceptibilities of E. coli to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin. Urinary E. coli isolates from male patients tended to exhibit increased antibiotic resistance than isolates from female patients. Despite the statistical significance of time trends and differences in age-specific susceptibilities, the magnitude of these differences was generally less than 5% and thus may not represent clinically meaningful differences. The exception was susceptibility to amoxicillin-clavulanate, where susceptibility was roughly 10% lower in males age 18 to 64 years than females in the same age group. Yet these differences should be interpreted cautiously because susceptibility to amoxicillin-clavulanate was only provided for ampicillin non-susceptible E. coli isolates in the current analysis. If all ampicillin susceptible isolates are assumed to be amoxicillin-clavulanate susceptible, then the magnitude of the differences between males and females would be less than 5% in all age categories (difference not statistically significant).\nWhile few other studies have explored the differences in antibiotic susceptibility of uropathogens isolated from male and female ambulatory patients, our findings are consistent with the trends observed in the literature. A recent 10-year study of community UTI in Portuguese patients also identified differences in antibiotic susceptibility by patient sex. The authors reported that urinary isolates of E. coli were significantly more resistant to fluoroquinolones, penicillins, nitrofurantoin, and first and second generation cephalosporins among men compared to women [13]. Another study focused on pediatric patients, also identified significantly higher resistance to TMP/SMX and ciprofloxacin in male versus female patients [14]. The NAUTICA surveillance study of outpatient UTIs reported greater antibiotic resistance to ciprofloxacin, levofloxacin, and TMP/SMX among all urinary isolates from U.S. and Canadian male patients [15]. In the CANWARD study, antibiotic susceptibility among all E. coli isolates (not limited to urine isolates) collected from Canadian tertiary medical centers were compared and resistance was also observed to be significantly higher to ciprofloxacin, levofloxacin, and TMP/SMX in isolates collected from male patients versus female patients [11].\nThe prevalence and susceptibilities of antibiotic-resistant bacteria varies widely by geographic region, thus these data may not be generalizable to all regions. Local surveillance data of antibiotic susceptibilities would be needed to validate these findings in different patient populations. It should be noted that in our study resistance to TMP/SMX did not exceed 20%, as it does in many other regions of the U.S., thus in this patient setting TMP/SMX may be a viable empiric treatment option in both males and female patients. Additionally, antibiotic susceptibilities may differ by other patient or infection characteristics (e.g., type of UTI). These data were not available for analysis in this study and further investigation in this area is needed. Also, because clinical signs and symptoms of infection were not available and because not all suspected infections are cultured, the use of all urinary microbiology from this population cultures may not represent all true infections.\nCurrently there exists insufficient data to inform the development of evidence-based guidelines for the treatment of community UTIs in men. UTIs in adult males are considered complicated infections according to most clinical definitions [16-18]. Treatment recommendations vary from longer durations of therapy to the selection of broad-spectrum agents such as fluoroquinolones [6]. These recommendations are driven by the high proportion of UTIs in men with prostate involvement and concerns surrounding antibiotic resistance to TMP/SMX [7,8]. Previous research among Swiss outpatients has demonstrated the selection of fluoroquinolones versus TMP/SMX for the treatment of UTI is often influenced by nonclinical factors [19]. In this study, TMP/SMX, a recommended first-line agent for UTIs in both children and adults, the differences in susceptibility between E. coli isolated from males and females were neither statistically nor clinically significant and resistance rates are below 20%. Consequently, in this population, there is no evidence that male sex alone should be an indication for empiric selection of a second-line broad-spectrum antibiotic agent for the treatment of UTI. While more research is needed on treatment effectiveness of different regimens used to treat community UTIs in men, population-specific antibiotic susceptibility data are a necessary component in the empiric antibiotic selection process.",
"In the era of increasing antibiotic resistance, prudent use of antibiotics is critical to prolong the clinical effectiveness of existing agents. Excessive use of broad-spectrum agents, such as fluoroquinolones, increases the evolutionary selective pressures that drive the increasing prevalence of resistance. These data suggest that first-line urinary anti-infectives such as TMP/SMX may be effective agents for treating UTIs in men. While more data are needed, clinicians should use local surveillance data to guide the prudent, empiric selection of antibiotic therapy for UTIs.",
"UTI: Urinary Tract Infection; KPNW: Kaiser Permanente Northwest; TMP/SMX: trimethoprim/sulfamethoxazole",
"The authors declare that they have no competing interests.",
"JCM participated in the conception and design of the study; the acquisition, statistical analysis, and interpretation of the data; and the drafting of the manuscript. JCM, MRE, DTB, and DHS assisted with the interpretation of the data and participated in the critical revision of the manuscript. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/14/25/prepub\n"
] | [
null,
"methods",
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null,
null
] | [
"Urinary tract infection",
"Urinary anti-infectives",
"Escherichia coli"
] | Introduction:
Urinary tract infections (UTIs) are one of the most commonly treated bacterial infections and account for over 10 million ambulatory care visits annually in the United States [1]. Antibiotic treatment is typically selected empirically, based on the patient clinical presentation, medical history and local patterns of antibiotic susceptibility [2]. Because the incidence of UTIs is significantly greater among women, much of the research has focused on women. Thus there exists a paucity of research on UTIs in men. As such, guidelines on the diagnosis, treatment, and management of UTIs focus largely on infection in women [2-5].
The incidence, presentation, and course of infection for UTIs in men and women differ in large part due to anatomical differences. For the treatment of acute uncomplicated cystitis in women, trimethoprim/sulfamethoxazole (TMP/SMX), nitrofurantoin, fosfomycin, and pivmecillinam are recommended first-line empiric therapies [2,6]. In men, because prostate involvement occurs in roughly 90% of cases, an empiric agent should be selected that achieves therapeutic concentrations in prostatic tissues (e.g., trimethoprim or ciprofloxacin) [7,8].
While historically it was believed that the causative organism in UTIs differed between men and women, more recent data has shown that for both sexes the primary causative pathogen is Escherichia coli, which accounts for 75-90% of UTIs [8,9]. With the increases in antibiotic resistance among E. coli and other Enterobacteriaceae over the past several decades, surveillance data have become critical for appropriate empiric selection of antibiotic therapy. U.S. guidelines specify that TMP/SMX should be avoided for empiric treatment of uncomplicated acute cystitis or pyelonephritis in populations where non-susceptibility to this agent exceeds 20% in uropathogens [2].
While some surveillance studies have identified significant differences in the frequency of susceptibility to common urinary anti-infectives between isolates collected from male and female patients, this has not been consistently observed [10,11]. The objective of this study was to describe age- and sex-specific antibiotic susceptibility patterns for common urinary anti-infectives among E. coli urine isolates using six years of data from a large ambulatory primary care patient population.
Methods:
Study design and patient population We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].
We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].
Data collection Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.
Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.
Data analysis The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.
The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.
Study design and patient population:
We conducted a cross-sectional study of urinary E. coli isolates from outpatients of Kaiser Permanente Northwest (KPNW) primary care clinics. KPNW is a regional health maintenance organization that serves over 485,000 members in northwest Oregon and southwest Washington. Primary care clinics were defined as non-specialty care clinics within the Family Practice, Internal Medicine, or Pediatrics departments; all primary care clinics were included. Urine cultures positive for E. coli that were drawn from patients with visits in the primary care clinics between January 1, 2005 and December 31, 2010 were eligible for inclusion in the analysis. Cultures in which less than 10,000 colonies/mL were identified or 3 or more organisms were isolated were excluded. The analysis dataset was then limited to the first isolate tested for antibiotic susceptibilities per patient and year to minimize potential bias resulting from repeat culturing [12].
Data collection:
Data were electronically extracted from the virtual data warehouse maintained by the Kaiser Permanente Center for Health Research. Collected data included patient demographics, department in which the clinic visit occurred, and all clinical microbiology data for urine cultures. Approval for this study was obtained from the KPNW institutional review board.
Data analysis:
The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for E. coli isolates stratified by patient sex and year. The Cochrane-Armitage test for trend was used to identify changes in the frequency of susceptibility to each antibiotic over time independently among males and females. Patient age at the time of culture was categorized as less than 18 years, 18–64 years, and 65 years and older. The frequency of susceptibility to each antibiotic was also compared across age categories among males and females using the Cochran-Mantel-Haenszel test. An alpha level less than or equal to 0.05 was the statistical significance level for all analyses and data were analyzed with SAS (version 9.2, SAS Corporation). Clinically significant differences were defined as differences of 10% or greater.
Results:
Description of study sample During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.
Patient characteristics based on first
E. coli
isolate from urine specimen in KPNW outpatients 2005-2010
a
aData are No. (%) unless otherwise indicated.
During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.
Patient characteristics based on first
E. coli
isolate from urine specimen in KPNW outpatients 2005-2010
a
aData are No. (%) unless otherwise indicated.
Antibiotic susceptibility of urinary Escherichia coli isolates by patient sex Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.
Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.
Trends in antibiotic susceptibility of urinary Escherichia coli isolates over time Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.
Susceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).
Susceptibility to urinary anti-infectives by sex and age for
E. coli
cultured from urine*
*Represents the first isolate for each patient and year.
CMH: Cochrane Maentel Haenszel correlation test.
Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.
Susceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).
Susceptibility to urinary anti-infectives by sex and age for
E. coli
cultured from urine*
*Represents the first isolate for each patient and year.
CMH: Cochrane Maentel Haenszel correlation test.
Description of study sample:
During the study time frame 190,396 urine cultures were performed at primary care clinic visits; 70,180 were positive for E. coli and 57,550 of those were tested for antibiotic susceptibilities. After restricting the data to the first isolate per patient per year, 43,493 isolates from 34,539 unique patients remained in the final analysis data set. Table 1 describes the demographics of these patients. Of the included isolates, 2,520 (5.8%) were from male patients.
Patient characteristics based on first
E. coli
isolate from urine specimen in KPNW outpatients 2005-2010
a
aData are No. (%) unless otherwise indicated.
Antibiotic susceptibility of urinary Escherichia coli isolates by patient sex:
Overall, 66.0% (1,664/2,520) of E. coli isolated from males and 66.3% (27,175/40,971) from females were susceptible to ampicillin. Amoxicillin-clavulanate susceptibility was 56.9% (484/850) among males and 67.3% (9,291/13,798) among females; however, it should be noted that testing for susceptibility to amoxicillin-clavulanate was routinely performed and reported only for ampicillin non-susceptible E. coli. Ciprofloxacin susceptibility for the E. coli isolates was 93.2% (2,292/2,458) among males and 95.9% (37,900/39,524) among females, and nitrofurantoin susceptibility was 96.4% (2,430/2,520) among males and 97.6% (39,979/40,968) among females. For TMP/SMX, 86.3% (2,176/2,520) of E. coli isolated from males were susceptible compared to 84.7% (34,674/40,958) among females.
Trends in antibiotic susceptibility of urinary Escherichia coli isolates over time:
Figure 1 presents the susceptibility of E. coli to each antibiotic by sex and year. Susceptibility to amoxicillin-clavulanate and TMP/SMX decreased significantly over time among both males and females. Ciprofloxacin susceptibility also decreased significantly over time among females, but not males. Table 2 presents the age stratified antibiotic susceptibilities for E. coli isolated from males and females. The age-specific susceptibilities differed significantly between males and females for all antibiotics except TMP/SMX.
Susceptibility to urinary anti-Infectives by sex and year for E. coli cultured from urine. For all figures, males shown as blue solid line, females as red dashed line. a, ampicillin susceptibility (test for trend: males, p=0.058; females, p=0.6256); b, amoxicillin clavulanate susceptibility (test for trend: males, p=0.0006; females, p<0.0001); c, ciprofloxacin susceptibility (test for trend: males, p=0.1445; females, p<0.0001); d, nitrofurantoin susceptibility (test for trend: males, p=0.4967; females, p=0.5508); e, trimethoprim/sulfamethoxazole susceptibility (test for trend: males, p=0.0004; females, p<0.0001).
Susceptibility to urinary anti-infectives by sex and age for
E. coli
cultured from urine*
*Represents the first isolate for each patient and year.
CMH: Cochrane Maentel Haenszel correlation test.
Discussion:
In this study, we observed statistically significant differences between males and females in the age-specific susceptibilities of E. coli to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin. Urinary E. coli isolates from male patients tended to exhibit increased antibiotic resistance than isolates from female patients. Despite the statistical significance of time trends and differences in age-specific susceptibilities, the magnitude of these differences was generally less than 5% and thus may not represent clinically meaningful differences. The exception was susceptibility to amoxicillin-clavulanate, where susceptibility was roughly 10% lower in males age 18 to 64 years than females in the same age group. Yet these differences should be interpreted cautiously because susceptibility to amoxicillin-clavulanate was only provided for ampicillin non-susceptible E. coli isolates in the current analysis. If all ampicillin susceptible isolates are assumed to be amoxicillin-clavulanate susceptible, then the magnitude of the differences between males and females would be less than 5% in all age categories (difference not statistically significant).
While few other studies have explored the differences in antibiotic susceptibility of uropathogens isolated from male and female ambulatory patients, our findings are consistent with the trends observed in the literature. A recent 10-year study of community UTI in Portuguese patients also identified differences in antibiotic susceptibility by patient sex. The authors reported that urinary isolates of E. coli were significantly more resistant to fluoroquinolones, penicillins, nitrofurantoin, and first and second generation cephalosporins among men compared to women [13]. Another study focused on pediatric patients, also identified significantly higher resistance to TMP/SMX and ciprofloxacin in male versus female patients [14]. The NAUTICA surveillance study of outpatient UTIs reported greater antibiotic resistance to ciprofloxacin, levofloxacin, and TMP/SMX among all urinary isolates from U.S. and Canadian male patients [15]. In the CANWARD study, antibiotic susceptibility among all E. coli isolates (not limited to urine isolates) collected from Canadian tertiary medical centers were compared and resistance was also observed to be significantly higher to ciprofloxacin, levofloxacin, and TMP/SMX in isolates collected from male patients versus female patients [11].
The prevalence and susceptibilities of antibiotic-resistant bacteria varies widely by geographic region, thus these data may not be generalizable to all regions. Local surveillance data of antibiotic susceptibilities would be needed to validate these findings in different patient populations. It should be noted that in our study resistance to TMP/SMX did not exceed 20%, as it does in many other regions of the U.S., thus in this patient setting TMP/SMX may be a viable empiric treatment option in both males and female patients. Additionally, antibiotic susceptibilities may differ by other patient or infection characteristics (e.g., type of UTI). These data were not available for analysis in this study and further investigation in this area is needed. Also, because clinical signs and symptoms of infection were not available and because not all suspected infections are cultured, the use of all urinary microbiology from this population cultures may not represent all true infections.
Currently there exists insufficient data to inform the development of evidence-based guidelines for the treatment of community UTIs in men. UTIs in adult males are considered complicated infections according to most clinical definitions [16-18]. Treatment recommendations vary from longer durations of therapy to the selection of broad-spectrum agents such as fluoroquinolones [6]. These recommendations are driven by the high proportion of UTIs in men with prostate involvement and concerns surrounding antibiotic resistance to TMP/SMX [7,8]. Previous research among Swiss outpatients has demonstrated the selection of fluoroquinolones versus TMP/SMX for the treatment of UTI is often influenced by nonclinical factors [19]. In this study, TMP/SMX, a recommended first-line agent for UTIs in both children and adults, the differences in susceptibility between E. coli isolated from males and females were neither statistically nor clinically significant and resistance rates are below 20%. Consequently, in this population, there is no evidence that male sex alone should be an indication for empiric selection of a second-line broad-spectrum antibiotic agent for the treatment of UTI. While more research is needed on treatment effectiveness of different regimens used to treat community UTIs in men, population-specific antibiotic susceptibility data are a necessary component in the empiric antibiotic selection process.
Conclusions:
In the era of increasing antibiotic resistance, prudent use of antibiotics is critical to prolong the clinical effectiveness of existing agents. Excessive use of broad-spectrum agents, such as fluoroquinolones, increases the evolutionary selective pressures that drive the increasing prevalence of resistance. These data suggest that first-line urinary anti-infectives such as TMP/SMX may be effective agents for treating UTIs in men. While more data are needed, clinicians should use local surveillance data to guide the prudent, empiric selection of antibiotic therapy for UTIs.
Abbreviations:
UTI: Urinary Tract Infection; KPNW: Kaiser Permanente Northwest; TMP/SMX: trimethoprim/sulfamethoxazole
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
JCM participated in the conception and design of the study; the acquisition, statistical analysis, and interpretation of the data; and the drafting of the manuscript. JCM, MRE, DTB, and DHS assisted with the interpretation of the data and participated in the critical revision of the manuscript. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2296/14/25/prepub
| Background:
Urinary tract infections (UTIs) are one of the most common infections treated in ambulatory care settings, however the epidemiology differs by age and sex. The incidence of UTI is far greater in females than males, and infection in pediatric patients is more often due to anatomical abnormalities. The purpose of this research was to describe age- and sex-specific trends in antibiotic susceptibility to common urinary anti-infectives among urinary isolates of Escherichia coli from ambulatory primary care patients in a regional health maintenance organization.
Methods:
Clinical microbiology data were collected for all urine cultures from patients with visits to primary care clinics in a regional health maintenance organization between 2005 and 2010. The first positive culture for E. coli tested for antibiotic susceptibilities per patient per year was included in the analysis dataset. The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for male and female patients. The Cochrane-Mantel-Haenzel test was used to test for differences in age-stratified susceptibility to each antibiotic between males and females.
Results:
A total of 43,493 E. coli isolates from 34,539 unique patients were identified for study inclusion. After stratifying by age, E. coli susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin differed significantly between males and females. However, the magnitude of the differences was less than 10% for all strata except amoxicillin-clavulanate susceptibility in E. coli isolated from males age 18-64 compared to females of the same age.
Conclusions:
We did not observe clinically meaningful differences in antibiotic susceptibility to common urinary anti-infectives among E. coli isolated from males versus females. These data suggest that male sex alone should not be used as an indication for empiric use of second-line broad-spectrum antibiotic agents for the treatment of UTIs. | Introduction:
Urinary tract infections (UTIs) are one of the most commonly treated bacterial infections and account for over 10 million ambulatory care visits annually in the United States [1]. Antibiotic treatment is typically selected empirically, based on the patient clinical presentation, medical history and local patterns of antibiotic susceptibility [2]. Because the incidence of UTIs is significantly greater among women, much of the research has focused on women. Thus there exists a paucity of research on UTIs in men. As such, guidelines on the diagnosis, treatment, and management of UTIs focus largely on infection in women [2-5].
The incidence, presentation, and course of infection for UTIs in men and women differ in large part due to anatomical differences. For the treatment of acute uncomplicated cystitis in women, trimethoprim/sulfamethoxazole (TMP/SMX), nitrofurantoin, fosfomycin, and pivmecillinam are recommended first-line empiric therapies [2,6]. In men, because prostate involvement occurs in roughly 90% of cases, an empiric agent should be selected that achieves therapeutic concentrations in prostatic tissues (e.g., trimethoprim or ciprofloxacin) [7,8].
While historically it was believed that the causative organism in UTIs differed between men and women, more recent data has shown that for both sexes the primary causative pathogen is Escherichia coli, which accounts for 75-90% of UTIs [8,9]. With the increases in antibiotic resistance among E. coli and other Enterobacteriaceae over the past several decades, surveillance data have become critical for appropriate empiric selection of antibiotic therapy. U.S. guidelines specify that TMP/SMX should be avoided for empiric treatment of uncomplicated acute cystitis or pyelonephritis in populations where non-susceptibility to this agent exceeds 20% in uropathogens [2].
While some surveillance studies have identified significant differences in the frequency of susceptibility to common urinary anti-infectives between isolates collected from male and female patients, this has not been consistently observed [10,11]. The objective of this study was to describe age- and sex-specific antibiotic susceptibility patterns for common urinary anti-infectives among E. coli urine isolates using six years of data from a large ambulatory primary care patient population.
Conclusions:
In the era of increasing antibiotic resistance, prudent use of antibiotics is critical to prolong the clinical effectiveness of existing agents. Excessive use of broad-spectrum agents, such as fluoroquinolones, increases the evolutionary selective pressures that drive the increasing prevalence of resistance. These data suggest that first-line urinary anti-infectives such as TMP/SMX may be effective agents for treating UTIs in men. While more data are needed, clinicians should use local surveillance data to guide the prudent, empiric selection of antibiotic therapy for UTIs. | Background:
Urinary tract infections (UTIs) are one of the most common infections treated in ambulatory care settings, however the epidemiology differs by age and sex. The incidence of UTI is far greater in females than males, and infection in pediatric patients is more often due to anatomical abnormalities. The purpose of this research was to describe age- and sex-specific trends in antibiotic susceptibility to common urinary anti-infectives among urinary isolates of Escherichia coli from ambulatory primary care patients in a regional health maintenance organization.
Methods:
Clinical microbiology data were collected for all urine cultures from patients with visits to primary care clinics in a regional health maintenance organization between 2005 and 2010. The first positive culture for E. coli tested for antibiotic susceptibilities per patient per year was included in the analysis dataset. The frequency of susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole (TMP/SMX) was calculated for male and female patients. The Cochrane-Mantel-Haenzel test was used to test for differences in age-stratified susceptibility to each antibiotic between males and females.
Results:
A total of 43,493 E. coli isolates from 34,539 unique patients were identified for study inclusion. After stratifying by age, E. coli susceptibility to ampicillin, amoxicillin-clavulanate, ciprofloxacin, and nitrofurantoin differed significantly between males and females. However, the magnitude of the differences was less than 10% for all strata except amoxicillin-clavulanate susceptibility in E. coli isolated from males age 18-64 compared to females of the same age.
Conclusions:
We did not observe clinically meaningful differences in antibiotic susceptibility to common urinary anti-infectives among E. coli isolated from males versus females. These data suggest that male sex alone should not be used as an indication for empiric use of second-line broad-spectrum antibiotic agents for the treatment of UTIs. | 4,269 | 355 | [
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[CONTENT] Adolescent | Adult | Age Factors | Aged | Ambulatory Care | Amoxicillin-Potassium Clavulanate Combination | Ampicillin | Anti-Bacterial Agents | Ciprofloxacin | Cross-Sectional Studies | Drug Resistance, Bacterial | Escherichia coli | Female | Humans | Male | Microbial Sensitivity Tests | Middle Aged | Nitrofurantoin | Sex Factors | Trimethoprim, Sulfamethoxazole Drug Combination | Urinary Tract Infections | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Factors | Aged | Ambulatory Care | Amoxicillin-Potassium Clavulanate Combination | Ampicillin | Anti-Bacterial Agents | Ciprofloxacin | Cross-Sectional Studies | Drug Resistance, Bacterial | Escherichia coli | Female | Humans | Male | Microbial Sensitivity Tests | Middle Aged | Nitrofurantoin | Sex Factors | Trimethoprim, Sulfamethoxazole Drug Combination | Urinary Tract Infections | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Factors | Aged | Ambulatory Care | Amoxicillin-Potassium Clavulanate Combination | Ampicillin | Anti-Bacterial Agents | Ciprofloxacin | Cross-Sectional Studies | Drug Resistance, Bacterial | Escherichia coli | Female | Humans | Male | Microbial Sensitivity Tests | Middle Aged | Nitrofurantoin | Sex Factors | Trimethoprim, Sulfamethoxazole Drug Combination | Urinary Tract Infections | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Factors | Aged | Ambulatory Care | Amoxicillin-Potassium Clavulanate Combination | Ampicillin | Anti-Bacterial Agents | Ciprofloxacin | Cross-Sectional Studies | Drug Resistance, Bacterial | Escherichia coli | Female | Humans | Male | Microbial Sensitivity Tests | Middle Aged | Nitrofurantoin | Sex Factors | Trimethoprim, Sulfamethoxazole Drug Combination | Urinary Tract Infections | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Factors | Aged | Ambulatory Care | Amoxicillin-Potassium Clavulanate Combination | Ampicillin | Anti-Bacterial Agents | Ciprofloxacin | Cross-Sectional Studies | Drug Resistance, Bacterial | Escherichia coli | Female | Humans | Male | Microbial Sensitivity Tests | Middle Aged | Nitrofurantoin | Sex Factors | Trimethoprim, Sulfamethoxazole Drug Combination | Urinary Tract Infections | Young Adult [SUMMARY] | [CONTENT] utis increases antibiotic | antibiotic susceptibility urinary | treatment management utis | treating utis men | urinary tract infections [SUMMARY] | [CONTENT] utis increases antibiotic | antibiotic susceptibility urinary | treatment management utis | treating utis men | urinary tract infections [SUMMARY] | [CONTENT] utis increases antibiotic | antibiotic susceptibility urinary | treatment management utis | treating utis men | urinary tract infections [SUMMARY] | [CONTENT] utis increases antibiotic | antibiotic susceptibility urinary | treatment management utis | treating utis men | urinary tract infections [SUMMARY] | [CONTENT] utis increases antibiotic | antibiotic susceptibility urinary | treatment management utis | treating utis men | urinary tract infections [SUMMARY] | [CONTENT] utis increases antibiotic | antibiotic susceptibility urinary | treatment management utis | treating utis men | urinary tract infections [SUMMARY] | [CONTENT] susceptibility | males | females | coli | antibiotic | data | patient | isolates | test | tmp smx [SUMMARY] | [CONTENT] susceptibility | males | females | coli | antibiotic | data | patient | isolates | test | tmp smx [SUMMARY] | [CONTENT] susceptibility | males | females | coli | antibiotic | data | patient | isolates | test | tmp smx [SUMMARY] | [CONTENT] susceptibility | males | females | coli | antibiotic | data | patient | isolates | test | tmp smx [SUMMARY] | [CONTENT] susceptibility | males | females | coli | antibiotic | data | patient | isolates | test | tmp smx [SUMMARY] | [CONTENT] susceptibility | males | females | coli | antibiotic | data | patient | isolates | test | tmp smx [SUMMARY] | [CONTENT] utis | women | treatment | men | empiric | antibiotic | susceptibility | selected | common urinary anti infectives | cystitis [SUMMARY] | [CONTENT] care clinics | clinics | primary care clinics | care | data | primary care | primary | frequency | frequency susceptibility | patient [SUMMARY] | [CONTENT] males | females | susceptibility | coli | susceptibility test trend | test trend males | trend males | susceptibility test | susceptibility test trend males | test [SUMMARY] | [CONTENT] use | agents | increasing | prudent | resistance | utis | data | prudent use antibiotics critical | effective | effective agents [SUMMARY] | [CONTENT] susceptibility | males | females | data | coli | antibiotic | test | patient | care | clinics [SUMMARY] | [CONTENT] susceptibility | males | females | data | coli | antibiotic | test | patient | care | clinics [SUMMARY] | [CONTENT] one ||| UTI ||| Escherichia [SUMMARY] | [CONTENT] between 2005 and 2010 ||| first ||| ||| [SUMMARY] | [CONTENT] 43,493 E. coli | 34,539 ||| ||| less than 10% | age 18-64 [SUMMARY] | [CONTENT] ||| second [SUMMARY] | [CONTENT] one ||| UTI ||| Escherichia ||| between 2005 and 2010 ||| first ||| ||| ||| 43,493 E. coli | 34,539 ||| ||| less than 10% | age 18-64 ||| ||| second [SUMMARY] | [CONTENT] one ||| UTI ||| Escherichia ||| between 2005 and 2010 ||| first ||| ||| ||| 43,493 E. coli | 34,539 ||| ||| less than 10% | age 18-64 ||| ||| second [SUMMARY] |
||||||
Different patterns of cortical excitability in major depression and vascular depression: a transcranial magnetic stimulation study. | 24206945 | Clinical and functional studies consider major depression (MD) and vascular depression (VD) as different neurobiological processes. Hypoexcitability of the left frontal cortex to transcranial magnetic stimulation (TMS) is frequently reported in MD, whereas little is known about the effects of TMS in VD. Thus, we aimed to assess and compare motor cortex excitability in patients with VD and MD. | BACKGROUND | Eleven VD patients, 11 recurrent drug-resistant MD patients, and 11 healthy controls underwent clinical, neuropsychological and neuroimaging evaluations in addition to bilateral resting motor threshold, cortical silent period, and paired-pulse TMS curves of intracortical excitability. All patients continued on psychotropic drugs, which were unchanged throughout the study. | METHODS | Scores on one of the tests evaluating frontal lobe abilities (Stroop Color-Word interference test) were worse in patients compared with controls. The resting motor threshold in patients with MD was significantly higher in the left hemisphere compared with the right (p < 0.05), and compared with the VD patients and controls. The cortical silent period was bilaterally prolonged in MD patients compared with VD patients and controls, with a statistically significant difference in the left hemisphere (p < 0.01). No differences were observed in the paired-pulse curves between patients and controls. | RESULTS | This study showed distinctive patterns of motor cortex excitability between late-onset depression with subcortical vascular disease and early-onset recurrent drug resistant MD. The data provide a TMS model of the different processes underlying VD and MD. Additionally, our results support the "Vascular depression hypothesis" at the neurophysiological level, and confirm the inter-hemispheric asymmetry to TMS in patients with MD. We were unable to support previous findings of impaired intracortical inhibitory mechanisms to TMS in patients with MD, although a drug-induced effect on our results cannot be excluded. This study may aid the understanding of the pathogenetic differences underlying the clinical spectrum of depressive disorders. | CONCLUSIONS | [
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] | 4226249 | Background | Recently, the finding that patients with late-onset depression had higher rates of brain magnetic resonance imaging (MRI) changes compared with patients with early onset major depression (MD), has led to the hypothesis that mood disorders in the elderly may be related to neurobiological abnormalities, such as cerebrovascular disease [1]. In 1997, Alexopoulos and co-workers [2] introduced the concept of “vascular depression” (VD) as a subtype of geriatric mood disorder characterised by a late age at onset or change in the course of early onset depressive symptoms, persistent symptoms, association with vascular disease or vascular risk factors and diffuse or multifocal cerebrovascular lesions. The “vascular depression hypothesis”, presenting clinically as a depression-executive dysfunction syndrome of late-life, states that disruption of fronto-striatal circuits by vascular lesions predisposes, precipitates, or perpetuates late-life depressive syndromes [3]. Indeed, numerous neuroimaging and neuropathological studies reported increased prevalence and severity of white matter lesions (WMLs) of vascular origin in individuals with elderly depression, especially in those with late-onset illness [4-6].
Nevertheless, depression in the elderly might also result from a recurrent form of MD with an early onset. However, compared with patients with recurrent MD, elderly patients with VD often exhibit a clinical presentation characterized by psychomotor retardation, lack of interest, limited depressive ideation and insight, and prominent disability [7,8]. Moreover, patients with late-onset MD disorder showed specific deficits in attention and executive function [9,10], whereas patients with recurrent MD exhibited deficits in episodic memory [11,12]. These neuropsychological differences are thought to be associated with prominent fronto-striatal dysfunction in late-onset MD, and with a reduction in hippocampal volume in recurrent geriatric MD. The rates of anhedonia and comorbid cardiovascular illness were higher in patients with late-onset MD [11].
Despite a body of literature on clinical, psychopathological, and neuroradiological features of both VD and MD, studies comparing their neurophysiological profiles are lacking. Previous electroencephalography studies in MD patients demonstrated decreased neural activity in the left frontal regions, as shown by an increased alpha power band [13,14]. Recently, changes in transcranial magnetic stimulation (TMS) related-measures of cortical excitability have been shown to be associated with depression. Specifically, most, but not all, TMS studies in patients with MD found reduced activation of both excitatory and inhibitory circuits in the left hemisphere [15-19]. Moreover, some functional neuroimaging studies have shown hypometabolism and hypoperfusion of the left dorsolateral prefrontal cortex (DLPFC) in patients with MD [16,17]. However, the applicability of the findings obtained with TMS on the primary motor cortex (M1) and those obtained with other techniques on the DLPFC requires further investigation.
TMS is a safe and non-invasive neurophysiological investigation technique used to evaluate the cortico-spinal tract, cortical motor areas [20], map motor and cognitive functions, study neural networks, and modulate brain function with a potential therapeutic aim [21-23]. The development of specific stimulation protocols, such as the cortical silent period (CSP) and paired-pulse paradigms, as well as the emerging concept that motor cortical output is influenced by non-primary motor areas, including the ventral and dorsal premotor cortex, supplementary motor area, and cingulate cortex [24], has allowed the use of TMS to explore inhibitory and excitatory interactions within motor cortical regions in several neuropsychiatric disorders [25,26]. A single TMS pulse applied over the M1 through the scalp elicits a motor evoked potential (MEP) in the contralateral target muscles [27,28]. The resting motor threshold (rMT) is believed to reflect membrane excitability of cortico-spinal motor neurons, which is mainly dependent on ion channel conductivity and on excitatory interneurons that project to these neurons [20]. The CSP refers to a suppression of electromyographic activity during a voluntary contraction of the target muscle and depends, at least in part, on inhibitory mechanisms at the level of the motor cortex, probably mediated by gamma-aminobutyric acid (GABA)-b receptors [29]. The paired-pulse TMS couples a suprathreshold magnetic stimulus with a preceding subthreshold stimulus, and the response to the paired stimuli may be increased (facilitation) or decreased (inhibition) depending on the interstimulus interval (ISI): at short ISIs (1–4 ms) the conditioning stimulus determines the intracortical inhibition (ICI) with respect to the test stimulus, whereas at longer ISIs (>5 ms) the effect is intracortical facilitation (ICF). ICI and ICF interactions are likley related to the balance of GABAergic, dopaminergic, and glutamatergic transmissions [29]. In a single study evaluating inhibitory/facilitatory intracortical circuit changes, Bella et al. [30] did not find significant differences between patients with VD and patients with subcortical vascular disease (SVD), suggesting that VD with or without depression might result in a similar neurophysiological profile of cortical excitability, probably as a consequence of cerebral small vessel disease.
In this study, we aimed to assess and compare single and paired-pulse TMS measures of cortical excitability in patients with VD versus MD and between hemispheres. We hypothesised that VD patients would show distinctive neurophysiological changes compared with MD, consistent with the involvement of different neurobiological substrates. | Methods | Participants A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).
VD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.
Drug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].
As shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.
Pharmacological treatment of MD patients
All patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.
A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).
VD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.
Drug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].
As shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.
Pharmacological treatment of MD patients
All patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.
Assessment All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].
All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].
Transcranial magnetic stimulation MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.
The rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).
MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.
The rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).
Statistical analysis The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.
The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant. | Results | The relevant demographic and clinical characteristics of the two patient groups are summarized in Tables 2 and 3. MD were younger than VD patients and controls. The depression rating was less severe in the VD group compared with the MD group. As expected, hypertension was more frequent in VD compared with MD patients, whereas personal history of depressive symptoms was the opposite, and scores at Stroop T were worse in patients compared with controls. WML severity was mild in 4, moderate in 5, and severe in 2 patients. Brain MRI of MD patients and controls was unremarkable (Fazekas 0), and the physical and mental state of controls was also unremarkable. As shown in Table 4, there was a significant increase in rMT in MD patients in the left hemisphere compared with the right (48.36 ± 9.64 vs. 45.00 ± 8.82; p < 0.05). No significant differences were found between the right and left hemispheres for the other measures of cortico-spinal excitability in VD patients, except for a trend toward an increase in ICF at the ISI of 15 ms from the right hemisphere (VD 2.08 ± 0.7 vs. MD 1.78 ± 0.87 vs. Controls 1.52 ± 0.75, p = 0.153) (Figures 1 and 2). No significant differences were found in rMT, central motor conduction time, amplitude ratio, and mean amplitude of the F wave between VD, MD, and controls. In MD patients, the duration of the CSP from both hemispheres was increased, which was significant from the left hemisphere (MD 115.00 ± 32.39 vs. VD 72.18 ± 26.57 vs. Controls 80.09 ± 19.19, p = 0.004), and there was a trend towards significance from the right hemisphere (MD 111.82 ± 42.09 vs. VD 85.45 ± 45.13 vs. Controls 79.18 ± 21.51, p = 0.105) (Table 4).
Demographic and neuropsychological characteristics of patients and controls
VD = vascular depression; MD = major depression; MMSE = Mini Mental State Examination; ADL = Activity of Daily Living; IADL = Instrumental Activity of Daily Living; HAM-D17 = 17-items Hamilton Depression Rating Scale; SCID-I = Structured Clinical Interview for DSM-IV Axis I Disorders; Stroop T = Stroop Color-Word Test interference score; Stroop E = Stroop Color-Word Test interference number of errors; FAB = Frontal Assessment Battery. Data are expressed as mean ± SD and were analyzed using Kruskal-Wallis ANOVA. * = p < 0.05.
Clinical characteristics of patients and controls
VD = vascular depression; MD = major depression. * = p < 0.05.
TMS parameters of the patient and the control groups obtained from both hemispheres
VD = vascular depression; MD = major depression; rMT = resting motor threshold (%); CSP = cortical silent period (ms); CMCT = central motor conduction time (ms); CMCTF = central motor conduction time calculated with F-waves (ms); A ratio = amplitude ratio; F wave A = amplitude of F-waves (mV). Data are expressed as mean ± SD. * = p < 0.05.
MEP amplitudes at different ISIs from the 3 groups with respect to the hemisphere. Data are expressed as mean ± SE. (whiskers). VD = vascular depression; MD = major depression; C = Controls; MEP = Motor Evoked Potential; ISI = Interstimulus Interval.
Curves of intracortical excitability obtained from both hemispheres in each group. | Conclusions | The current study showed distinctive patterns of motor cortex excitability between late-onset depression with SVD and early-onset recurrent MD, providing a potential TMS model of the different processes underlying them. These results support the “Vascular depression hypothesis” at the neurophysiological level and confirm inter-hemispheric asymmetry to TMS in MD. Further research and comparison studies with homogeneous groups of patients and other methodologies are needed to confirm the present findings, as well as their modifications over time and clinical correlates. | [
"Background",
"Participants",
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"Statistical analysis",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Recently, the finding that patients with late-onset depression had higher rates of brain magnetic resonance imaging (MRI) changes compared with patients with early onset major depression (MD), has led to the hypothesis that mood disorders in the elderly may be related to neurobiological abnormalities, such as cerebrovascular disease [1]. In 1997, Alexopoulos and co-workers [2] introduced the concept of “vascular depression” (VD) as a subtype of geriatric mood disorder characterised by a late age at onset or change in the course of early onset depressive symptoms, persistent symptoms, association with vascular disease or vascular risk factors and diffuse or multifocal cerebrovascular lesions. The “vascular depression hypothesis”, presenting clinically as a depression-executive dysfunction syndrome of late-life, states that disruption of fronto-striatal circuits by vascular lesions predisposes, precipitates, or perpetuates late-life depressive syndromes [3]. Indeed, numerous neuroimaging and neuropathological studies reported increased prevalence and severity of white matter lesions (WMLs) of vascular origin in individuals with elderly depression, especially in those with late-onset illness [4-6].\nNevertheless, depression in the elderly might also result from a recurrent form of MD with an early onset. However, compared with patients with recurrent MD, elderly patients with VD often exhibit a clinical presentation characterized by psychomotor retardation, lack of interest, limited depressive ideation and insight, and prominent disability [7,8]. Moreover, patients with late-onset MD disorder showed specific deficits in attention and executive function [9,10], whereas patients with recurrent MD exhibited deficits in episodic memory [11,12]. These neuropsychological differences are thought to be associated with prominent fronto-striatal dysfunction in late-onset MD, and with a reduction in hippocampal volume in recurrent geriatric MD. The rates of anhedonia and comorbid cardiovascular illness were higher in patients with late-onset MD [11].\nDespite a body of literature on clinical, psychopathological, and neuroradiological features of both VD and MD, studies comparing their neurophysiological profiles are lacking. Previous electroencephalography studies in MD patients demonstrated decreased neural activity in the left frontal regions, as shown by an increased alpha power band [13,14]. Recently, changes in transcranial magnetic stimulation (TMS) related-measures of cortical excitability have been shown to be associated with depression. Specifically, most, but not all, TMS studies in patients with MD found reduced activation of both excitatory and inhibitory circuits in the left hemisphere [15-19]. Moreover, some functional neuroimaging studies have shown hypometabolism and hypoperfusion of the left dorsolateral prefrontal cortex (DLPFC) in patients with MD [16,17]. However, the applicability of the findings obtained with TMS on the primary motor cortex (M1) and those obtained with other techniques on the DLPFC requires further investigation.\nTMS is a safe and non-invasive neurophysiological investigation technique used to evaluate the cortico-spinal tract, cortical motor areas [20], map motor and cognitive functions, study neural networks, and modulate brain function with a potential therapeutic aim [21-23]. The development of specific stimulation protocols, such as the cortical silent period (CSP) and paired-pulse paradigms, as well as the emerging concept that motor cortical output is influenced by non-primary motor areas, including the ventral and dorsal premotor cortex, supplementary motor area, and cingulate cortex [24], has allowed the use of TMS to explore inhibitory and excitatory interactions within motor cortical regions in several neuropsychiatric disorders [25,26]. A single TMS pulse applied over the M1 through the scalp elicits a motor evoked potential (MEP) in the contralateral target muscles [27,28]. The resting motor threshold (rMT) is believed to reflect membrane excitability of cortico-spinal motor neurons, which is mainly dependent on ion channel conductivity and on excitatory interneurons that project to these neurons [20]. The CSP refers to a suppression of electromyographic activity during a voluntary contraction of the target muscle and depends, at least in part, on inhibitory mechanisms at the level of the motor cortex, probably mediated by gamma-aminobutyric acid (GABA)-b receptors [29]. The paired-pulse TMS couples a suprathreshold magnetic stimulus with a preceding subthreshold stimulus, and the response to the paired stimuli may be increased (facilitation) or decreased (inhibition) depending on the interstimulus interval (ISI): at short ISIs (1–4 ms) the conditioning stimulus determines the intracortical inhibition (ICI) with respect to the test stimulus, whereas at longer ISIs (>5 ms) the effect is intracortical facilitation (ICF). ICI and ICF interactions are likley related to the balance of GABAergic, dopaminergic, and glutamatergic transmissions [29]. In a single study evaluating inhibitory/facilitatory intracortical circuit changes, Bella et al. [30] did not find significant differences between patients with VD and patients with subcortical vascular disease (SVD), suggesting that VD with or without depression might result in a similar neurophysiological profile of cortical excitability, probably as a consequence of cerebral small vessel disease.\nIn this study, we aimed to assess and compare single and paired-pulse TMS measures of cortical excitability in patients with VD versus MD and between hemispheres. We hypothesised that VD patients would show distinctive neurophysiological changes compared with MD, consistent with the involvement of different neurobiological substrates.",
"A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).\nVD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.\nDrug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].\nAs shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.\nPharmacological treatment of MD patients\nAll patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.",
"All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].",
"MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.\nThe rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).",
"The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.",
"The authors declare that they do not have any financial or non-financial competing interests. There was no study sponsor involved in the funding or write-up of this research.",
"CC and EA conceived and designed the study and participated in writing the protocol. GL and RR performed clinical and neuropsychological assessments, managed literature searches, and helped to draft the manuscript. MC and MP carried out transcranial magnetic stimulation procedures and analysed the data. DG and CS performed the statistical analysis. GP and RB reviewed the neuroradiological images, drafted the manuscript, and participated in its design and coordination. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-244X/13/300/prepub\n"
] | [
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"Background",
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"Transcranial magnetic stimulation",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
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"Recently, the finding that patients with late-onset depression had higher rates of brain magnetic resonance imaging (MRI) changes compared with patients with early onset major depression (MD), has led to the hypothesis that mood disorders in the elderly may be related to neurobiological abnormalities, such as cerebrovascular disease [1]. In 1997, Alexopoulos and co-workers [2] introduced the concept of “vascular depression” (VD) as a subtype of geriatric mood disorder characterised by a late age at onset or change in the course of early onset depressive symptoms, persistent symptoms, association with vascular disease or vascular risk factors and diffuse or multifocal cerebrovascular lesions. The “vascular depression hypothesis”, presenting clinically as a depression-executive dysfunction syndrome of late-life, states that disruption of fronto-striatal circuits by vascular lesions predisposes, precipitates, or perpetuates late-life depressive syndromes [3]. Indeed, numerous neuroimaging and neuropathological studies reported increased prevalence and severity of white matter lesions (WMLs) of vascular origin in individuals with elderly depression, especially in those with late-onset illness [4-6].\nNevertheless, depression in the elderly might also result from a recurrent form of MD with an early onset. However, compared with patients with recurrent MD, elderly patients with VD often exhibit a clinical presentation characterized by psychomotor retardation, lack of interest, limited depressive ideation and insight, and prominent disability [7,8]. Moreover, patients with late-onset MD disorder showed specific deficits in attention and executive function [9,10], whereas patients with recurrent MD exhibited deficits in episodic memory [11,12]. These neuropsychological differences are thought to be associated with prominent fronto-striatal dysfunction in late-onset MD, and with a reduction in hippocampal volume in recurrent geriatric MD. The rates of anhedonia and comorbid cardiovascular illness were higher in patients with late-onset MD [11].\nDespite a body of literature on clinical, psychopathological, and neuroradiological features of both VD and MD, studies comparing their neurophysiological profiles are lacking. Previous electroencephalography studies in MD patients demonstrated decreased neural activity in the left frontal regions, as shown by an increased alpha power band [13,14]. Recently, changes in transcranial magnetic stimulation (TMS) related-measures of cortical excitability have been shown to be associated with depression. Specifically, most, but not all, TMS studies in patients with MD found reduced activation of both excitatory and inhibitory circuits in the left hemisphere [15-19]. Moreover, some functional neuroimaging studies have shown hypometabolism and hypoperfusion of the left dorsolateral prefrontal cortex (DLPFC) in patients with MD [16,17]. However, the applicability of the findings obtained with TMS on the primary motor cortex (M1) and those obtained with other techniques on the DLPFC requires further investigation.\nTMS is a safe and non-invasive neurophysiological investigation technique used to evaluate the cortico-spinal tract, cortical motor areas [20], map motor and cognitive functions, study neural networks, and modulate brain function with a potential therapeutic aim [21-23]. The development of specific stimulation protocols, such as the cortical silent period (CSP) and paired-pulse paradigms, as well as the emerging concept that motor cortical output is influenced by non-primary motor areas, including the ventral and dorsal premotor cortex, supplementary motor area, and cingulate cortex [24], has allowed the use of TMS to explore inhibitory and excitatory interactions within motor cortical regions in several neuropsychiatric disorders [25,26]. A single TMS pulse applied over the M1 through the scalp elicits a motor evoked potential (MEP) in the contralateral target muscles [27,28]. The resting motor threshold (rMT) is believed to reflect membrane excitability of cortico-spinal motor neurons, which is mainly dependent on ion channel conductivity and on excitatory interneurons that project to these neurons [20]. The CSP refers to a suppression of electromyographic activity during a voluntary contraction of the target muscle and depends, at least in part, on inhibitory mechanisms at the level of the motor cortex, probably mediated by gamma-aminobutyric acid (GABA)-b receptors [29]. The paired-pulse TMS couples a suprathreshold magnetic stimulus with a preceding subthreshold stimulus, and the response to the paired stimuli may be increased (facilitation) or decreased (inhibition) depending on the interstimulus interval (ISI): at short ISIs (1–4 ms) the conditioning stimulus determines the intracortical inhibition (ICI) with respect to the test stimulus, whereas at longer ISIs (>5 ms) the effect is intracortical facilitation (ICF). ICI and ICF interactions are likley related to the balance of GABAergic, dopaminergic, and glutamatergic transmissions [29]. In a single study evaluating inhibitory/facilitatory intracortical circuit changes, Bella et al. [30] did not find significant differences between patients with VD and patients with subcortical vascular disease (SVD), suggesting that VD with or without depression might result in a similar neurophysiological profile of cortical excitability, probably as a consequence of cerebral small vessel disease.\nIn this study, we aimed to assess and compare single and paired-pulse TMS measures of cortical excitability in patients with VD versus MD and between hemispheres. We hypothesised that VD patients would show distinctive neurophysiological changes compared with MD, consistent with the involvement of different neurobiological substrates.",
" Participants A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).\nVD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.\nDrug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].\nAs shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.\nPharmacological treatment of MD patients\nAll patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.\nA sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).\nVD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.\nDrug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].\nAs shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.\nPharmacological treatment of MD patients\nAll patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.\n Assessment All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].\nAll participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].\n Transcranial magnetic stimulation MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.\nThe rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).\nMEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.\nThe rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).\n Statistical analysis The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.\nThe non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.",
"A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).\nVD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.\nDrug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].\nAs shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.\nPharmacological treatment of MD patients\nAll patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.",
"All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].",
"MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.\nThe rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).",
"The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.",
"The relevant demographic and clinical characteristics of the two patient groups are summarized in Tables 2 and 3. MD were younger than VD patients and controls. The depression rating was less severe in the VD group compared with the MD group. As expected, hypertension was more frequent in VD compared with MD patients, whereas personal history of depressive symptoms was the opposite, and scores at Stroop T were worse in patients compared with controls. WML severity was mild in 4, moderate in 5, and severe in 2 patients. Brain MRI of MD patients and controls was unremarkable (Fazekas 0), and the physical and mental state of controls was also unremarkable. As shown in Table 4, there was a significant increase in rMT in MD patients in the left hemisphere compared with the right (48.36 ± 9.64 vs. 45.00 ± 8.82; p < 0.05). No significant differences were found between the right and left hemispheres for the other measures of cortico-spinal excitability in VD patients, except for a trend toward an increase in ICF at the ISI of 15 ms from the right hemisphere (VD 2.08 ± 0.7 vs. MD 1.78 ± 0.87 vs. Controls 1.52 ± 0.75, p = 0.153) (Figures 1 and 2). No significant differences were found in rMT, central motor conduction time, amplitude ratio, and mean amplitude of the F wave between VD, MD, and controls. In MD patients, the duration of the CSP from both hemispheres was increased, which was significant from the left hemisphere (MD 115.00 ± 32.39 vs. VD 72.18 ± 26.57 vs. Controls 80.09 ± 19.19, p = 0.004), and there was a trend towards significance from the right hemisphere (MD 111.82 ± 42.09 vs. VD 85.45 ± 45.13 vs. Controls 79.18 ± 21.51, p = 0.105) (Table 4).\nDemographic and neuropsychological characteristics of patients and controls\nVD = vascular depression; MD = major depression; MMSE = Mini Mental State Examination; ADL = Activity of Daily Living; IADL = Instrumental Activity of Daily Living; HAM-D17 = 17-items Hamilton Depression Rating Scale; SCID-I = Structured Clinical Interview for DSM-IV Axis I Disorders; Stroop T = Stroop Color-Word Test interference score; Stroop E = Stroop Color-Word Test interference number of errors; FAB = Frontal Assessment Battery. Data are expressed as mean ± SD and were analyzed using Kruskal-Wallis ANOVA. * = p < 0.05.\nClinical characteristics of patients and controls\nVD = vascular depression; MD = major depression. * = p < 0.05.\nTMS parameters of the patient and the control groups obtained from both hemispheres\nVD = vascular depression; MD = major depression; rMT = resting motor threshold (%); CSP = cortical silent period (ms); CMCT = central motor conduction time (ms); CMCTF = central motor conduction time calculated with F-waves (ms); A ratio = amplitude ratio; F wave A = amplitude of F-waves (mV). Data are expressed as mean ± SD. * = p < 0.05.\nMEP amplitudes at different ISIs from the 3 groups with respect to the hemisphere. Data are expressed as mean ± SE. (whiskers). VD = vascular depression; MD = major depression; C = Controls; MEP = Motor Evoked Potential; ISI = Interstimulus Interval.\nCurves of intracortical excitability obtained from both hemispheres in each group.",
"This is the first report comparing motor cortex excitability between VD and MD patients. The main finding is the observation of potentially distinctive neurophysiological profiles elicited by TMS, adding support to the hypothesis that late onset VD is a different syndrome with respect to early onset recurrent MD. Despite the abundant literature on TMS and depression, only one study has been conducted in VD patients, where VD patients were compared with patients with SVD patients and controls [30]. The results of this study confirm previous findings on VD, and are consistent with most TMS reports investigating MD. An interhemispheric imbalance in frontal cortical activities, as indexed by a higher rMT, was observed in our MD patients and is consistent with an overall hypoexcitability of the left hemisphere [15-17].\nIn line with our finding of laterality in MD, Soares and Mann [40] described structural asymmetries in cortical and subcortical brain regions crucial in the neuroanatomical model of mood disorders. More recently, it has been reported that decreased relative left-frontal brain electrical activity may be a trait-like marker of depression, suggesting that frontal asymmetry could also be a shared factor predicting first depression onset [41]. However, it remains difficult to infer functional asymmetry between the left and right prefrontal cortices by examining differences in motor responses to TMS only. Findings from previous studies conclude that both glutamatergic and GABAergic pathways may be defective in the left hemisphere of patients with MD. The effect of laterality has also been reported in patients with late-life depression and MRI signal hyperintensities, but it is still controversial as some reports found that left-sided WMLs were significantly associated with older age of depression onset [42], whereas others did not find any laterality effect [43].\nAlthough many, but not all, TMS studies in MD have shown a significant reduction in the amount of both inhibitory (shortened CSP and decreased ICI) and facilitatory (reduced ICF) inputs regarding the left frontal cortex compared with the contralateral hemisphere [17-19], we were not able to find substantial differences. On the contrary, we observed a bilateral increase in CSP duration. The reason for this dissociation remains unclear. Previous studies have obtained conflicting results, probably due to the heterogeneous methods employed, differences in clinical presentation and severity as well as the medication status. For example, Bajbouj et al. [17] and Lefaucheur et al. [18] found reduced CSP and ICI in medication-free and treated MD patients, respectively. Additionally, Fitzgerald et al. [16] did not observe consistent differences in hemispheric activity in drug-resistant MD patients, whereas Levinson et al. [19] demonstrated significantly shortened CSP duration in all MD patient groups and decreased ICI in the drug-resistant group only. In contrast, one study observed an increased CSP in MD patients compared with age-matched controls, suggesting increased cortical inhibition [44]. In our study, however, we cannot exclude the possibility of a drug-induced effect on CSP duration, related to zolpidem administration in many MD patients (but not in VD group or controls), highlighting the role of specific subtypes of GABA receptors in the control of inhibitory neuronal loops within the M1 [45]. It is more difficult to explain a drug effect on ICI since it has been shown to be affected by benzodiazepine administration but not zolpidem, and this is consistent with the different segregation of the two inhibitory circuits in the motor cortex at the level of GABA receptor subtypes [46].\nRegarding the facilitatory component of intracortical excitability, we observed a trend toward an increase in ICF in VD patients, supporting previous results [30]. A significant hyperfacilitation was found in patients with SVD and clinical features of vascular cognitive impairment-no dementia (VCI-ND) [47], although it was not observed at follow-up [48], supporting the concept that specific TMS measures of cortical excitability can be considered indices of motor cortex plasticity [49]. SVD is indeed a potential aetiology of both VD and VCI and the accumulation of microvascular lesions constitutes a common neuropathological platform for both cognitive decline and depressive episodes in old age [6].\nA growing body of evidence indicates that glutamate-mediated compensatory plastic events might also occur in MD [50]. In a recent paper, Spampinato et al. [34] demonstrated that repetitive TMS (rTMS) improved performance in a test evaluating frontal lobe abilities and was able to restore inter-hemispheric asymmetry of the ICF and rMT. These results suggested that the TMS changes observed before treatment might be the expression of disruption of glutamatergic receptor plasticity-related processes, and that this neurophysiological behaviour might correlate with executive functions.\nTaken together, the data presented here may help to further understanding of the pathogenetic differences underlying the clinical spectrum of depressive disorders. However, relating the distinctive electrophysiological profile between VD and MD patients to what is clinically observed is challenging. Currently, it is difficult to relate the subtle TMS changes with the clinical picture of psychomotor retardation, persistent symptoms, and prominent disability seen in patients with VD. It has been proposed that vascular damage to the reciprocal connections between the prefrontal cortex, basal ganglia, and cerebellum may affect the presentation and the course of late-life VD, although the atrophy of frontal grey matter due to deafferentation could also contribute [10]. Finally, although not statistically significant, we did observe some trends in this study that may be of interest in future studies. For example, the MMSE scores obtained in patients with VD were lower compared with the MD group and controls (p = 0.064), although all values were within normal limits. This could be related to poorer educational level in the VD group, and the clinical presentation and vascular burden. The significance of other trend-level differences, such as for the CMCT from the right hemisphere among the three groups (p = 0.068), and for the CMCT between the left and right side in controls (p = 0.075), is unclear although they may not have a significant clinical relevance, and are likely arising from procedural variability or related to the small samples size in this study.\nThe findings of this study must be viewed in light of some important limitations. The main limitation is the relatively small number of patients, although they were matched with controls without WMLs that are strikingly prevalent among the elderly. Secondly, given the well-known effect of neuroactive drugs on TMS measures of cortical excitability, we cannot exclude a drug-induced effect on the results. Nevertheless, taking into account these possible interactions, we selected patients assuming psychotropic drugs minimally affected cortico-spinal excitability [51-54], and who were not withdrawn from psychotropic drugs that were unchanged and unmodified in terms of the daily dosage throughout the course of the study. Moreover, medications taken by patients with MD belonged to relatively few classes whose members all share a common mode of action, thus providing more consistent results on the TMS excitability measure [53]. Furthermore, the within- and between-subject variability was minimized, as stated above. The medicaments taken by the VD group for treatment of vascular risk factors (anti-thrombosis agents, anti-hypertensive drugs, statins, oral anti-diabetes therapy) have no supporting data with respect to the possible influence on motor excitability parameters. Motor cortex excitability is unaffected in insulin-dependent diabetic patients when compared with normo- and hyperglycaemic subjects [55], except for a single study reporting a lack of facilitation at an ISI of 30 ms in diabetic patients compared with controls (ISI not explored in the present paper) [56]. Thirdly, transfer TMS findings obtained from stimulation of the M1 to other brain regions, such as those involved in the pathophysiology of depressive disorders, is challenging, and requires further investigation. Finally, although this sample of drug-resistant depressed patients is not likely to be representative of the whole population of patients with recurrent MD, it is homogeneous in the terms of the clinical features and neuropsychological profile.",
"The current study showed distinctive patterns of motor cortex excitability between late-onset depression with SVD and early-onset recurrent MD, providing a potential TMS model of the different processes underlying them. These results support the “Vascular depression hypothesis” at the neurophysiological level and confirm inter-hemispheric asymmetry to TMS in MD. Further research and comparison studies with homogeneous groups of patients and other methodologies are needed to confirm the present findings, as well as their modifications over time and clinical correlates.",
"The authors declare that they do not have any financial or non-financial competing interests. There was no study sponsor involved in the funding or write-up of this research.",
"CC and EA conceived and designed the study and participated in writing the protocol. GL and RR performed clinical and neuropsychological assessments, managed literature searches, and helped to draft the manuscript. MC and MP carried out transcranial magnetic stimulation procedures and analysed the data. DG and CS performed the statistical analysis. GP and RB reviewed the neuroradiological images, drafted the manuscript, and participated in its design and coordination. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-244X/13/300/prepub\n"
] | [
null,
"methods",
null,
null,
null,
null,
"results",
"discussion",
"conclusions",
null,
null,
null
] | [
"Late-onset depression",
"Cortical excitability",
"Subcortical vascular disease",
"Neuroplasticity"
] | Background:
Recently, the finding that patients with late-onset depression had higher rates of brain magnetic resonance imaging (MRI) changes compared with patients with early onset major depression (MD), has led to the hypothesis that mood disorders in the elderly may be related to neurobiological abnormalities, such as cerebrovascular disease [1]. In 1997, Alexopoulos and co-workers [2] introduced the concept of “vascular depression” (VD) as a subtype of geriatric mood disorder characterised by a late age at onset or change in the course of early onset depressive symptoms, persistent symptoms, association with vascular disease or vascular risk factors and diffuse or multifocal cerebrovascular lesions. The “vascular depression hypothesis”, presenting clinically as a depression-executive dysfunction syndrome of late-life, states that disruption of fronto-striatal circuits by vascular lesions predisposes, precipitates, or perpetuates late-life depressive syndromes [3]. Indeed, numerous neuroimaging and neuropathological studies reported increased prevalence and severity of white matter lesions (WMLs) of vascular origin in individuals with elderly depression, especially in those with late-onset illness [4-6].
Nevertheless, depression in the elderly might also result from a recurrent form of MD with an early onset. However, compared with patients with recurrent MD, elderly patients with VD often exhibit a clinical presentation characterized by psychomotor retardation, lack of interest, limited depressive ideation and insight, and prominent disability [7,8]. Moreover, patients with late-onset MD disorder showed specific deficits in attention and executive function [9,10], whereas patients with recurrent MD exhibited deficits in episodic memory [11,12]. These neuropsychological differences are thought to be associated with prominent fronto-striatal dysfunction in late-onset MD, and with a reduction in hippocampal volume in recurrent geriatric MD. The rates of anhedonia and comorbid cardiovascular illness were higher in patients with late-onset MD [11].
Despite a body of literature on clinical, psychopathological, and neuroradiological features of both VD and MD, studies comparing their neurophysiological profiles are lacking. Previous electroencephalography studies in MD patients demonstrated decreased neural activity in the left frontal regions, as shown by an increased alpha power band [13,14]. Recently, changes in transcranial magnetic stimulation (TMS) related-measures of cortical excitability have been shown to be associated with depression. Specifically, most, but not all, TMS studies in patients with MD found reduced activation of both excitatory and inhibitory circuits in the left hemisphere [15-19]. Moreover, some functional neuroimaging studies have shown hypometabolism and hypoperfusion of the left dorsolateral prefrontal cortex (DLPFC) in patients with MD [16,17]. However, the applicability of the findings obtained with TMS on the primary motor cortex (M1) and those obtained with other techniques on the DLPFC requires further investigation.
TMS is a safe and non-invasive neurophysiological investigation technique used to evaluate the cortico-spinal tract, cortical motor areas [20], map motor and cognitive functions, study neural networks, and modulate brain function with a potential therapeutic aim [21-23]. The development of specific stimulation protocols, such as the cortical silent period (CSP) and paired-pulse paradigms, as well as the emerging concept that motor cortical output is influenced by non-primary motor areas, including the ventral and dorsal premotor cortex, supplementary motor area, and cingulate cortex [24], has allowed the use of TMS to explore inhibitory and excitatory interactions within motor cortical regions in several neuropsychiatric disorders [25,26]. A single TMS pulse applied over the M1 through the scalp elicits a motor evoked potential (MEP) in the contralateral target muscles [27,28]. The resting motor threshold (rMT) is believed to reflect membrane excitability of cortico-spinal motor neurons, which is mainly dependent on ion channel conductivity and on excitatory interneurons that project to these neurons [20]. The CSP refers to a suppression of electromyographic activity during a voluntary contraction of the target muscle and depends, at least in part, on inhibitory mechanisms at the level of the motor cortex, probably mediated by gamma-aminobutyric acid (GABA)-b receptors [29]. The paired-pulse TMS couples a suprathreshold magnetic stimulus with a preceding subthreshold stimulus, and the response to the paired stimuli may be increased (facilitation) or decreased (inhibition) depending on the interstimulus interval (ISI): at short ISIs (1–4 ms) the conditioning stimulus determines the intracortical inhibition (ICI) with respect to the test stimulus, whereas at longer ISIs (>5 ms) the effect is intracortical facilitation (ICF). ICI and ICF interactions are likley related to the balance of GABAergic, dopaminergic, and glutamatergic transmissions [29]. In a single study evaluating inhibitory/facilitatory intracortical circuit changes, Bella et al. [30] did not find significant differences between patients with VD and patients with subcortical vascular disease (SVD), suggesting that VD with or without depression might result in a similar neurophysiological profile of cortical excitability, probably as a consequence of cerebral small vessel disease.
In this study, we aimed to assess and compare single and paired-pulse TMS measures of cortical excitability in patients with VD versus MD and between hemispheres. We hypothesised that VD patients would show distinctive neurophysiological changes compared with MD, consistent with the involvement of different neurobiological substrates.
Methods:
Participants A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).
VD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.
Drug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].
As shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.
Pharmacological treatment of MD patients
All patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.
A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).
VD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.
Drug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].
As shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.
Pharmacological treatment of MD patients
All patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.
Assessment All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].
All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].
Transcranial magnetic stimulation MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.
The rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).
MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.
The rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).
Statistical analysis The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.
The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.
Participants:
A sample of 11 patients with VD (6 males and 5 females; mean age, 67.72 ± 3.29 years; mean education, 7.91 ± 5.75 years), 11 drug-resistant recurrent patients with MD without SVD at MRI (5 males and 6 females; mean age, 57.18 ± 7.12; mean education, 12.18 ± 5.98) and 11 age-matched controls (6 males and 5 females; mean age, 67.36 ± 3.75 years; mean education, 9.64 ± 5.08) were consecutively recruited from the Cerebrovascular Disease Center and from the Department of Psychiatry of the University of Catania (Italy).
VD was defined according to the proposed clinical and neuroradiological diagnostic criteria as follows: evidence of vascular risk factors; depression onset after the age of 65 or change in course of depression after vascular disease in people with early-onset depression; presence of some of the following features: cognitive impairment (consisting of, but not limited to, disturbance of executive functions), psychomotor retardation, limited depressive ideation, poor insight, disability, absence of family history of mood disorders, and presence of cerebrovascular disease on neuroimaging [2,31]. VD patients fulfilled the brain MRI criteria for SVD [32], including extending periventricular and deep WMLs or multiple lacunes in deep grey matter, and at least moderate WMLs. VD patients had a primary diagnosis of a current major depressive episode as assessed by the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I). They were treated for their vascular risk factors with anti-platelet or anticoagulant medications (aspirin, clopidogrel, warfarin), anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonist, diuretics, dihydropyridine calcium channel blockers), cholesterol lowering medications (statins), and oral antidiabetic drugs or insulin. No patients had focal motor deficits, but a slight reflex asymmetry was present in three patients. Mean age at depression onset in the VD patients was 62.27 ± 5.04 years. No patients were on antidepressant treatment or other psychotropic medicaments.
Drug-resistant MD patients met the DSM-IV-TR clinical diagnostic criteria for recurrent MD as assessed by the SCID-I, and showed a poor response in the course of the current depressive episode (mean duration, 4.33 ± 2.50 months). We defined treatment resistance as drug-resistance to three adequate courses of antidepressants from at least two different classes during the current major depressive episode [33,34].
As shown in Table 1, the pharmacological regimen at the time of the study was: Selective Serotonin Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs; Serotonin Noradrenaline Reuptake Inhibitors, Tricyclic Antidepressant, Atypical Antipsychotic Drugs. Seven MD patients were on zolpidem. The pharmacotherapy was unchanged throughout the course of the study. Mean age at depression onset in MD patients was 27.82 ± 7.32 years and, including the current episode, 6 patients experienced four major depressive episodes, whereas the remaining 5 patients were diagnosed with five or more episodes.
Pharmacological treatment of MD patients
All patients were right-handed, with no history of brain trauma or seizures, and their general and neurological examination was unremarkable. Exclusion criteria were as follows: any non-mood psychotic disorder, chronic medical illness, endocrinopathies other than diabetes associated with depression or affecting cognitive functions (such as thyroid diseases), alcohol or drug abuse, use of drugs causing depressive symptoms (i.e. steroids, beta-blockers, clonidine), Mini Mental State Examination (MMSE) score <24, cases with cortical and/or cortico-subcortical non-lacunar territorial infarcts, borderzone infarcts, haemorrhages, signs of normal pressure hydrocephalus and specific causes of WMLs, and any condition precluding MRI or TMS execution. This study was approved by the local ethics committee based at the “Policlinico-Vittorio Emanuele” University Hospital of Catania (Italy), and all patients provided written informed consent.
Assessment:
All participants underwent a neuropsychological battery including the MMSE as a screening test for overall cognitive impairment, Clinical Dementia Rating Scale for the global cognitive and functional status, Frontal Assessment Battery and the Stroop Color-Word Test interference (normative values were collected from an Italian population sample, Stroop T score, ≤36.92 s; Stroop E errors, ≤4.24) [35] for the evaluation of different frontal lobe abilities, and the 17-item Hamilton Rating Scale for Depression (HRSD-17) for the rating of depressive symptoms. Functional status was evaluated by basic and instrumental activities of daily living (Activity of Daily Living; Instrumental Activity of Daily Living). The physical state of the control subjects was evaluated by general and neurological examinations; their mental state, assessed by means of the SCID-I, was unremarkable. All patients and controls underwent brain MRI scans, acquired using a 1.5 T General Electric system, before inclusion into the TMS study. The protocol included T1-, T2-, proton density-weighted and fluid-attenuated inversion recovery scans; slice thickness was 5 mm with a 0.5 mm slice gap. In the VD group, the severity of deep WMLs was graded according to the visual scale of Fazekas: 0 = absence; 1 = punctuate foci; 2 = beginning confluence of foci; 3 = large confluent areas [36].
Transcranial magnetic stimulation:
MEPs of the right and left first dorsal interosseous (FDI) muscles as well as single-pulse TMS measures of cortical excitability were elicited using a Magstim 200 stimulator (The Magstim Company, Whitland, Dyfed, UK) connected to a 70 mm figure-of-eight coil. The coil was applied with the handle pointing backwards and laterally, at an angle of 45° to the sagittal plane, on the optimum site of stimulation that consistently yielded the largest MEP (“hot spot”). Electromyographic activity was recorded from silver/silverchloride surface active electrodes placed over the motor point of the target muscle, with the reference electrode placed distally at the metacarpophalangeal joint of the index finger. Motor responses were amplified and filtered (bandwidth 3–3000 Hz) with gains of 100 μV and 5 mV/div.
The rMT was defined, according to the IFCN Committee recommendation [37], as the lowest stimulus intensity able to elicit MEPs of an amplitude >50 μV in at least 5 out of 10 trials, with the muscle at rest. The CSP was determined with an approximately 50% of maximum tonic voluntary contraction of the FDI muscles, induced by single TMS pulses delivered at 130% of rMT. The mean CSP duration of five rectified trials was calculated. Central motor conduction time (CMCT) was calculated by subtracting the conduction time in peripheral nerves, estimated by conventional F-wave techniques, from MEP latency obtained during moderate active muscle contraction (10–20% of maximum background force), at a stimulus intensity set at 130% of the rMT [37]. M and F waves were elicited by applying supramaximal electrical stimulation (constant current square-wave pulse of 0.2 ms) to the ulnar nerve at the wrist. The size of MEPs was expressed as a percentage of the supramaximal M wave amplitude (Amplitude ratio). Moreover, to assess spinal motor excitability, the mean amplitude of the F wave was measured in the target muscle. ICI and ICF were studied using the conditioning-test paradigm described by Kujirai et al. [38] through a Bistim module (The Magstim Company) connected to a Cambridge Electronic DesignMicro 1401 interface (Cambridge, UK). The procedure consisted of applying two magnetic stimuli in rapid succession through two magnetic stimulators connected to each other. The conditioning stimulus was applied at 80% of the subject’s rMT, and the intensity of the test stimulus was set at 130% of the rMT. The ISIs tested were 1, 3, 5, 7, 10, and 15 ms. Ten trials for each ISI were recorded randomly with an 8-second interval between each trial. The responses were expressed as the ratio of the MEP amplitude produced by paired stimulation to that produced by test stimulation alone. The use of a Bistim module was limited to the paired-pulse TMS measurements only, whereas all other investigations were performed using the single-pulse technique. All measurements were conducted while subjects were seated in a comfortable chair with continuous electromyographic monitoring to ensure either a constant level of electromyographic activity during tonic contraction or complete relaxation at rest. Data were collected on a computer and stored with software ad hoc for off-line analysis [39]. All procedures described above were performed in the same laboratory and situation, by the same operators for each subject at the same time during the day (approximately 3–5 pm).
Statistical analysis:
The non-parametric Kruskal-Wallis ANOVA test was used for comparison of clinical, neuropsychological, and neurophysiological variables obtained from patients and controls (followed by the Mann–Whitney test for post-hoc analysis for the comparison between pairs of groups), and the χ2 test was used for categorical variables. The Wilcoxon test for paired data sets was used for the comparison between hemispheres of patients and controls. Nonparametric statistics were used because of the categorical nature of the neuropsychological testing results, and the non-Gaussian distribution of the results of the TMS studies. A p value lower than 0.05 was considered statistically significant.
Results:
The relevant demographic and clinical characteristics of the two patient groups are summarized in Tables 2 and 3. MD were younger than VD patients and controls. The depression rating was less severe in the VD group compared with the MD group. As expected, hypertension was more frequent in VD compared with MD patients, whereas personal history of depressive symptoms was the opposite, and scores at Stroop T were worse in patients compared with controls. WML severity was mild in 4, moderate in 5, and severe in 2 patients. Brain MRI of MD patients and controls was unremarkable (Fazekas 0), and the physical and mental state of controls was also unremarkable. As shown in Table 4, there was a significant increase in rMT in MD patients in the left hemisphere compared with the right (48.36 ± 9.64 vs. 45.00 ± 8.82; p < 0.05). No significant differences were found between the right and left hemispheres for the other measures of cortico-spinal excitability in VD patients, except for a trend toward an increase in ICF at the ISI of 15 ms from the right hemisphere (VD 2.08 ± 0.7 vs. MD 1.78 ± 0.87 vs. Controls 1.52 ± 0.75, p = 0.153) (Figures 1 and 2). No significant differences were found in rMT, central motor conduction time, amplitude ratio, and mean amplitude of the F wave between VD, MD, and controls. In MD patients, the duration of the CSP from both hemispheres was increased, which was significant from the left hemisphere (MD 115.00 ± 32.39 vs. VD 72.18 ± 26.57 vs. Controls 80.09 ± 19.19, p = 0.004), and there was a trend towards significance from the right hemisphere (MD 111.82 ± 42.09 vs. VD 85.45 ± 45.13 vs. Controls 79.18 ± 21.51, p = 0.105) (Table 4).
Demographic and neuropsychological characteristics of patients and controls
VD = vascular depression; MD = major depression; MMSE = Mini Mental State Examination; ADL = Activity of Daily Living; IADL = Instrumental Activity of Daily Living; HAM-D17 = 17-items Hamilton Depression Rating Scale; SCID-I = Structured Clinical Interview for DSM-IV Axis I Disorders; Stroop T = Stroop Color-Word Test interference score; Stroop E = Stroop Color-Word Test interference number of errors; FAB = Frontal Assessment Battery. Data are expressed as mean ± SD and were analyzed using Kruskal-Wallis ANOVA. * = p < 0.05.
Clinical characteristics of patients and controls
VD = vascular depression; MD = major depression. * = p < 0.05.
TMS parameters of the patient and the control groups obtained from both hemispheres
VD = vascular depression; MD = major depression; rMT = resting motor threshold (%); CSP = cortical silent period (ms); CMCT = central motor conduction time (ms); CMCTF = central motor conduction time calculated with F-waves (ms); A ratio = amplitude ratio; F wave A = amplitude of F-waves (mV). Data are expressed as mean ± SD. * = p < 0.05.
MEP amplitudes at different ISIs from the 3 groups with respect to the hemisphere. Data are expressed as mean ± SE. (whiskers). VD = vascular depression; MD = major depression; C = Controls; MEP = Motor Evoked Potential; ISI = Interstimulus Interval.
Curves of intracortical excitability obtained from both hemispheres in each group.
Discussion:
This is the first report comparing motor cortex excitability between VD and MD patients. The main finding is the observation of potentially distinctive neurophysiological profiles elicited by TMS, adding support to the hypothesis that late onset VD is a different syndrome with respect to early onset recurrent MD. Despite the abundant literature on TMS and depression, only one study has been conducted in VD patients, where VD patients were compared with patients with SVD patients and controls [30]. The results of this study confirm previous findings on VD, and are consistent with most TMS reports investigating MD. An interhemispheric imbalance in frontal cortical activities, as indexed by a higher rMT, was observed in our MD patients and is consistent with an overall hypoexcitability of the left hemisphere [15-17].
In line with our finding of laterality in MD, Soares and Mann [40] described structural asymmetries in cortical and subcortical brain regions crucial in the neuroanatomical model of mood disorders. More recently, it has been reported that decreased relative left-frontal brain electrical activity may be a trait-like marker of depression, suggesting that frontal asymmetry could also be a shared factor predicting first depression onset [41]. However, it remains difficult to infer functional asymmetry between the left and right prefrontal cortices by examining differences in motor responses to TMS only. Findings from previous studies conclude that both glutamatergic and GABAergic pathways may be defective in the left hemisphere of patients with MD. The effect of laterality has also been reported in patients with late-life depression and MRI signal hyperintensities, but it is still controversial as some reports found that left-sided WMLs were significantly associated with older age of depression onset [42], whereas others did not find any laterality effect [43].
Although many, but not all, TMS studies in MD have shown a significant reduction in the amount of both inhibitory (shortened CSP and decreased ICI) and facilitatory (reduced ICF) inputs regarding the left frontal cortex compared with the contralateral hemisphere [17-19], we were not able to find substantial differences. On the contrary, we observed a bilateral increase in CSP duration. The reason for this dissociation remains unclear. Previous studies have obtained conflicting results, probably due to the heterogeneous methods employed, differences in clinical presentation and severity as well as the medication status. For example, Bajbouj et al. [17] and Lefaucheur et al. [18] found reduced CSP and ICI in medication-free and treated MD patients, respectively. Additionally, Fitzgerald et al. [16] did not observe consistent differences in hemispheric activity in drug-resistant MD patients, whereas Levinson et al. [19] demonstrated significantly shortened CSP duration in all MD patient groups and decreased ICI in the drug-resistant group only. In contrast, one study observed an increased CSP in MD patients compared with age-matched controls, suggesting increased cortical inhibition [44]. In our study, however, we cannot exclude the possibility of a drug-induced effect on CSP duration, related to zolpidem administration in many MD patients (but not in VD group or controls), highlighting the role of specific subtypes of GABA receptors in the control of inhibitory neuronal loops within the M1 [45]. It is more difficult to explain a drug effect on ICI since it has been shown to be affected by benzodiazepine administration but not zolpidem, and this is consistent with the different segregation of the two inhibitory circuits in the motor cortex at the level of GABA receptor subtypes [46].
Regarding the facilitatory component of intracortical excitability, we observed a trend toward an increase in ICF in VD patients, supporting previous results [30]. A significant hyperfacilitation was found in patients with SVD and clinical features of vascular cognitive impairment-no dementia (VCI-ND) [47], although it was not observed at follow-up [48], supporting the concept that specific TMS measures of cortical excitability can be considered indices of motor cortex plasticity [49]. SVD is indeed a potential aetiology of both VD and VCI and the accumulation of microvascular lesions constitutes a common neuropathological platform for both cognitive decline and depressive episodes in old age [6].
A growing body of evidence indicates that glutamate-mediated compensatory plastic events might also occur in MD [50]. In a recent paper, Spampinato et al. [34] demonstrated that repetitive TMS (rTMS) improved performance in a test evaluating frontal lobe abilities and was able to restore inter-hemispheric asymmetry of the ICF and rMT. These results suggested that the TMS changes observed before treatment might be the expression of disruption of glutamatergic receptor plasticity-related processes, and that this neurophysiological behaviour might correlate with executive functions.
Taken together, the data presented here may help to further understanding of the pathogenetic differences underlying the clinical spectrum of depressive disorders. However, relating the distinctive electrophysiological profile between VD and MD patients to what is clinically observed is challenging. Currently, it is difficult to relate the subtle TMS changes with the clinical picture of psychomotor retardation, persistent symptoms, and prominent disability seen in patients with VD. It has been proposed that vascular damage to the reciprocal connections between the prefrontal cortex, basal ganglia, and cerebellum may affect the presentation and the course of late-life VD, although the atrophy of frontal grey matter due to deafferentation could also contribute [10]. Finally, although not statistically significant, we did observe some trends in this study that may be of interest in future studies. For example, the MMSE scores obtained in patients with VD were lower compared with the MD group and controls (p = 0.064), although all values were within normal limits. This could be related to poorer educational level in the VD group, and the clinical presentation and vascular burden. The significance of other trend-level differences, such as for the CMCT from the right hemisphere among the three groups (p = 0.068), and for the CMCT between the left and right side in controls (p = 0.075), is unclear although they may not have a significant clinical relevance, and are likely arising from procedural variability or related to the small samples size in this study.
The findings of this study must be viewed in light of some important limitations. The main limitation is the relatively small number of patients, although they were matched with controls without WMLs that are strikingly prevalent among the elderly. Secondly, given the well-known effect of neuroactive drugs on TMS measures of cortical excitability, we cannot exclude a drug-induced effect on the results. Nevertheless, taking into account these possible interactions, we selected patients assuming psychotropic drugs minimally affected cortico-spinal excitability [51-54], and who were not withdrawn from psychotropic drugs that were unchanged and unmodified in terms of the daily dosage throughout the course of the study. Moreover, medications taken by patients with MD belonged to relatively few classes whose members all share a common mode of action, thus providing more consistent results on the TMS excitability measure [53]. Furthermore, the within- and between-subject variability was minimized, as stated above. The medicaments taken by the VD group for treatment of vascular risk factors (anti-thrombosis agents, anti-hypertensive drugs, statins, oral anti-diabetes therapy) have no supporting data with respect to the possible influence on motor excitability parameters. Motor cortex excitability is unaffected in insulin-dependent diabetic patients when compared with normo- and hyperglycaemic subjects [55], except for a single study reporting a lack of facilitation at an ISI of 30 ms in diabetic patients compared with controls (ISI not explored in the present paper) [56]. Thirdly, transfer TMS findings obtained from stimulation of the M1 to other brain regions, such as those involved in the pathophysiology of depressive disorders, is challenging, and requires further investigation. Finally, although this sample of drug-resistant depressed patients is not likely to be representative of the whole population of patients with recurrent MD, it is homogeneous in the terms of the clinical features and neuropsychological profile.
Conclusions:
The current study showed distinctive patterns of motor cortex excitability between late-onset depression with SVD and early-onset recurrent MD, providing a potential TMS model of the different processes underlying them. These results support the “Vascular depression hypothesis” at the neurophysiological level and confirm inter-hemispheric asymmetry to TMS in MD. Further research and comparison studies with homogeneous groups of patients and other methodologies are needed to confirm the present findings, as well as their modifications over time and clinical correlates.
Competing interests:
The authors declare that they do not have any financial or non-financial competing interests. There was no study sponsor involved in the funding or write-up of this research.
Authors’ contributions:
CC and EA conceived and designed the study and participated in writing the protocol. GL and RR performed clinical and neuropsychological assessments, managed literature searches, and helped to draft the manuscript. MC and MP carried out transcranial magnetic stimulation procedures and analysed the data. DG and CS performed the statistical analysis. GP and RB reviewed the neuroradiological images, drafted the manuscript, and participated in its design and coordination. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-244X/13/300/prepub
| Background:
Clinical and functional studies consider major depression (MD) and vascular depression (VD) as different neurobiological processes. Hypoexcitability of the left frontal cortex to transcranial magnetic stimulation (TMS) is frequently reported in MD, whereas little is known about the effects of TMS in VD. Thus, we aimed to assess and compare motor cortex excitability in patients with VD and MD.
Methods:
Eleven VD patients, 11 recurrent drug-resistant MD patients, and 11 healthy controls underwent clinical, neuropsychological and neuroimaging evaluations in addition to bilateral resting motor threshold, cortical silent period, and paired-pulse TMS curves of intracortical excitability. All patients continued on psychotropic drugs, which were unchanged throughout the study.
Results:
Scores on one of the tests evaluating frontal lobe abilities (Stroop Color-Word interference test) were worse in patients compared with controls. The resting motor threshold in patients with MD was significantly higher in the left hemisphere compared with the right (p < 0.05), and compared with the VD patients and controls. The cortical silent period was bilaterally prolonged in MD patients compared with VD patients and controls, with a statistically significant difference in the left hemisphere (p < 0.01). No differences were observed in the paired-pulse curves between patients and controls.
Conclusions:
This study showed distinctive patterns of motor cortex excitability between late-onset depression with subcortical vascular disease and early-onset recurrent drug resistant MD. The data provide a TMS model of the different processes underlying VD and MD. Additionally, our results support the "Vascular depression hypothesis" at the neurophysiological level, and confirm the inter-hemispheric asymmetry to TMS in patients with MD. We were unable to support previous findings of impaired intracortical inhibitory mechanisms to TMS in patients with MD, although a drug-induced effect on our results cannot be excluded. This study may aid the understanding of the pathogenetic differences underlying the clinical spectrum of depressive disorders. | Background:
Recently, the finding that patients with late-onset depression had higher rates of brain magnetic resonance imaging (MRI) changes compared with patients with early onset major depression (MD), has led to the hypothesis that mood disorders in the elderly may be related to neurobiological abnormalities, such as cerebrovascular disease [1]. In 1997, Alexopoulos and co-workers [2] introduced the concept of “vascular depression” (VD) as a subtype of geriatric mood disorder characterised by a late age at onset or change in the course of early onset depressive symptoms, persistent symptoms, association with vascular disease or vascular risk factors and diffuse or multifocal cerebrovascular lesions. The “vascular depression hypothesis”, presenting clinically as a depression-executive dysfunction syndrome of late-life, states that disruption of fronto-striatal circuits by vascular lesions predisposes, precipitates, or perpetuates late-life depressive syndromes [3]. Indeed, numerous neuroimaging and neuropathological studies reported increased prevalence and severity of white matter lesions (WMLs) of vascular origin in individuals with elderly depression, especially in those with late-onset illness [4-6].
Nevertheless, depression in the elderly might also result from a recurrent form of MD with an early onset. However, compared with patients with recurrent MD, elderly patients with VD often exhibit a clinical presentation characterized by psychomotor retardation, lack of interest, limited depressive ideation and insight, and prominent disability [7,8]. Moreover, patients with late-onset MD disorder showed specific deficits in attention and executive function [9,10], whereas patients with recurrent MD exhibited deficits in episodic memory [11,12]. These neuropsychological differences are thought to be associated with prominent fronto-striatal dysfunction in late-onset MD, and with a reduction in hippocampal volume in recurrent geriatric MD. The rates of anhedonia and comorbid cardiovascular illness were higher in patients with late-onset MD [11].
Despite a body of literature on clinical, psychopathological, and neuroradiological features of both VD and MD, studies comparing their neurophysiological profiles are lacking. Previous electroencephalography studies in MD patients demonstrated decreased neural activity in the left frontal regions, as shown by an increased alpha power band [13,14]. Recently, changes in transcranial magnetic stimulation (TMS) related-measures of cortical excitability have been shown to be associated with depression. Specifically, most, but not all, TMS studies in patients with MD found reduced activation of both excitatory and inhibitory circuits in the left hemisphere [15-19]. Moreover, some functional neuroimaging studies have shown hypometabolism and hypoperfusion of the left dorsolateral prefrontal cortex (DLPFC) in patients with MD [16,17]. However, the applicability of the findings obtained with TMS on the primary motor cortex (M1) and those obtained with other techniques on the DLPFC requires further investigation.
TMS is a safe and non-invasive neurophysiological investigation technique used to evaluate the cortico-spinal tract, cortical motor areas [20], map motor and cognitive functions, study neural networks, and modulate brain function with a potential therapeutic aim [21-23]. The development of specific stimulation protocols, such as the cortical silent period (CSP) and paired-pulse paradigms, as well as the emerging concept that motor cortical output is influenced by non-primary motor areas, including the ventral and dorsal premotor cortex, supplementary motor area, and cingulate cortex [24], has allowed the use of TMS to explore inhibitory and excitatory interactions within motor cortical regions in several neuropsychiatric disorders [25,26]. A single TMS pulse applied over the M1 through the scalp elicits a motor evoked potential (MEP) in the contralateral target muscles [27,28]. The resting motor threshold (rMT) is believed to reflect membrane excitability of cortico-spinal motor neurons, which is mainly dependent on ion channel conductivity and on excitatory interneurons that project to these neurons [20]. The CSP refers to a suppression of electromyographic activity during a voluntary contraction of the target muscle and depends, at least in part, on inhibitory mechanisms at the level of the motor cortex, probably mediated by gamma-aminobutyric acid (GABA)-b receptors [29]. The paired-pulse TMS couples a suprathreshold magnetic stimulus with a preceding subthreshold stimulus, and the response to the paired stimuli may be increased (facilitation) or decreased (inhibition) depending on the interstimulus interval (ISI): at short ISIs (1–4 ms) the conditioning stimulus determines the intracortical inhibition (ICI) with respect to the test stimulus, whereas at longer ISIs (>5 ms) the effect is intracortical facilitation (ICF). ICI and ICF interactions are likley related to the balance of GABAergic, dopaminergic, and glutamatergic transmissions [29]. In a single study evaluating inhibitory/facilitatory intracortical circuit changes, Bella et al. [30] did not find significant differences between patients with VD and patients with subcortical vascular disease (SVD), suggesting that VD with or without depression might result in a similar neurophysiological profile of cortical excitability, probably as a consequence of cerebral small vessel disease.
In this study, we aimed to assess and compare single and paired-pulse TMS measures of cortical excitability in patients with VD versus MD and between hemispheres. We hypothesised that VD patients would show distinctive neurophysiological changes compared with MD, consistent with the involvement of different neurobiological substrates.
Conclusions:
The current study showed distinctive patterns of motor cortex excitability between late-onset depression with SVD and early-onset recurrent MD, providing a potential TMS model of the different processes underlying them. These results support the “Vascular depression hypothesis” at the neurophysiological level and confirm inter-hemispheric asymmetry to TMS in MD. Further research and comparison studies with homogeneous groups of patients and other methodologies are needed to confirm the present findings, as well as their modifications over time and clinical correlates. | Background:
Clinical and functional studies consider major depression (MD) and vascular depression (VD) as different neurobiological processes. Hypoexcitability of the left frontal cortex to transcranial magnetic stimulation (TMS) is frequently reported in MD, whereas little is known about the effects of TMS in VD. Thus, we aimed to assess and compare motor cortex excitability in patients with VD and MD.
Methods:
Eleven VD patients, 11 recurrent drug-resistant MD patients, and 11 healthy controls underwent clinical, neuropsychological and neuroimaging evaluations in addition to bilateral resting motor threshold, cortical silent period, and paired-pulse TMS curves of intracortical excitability. All patients continued on psychotropic drugs, which were unchanged throughout the study.
Results:
Scores on one of the tests evaluating frontal lobe abilities (Stroop Color-Word interference test) were worse in patients compared with controls. The resting motor threshold in patients with MD was significantly higher in the left hemisphere compared with the right (p < 0.05), and compared with the VD patients and controls. The cortical silent period was bilaterally prolonged in MD patients compared with VD patients and controls, with a statistically significant difference in the left hemisphere (p < 0.01). No differences were observed in the paired-pulse curves between patients and controls.
Conclusions:
This study showed distinctive patterns of motor cortex excitability between late-onset depression with subcortical vascular disease and early-onset recurrent drug resistant MD. The data provide a TMS model of the different processes underlying VD and MD. Additionally, our results support the "Vascular depression hypothesis" at the neurophysiological level, and confirm the inter-hemispheric asymmetry to TMS in patients with MD. We were unable to support previous findings of impaired intracortical inhibitory mechanisms to TMS in patients with MD, although a drug-induced effect on our results cannot be excluded. This study may aid the understanding of the pathogenetic differences underlying the clinical spectrum of depressive disorders. | 9,002 | 374 | [
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] | [CONTENT] Late-onset depression | Cortical excitability | Subcortical vascular disease | Neuroplasticity [SUMMARY] | [CONTENT] Late-onset depression | Cortical excitability | Subcortical vascular disease | Neuroplasticity [SUMMARY] | [CONTENT] Late-onset depression | Cortical excitability | Subcortical vascular disease | Neuroplasticity [SUMMARY] | [CONTENT] Late-onset depression | Cortical excitability | Subcortical vascular disease | Neuroplasticity [SUMMARY] | [CONTENT] Late-onset depression | Cortical excitability | Subcortical vascular disease | Neuroplasticity [SUMMARY] | [CONTENT] Late-onset depression | Cortical excitability | Subcortical vascular disease | Neuroplasticity [SUMMARY] | [CONTENT] Aged | Depressive Disorder | Depressive Disorder, Major | Female | Humans | Male | Middle Aged | Motor Cortex | Neuropsychological Tests | Transcranial Magnetic Stimulation [SUMMARY] | [CONTENT] Aged | Depressive Disorder | Depressive Disorder, Major | Female | Humans | Male | Middle Aged | Motor Cortex | Neuropsychological Tests | Transcranial Magnetic Stimulation [SUMMARY] | [CONTENT] Aged | Depressive Disorder | Depressive Disorder, Major | Female | Humans | Male | Middle Aged | Motor Cortex | Neuropsychological Tests | Transcranial Magnetic Stimulation [SUMMARY] | [CONTENT] Aged | Depressive Disorder | Depressive Disorder, Major | Female | Humans | Male | Middle Aged | Motor Cortex | Neuropsychological Tests | Transcranial Magnetic Stimulation [SUMMARY] | [CONTENT] Aged | Depressive Disorder | Depressive Disorder, Major | Female | Humans | Male | Middle Aged | Motor Cortex | Neuropsychological Tests | Transcranial Magnetic Stimulation [SUMMARY] | [CONTENT] Aged | Depressive Disorder | Depressive Disorder, Major | Female | Humans | Male | Middle Aged | Motor Cortex | Neuropsychological Tests | Transcranial Magnetic Stimulation [SUMMARY] | [CONTENT] vascular depression hypothesis | depression elderly result | depression onset age | lesions vascular depression | late onset depression [SUMMARY] | [CONTENT] vascular depression hypothesis | depression elderly result | depression onset age | lesions vascular depression | late onset depression [SUMMARY] | [CONTENT] vascular depression hypothesis | depression elderly result | depression onset age | lesions vascular depression | late onset depression [SUMMARY] | [CONTENT] vascular depression hypothesis | depression elderly result | depression onset age | lesions vascular depression | late onset depression [SUMMARY] | [CONTENT] vascular depression hypothesis | depression elderly result | depression onset age | lesions vascular depression | late onset depression [SUMMARY] | [CONTENT] vascular depression hypothesis | depression elderly result | depression onset age | lesions vascular depression | late onset depression [SUMMARY] | [CONTENT] patients | md | vd | depression | tms | motor | mean | test | controls | clinical [SUMMARY] | [CONTENT] patients | md | vd | depression | tms | motor | mean | test | controls | clinical [SUMMARY] | [CONTENT] patients | md | vd | depression | tms | motor | mean | test | controls | clinical [SUMMARY] | [CONTENT] patients | md | vd | depression | tms | motor | mean | test | controls | clinical [SUMMARY] | [CONTENT] patients | md | vd | depression | tms | motor | mean | test | controls | clinical [SUMMARY] | [CONTENT] patients | md | vd | depression | tms | motor | mean | test | controls | clinical [SUMMARY] | [CONTENT] md | patients | late | motor | onset | depression | vd | vascular | cortical | cortex [SUMMARY] | [CONTENT] patients | mean | age | test | mean age | years | depressive | drugs | amplitude | depression [SUMMARY] | [CONTENT] md | vs | vd | controls | depression | patients | vascular depression md | vd vascular depression | vd vascular depression md | vd vascular [SUMMARY] | [CONTENT] confirm | onset | md | depression | confirm present findings | model different processes underlying | hemispheric asymmetry tms | hemispheric asymmetry tms md | support vascular depression hypothesis | support vascular depression [SUMMARY] | [CONTENT] patients | md | vd | depression | mean | tms | motor | test | controls | onset [SUMMARY] | [CONTENT] patients | md | vd | depression | mean | tms | motor | test | controls | onset [SUMMARY] | [CONTENT] MD ||| TMS | MD | TMS | VD ||| VD | MD [SUMMARY] | [CONTENT] Eleven VD | 11 | MD | 11 | TMS ||| [SUMMARY] | [CONTENT] one ||| MD | VD ||| MD | VD ||| [SUMMARY] | [CONTENT] MD ||| TMS | VD | MD ||| Vascular | TMS | MD ||| TMS | MD ||| [SUMMARY] | [CONTENT] MD ||| TMS | MD | TMS | VD ||| VD | MD ||| 11 | MD | 11 | TMS ||| ||| ||| one ||| MD | VD ||| MD | VD ||| ||| MD ||| TMS | VD | MD ||| Vascular | TMS | MD ||| TMS | MD ||| [SUMMARY] | [CONTENT] MD ||| TMS | MD | TMS | VD ||| VD | MD ||| 11 | MD | 11 | TMS ||| ||| ||| one ||| MD | VD ||| MD | VD ||| ||| MD ||| TMS | VD | MD ||| Vascular | TMS | MD ||| TMS | MD ||| [SUMMARY] |
||||||
Dietary animal and plant protein intakes and their associations with obesity and cardio-metabolic indicators in European adolescents: the HELENA cross-sectional study. | 25609179 | Previous studies suggest that dietary protein might play a beneficial role in combating obesity and its related chronic diseases. Total, animal and plant protein intakes and their associations with anthropometry and serum biomarkers in European adolescents using one standardised methodology across European countries are not well documented. | BACKGROUND | The current analysis included 1804 randomly selected adolescents participating in the HELENA study (conducted in 2006-2007) aged 12.5-17.5 y (47% males) who completed two non-consecutive computerised 24-h dietary recalls. Associations between animal and plant protein intakes, and anthropometry and serum biomarkers were examined with General linear Model multivariate analysis. | METHODS | Average total protein intake exceeded the recommendations of World Health Organization and European Food Safety Authority. Mean total protein intake was 96 g/d (59% derived from animal protein). Total, animal and plant protein intakes (g/d) were significantly lower in females than in males and total and plant protein intakes were lower in younger participants (12.5-14.9 y). Protein intake was significantly lower in underweight subjects and higher in obese ones; the direction of the relationship was reversed after adjustments for body weight (g/(kg.d)). The inverse association of plant protein intakes was stronger with BMI z-score and body fat percentage (BF%) compared to animal protein intakes. Additionally, BMI and BF% were positively associated with energy percentage of animal protein. | RESULTS | This sample of European adolescents appeared to have adequate total protein intake. Our findings suggest that plant protein intakes may play a role in preventing obesity among European adolescents. Further longitudinal studies are needed to investigate the potential beneficial effects observed in this study in the prevention of obesity and related chronic diseases. | CONCLUSIONS | [
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] | 4334414 | Introduction | The prevalence of overweight (OW) and obesity (OB) in adolescents, defined on the basis of body mass
[1], has increased rapidly worldwide. In 2010, the estimated prevalence of OW and OB in European children and adolescents was approximately 38%, including 10% OB
[2]. As a consequence of OB-related co-morbidities, over 20000 children suffer from type 2 diabetes and more than 400000 have impaired glucose levels
[2]. Childhood OW and OB both influence long-term health and evidence suggest an association with coronary events and mortality later in life
[3, 4].
Nutrition during the early years of life is a critical factor of OB in adolescence further impacting on adulthood OW and OB, and the consequences of chronic diseases
[5, 6]. High protein intakes were reported to improve cardiovascular risk factors including abdominal OB, dyslipidemia, glucose intolerance, and hypertension in European children (5–18 y)
[7]. Previous randomised trials
[8, 9] suggest that a high-protein diet defined as ≥20% of total energy lowers the risk of OW and promotes weight maintenance among adolescents
[10]. The association between dietary protein intake and adolescent OW and OB has mainly been investigated in relation to its increased thermic effect and satiety when compared to fats and carbohydrates
[9, 11]. Others, however, have reported that higher protein content in the diet did not confer any benefit in the treatment of OB among children 9–18 y old
[12].
The debate on protein sources is still ongoing, addressing the nutritional quality of dietary proteins based on their amino acids composition. The protein quality or biological value of proteins from animal sources is high, whereas most plant proteins lack one or more essential amino acids and are therefore considered as incomplete proteins. What some seem to be concerned with is that the majority of high-protein foods are significant sources of fat and/or sugar as well (such as meat and meat products, cheese, and dairy desserts), and should therefore be carefully selected. Hermanussen et al. reported a positive correlation between the energy contribution of animal proteins to the diet and the body mass index (BMI) in adolescents
[13]. On the other hand, Bradlee et al. found no association between OB and meat consumption among adolescents
[14], while, plant-based diets were inversely associated with normal BMI in children in Hermanussen’s study
[13]. A Western dietary pattern high in animal sources is associated with an increased risk of metabolic syndrome (MetS)
[15, 16], whereas diets high in fruits, vegetables and whole grains are associated with a decreased risk
[17]. Evidence showed that plant protein, soy in particular, can bind phytoestrogen compounds to stimulate lipid metabolism resulting in a better blood profile, by lowering total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and reducing insulin resistance
[18, 19].
The aim of the current study was to evaluate total, animal and plant protein intakes in European adolescents and to investigate their association with cardio-metabolic indicators (anthropometry: BMI z-score and body fat percentage (BF%); and biomarkers: TC, TG, LDL-C, very LDL-C (VLDL-C), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP), glucose, insulin and leptin). | Methods | Survey population The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.
Male and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere
[20, 21].
The study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves
[22].
The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.
Male and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere
[20, 21].
The study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves
[22].
Dietary intake assessment Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.
HELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)
[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C
[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions
[24, 25].
Dietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)
[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL
[27], the Dutch NEVO
[28] and the USDA
[29] food composition databases which used the Kjeldahl method for analysing protein
[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).
Under-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96
[31].
Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.
HELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)
[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C
[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions
[24, 25].
Dietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)
[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL
[27], the Dutch NEVO
[28] and the USDA
[29] food composition databases which used the Kjeldahl method for analysing protein
[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).
Under-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96
[31].
Anthropometric measurements Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents
[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method
[32]. The cut-off of BMI z-score
[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate
[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations
[35]. More details about the anthropometric measurements are given in a previous manuscript
[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages
[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).
Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents
[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method
[32]. The cut-off of BMI z-score
[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate
[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations
[35]. More details about the anthropometric measurements are given in a previous manuscript
[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages
[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).
Blood samples Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere
[37].
Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere
[37].
Physical activity Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously
[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.
Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously
[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.
Statistical analysis Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes
[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.
GLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.
All statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.
Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes
[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.
GLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.
All statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05. | Results | A total of 1804 out of 3528 adolescents (47% males) from 8 centres with valid and complete dietary data and measurements of weight and height were included in the analysis (Table
1). 74% participants were classified in tanner stage 2–4, including 7% in tanner 2, 24% in tanner 3 and 41% in tanner 4. In total 279 adolescents were classified as OW and OB. Mean BMI z-score for both genders was in the NW range. Females had higher BF %, but lower BMI z-score compared to males. Furthermore, higher serum lipid profiles and leptin levels were found in females.Table 1
Anthropometric characteristics and levels of obesity-related biomarkers in adolescents participating in the HELENA-CSS
TotalMalesFemalesTotal participants (n)1804855949Age (y) (mean (range))14.7 (12.5-17.4)14.8 (12.5-17.4)14.7 (12.5-17.4) 12.5-14.9 y (n)1032481551 15.0-17.5 y (n)772374398Tanner Stage (n = 1752)n (%) Tanner 19 (0.514)9 (1.1)0 (0.0) Tanner 2-41294 (73.9)614 (74.2)680 (73.5) Tanner 5449 (25.6)204 (24.7)245 (26.5)Weight status (n = 1804)μ
Underweight142 (7.9)58 (6.8)84 (8.9) Normal weight1383 (76.7)649 (75.9)734 (77.3) Overweight222 (12.3)114 (13.3)108 (11.4) Obesity57 (3.2)34 (4.0)23 (2.4)Mean (SD)Anthropometry BMI z-score (n = 1804)0.270 (1.1)0.358 (1.1)0.190 (1.0) BF% (n = 1764)22.0 (8.6)18.4 (9.1)25.1 (6.8)Biomarkers TC (mg/dL) (n = 552)159.1 (27)151.9 (24.9)165.8 (27.1) TG (mg/dL) (n = 552)67.6 (31.1)64.5 (31.5)70.5 (30.5) LDL-C (mg/dL) (n = 552)92.6 (24.2)89.0 (23.2)96.0 (24.7) VLDL-C (mg/dL) (n = 552)13.5 (6.2)12.9 (6.3)14.1 (6.1) HDL-C (mg/dL) (n = 552)55.6 (10.3)53.3 (9.3)57.8 (10.7) CRP (mg/L) (n = 524)1.2 (4.0)1.5 (5.5)0.841 (1.3) Glucose (mg/dL) (n = 552)90.1 (7.0)91.9 (7.2)88.5 (6.4) Insulin (μlU/mL) (n = 545)9.5 (6.0)9.0 (6.6)10.0 (6.6) Leptin (ng/mL) (n = 518)18.5 (21.9)8.1 (12.9)27.5 (24.1)SD, standard deviation; BMI, body mass index; BF%, body fat percentage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein- cholesterol; VLDL-C, very low-density lipoprotein- cholesterol; HDL-C, high-density lipoprotein- cholesterol; CRP, c-reactive protein.
μBMI categories is classified based on the International Obesity Task Force cut-offs, underweight: <18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25.0-29.9 kg/m2, obesity: ≥30.0 kg/m2.
Anthropometric characteristics and levels of obesity-related biomarkers in adolescents participating in the HELENA-CSS
SD, standard deviation; BMI, body mass index; BF%, body fat percentage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein- cholesterol; VLDL-C, very low-density lipoprotein- cholesterol; HDL-C, high-density lipoprotein- cholesterol; CRP, c-reactive protein.
μBMI categories is classified based on the International Obesity Task Force cut-offs, underweight: <18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25.0-29.9 kg/m2, obesity: ≥30.0 kg/m2.
Total energy and total, animal and plant protein intakes Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)
[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))
[41] (Table
2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
CharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR
μ
PRI
μ
Total180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Mean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table
3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
CharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a
1.8 (0.6)a
58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab
1.4 (0.4)ab
59 (24)37 (11)16.2 (3.0)b
6.2 (1.3) Obesity572476 (701)102 (33)abc
1.2 (0.4)ab
63 (27)38 (12)16.5 (3.1)b
6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
SD, standard deviation.
*Mean value was significantly different between males and females by Student T- test (P < 0.05).
**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)
[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))
[41] (Table
2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
CharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR
μ
PRI
μ
Total180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Mean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table
3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
CharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a
1.8 (0.6)a
58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab
1.4 (0.4)ab
59 (24)37 (11)16.2 (3.0)b
6.2 (1.3) Obesity572476 (701)102 (33)abc
1.2 (0.4)ab
63 (27)38 (12)16.5 (3.1)b
6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
SD, standard deviation.
*Mean value was significantly different between males and females by Student T- test (P < 0.05).
**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Associations between total, animal and plant protein intakes and cardio-metabolic indicators Figure
1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table
4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Associations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)
SE, standard error of coefficient β; CI, confidence interval.
μModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).
Figure
1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table
4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Associations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)
SE, standard error of coefficient β; CI, confidence interval.
μModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model). | Conclusion | The total protein intake of European adolescents exceeded the recommendations and animal proteins contribute most to the energy intake derived from total protein intake. Total and animal protein intake and E% derived from protein intake were higher in obese subjects. A negative association of total protein intake was found with BF%. GLM multivariate analysis indicates inverse associations, on one hand, between BMI z-score and plant protein intake, and on the other hand between BF% and animal and plant protein intakes. Both BMI z-score and BF% were positively associated with animal protein (E%). In conclusion our findings suggest that plant protein intakes may play a role in preventing OB among European adolescents. Further longitudinal studies should be conducted to investigate these potential beneficial effects of plant protein intakes in the prevention of OB and related chronic diseases. | [
"Survey population",
"Dietary intake assessment",
"Anthropometric measurements",
"Blood samples",
"Physical activity",
"Statistical analysis",
"Total energy and total, animal and plant protein intakes",
"Associations between total, animal and plant protein intakes and cardio-metabolic indicators",
"Total energy and total, animal and plant protein intakes",
"Associations between total, animal and plant protein intakes and cardio-metabolic indicators",
"Strengths and limitations",
"Recommendations"
] | [
"The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.\nMale and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere\n[20, 21].\nThe study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves\n[22].",
"Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.\nHELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)\n[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C\n[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions\n[24, 25].\nDietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)\n[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL\n[27], the Dutch NEVO\n[28] and the USDA\n[29] food composition databases which used the Kjeldahl method for analysing protein\n[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).\nUnder-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96\n[31].",
"Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents\n[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method\n[32]. The cut-off of BMI z-score\n[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate\n[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations\n[35]. More details about the anthropometric measurements are given in a previous manuscript\n[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages\n[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).",
"Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere\n[37].",
"Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously\n[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.",
"Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes\n[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.\nGLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.\nAll statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.",
"Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)\n[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))\n[41] (Table \n2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\nCharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR\nμ\nPRI\nμ\nTotal180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\n\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\n\nEAR: estimated average requirement; PRI: population reference intake.\n\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\nMean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table \n3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\nCharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a\n1.8 (0.6)a\n58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab\n1.4 (0.4)ab\n59 (24)37 (11)16.2 (3.0)b\n6.2 (1.3) Obesity572476 (701)102 (33)abc\n1.2 (0.4)ab\n63 (27)38 (12)16.5 (3.1)b\n6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).\n\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\n\nSD, standard deviation.\n*Mean value was significantly different between males and females by Student T- test (P < 0.05).\n**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\n\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\n\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\n\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).",
"Figure \n1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table \n4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nAssociations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)\n\nSE, standard error of coefficient β; CI, confidence interval.\n\nμModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).",
"The contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)\n[42], but higher than adolescents in review studies of Western, Central and Eastern European countries\n[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)\n[46], Spanish males (male: 105 g/d, female: 86 g/d)\n[42], and Western European adolescents\n[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)\n[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).",
"Obese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources\n[44, 47] to total protein and lower from plant protein consumptions\n[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition\n[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass\n[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB\n[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females\n[50]. Serum leptin is proven to be related to BF%\n[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.\nEvidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein\n[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents\n[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption\n[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty\n[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB\n[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake\n[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.\nEvidence also shows that increasing protein intake results in improvement of serum lipids\n[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood\n[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents\n[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels\n[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS\n[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term\n[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.",
"This European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.\nThe current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.",
"Protein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
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] | [
"Introduction",
"Methods",
"Survey population",
"Dietary intake assessment",
"Anthropometric measurements",
"Blood samples",
"Physical activity",
"Statistical analysis",
"Results",
"Total energy and total, animal and plant protein intakes",
"Associations between total, animal and plant protein intakes and cardio-metabolic indicators",
"Discussion",
"Total energy and total, animal and plant protein intakes",
"Associations between total, animal and plant protein intakes and cardio-metabolic indicators",
"Strengths and limitations",
"Recommendations",
"Conclusion"
] | [
"The prevalence of overweight (OW) and obesity (OB) in adolescents, defined on the basis of body mass\n[1], has increased rapidly worldwide. In 2010, the estimated prevalence of OW and OB in European children and adolescents was approximately 38%, including 10% OB\n[2]. As a consequence of OB-related co-morbidities, over 20000 children suffer from type 2 diabetes and more than 400000 have impaired glucose levels\n[2]. Childhood OW and OB both influence long-term health and evidence suggest an association with coronary events and mortality later in life\n[3, 4].\nNutrition during the early years of life is a critical factor of OB in adolescence further impacting on adulthood OW and OB, and the consequences of chronic diseases\n[5, 6]. High protein intakes were reported to improve cardiovascular risk factors including abdominal OB, dyslipidemia, glucose intolerance, and hypertension in European children (5–18 y)\n[7]. Previous randomised trials\n[8, 9] suggest that a high-protein diet defined as ≥20% of total energy lowers the risk of OW and promotes weight maintenance among adolescents\n[10]. The association between dietary protein intake and adolescent OW and OB has mainly been investigated in relation to its increased thermic effect and satiety when compared to fats and carbohydrates\n[9, 11]. Others, however, have reported that higher protein content in the diet did not confer any benefit in the treatment of OB among children 9–18 y old\n[12].\nThe debate on protein sources is still ongoing, addressing the nutritional quality of dietary proteins based on their amino acids composition. The protein quality or biological value of proteins from animal sources is high, whereas most plant proteins lack one or more essential amino acids and are therefore considered as incomplete proteins. What some seem to be concerned with is that the majority of high-protein foods are significant sources of fat and/or sugar as well (such as meat and meat products, cheese, and dairy desserts), and should therefore be carefully selected. Hermanussen et al. reported a positive correlation between the energy contribution of animal proteins to the diet and the body mass index (BMI) in adolescents\n[13]. On the other hand, Bradlee et al. found no association between OB and meat consumption among adolescents\n[14], while, plant-based diets were inversely associated with normal BMI in children in Hermanussen’s study\n[13]. A Western dietary pattern high in animal sources is associated with an increased risk of metabolic syndrome (MetS)\n[15, 16], whereas diets high in fruits, vegetables and whole grains are associated with a decreased risk\n[17]. Evidence showed that plant protein, soy in particular, can bind phytoestrogen compounds to stimulate lipid metabolism resulting in a better blood profile, by lowering total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and reducing insulin resistance\n[18, 19].\nThe aim of the current study was to evaluate total, animal and plant protein intakes in European adolescents and to investigate their association with cardio-metabolic indicators (anthropometry: BMI z-score and body fat percentage (BF%); and biomarkers: TC, TG, LDL-C, very LDL-C (VLDL-C), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP), glucose, insulin and leptin).",
" Survey population The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.\nMale and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere\n[20, 21].\nThe study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves\n[22].\nThe Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.\nMale and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere\n[20, 21].\nThe study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves\n[22].\n Dietary intake assessment Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.\nHELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)\n[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C\n[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions\n[24, 25].\nDietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)\n[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL\n[27], the Dutch NEVO\n[28] and the USDA\n[29] food composition databases which used the Kjeldahl method for analysing protein\n[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).\nUnder-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96\n[31].\nTwo non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.\nHELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)\n[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C\n[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions\n[24, 25].\nDietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)\n[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL\n[27], the Dutch NEVO\n[28] and the USDA\n[29] food composition databases which used the Kjeldahl method for analysing protein\n[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).\nUnder-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96\n[31].\n Anthropometric measurements Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents\n[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method\n[32]. The cut-off of BMI z-score\n[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate\n[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations\n[35]. More details about the anthropometric measurements are given in a previous manuscript\n[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages\n[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).\nWeight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents\n[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method\n[32]. The cut-off of BMI z-score\n[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate\n[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations\n[35]. More details about the anthropometric measurements are given in a previous manuscript\n[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages\n[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).\n Blood samples Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere\n[37].\nBlood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere\n[37].\n Physical activity Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously\n[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.\nPhysical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously\n[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.\n Statistical analysis Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes\n[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.\nGLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.\nAll statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.\nDescriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes\n[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.\nGLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.\nAll statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.",
"The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.\nMale and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere\n[20, 21].\nThe study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves\n[22].",
"Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.\nHELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)\n[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C\n[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions\n[24, 25].\nDietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)\n[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL\n[27], the Dutch NEVO\n[28] and the USDA\n[29] food composition databases which used the Kjeldahl method for analysing protein\n[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).\nUnder-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96\n[31].",
"Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents\n[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method\n[32]. The cut-off of BMI z-score\n[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate\n[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations\n[35]. More details about the anthropometric measurements are given in a previous manuscript\n[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages\n[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).",
"Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere\n[37].",
"Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously\n[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.",
"Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes\n[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.\nGLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.\nAll statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.",
"A total of 1804 out of 3528 adolescents (47% males) from 8 centres with valid and complete dietary data and measurements of weight and height were included in the analysis (Table \n1). 74% participants were classified in tanner stage 2–4, including 7% in tanner 2, 24% in tanner 3 and 41% in tanner 4. In total 279 adolescents were classified as OW and OB. Mean BMI z-score for both genders was in the NW range. Females had higher BF %, but lower BMI z-score compared to males. Furthermore, higher serum lipid profiles and leptin levels were found in females.Table 1\nAnthropometric characteristics and levels of obesity-related biomarkers in adolescents participating in the HELENA-CSS\nTotalMalesFemalesTotal participants (n)1804855949Age (y) (mean (range))14.7 (12.5-17.4)14.8 (12.5-17.4)14.7 (12.5-17.4) 12.5-14.9 y (n)1032481551 15.0-17.5 y (n)772374398Tanner Stage (n = 1752)n (%) Tanner 19 (0.514)9 (1.1)0 (0.0) Tanner 2-41294 (73.9)614 (74.2)680 (73.5) Tanner 5449 (25.6)204 (24.7)245 (26.5)Weight status (n = 1804)μ\n Underweight142 (7.9)58 (6.8)84 (8.9) Normal weight1383 (76.7)649 (75.9)734 (77.3) Overweight222 (12.3)114 (13.3)108 (11.4) Obesity57 (3.2)34 (4.0)23 (2.4)Mean (SD)Anthropometry BMI z-score (n = 1804)0.270 (1.1)0.358 (1.1)0.190 (1.0) BF% (n = 1764)22.0 (8.6)18.4 (9.1)25.1 (6.8)Biomarkers TC (mg/dL) (n = 552)159.1 (27)151.9 (24.9)165.8 (27.1) TG (mg/dL) (n = 552)67.6 (31.1)64.5 (31.5)70.5 (30.5) LDL-C (mg/dL) (n = 552)92.6 (24.2)89.0 (23.2)96.0 (24.7) VLDL-C (mg/dL) (n = 552)13.5 (6.2)12.9 (6.3)14.1 (6.1) HDL-C (mg/dL) (n = 552)55.6 (10.3)53.3 (9.3)57.8 (10.7) CRP (mg/L) (n = 524)1.2 (4.0)1.5 (5.5)0.841 (1.3) Glucose (mg/dL) (n = 552)90.1 (7.0)91.9 (7.2)88.5 (6.4) Insulin (μlU/mL) (n = 545)9.5 (6.0)9.0 (6.6)10.0 (6.6) Leptin (ng/mL) (n = 518)18.5 (21.9)8.1 (12.9)27.5 (24.1)SD, standard deviation; BMI, body mass index; BF%, body fat percentage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein- cholesterol; VLDL-C, very low-density lipoprotein- cholesterol; HDL-C, high-density lipoprotein- cholesterol; CRP, c-reactive protein.\nμBMI categories is classified based on the International Obesity Task Force cut-offs, underweight: <18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25.0-29.9 kg/m2, obesity: ≥30.0 kg/m2.\n\nAnthropometric characteristics and levels of obesity-related biomarkers in adolescents participating in the HELENA-CSS\n\nSD, standard deviation; BMI, body mass index; BF%, body fat percentage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein- cholesterol; VLDL-C, very low-density lipoprotein- cholesterol; HDL-C, high-density lipoprotein- cholesterol; CRP, c-reactive protein.\n\nμBMI categories is classified based on the International Obesity Task Force cut-offs, underweight: <18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25.0-29.9 kg/m2, obesity: ≥30.0 kg/m2.\n Total energy and total, animal and plant protein intakes Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)\n[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))\n[41] (Table \n2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\nCharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR\nμ\nPRI\nμ\nTotal180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\n\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\n\nEAR: estimated average requirement; PRI: population reference intake.\n\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\nMean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table \n3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\nCharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a\n1.8 (0.6)a\n58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab\n1.4 (0.4)ab\n59 (24)37 (11)16.2 (3.0)b\n6.2 (1.3) Obesity572476 (701)102 (33)abc\n1.2 (0.4)ab\n63 (27)38 (12)16.5 (3.1)b\n6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).\n\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\n\nSD, standard deviation.\n*Mean value was significantly different between males and females by Student T- test (P < 0.05).\n**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\n\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\n\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\n\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).\nMedian total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)\n[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))\n[41] (Table \n2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\nCharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR\nμ\nPRI\nμ\nTotal180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\n\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\n\nEAR: estimated average requirement; PRI: population reference intake.\n\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\nMean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table \n3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\nCharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a\n1.8 (0.6)a\n58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab\n1.4 (0.4)ab\n59 (24)37 (11)16.2 (3.0)b\n6.2 (1.3) Obesity572476 (701)102 (33)abc\n1.2 (0.4)ab\n63 (27)38 (12)16.5 (3.1)b\n6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).\n\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\n\nSD, standard deviation.\n*Mean value was significantly different between males and females by Student T- test (P < 0.05).\n**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\n\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\n\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\n\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).\n Associations between total, animal and plant protein intakes and cardio-metabolic indicators Figure \n1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table \n4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nAssociations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)\n\nSE, standard error of coefficient β; CI, confidence interval.\n\nμModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).\nFigure \n1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table \n4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nAssociations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)\n\nSE, standard error of coefficient β; CI, confidence interval.\n\nμModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).",
"Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)\n[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))\n[41] (Table \n2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\nCharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR\nμ\nPRI\nμ\nTotal180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\n\nPercentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents\n\nEAR: estimated average requirement; PRI: population reference intake.\n\nμEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).\nMean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table \n3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\nCharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a\n1.8 (0.6)a\n58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab\n1.4 (0.4)ab\n59 (24)37 (11)16.2 (3.0)b\n6.2 (1.3) Obesity572476 (701)102 (33)abc\n1.2 (0.4)ab\n63 (27)38 (12)16.5 (3.1)b\n6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).\n\nEstimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category\n\nSD, standard deviation.\n*Mean value was significantly different between males and females by Student T- test (P < 0.05).\n**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).\n\naMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.\n\nbMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).\n\ncMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).",
"Figure \n1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table \n4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nTertiles\nμ\nof total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).\nμTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.\n\nAssociations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)\n\nSE, standard error of coefficient β; CI, confidence interval.\n\nμModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).",
"The HELENA study is the first large-scale European adolescent population-based dietary survey of 8 European countries providing data on the nutritional intake, status, main determinants of food choices and preferences among European adolescents. The current study is the first to provide information on intakes of total, animal and plant proteins and their associations with OB and cardio-metabolic indicators.\n Total energy and total, animal and plant protein intakes The contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)\n[42], but higher than adolescents in review studies of Western, Central and Eastern European countries\n[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)\n[46], Spanish males (male: 105 g/d, female: 86 g/d)\n[42], and Western European adolescents\n[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)\n[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).\nThe contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)\n[42], but higher than adolescents in review studies of Western, Central and Eastern European countries\n[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)\n[46], Spanish males (male: 105 g/d, female: 86 g/d)\n[42], and Western European adolescents\n[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)\n[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).\n Associations between total, animal and plant protein intakes and cardio-metabolic indicators Obese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources\n[44, 47] to total protein and lower from plant protein consumptions\n[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition\n[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass\n[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB\n[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females\n[50]. Serum leptin is proven to be related to BF%\n[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.\nEvidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein\n[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents\n[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption\n[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty\n[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB\n[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake\n[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.\nEvidence also shows that increasing protein intake results in improvement of serum lipids\n[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood\n[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents\n[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels\n[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS\n[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term\n[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.\nObese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources\n[44, 47] to total protein and lower from plant protein consumptions\n[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition\n[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass\n[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB\n[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females\n[50]. Serum leptin is proven to be related to BF%\n[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.\nEvidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein\n[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents\n[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption\n[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty\n[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB\n[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake\n[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.\nEvidence also shows that increasing protein intake results in improvement of serum lipids\n[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood\n[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents\n[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels\n[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS\n[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term\n[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.\n Strengths and limitations This European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.\nThe current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.\nThis European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.\nThe current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.\n Recommendations Protein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables.\nProtein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables.",
"The contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)\n[42], but higher than adolescents in review studies of Western, Central and Eastern European countries\n[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)\n[46], Spanish males (male: 105 g/d, female: 86 g/d)\n[42], and Western European adolescents\n[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)\n[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).",
"Obese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources\n[44, 47] to total protein and lower from plant protein consumptions\n[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition\n[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass\n[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB\n[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females\n[50]. Serum leptin is proven to be related to BF%\n[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.\nEvidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein\n[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents\n[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption\n[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty\n[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB\n[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake\n[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.\nEvidence also shows that increasing protein intake results in improvement of serum lipids\n[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood\n[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents\n[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels\n[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS\n[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term\n[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.",
"This European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.\nThe current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.",
"Protein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables.",
"The total protein intake of European adolescents exceeded the recommendations and animal proteins contribute most to the energy intake derived from total protein intake. Total and animal protein intake and E% derived from protein intake were higher in obese subjects. A negative association of total protein intake was found with BF%. GLM multivariate analysis indicates inverse associations, on one hand, between BMI z-score and plant protein intake, and on the other hand between BF% and animal and plant protein intakes. Both BMI z-score and BF% were positively associated with animal protein (E%). In conclusion our findings suggest that plant protein intakes may play a role in preventing OB among European adolescents. Further longitudinal studies should be conducted to investigate these potential beneficial effects of plant protein intakes in the prevention of OB and related chronic diseases."
] | [
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"methods",
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"results",
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"discussion",
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] | [
"Protein intake",
"Adolescence",
"Body composition",
"Biomarkers",
"HELENA study"
] | Introduction:
The prevalence of overweight (OW) and obesity (OB) in adolescents, defined on the basis of body mass
[1], has increased rapidly worldwide. In 2010, the estimated prevalence of OW and OB in European children and adolescents was approximately 38%, including 10% OB
[2]. As a consequence of OB-related co-morbidities, over 20000 children suffer from type 2 diabetes and more than 400000 have impaired glucose levels
[2]. Childhood OW and OB both influence long-term health and evidence suggest an association with coronary events and mortality later in life
[3, 4].
Nutrition during the early years of life is a critical factor of OB in adolescence further impacting on adulthood OW and OB, and the consequences of chronic diseases
[5, 6]. High protein intakes were reported to improve cardiovascular risk factors including abdominal OB, dyslipidemia, glucose intolerance, and hypertension in European children (5–18 y)
[7]. Previous randomised trials
[8, 9] suggest that a high-protein diet defined as ≥20% of total energy lowers the risk of OW and promotes weight maintenance among adolescents
[10]. The association between dietary protein intake and adolescent OW and OB has mainly been investigated in relation to its increased thermic effect and satiety when compared to fats and carbohydrates
[9, 11]. Others, however, have reported that higher protein content in the diet did not confer any benefit in the treatment of OB among children 9–18 y old
[12].
The debate on protein sources is still ongoing, addressing the nutritional quality of dietary proteins based on their amino acids composition. The protein quality or biological value of proteins from animal sources is high, whereas most plant proteins lack one or more essential amino acids and are therefore considered as incomplete proteins. What some seem to be concerned with is that the majority of high-protein foods are significant sources of fat and/or sugar as well (such as meat and meat products, cheese, and dairy desserts), and should therefore be carefully selected. Hermanussen et al. reported a positive correlation between the energy contribution of animal proteins to the diet and the body mass index (BMI) in adolescents
[13]. On the other hand, Bradlee et al. found no association between OB and meat consumption among adolescents
[14], while, plant-based diets were inversely associated with normal BMI in children in Hermanussen’s study
[13]. A Western dietary pattern high in animal sources is associated with an increased risk of metabolic syndrome (MetS)
[15, 16], whereas diets high in fruits, vegetables and whole grains are associated with a decreased risk
[17]. Evidence showed that plant protein, soy in particular, can bind phytoestrogen compounds to stimulate lipid metabolism resulting in a better blood profile, by lowering total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and reducing insulin resistance
[18, 19].
The aim of the current study was to evaluate total, animal and plant protein intakes in European adolescents and to investigate their association with cardio-metabolic indicators (anthropometry: BMI z-score and body fat percentage (BF%); and biomarkers: TC, TG, LDL-C, very LDL-C (VLDL-C), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP), glucose, insulin and leptin).
Methods:
Survey population The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.
Male and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere
[20, 21].
The study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves
[22].
The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.
Male and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere
[20, 21].
The study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves
[22].
Dietary intake assessment Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.
HELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)
[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C
[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions
[24, 25].
Dietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)
[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL
[27], the Dutch NEVO
[28] and the USDA
[29] food composition databases which used the Kjeldahl method for analysing protein
[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).
Under-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96
[31].
Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.
HELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)
[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C
[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions
[24, 25].
Dietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)
[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL
[27], the Dutch NEVO
[28] and the USDA
[29] food composition databases which used the Kjeldahl method for analysing protein
[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).
Under-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96
[31].
Anthropometric measurements Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents
[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method
[32]. The cut-off of BMI z-score
[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate
[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations
[35]. More details about the anthropometric measurements are given in a previous manuscript
[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages
[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).
Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents
[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method
[32]. The cut-off of BMI z-score
[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate
[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations
[35]. More details about the anthropometric measurements are given in a previous manuscript
[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages
[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).
Blood samples Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere
[37].
Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere
[37].
Physical activity Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously
[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.
Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously
[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.
Statistical analysis Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes
[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.
GLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.
All statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.
Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes
[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.
GLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.
All statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.
Survey population:
The Healthy Lifestyle in Europe by Nutrition in Adolescence-Cross Sectional Study (HELENA-CSS) is a European Commission funded project on lifestyle and nutrition among adolescents from 10 cities of European countries: Stockholm, Athens, Heraklion, Rome, Zaragoza, Ghent, Lille, Dortmund,Vienna, and Pecs that ran between October 2006 and December 2007. Due to logistical reasons, adolescents from Heraklion and Pecs were excluded for the dietary intake assessments. A multi-stage random cluster sampling procedure was used to select 3528 adolescents, stratified by geographical location, age and socioeconomic status (SES). Schools were randomly selected after stratification to guarantee diversity of the sample in culture and SES.
Male and female adolescents, aged 12.5-17.5 y, not participating simultaneously in a clinical trial, free of any acute infection lasting less than 1 week before inclusion year, and who provided two 24-h recall interviews with valid information and complete anthropometric measurements, were included in the final analysis of the current study. Details on sampling procedures, study design and non-respondents have been reported elsewhere
[20, 21].
The study was approved by the Research Ethics Committees of each city involved. Written informed consent was obtained from the adolescents’ parents and the adolescents themselves
[22].
Dietary intake assessment:
Two non-consecutive computerised 24-h dietary recalls (HELENA-DIAT), instructed by dieticians/researchers, were used to collect food consumption data. During interviews, adolescents were allowed to ask questions and following completion the recall was checked for completeness. Each participant was asked to complete the recall twice in a time-span of 2 weeks during the school time.
HELENA-DIAT is a self-administered computer program based on the Young Adolescents’ Nutrition Assessment on Computer (YANA-C)
[23], consisting of a single computerised 24-h recall with a structured program based on six meal occasions. The validated YANA-C
[23], was designed to obtain a detailed description and quantification of foods consumed, and eventually included about 800 food items hierarchically organized in 25 food groups, and about 300 colored photograph sets of foods in different portions
[24, 25].
Dietary intakes were linked to the German Food Code and Nutrient DataBase (BLS (Bundeslebensmittelschlüssel), version II.3.1, 2011)
[26]. However, the estimated percentage of animal and plant protein intakes were calculated by linking the 24-h recall food consumption data to the Belgian NUBEL
[27], the Dutch NEVO
[28] and the USDA
[29] food composition databases which used the Kjeldahl method for analysing protein
[30], because no differentiation was made between plant and animal proteins in the BLS database. Protein intakes were calculated in absolute terms (g/d) and relative terms (energy percentages (E%); per kg body weight).
Under-reporters, excluded in the current study, were considered as individuals with a ratio of energy intake over estimated basal metabolic rate lower than 0.96
[31].
Anthropometric measurements:
Weight (kg) and height (m) were measured in underwear and barefoot to the nearest 0.1 kg and 0.1 cm, respectively, by trained researchers. BMI was calculated as weight (kg)/height (m2). Participants were classified into four BMI categories according to the International Obesity Task Force (IOTF) cut-offs for adolescents
[1]: equivalent to underweight (UW) (<18.5 kg/m2), normal weight (NW) (18.5-24.9 kg/m2), OW (25.0-29.9 kg/m2), and OB (≥30.0 kg/m2). Standard deviation score of BMI (BMI z-score) was calculated using the lmsGrowth method
[32]. The cut-off of BMI z-score
[33]: UW (<-2), NW (-2 -1), OW (>1) and OB (>2). Skinfold thickness was measured to the nearest 0.2 mm in triplicate
[34]. The same trained investigators made all measurements (inter-rater reliability >95 %). BF% was calculated using Slaughter’s equations
[35]. More details about the anthropometric measurements are given in a previous manuscript
[34]. Physical maturations were examined by a physician during a medical examination to determine the pubertal status based on Tanner stages
[36]. The final physical maturations were classified into three categories: pre-pubertal (stage 1); pubertal (stage 2 to 4) and post-pubertal (stage 5).
Blood samples:
Blood samples were collected in a randomly selected subsample of the total HELENA-CSS. Adolescents who agreed to be involved in the blood sampling were asked to fast after 8 pm on the previous day. Fasting blood samples, information of adolescents’ medical history and recent acute diseases were collected by venipuncture between 8–10 a.m. at schools or hospitals by a medical doctor, A blood sampling questionnaire was completed by the participants for the purposes of assessing fasting status, acute infection, allergies, smoking, vitamin and mineral supplements, and medication. A specific handling, transport and traceability system for biological samples was developed for the HELENA study. All samples were analyzed centrally. The blood sampling procedure has been described elsewhere
[37].
Physical activity:
Physical activity (PA) was assessed for 7 days by an uniaxial accelerometer (Actigraph GT1M), described previously
[38]. At least 3 days of recording with a minimum of 8 hours’ registration per day was set as an inclusion criterion. PA, used in the current study, was categorized in the following categories: at least 1 hour of PA per day, no PA or less than 1 hour of PA per day.
Statistical analysis:
Descriptive data is presented as means with standard deviation or frequency distributions. Energy and total, animal and plant protein intakes were corrected for within-person variation using the Multiple Source Method (MSM), which is suitable for estimating population’s usual intakes
[39]. Statistical differences for total energy and total, animal and plant protein intakes between subgroups (gender and age) were assessed using the Student T-test and ANOVA.
GLM multivariate analysis was used to investigate the associations of indicators (dependent variables) with animal and plant protein intakes, and animal (E%) and plant (E%) energy percentages (independent variables) through three models (stepwise approach): (1) model 1 = unadjusted model; (2) model 2 = model 1 + adjusted for fat intake; (3) model 3 = model 2 + further adjusted for PA, confounding factors and interactions, and controlling for the country clustering effect. Potential confounding factors including age (younger group (12.5-14.9 y) and older group (15.0-17.5 y)), gender, tanner stage (pre-puberty, puberty and post-puberty) and two-way interactions between potential confounding factors and independent variables were included in the model 3. Anthropometry and serum biomarkers were investigated separately. In addition, animal and plant protein intakes, and the energy percentage (E%) from animal and plant protein were examined in a separate model due to colinearity.
All statistical analysis were performed using the statistical software SPSS for Windows version 18 (SPSS Inc, Chicago, IL, USA). Results were considered statistically significant at α two-tailed level of 0.05.
Results:
A total of 1804 out of 3528 adolescents (47% males) from 8 centres with valid and complete dietary data and measurements of weight and height were included in the analysis (Table
1). 74% participants were classified in tanner stage 2–4, including 7% in tanner 2, 24% in tanner 3 and 41% in tanner 4. In total 279 adolescents were classified as OW and OB. Mean BMI z-score for both genders was in the NW range. Females had higher BF %, but lower BMI z-score compared to males. Furthermore, higher serum lipid profiles and leptin levels were found in females.Table 1
Anthropometric characteristics and levels of obesity-related biomarkers in adolescents participating in the HELENA-CSS
TotalMalesFemalesTotal participants (n)1804855949Age (y) (mean (range))14.7 (12.5-17.4)14.8 (12.5-17.4)14.7 (12.5-17.4) 12.5-14.9 y (n)1032481551 15.0-17.5 y (n)772374398Tanner Stage (n = 1752)n (%) Tanner 19 (0.514)9 (1.1)0 (0.0) Tanner 2-41294 (73.9)614 (74.2)680 (73.5) Tanner 5449 (25.6)204 (24.7)245 (26.5)Weight status (n = 1804)μ
Underweight142 (7.9)58 (6.8)84 (8.9) Normal weight1383 (76.7)649 (75.9)734 (77.3) Overweight222 (12.3)114 (13.3)108 (11.4) Obesity57 (3.2)34 (4.0)23 (2.4)Mean (SD)Anthropometry BMI z-score (n = 1804)0.270 (1.1)0.358 (1.1)0.190 (1.0) BF% (n = 1764)22.0 (8.6)18.4 (9.1)25.1 (6.8)Biomarkers TC (mg/dL) (n = 552)159.1 (27)151.9 (24.9)165.8 (27.1) TG (mg/dL) (n = 552)67.6 (31.1)64.5 (31.5)70.5 (30.5) LDL-C (mg/dL) (n = 552)92.6 (24.2)89.0 (23.2)96.0 (24.7) VLDL-C (mg/dL) (n = 552)13.5 (6.2)12.9 (6.3)14.1 (6.1) HDL-C (mg/dL) (n = 552)55.6 (10.3)53.3 (9.3)57.8 (10.7) CRP (mg/L) (n = 524)1.2 (4.0)1.5 (5.5)0.841 (1.3) Glucose (mg/dL) (n = 552)90.1 (7.0)91.9 (7.2)88.5 (6.4) Insulin (μlU/mL) (n = 545)9.5 (6.0)9.0 (6.6)10.0 (6.6) Leptin (ng/mL) (n = 518)18.5 (21.9)8.1 (12.9)27.5 (24.1)SD, standard deviation; BMI, body mass index; BF%, body fat percentage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein- cholesterol; VLDL-C, very low-density lipoprotein- cholesterol; HDL-C, high-density lipoprotein- cholesterol; CRP, c-reactive protein.
μBMI categories is classified based on the International Obesity Task Force cut-offs, underweight: <18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25.0-29.9 kg/m2, obesity: ≥30.0 kg/m2.
Anthropometric characteristics and levels of obesity-related biomarkers in adolescents participating in the HELENA-CSS
SD, standard deviation; BMI, body mass index; BF%, body fat percentage; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein- cholesterol; VLDL-C, very low-density lipoprotein- cholesterol; HDL-C, high-density lipoprotein- cholesterol; CRP, c-reactive protein.
μBMI categories is classified based on the International Obesity Task Force cut-offs, underweight: <18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25.0-29.9 kg/m2, obesity: ≥30.0 kg/m2.
Total energy and total, animal and plant protein intakes Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)
[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))
[41] (Table
2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
CharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR
μ
PRI
μ
Total180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Mean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table
3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
CharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a
1.8 (0.6)a
58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab
1.4 (0.4)ab
59 (24)37 (11)16.2 (3.0)b
6.2 (1.3) Obesity572476 (701)102 (33)abc
1.2 (0.4)ab
63 (27)38 (12)16.5 (3.1)b
6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
SD, standard deviation.
*Mean value was significantly different between males and females by Student T- test (P < 0.05).
**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)
[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))
[41] (Table
2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
CharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR
μ
PRI
μ
Total180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Mean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table
3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
CharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a
1.8 (0.6)a
58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab
1.4 (0.4)ab
59 (24)37 (11)16.2 (3.0)b
6.2 (1.3) Obesity572476 (701)102 (33)abc
1.2 (0.4)ab
63 (27)38 (12)16.5 (3.1)b
6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
SD, standard deviation.
*Mean value was significantly different between males and females by Student T- test (P < 0.05).
**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Associations between total, animal and plant protein intakes and cardio-metabolic indicators Figure
1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table
4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Associations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)
SE, standard error of coefficient β; CI, confidence interval.
μModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).
Figure
1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table
4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Associations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)
SE, standard error of coefficient β; CI, confidence interval.
μModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).
Total energy and total, animal and plant protein intakes:
Median total protein contributing to energy intake was 15.5%. Average total protein intakes exceeded the World Health Organization (WHO) recommendations (10.0 – 15.0% of the total energy intake)
[40] and the estimated average requirements (EAR) and population reference intake (PRI) of the European Food Safety Authority (EFSA) (EAR: 0.66 g/(kg.d) for both genders; PRI: males, 0.70-0.74 (g/(kg.d), and females, 0.67-0.72 g/(kg.d))
[41] (Table
2). All but one adolescent met the EAR, while, fourteen and two adolescents did not reach the WHO recommendations for protein intakes and the PRI, respectively.Table 2
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
CharacteristicsNTotal protein (g/d)Total protein (g/(kg.d))The number of subjects below the recommendations25%50%75%25%50%75%EAR
μ
PRI
μ
Total180476911091.31.62.012Gender Males855901061271.51.82.300 Females9496880941.21.51.812Age 12.5-14.9 y103274901081.41.72.111 15.0-17.5 y77277941121.31.61.901EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Percentile of total protein intakes and the number of the subjects below the recommendations of European food safety authority in the European adolescents
EAR: estimated average requirement; PRI: population reference intake.
μEAR: 0.66 g/(kg.d) for both genders; PRI : males, 0.70-0.74 g/(kg.d) and females, 0.67-0.72 g/(kg.d).
Mean total protein intake (384 kcal/d) contributed 15.8% to total energy intake. Mean animal protein intakes were the main contributor (59%) to total protein intakes, as opposed to mean plant protein (Table
3). Total and plant protein intakes were significantly lower in females and the younger group. Body weight adjusted total protein intakes and E% from total protein were significantly lower in the older group. Total energy, total and animal protein intakes and total protein (E%) were higher in obese adolescents than non-obese ones. More specifically, body weight adjusted total protein intake (g/(kg.d)) was significantly lower in OB, and higher in UW peers.Table 3
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
CharacteristicsNEnergy (kcal/d)Total protein (g/d)Total protein (g/(kg.d))Animal protein (g/d)Plant protein (g/d)Total proteinPlant protein% energy contributing to total energy intakeMean intake (SD)Total18042450 (637)96 (28)1.7 (0.6)58 (23)38 (13)15.8 (2.8)6.2 (1.3)Gender Males8552792 (655)110 (29)1.9 (0.6)66 (24)43 (13)15.9 (3.0)6.2 (1.3) Females9492141 (428)*83 (20)*1.6 (0.5)*50 (18)*33 (10)*15.6 (2.7)6.3 (1.3)Age 12.5-14.9 y10322358 (637)94 (28)1.8 (0.6)57 (22)37 (12)16.1 (2.9)6.2 (1.4) 15.0-17.5 y7722752 (713)**98 (29)**1.6 (0.5)**58 (23)39 (12)**15.4 (2.8)**6.2 (1.2)Weight status Underweight1422443 (631)94 (28)2.2 (0.7)56 (21)39 (12)15.5 (2.7)6.3 (1.2) Normal weight13832458 (635)96 (28)a
1.8 (0.6)a
58 (22)38 (13)15.7 (2.8)6.2 (1.3) Overweight2222397 (636)96 (29)ab
1.4 (0.4)ab
59 (24)37 (11)16.2 (3.0)b
6.2 (1.3) Obesity572476 (701)102 (33)abc
1.2 (0.4)ab
63 (27)38 (12)16.5 (3.1)b
6.2 (1.2)SD, standard deviation.*Mean value was significantly different between males and females by Student T- test (P < 0.05).**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Estimated means of energy, total, animal and plant protein intakes, and energy percentage of protein intakes of adolescents participating in the in HELENA-CSS stratified by gender, age, tanner and BMI category
SD, standard deviation.
*Mean value was significantly different between males and females by Student T- test (P < 0.05).
**Mean value was significantly different from the young group (12.5-14.9 y) by Student T- test (P < 0.05).
aMean value was significantly different from underweight by ANOVA, (P < 0.05, Bonferroni correction.
bMean value was significantly different from normal weight by ANOVA, (P < 0.05, Bonferroni correction).
cMean value was significantly different from overweight by ANOVA, (P < 0.05, Bonferroni correction).
Associations between total, animal and plant protein intakes and cardio-metabolic indicators:
Figure
1 shows a significant decline in BF% across the total protein tertiles (P < 0.001) by age. But no significance was observed in males and females. The results of the GLM multivariate analysis showed that crude BF% was inversely associated with absolute animal and plant protein in model 1, but crude BMI z-score and BF% were positively associated with animal protein (E%) (Table
4). Absolute animal protein intake was inversely associated with crude serum biomarkers including TC, TG, VLDL-C and leptin, but positively with serum fasting glucose. While absolute plant protein intake was inversely associated with crude TC, HDL-C, and leptin, but positively with serum fasting glucose. After adjustments for fat intake (Model 2), BMI z-score became positively associated with absolute animal protein intake, but several significant associations found in model 1 disappeared. Leptin kept to be inversely associated with absolute animal protein intake in model 2, and BF%, TC and HDL-C with absolute plant protein intake. Only serum HDL-C became positively associated with absolute animal protein intake, after further adjusting for confounding factors, PA and interaction factors (Model 3). Inverse associations were observed between BMI z-scores and BF%, and absolute plant protein intake. Whereas both BMI z-scores and BF% were positively associated with animal protein (E%). No biomarker was associated with percentage of energy intake derived from animal and plant protein (data not shown).Figure 1
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Tertiles
μ
of total protein intake (g/d) and anthropometric indicators in adolescents participating in HELENA-CSS (n = 1804).
μTertile 1 (T1): <81 g/d; tertile 2 (T2): 81 g/d to 103 g/d; tertile 3 (T3): ≥103 g/d.
Associations between dietary animal and plant protein intakes (g/d and E%) and body composition of adolescents participating in the HELENA-CSS (n = 1804)
SE, standard error of coefficient β; CI, confidence interval.
μModel 1, unadjusted; model 2, adjusted for fat intake; model 3, model 2 further adjusted for age, sex, tanner stage, physical activity, country cluster, and interactions between potential confounding factors and animal / plant protein (separate model).
Discussion:
The HELENA study is the first large-scale European adolescent population-based dietary survey of 8 European countries providing data on the nutritional intake, status, main determinants of food choices and preferences among European adolescents. The current study is the first to provide information on intakes of total, animal and plant proteins and their associations with OB and cardio-metabolic indicators.
Total energy and total, animal and plant protein intakes The contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)
[42], but higher than adolescents in review studies of Western, Central and Eastern European countries
[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)
[46], Spanish males (male: 105 g/d, female: 86 g/d)
[42], and Western European adolescents
[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)
[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).
The contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)
[42], but higher than adolescents in review studies of Western, Central and Eastern European countries
[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)
[46], Spanish males (male: 105 g/d, female: 86 g/d)
[42], and Western European adolescents
[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)
[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).
Associations between total, animal and plant protein intakes and cardio-metabolic indicators Obese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources
[44, 47] to total protein and lower from plant protein consumptions
[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition
[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass
[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB
[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females
[50]. Serum leptin is proven to be related to BF%
[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.
Evidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein
[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents
[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption
[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty
[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB
[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake
[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.
Evidence also shows that increasing protein intake results in improvement of serum lipids
[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood
[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents
[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels
[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS
[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term
[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.
Obese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources
[44, 47] to total protein and lower from plant protein consumptions
[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition
[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass
[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB
[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females
[50]. Serum leptin is proven to be related to BF%
[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.
Evidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein
[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents
[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption
[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty
[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB
[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake
[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.
Evidence also shows that increasing protein intake results in improvement of serum lipids
[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood
[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents
[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels
[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS
[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term
[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.
Strengths and limitations This European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.
The current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.
This European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.
The current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.
Recommendations Protein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables.
Protein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables.
Total energy and total, animal and plant protein intakes:
The contribution of protein to energy intake in our study was similar to that reported in Greek and Italian adolescents, lower than that of Spanish peers (male: 17.2%, female: 17.8%)
[42], but higher than adolescents in review studies of Western, Central and Eastern European countries
[43–45]. In addition, total protein intake was reported to be slightly lower in Italian peers (male: 99 g/d, female: 82 g/d)
[46], Spanish males (male: 105 g/d, female: 86 g/d)
[42], and Western European adolescents
[43, 45]. The adolescents in this study had much higher animal and plant protein intakes than those of Belgian peers (male: 52 g/d, female: 37 g/d; male: 30 g/d, female: 24 g/d, respectively)
[43] and higher plant protein intake (male: 30 g/d, female: 25 g/d), but lower animal protein intake than Spanish peers (male: 74 g/d, female: 60 g/d).
Associations between total, animal and plant protein intakes and cardio-metabolic indicators:
Obese HELENA participants consumed more total protein than non-obese participants. Evidence from other European studies indicate higher contribution of animal sources
[44, 47] to total protein and lower from plant protein consumptions
[45], which might point to a relationship between increasing prevalence of OB in European adolescents. Our results suggest that increasing total protein intakes may be inversely associated with adolescents’ BF%, which can be explained by plant protein intakes being significantly inversely associated with BMI z-score and BF%, after adjustment for fat intake, PA and confounding factors. Consistent with our findings, observed benefits of increasing total and plant protein intakes on body composition
[14, 48] could be attributed to the protein effect on increasing stimulated fat oxidation and building of lean body mass
[49]. Conversely, the results of a previous randomized trial on obese adolescents (11–16 y) demonstrated that increasing protein consumption conferred no benefit on weight loss and body composition in the treatment of adolescent OB
[12]. The different study design and target population might partly explain differences observed. Remarkably, the level of serum leptin was found to be extremely low among males in our study. High levels of leptin can easily be observed in female adolescents, because leptin was reported to play a critical role in the regulation of puberty, especially in females
[50]. Serum leptin is proven to be related to BF%
[51], and this might partly explain our finding on why females kept high BF% when increasing total protein intake, whereas BF% in males decreased gradually.
Evidence shows that plant protein from vegetables, fruits, and legumes not only improves body composition, but also results in lower body weight compared to animal protein
[13, 52]. In our study, although animal protein intake was found to be weakly inversely associated with BF%, animal protein (E%) was observed to be positively associated with BF%. Previous studies concluded that total and animal protein intakes might be responsible for increasing body weight and BMI in adolescents
[12, 13]. Mirkopoulou et al. suggested that extremely high protein intakes, animal protein in particular, might increase the risk of adolescents’ OB due to higher energy consumption
[53]. Furthermore, the results of a longitudinal study suggested that a high animal protein intake in mid-childhood might be associated with an earlier pubertal growth and spurt peak height velocity, whereas a higher plant protein intake could delay puberty
[54]. On the contrary, some studies disagreed the above hypothesis of increased intake of total and animal protein resulting in decreasing the risk of OW and OB
[55, 56] by affecting the appetite. A randomized 8-weeks parallel intervention trial suggested that seafood protein sources from cod and salmon were efficient to treat OB because of caloric restriction and lower saturated fatty acids intake
[55]. Therefore, the amount of total, animal and plant proteins in the diet may be a critical factor on prevention against OW and OB.
Evidence also shows that increasing protein intake results in improvement of serum lipids
[57]. Plant protein based diets in childhood could be responsible for lowering the risk of MetS and its consequence in the adulthood
[58]. In the current study, only serum HDL-C was found to be weakly positively associated with animal protein intake. The increases in HDL-C might possibly be explained by the inverse association of animal protein intake with BF%. Mirkopoulou et al. reported that no association with blood lipid profile was observed in Greek adolescents
[53], supporting most of our results, as similarities in the study design and target population might explain similarities in observations. Some cross-sectional studies showed that plant based diets were associated with more favourable lipid levels in adolescents by lowering TC and LDL-C, but increasing the HDL-C levels
[17, 59], whereas high intakes derived from animal sources were associated with an increased risk of MetS
[15]. However, it has to be considered that adolescence is a critical period with inevitable increases in energy and nutrient intakes to regulate hormone balances resulting in physical, behaviour and social development. Leptin is a protein hormone that has a key role in regulating energy intake and energy expenditure, including appetite in the longer term
[60, 61]. In the current study, no significance of serum leptin was found in model 3, but it was negatively associated with animal and plant protein intakes in model 1 and model 2, respectively. The status of statistical significance between serum leptin and plant protein intake changed in the model 2 compared to model 1 due to fat intake. In addition, fat intake can be a critical factor for the serum lipid profile and plant protein intake. No study has provided evidence on clear mechanisms, though it is possible that plant protein intake might stimulate serum leptin via homeostasis impacting on body weight and BF%. In addition, female, OW and obese adolescents in particular, during puberty might most likely underestimate energy and dietary intakes, which may bias the associations. Confounding factors, such as gender, age, Tanner stage and region, may account for some unexpected findings, serum biomarkers in particular.
Strengths and limitations:
This European nutrition survey is the first large-scale study among European adolescents that used a standardized approach accross 8 participating centers. Additionally, it is the first study evaluating total, animal and plant protein intakes in European adolescents stratified by gender and age, and investigating associations with anthropometry and serum biomarkers as studies with the same standardised methodology across European countries are limited.
The current study has also some limitations including the dietary assessment method used to assess diet that only included dietary information of two non-consecutive days. The 24-h dietary recall method does not allow quantifying proportions of non-consumers for particular food items, especially for those less frequently consumed. In order to decrease the influence of such limitation, nutrient intakes were corrected for within- person variability by applying the MSM method. Moreover, accuracy of collected data relies on the individual’s ability to remember foods and beverages consumed in the past 24 hours, and might, therefore, be biased towards misreporting. In this respect, the 24-h dietary recalls were performed through computer-assisted HELENA-DIAT software to standardize the recall procedures as much as possible. Food pictures, showing daily foods consumed by European adolescents, were used in order to facilitate the participants to recall the potion size of the foods consumed in the previous days, which assisted participants and interviewers in accurately assessing the consumed amounts. The same food composition table for conversion of food intake data to estimated nutrient intakes was used for all survey centres. In this way, differences in definitions, analytical methods, units and modes of expression were overcome. However, missing foods of protein contents in the BLS table were calculated via recipes or taken from local food composition tables. In addition, the small sample size of serum biomarkers may also be a potential influencing factor leading to weak linear relationship between animal and plant protein intakes and serum biomarkers. Furthermore, the cross-sectional study design of this study cannot assess causality between health outcomes and dietary intakes.
Recommendations:
Protein is critical for the development of bone and muscle mass, and health in adolescents. An increased protein intake is one of the most common approaches to the dietary management of OB and related chronic diseases. However, extra high protein intake can result in side-effects due to imbalance in energy intake and food consumption. The findings of current study indicate that plant protein had more protective effect against OB compared to animal protein, although HDL-C was found to be weakly positively associated with absolute animal protein intake. We noticed that participants exceeded protein intake based on WHO requirement, and almost 2/3 sources were from animal origin rather than from plants, which may influence body weight and body composition. The findings of our study highlight that future public health policies and school policies need to be developed and implemented to help establishing healthy food preferences, and adjusting food concepts and dietary behaviors in adolescents. Possible prevention strategies could include the development of multicomponent school-based interventions combining education and environmental changes towards increased intakes of plant proteins from legumes and vegetables.
Conclusion:
The total protein intake of European adolescents exceeded the recommendations and animal proteins contribute most to the energy intake derived from total protein intake. Total and animal protein intake and E% derived from protein intake were higher in obese subjects. A negative association of total protein intake was found with BF%. GLM multivariate analysis indicates inverse associations, on one hand, between BMI z-score and plant protein intake, and on the other hand between BF% and animal and plant protein intakes. Both BMI z-score and BF% were positively associated with animal protein (E%). In conclusion our findings suggest that plant protein intakes may play a role in preventing OB among European adolescents. Further longitudinal studies should be conducted to investigate these potential beneficial effects of plant protein intakes in the prevention of OB and related chronic diseases.
| Background:
Previous studies suggest that dietary protein might play a beneficial role in combating obesity and its related chronic diseases. Total, animal and plant protein intakes and their associations with anthropometry and serum biomarkers in European adolescents using one standardised methodology across European countries are not well documented.
Methods:
The current analysis included 1804 randomly selected adolescents participating in the HELENA study (conducted in 2006-2007) aged 12.5-17.5 y (47% males) who completed two non-consecutive computerised 24-h dietary recalls. Associations between animal and plant protein intakes, and anthropometry and serum biomarkers were examined with General linear Model multivariate analysis.
Results:
Average total protein intake exceeded the recommendations of World Health Organization and European Food Safety Authority. Mean total protein intake was 96 g/d (59% derived from animal protein). Total, animal and plant protein intakes (g/d) were significantly lower in females than in males and total and plant protein intakes were lower in younger participants (12.5-14.9 y). Protein intake was significantly lower in underweight subjects and higher in obese ones; the direction of the relationship was reversed after adjustments for body weight (g/(kg.d)). The inverse association of plant protein intakes was stronger with BMI z-score and body fat percentage (BF%) compared to animal protein intakes. Additionally, BMI and BF% were positively associated with energy percentage of animal protein.
Conclusions:
This sample of European adolescents appeared to have adequate total protein intake. Our findings suggest that plant protein intakes may play a role in preventing obesity among European adolescents. Further longitudinal studies are needed to investigate the potential beneficial effects observed in this study in the prevention of obesity and related chronic diseases. | Introduction:
The prevalence of overweight (OW) and obesity (OB) in adolescents, defined on the basis of body mass
[1], has increased rapidly worldwide. In 2010, the estimated prevalence of OW and OB in European children and adolescents was approximately 38%, including 10% OB
[2]. As a consequence of OB-related co-morbidities, over 20000 children suffer from type 2 diabetes and more than 400000 have impaired glucose levels
[2]. Childhood OW and OB both influence long-term health and evidence suggest an association with coronary events and mortality later in life
[3, 4].
Nutrition during the early years of life is a critical factor of OB in adolescence further impacting on adulthood OW and OB, and the consequences of chronic diseases
[5, 6]. High protein intakes were reported to improve cardiovascular risk factors including abdominal OB, dyslipidemia, glucose intolerance, and hypertension in European children (5–18 y)
[7]. Previous randomised trials
[8, 9] suggest that a high-protein diet defined as ≥20% of total energy lowers the risk of OW and promotes weight maintenance among adolescents
[10]. The association between dietary protein intake and adolescent OW and OB has mainly been investigated in relation to its increased thermic effect and satiety when compared to fats and carbohydrates
[9, 11]. Others, however, have reported that higher protein content in the diet did not confer any benefit in the treatment of OB among children 9–18 y old
[12].
The debate on protein sources is still ongoing, addressing the nutritional quality of dietary proteins based on their amino acids composition. The protein quality or biological value of proteins from animal sources is high, whereas most plant proteins lack one or more essential amino acids and are therefore considered as incomplete proteins. What some seem to be concerned with is that the majority of high-protein foods are significant sources of fat and/or sugar as well (such as meat and meat products, cheese, and dairy desserts), and should therefore be carefully selected. Hermanussen et al. reported a positive correlation between the energy contribution of animal proteins to the diet and the body mass index (BMI) in adolescents
[13]. On the other hand, Bradlee et al. found no association between OB and meat consumption among adolescents
[14], while, plant-based diets were inversely associated with normal BMI in children in Hermanussen’s study
[13]. A Western dietary pattern high in animal sources is associated with an increased risk of metabolic syndrome (MetS)
[15, 16], whereas diets high in fruits, vegetables and whole grains are associated with a decreased risk
[17]. Evidence showed that plant protein, soy in particular, can bind phytoestrogen compounds to stimulate lipid metabolism resulting in a better blood profile, by lowering total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and reducing insulin resistance
[18, 19].
The aim of the current study was to evaluate total, animal and plant protein intakes in European adolescents and to investigate their association with cardio-metabolic indicators (anthropometry: BMI z-score and body fat percentage (BF%); and biomarkers: TC, TG, LDL-C, very LDL-C (VLDL-C), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP), glucose, insulin and leptin).
Conclusion:
The total protein intake of European adolescents exceeded the recommendations and animal proteins contribute most to the energy intake derived from total protein intake. Total and animal protein intake and E% derived from protein intake were higher in obese subjects. A negative association of total protein intake was found with BF%. GLM multivariate analysis indicates inverse associations, on one hand, between BMI z-score and plant protein intake, and on the other hand between BF% and animal and plant protein intakes. Both BMI z-score and BF% were positively associated with animal protein (E%). In conclusion our findings suggest that plant protein intakes may play a role in preventing OB among European adolescents. Further longitudinal studies should be conducted to investigate these potential beneficial effects of plant protein intakes in the prevention of OB and related chronic diseases. | Background:
Previous studies suggest that dietary protein might play a beneficial role in combating obesity and its related chronic diseases. Total, animal and plant protein intakes and their associations with anthropometry and serum biomarkers in European adolescents using one standardised methodology across European countries are not well documented.
Methods:
The current analysis included 1804 randomly selected adolescents participating in the HELENA study (conducted in 2006-2007) aged 12.5-17.5 y (47% males) who completed two non-consecutive computerised 24-h dietary recalls. Associations between animal and plant protein intakes, and anthropometry and serum biomarkers were examined with General linear Model multivariate analysis.
Results:
Average total protein intake exceeded the recommendations of World Health Organization and European Food Safety Authority. Mean total protein intake was 96 g/d (59% derived from animal protein). Total, animal and plant protein intakes (g/d) were significantly lower in females than in males and total and plant protein intakes were lower in younger participants (12.5-14.9 y). Protein intake was significantly lower in underweight subjects and higher in obese ones; the direction of the relationship was reversed after adjustments for body weight (g/(kg.d)). The inverse association of plant protein intakes was stronger with BMI z-score and body fat percentage (BF%) compared to animal protein intakes. Additionally, BMI and BF% were positively associated with energy percentage of animal protein.
Conclusions:
This sample of European adolescents appeared to have adequate total protein intake. Our findings suggest that plant protein intakes may play a role in preventing obesity among European adolescents. Further longitudinal studies are needed to investigate the potential beneficial effects observed in this study in the prevention of obesity and related chronic diseases. | 16,155 | 333 | [
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] | [CONTENT] Protein intake | Adolescence | Body composition | Biomarkers | HELENA study [SUMMARY] | [CONTENT] Protein intake | Adolescence | Body composition | Biomarkers | HELENA study [SUMMARY] | [CONTENT] Protein intake | Adolescence | Body composition | Biomarkers | HELENA study [SUMMARY] | [CONTENT] Protein intake | Adolescence | Body composition | Biomarkers | HELENA study [SUMMARY] | [CONTENT] Protein intake | Adolescence | Body composition | Biomarkers | HELENA study [SUMMARY] | [CONTENT] Protein intake | Adolescence | Body composition | Biomarkers | HELENA study [SUMMARY] | [CONTENT] Adolescent | Age Factors | Animals | Anthropometry | Body Composition | Body Mass Index | Child | Cross-Sectional Studies | Diet | Diet Records | Dietary Proteins | Energy Intake | Europe | Exercise | Female | Humans | Lipids | Male | Meat | Nutrition Assessment | Obesity | Plant Proteins | Sex Factors | Thinness [SUMMARY] | [CONTENT] Adolescent | Age Factors | Animals | Anthropometry | Body Composition | Body Mass Index | Child | Cross-Sectional Studies | Diet | Diet Records | Dietary Proteins | Energy Intake | Europe | Exercise | Female | Humans | Lipids | Male | Meat | Nutrition Assessment | Obesity | Plant Proteins | Sex Factors | Thinness [SUMMARY] | [CONTENT] Adolescent | Age Factors | Animals | Anthropometry | Body Composition | Body Mass Index | Child | Cross-Sectional Studies | Diet | Diet Records | Dietary Proteins | Energy Intake | Europe | Exercise | Female | Humans | Lipids | Male | Meat | Nutrition Assessment | Obesity | Plant Proteins | Sex Factors | Thinness [SUMMARY] | [CONTENT] Adolescent | Age Factors | Animals | Anthropometry | Body Composition | Body Mass Index | Child | Cross-Sectional Studies | Diet | Diet Records | Dietary Proteins | Energy Intake | Europe | Exercise | Female | Humans | Lipids | Male | Meat | Nutrition Assessment | Obesity | Plant Proteins | Sex Factors | Thinness [SUMMARY] | [CONTENT] Adolescent | Age Factors | Animals | Anthropometry | Body Composition | Body Mass Index | Child | Cross-Sectional Studies | Diet | Diet Records | Dietary Proteins | Energy Intake | Europe | Exercise | Female | Humans | Lipids | Male | Meat | Nutrition Assessment | Obesity | Plant Proteins | Sex Factors | Thinness [SUMMARY] | [CONTENT] Adolescent | Age Factors | Animals | Anthropometry | Body Composition | Body Mass Index | Child | Cross-Sectional Studies | Diet | Diet Records | Dietary Proteins | Energy Intake | Europe | Exercise | Female | Humans | Lipids | Male | Meat | Nutrition Assessment | Obesity | Plant Proteins | Sex Factors | Thinness [SUMMARY] | [CONTENT] obesity ob adolescents | adolescents nutrition assessment | obese adolescents non | protein intake adolescent | protein intakes adolescents [SUMMARY] | [CONTENT] obesity ob adolescents | adolescents nutrition assessment | obese adolescents non | protein intake adolescent | protein intakes adolescents [SUMMARY] | [CONTENT] obesity ob adolescents | adolescents nutrition assessment | obese adolescents non | protein intake adolescent | protein intakes adolescents [SUMMARY] | [CONTENT] obesity ob adolescents | adolescents nutrition assessment | obese adolescents non | protein intake adolescent | protein intakes adolescents [SUMMARY] | [CONTENT] obesity ob adolescents | adolescents nutrition assessment | obese adolescents non | protein intake adolescent | protein intakes adolescents [SUMMARY] | [CONTENT] obesity ob adolescents | adolescents nutrition assessment | obese adolescents non | protein intake adolescent | protein intakes adolescents [SUMMARY] | [CONTENT] protein | intake | animal | total | plant | intakes | adolescents | plant protein | protein intakes | protein intake [SUMMARY] | [CONTENT] protein | intake | animal | total | plant | intakes | adolescents | plant protein | protein intakes | protein intake [SUMMARY] | [CONTENT] protein | intake | animal | total | plant | intakes | adolescents | plant protein | protein intakes | protein intake [SUMMARY] | [CONTENT] protein | intake | animal | total | plant | intakes | adolescents | plant protein | protein intakes | protein intake [SUMMARY] | [CONTENT] protein | intake | animal | total | plant | intakes | adolescents | plant protein | protein intakes | protein intake [SUMMARY] | [CONTENT] protein | intake | animal | total | plant | intakes | adolescents | plant protein | protein intakes | protein intake [SUMMARY] | [CONTENT] ob | children | high | protein | ow | risk | meat | sources | association | proteins [SUMMARY] | [CONTENT] kg | model | samples | m2 | sampling | adolescents | blood | food | pa | calculated [SUMMARY] | [CONTENT] protein | total | kg | significantly | total protein | value significantly different | significantly different | value significantly | value | intake [SUMMARY] | [CONTENT] protein | protein intake | intake | hand | total protein | total protein intake | total | bf | intake derived | animal [SUMMARY] | [CONTENT] protein | intake | animal | plant | protein intake | adolescents | total | intakes | plant protein | model [SUMMARY] | [CONTENT] protein | intake | animal | plant | protein intake | adolescents | total | intakes | plant protein | model [SUMMARY] | [CONTENT] ||| European | one | European [SUMMARY] | [CONTENT] 1804 | 2006-2007 | 12.5-17.5 | 47% | two | 24 ||| Model [SUMMARY] | [CONTENT] World Health Organization and European Food Safety Authority ||| 96 | 59% ||| 12.5-14.9 ||| obese ||| BMI ||| BMI | BF% [SUMMARY] | [CONTENT] European ||| European ||| [SUMMARY] | [CONTENT] ||| European | one | European ||| 1804 | 2006-2007 | 12.5-17.5 | 47% | two | 24 ||| Model ||| World Health Organization and European Food Safety Authority ||| 96 | 59% ||| 12.5-14.9 ||| obese ||| BMI ||| BMI | BF% ||| European ||| European ||| [SUMMARY] | [CONTENT] ||| European | one | European ||| 1804 | 2006-2007 | 12.5-17.5 | 47% | two | 24 ||| Model ||| World Health Organization and European Food Safety Authority ||| 96 | 59% ||| 12.5-14.9 ||| obese ||| BMI ||| BMI | BF% ||| European ||| European ||| [SUMMARY] |
||||||
Fresh and Cryopreserved Human Umbilical-Cord-Derived Mesenchymal Stromal Cells Attenuate Injury and Enhance Resolution and Repair following Ventilation-Induced Lung Injury. | 34884645 | Ventilator-induced lung injury (VILI) frequently worsens acute respiratory distress syndrome (ARDS) severity. Human mesenchymal stem/stromal cells (MSCs) offer considerable therapeutic promise, but the key impediments of clinical translation stem from limitations due to cell source and availability, and concerns regarding the loss of efficacy following cryopreservation. These experiments compared the efficacy of umbilical-cord-derived MSCs (UC-MSCs), a readily available and homogenous tissue source, to the previously more widely utilised bone-marrow-derived MSCs (BM-MSCs). We assessed their capacity to limit inflammation, resolve injury and enhance repair in relevant lung mechanical stretch models, and the impact of cryopreservation on therapeutic efficacy. | BACKGROUND | In series 1, confluent alveolar epithelial layers were subjected to cyclic mechanical stretch (22% equibiaxial strain) and wound injury, and the potential of the secretome from BM- and UC-derived MSCs to attenuate epithelial inflammation and cell death, and enhance wound repair was determined. In series 2, anesthetized rats underwent VILI, and later received, in a randomised manner, 1 × 107 MSCs/kg intravenously, that were: (i) fresh BM-MSCs, (ii) fresh UC-MSCs or (iii) cryopreserved UC-MSCs. Control animals received a vehicle (PBS). The extent of the resolution of inflammation and injury, and repair was measured at 24 h. | METHODS | Conditioned medium from BM-MSCs and UC-MSCs comparably decreased stretch-induced pulmonary epithelial inflammation and cell death. BM-MSCs and UC-MSCs comparably enhanced wound resolution. In animals subjected to VILI, both fresh BM-MSCs and UC-MSCs enhanced injury resolution and repair, while cryopreserved UC-MSCs comparably retained their efficacy. | RESULTS | Cryopreserved UC-MSCs can reduce stretch-induced inflammation and cell death, enhance wound resolution, and enhance injury resolution and repair following VILI. Cryopreserved UC-MSCs represent a more abundant, cost-efficient, less variable and equally efficacious source of therapeutic MSC product. | CONCLUSIONS | [
"Animals",
"Cell Line, Tumor",
"Cells, Cultured",
"Cryopreservation",
"Culture Media, Conditioned",
"Humans",
"Lung",
"Male",
"Mesenchymal Stem Cell Transplantation",
"Mesenchymal Stem Cells",
"Rats",
"Rats, Sprague-Dawley",
"Respiratory Distress Syndrome",
"Umbilical Cord",
"Ventilator-Induced Lung Injury"
] | 8657992 | 1. Introduction | Mesenchymal stem/stromal cells (MSCs) show promise as a therapeutic strategy for sepsis and acute respiratory distress syndrome (ARDS). MSCs were observed to modulate the inflammatory process, improve alveolar epithelial barrier function, attenuate lung injury and reduce the overall mortality in diverse preclinical sepsis and ARDS animal models [1,2,3,4,5,6,7], including our model of E. coli-induced pneumonia [8,9] and repair from ventilator-induced lung injury (VILI) [10]. VILI is an inflammatory process that can result from certain mechanical ventilation strategies, which are necessary to ensure adequate oxygenation but often worsen ARDS. The mechanisms of action of MSCs in VILI are numerous, including the secretion of a range of paracrine mediators and microparticles that can ameliorate the evolution of injury and inflammation, as well as promote tissue repair and recovery [11]. Importantly, MSCs demonstrate utility in human lungs ex vivo [12], and MSCs are well tolerated, showing promising efficacy in a number of phase I/phase II clinical trials [13].
As early phase trials progress, three key issues in relation to the clinical translation of MSCs have been highlighted which are the source of the cell, the protocol of cell expansion, and the mechanism of storage and transport. The industrialization of bone marrow (BM) MSC production for large-scale phase III clinical trials necessitates extensive culture expansion from each bone marrow donation in order to secure suitable doses for systemic delivery [14]. MSCs, which are expanded in cultures, exhibit telomere shortening, which is one factor causative of senescence and an eventual form of cell exhaustion and cessation of proliferation. [15,16]. Other expansion protocol factors can also contribute to phenotypic changes in the cell therapy which could diminish efficacy, reduce cell longevity after engraftment and hamper their regenerative and immunomodulatory properties [16].
The human umbilical cord is a rich source of MSCs and preparing them from this tissue has many obvious advantages when compared to the traditional alternatives. These advantages are particularly clear with regard to the production volumes of the MSCs without introducing senescence. The umbilical cord is also easily procured, as it is a waste tissue where harvesting does not require any sort of invasive procedure as with bone marrow or adipose MSCs. From one donor, the umbilical cord can produce more early-passage MSCs (by one order of magnitude) than a typical bone marrow donation, instead facilitating the manufacturing of large quantities of the lower-passage MSCs to provide the greatest immunomodulatory capacity [17]. Due to the age of their source, all UC-MSCs are essentially the same age, which increases the homogeneity of the final cell product, enhancing reproducibility in the recipient [18].
Finally, the majority of previous studies using MSCs use them as harvested directly from culture, but this approach may only be of utility for chronic diseases and even then is of reduced practicality. It remains to be conclusively proven whether efficacy is retained when MSCs are administered immediately following cryopreservation; therefore, this is a critical step on the translational path. Differences in MSC characteristics, particularly their immune modulation capabilities, were linked to their source of origin and cryopreservation status and, as such, direct comparison studies are required [19,20,21,22].
We hypothesised that UC-MSCs would modulate inflammation and enhance repair after VILI, in a comparable manner to the “gold standard” BM-MSCs, and that cryopreservation would not alter MSC efficacy. | null | null | 2. Results | 2.1. Cyclic Stretch-Induced Epithelial Injury In Vitro Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).
Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).
2.2. Pulmonary Epithelial Wound Healing In Vitro Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).
Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).
2.3. Injury Resolution Following In Vivo Ventilation-Induced ARDS Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.
Modulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).
Restoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).
Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.
Modulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).
Restoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B). | 5. Conclusions | The study confirms that cryopreservation has no deleterious effect on the therapeutic efficacy of UC-MSCs in this injury and repair model, and this further supports the use of MSCs for different clinical presentations of ARDS. UC-MSCs represent a more readily available and cost-effective source of MSCs that is suitable for large-scale expansion and industrial production. The integrity and consistency of this cell product can be further maintained with cryopreservation without the loss of therapeutic efficacy, and signifies a huge advantage for the use of MSCs as a therapy for patients with ARDS. | [
"2.1. Cyclic Stretch-Induced Epithelial Injury In Vitro",
"2.2. Pulmonary Epithelial Wound Healing In Vitro",
"2.3. Injury Resolution Following In Vivo Ventilation-Induced ARDS",
"3.1. The Secretome of UC-MCS and BM-MSCs Comparably Rescues the Injured Lung Epithelium",
"3.2. UC-MSCs and BM-MSCs Comparably Restore Lung Function",
"3.3. UC-MSCs and BM-MSCs Comparably Modulate the Inflammatory Response",
"3.4. UC-MSCs and BM-MSCs Comparably Restore Lung Structure",
"4. Materials and Methods",
"4.1. Cell Culture",
"4.2. Cyclic Mechanical Cell Stretch",
"4.3. Stretch Injury Assessment",
"4.4. In Vitro Scratch Wounds",
"4.5. Rodent Model of Resolution Post Ventilation-Induced Lung Injury",
"4.6. Assessment of Lung Injury and Recovery",
"4.7. Statistical Analysis"
] | [
"Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).",
"Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).",
"Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.\nModulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).\nRestoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).",
"Treatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.",
"This study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.\nAdministration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.",
"The delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.\nThe administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].",
"Alveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].",
"4.1. Cell Culture A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).\nMSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.\nPreparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.\nA549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).\nMSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.\nPreparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.\n4.2. Cyclic Mechanical Cell Stretch As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.\nAs previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.\n4.3. Stretch Injury Assessment Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).\nViability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.\nLuciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).\nViability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.\n4.4. In Vitro Scratch Wounds A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).\nA549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).\n4.5. Rodent Model of Resolution Post Ventilation-Induced Lung Injury All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.\nInduction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].\nExperimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.\nAll work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.\nInduction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].\nExperimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.\n4.6. Assessment of Lung Injury and Recovery In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].\nEx Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.\nIn Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].\nEx Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.\n4.7. Statistical Analysis The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.\nThe distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.",
"A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).\nMSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.\nPreparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.",
"As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.",
"Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).\nViability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.",
"A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).",
"All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.\nInduction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].\nExperimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.",
"In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].\nEx Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.",
"The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80."
] | [
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] | [
"1. Introduction",
"2. Results",
"2.1. Cyclic Stretch-Induced Epithelial Injury In Vitro",
"2.2. Pulmonary Epithelial Wound Healing In Vitro",
"2.3. Injury Resolution Following In Vivo Ventilation-Induced ARDS",
"3. Discussion",
"3.1. The Secretome of UC-MCS and BM-MSCs Comparably Rescues the Injured Lung Epithelium",
"3.2. UC-MSCs and BM-MSCs Comparably Restore Lung Function",
"3.3. UC-MSCs and BM-MSCs Comparably Modulate the Inflammatory Response",
"3.4. UC-MSCs and BM-MSCs Comparably Restore Lung Structure",
"4. Materials and Methods",
"4.1. Cell Culture",
"4.2. Cyclic Mechanical Cell Stretch",
"4.3. Stretch Injury Assessment",
"4.4. In Vitro Scratch Wounds",
"4.5. Rodent Model of Resolution Post Ventilation-Induced Lung Injury",
"4.6. Assessment of Lung Injury and Recovery",
"4.7. Statistical Analysis",
"5. Conclusions"
] | [
"Mesenchymal stem/stromal cells (MSCs) show promise as a therapeutic strategy for sepsis and acute respiratory distress syndrome (ARDS). MSCs were observed to modulate the inflammatory process, improve alveolar epithelial barrier function, attenuate lung injury and reduce the overall mortality in diverse preclinical sepsis and ARDS animal models [1,2,3,4,5,6,7], including our model of E. coli-induced pneumonia [8,9] and repair from ventilator-induced lung injury (VILI) [10]. VILI is an inflammatory process that can result from certain mechanical ventilation strategies, which are necessary to ensure adequate oxygenation but often worsen ARDS. The mechanisms of action of MSCs in VILI are numerous, including the secretion of a range of paracrine mediators and microparticles that can ameliorate the evolution of injury and inflammation, as well as promote tissue repair and recovery [11]. Importantly, MSCs demonstrate utility in human lungs ex vivo [12], and MSCs are well tolerated, showing promising efficacy in a number of phase I/phase II clinical trials [13].\nAs early phase trials progress, three key issues in relation to the clinical translation of MSCs have been highlighted which are the source of the cell, the protocol of cell expansion, and the mechanism of storage and transport. The industrialization of bone marrow (BM) MSC production for large-scale phase III clinical trials necessitates extensive culture expansion from each bone marrow donation in order to secure suitable doses for systemic delivery [14]. MSCs, which are expanded in cultures, exhibit telomere shortening, which is one factor causative of senescence and an eventual form of cell exhaustion and cessation of proliferation. [15,16]. Other expansion protocol factors can also contribute to phenotypic changes in the cell therapy which could diminish efficacy, reduce cell longevity after engraftment and hamper their regenerative and immunomodulatory properties [16].\nThe human umbilical cord is a rich source of MSCs and preparing them from this tissue has many obvious advantages when compared to the traditional alternatives. These advantages are particularly clear with regard to the production volumes of the MSCs without introducing senescence. The umbilical cord is also easily procured, as it is a waste tissue where harvesting does not require any sort of invasive procedure as with bone marrow or adipose MSCs. From one donor, the umbilical cord can produce more early-passage MSCs (by one order of magnitude) than a typical bone marrow donation, instead facilitating the manufacturing of large quantities of the lower-passage MSCs to provide the greatest immunomodulatory capacity [17]. Due to the age of their source, all UC-MSCs are essentially the same age, which increases the homogeneity of the final cell product, enhancing reproducibility in the recipient [18].\nFinally, the majority of previous studies using MSCs use them as harvested directly from culture, but this approach may only be of utility for chronic diseases and even then is of reduced practicality. It remains to be conclusively proven whether efficacy is retained when MSCs are administered immediately following cryopreservation; therefore, this is a critical step on the translational path. Differences in MSC characteristics, particularly their immune modulation capabilities, were linked to their source of origin and cryopreservation status and, as such, direct comparison studies are required [19,20,21,22].\nWe hypothesised that UC-MSCs would modulate inflammation and enhance repair after VILI, in a comparable manner to the “gold standard” BM-MSCs, and that cryopreservation would not alter MSC efficacy.",
"2.1. Cyclic Stretch-Induced Epithelial Injury In Vitro Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).\nResolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).\n2.2. Pulmonary Epithelial Wound Healing In Vitro Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).\nWound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).\n2.3. Injury Resolution Following In Vivo Ventilation-Induced ARDS Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.\nModulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).\nRestoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).\nRecovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.\nModulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).\nRestoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).",
"Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).",
"Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).",
"Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.\nModulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).\nRestoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).",
"This study demonstrates several novel findings and concludes that UC-MSCs are comparably effective to the “gold standard” BM-MSCs for enhancing repair in the injured epithelium and lung and that UC-MSCs retain efficacy when delivered thawed, post cryopreservation. These findings have important implications for the translation and production of MSC therapies.\n3.1. The Secretome of UC-MCS and BM-MSCs Comparably Rescues the Injured Lung Epithelium Treatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.\nTreatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.\n3.2. UC-MSCs and BM-MSCs Comparably Restore Lung Function This study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.\nAdministration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.\nThis study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.\nAdministration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.\n3.3. UC-MSCs and BM-MSCs Comparably Modulate the Inflammatory Response The delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.\nThe administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].\nThe delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.\nThe administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].\n3.4. UC-MSCs and BM-MSCs Comparably Restore Lung Structure Alveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].\nAlveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].",
"Treatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.",
"This study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.\nAdministration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.",
"The delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.\nThe administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].",
"Alveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].",
"4.1. Cell Culture A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).\nMSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.\nPreparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.\nA549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).\nMSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.\nPreparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.\n4.2. Cyclic Mechanical Cell Stretch As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.\nAs previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.\n4.3. Stretch Injury Assessment Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).\nViability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.\nLuciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).\nViability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.\n4.4. In Vitro Scratch Wounds A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).\nA549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).\n4.5. Rodent Model of Resolution Post Ventilation-Induced Lung Injury All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.\nInduction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].\nExperimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.\nAll work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.\nInduction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].\nExperimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.\n4.6. Assessment of Lung Injury and Recovery In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].\nEx Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.\nIn Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].\nEx Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.\n4.7. Statistical Analysis The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.\nThe distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.",
"A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).\nMSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.\nPreparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.",
"As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.",
"Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).\nViability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.",
"A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).",
"All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.\nInduction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].\nExperimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.",
"In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].\nEx Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.",
"The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.",
"The study confirms that cryopreservation has no deleterious effect on the therapeutic efficacy of UC-MSCs in this injury and repair model, and this further supports the use of MSCs for different clinical presentations of ARDS. UC-MSCs represent a more readily available and cost-effective source of MSCs that is suitable for large-scale expansion and industrial production. The integrity and consistency of this cell product can be further maintained with cryopreservation without the loss of therapeutic efficacy, and signifies a huge advantage for the use of MSCs as a therapy for patients with ARDS."
] | [
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"results",
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"conclusions"
] | [
"acute respiratory distress syndrome",
"ventilation-induced lung injury",
"injury",
"mesenchymal stem/stromal cells",
"cryopreservation",
"tissue source"
] | 1. Introduction:
Mesenchymal stem/stromal cells (MSCs) show promise as a therapeutic strategy for sepsis and acute respiratory distress syndrome (ARDS). MSCs were observed to modulate the inflammatory process, improve alveolar epithelial barrier function, attenuate lung injury and reduce the overall mortality in diverse preclinical sepsis and ARDS animal models [1,2,3,4,5,6,7], including our model of E. coli-induced pneumonia [8,9] and repair from ventilator-induced lung injury (VILI) [10]. VILI is an inflammatory process that can result from certain mechanical ventilation strategies, which are necessary to ensure adequate oxygenation but often worsen ARDS. The mechanisms of action of MSCs in VILI are numerous, including the secretion of a range of paracrine mediators and microparticles that can ameliorate the evolution of injury and inflammation, as well as promote tissue repair and recovery [11]. Importantly, MSCs demonstrate utility in human lungs ex vivo [12], and MSCs are well tolerated, showing promising efficacy in a number of phase I/phase II clinical trials [13].
As early phase trials progress, three key issues in relation to the clinical translation of MSCs have been highlighted which are the source of the cell, the protocol of cell expansion, and the mechanism of storage and transport. The industrialization of bone marrow (BM) MSC production for large-scale phase III clinical trials necessitates extensive culture expansion from each bone marrow donation in order to secure suitable doses for systemic delivery [14]. MSCs, which are expanded in cultures, exhibit telomere shortening, which is one factor causative of senescence and an eventual form of cell exhaustion and cessation of proliferation. [15,16]. Other expansion protocol factors can also contribute to phenotypic changes in the cell therapy which could diminish efficacy, reduce cell longevity after engraftment and hamper their regenerative and immunomodulatory properties [16].
The human umbilical cord is a rich source of MSCs and preparing them from this tissue has many obvious advantages when compared to the traditional alternatives. These advantages are particularly clear with regard to the production volumes of the MSCs without introducing senescence. The umbilical cord is also easily procured, as it is a waste tissue where harvesting does not require any sort of invasive procedure as with bone marrow or adipose MSCs. From one donor, the umbilical cord can produce more early-passage MSCs (by one order of magnitude) than a typical bone marrow donation, instead facilitating the manufacturing of large quantities of the lower-passage MSCs to provide the greatest immunomodulatory capacity [17]. Due to the age of their source, all UC-MSCs are essentially the same age, which increases the homogeneity of the final cell product, enhancing reproducibility in the recipient [18].
Finally, the majority of previous studies using MSCs use them as harvested directly from culture, but this approach may only be of utility for chronic diseases and even then is of reduced practicality. It remains to be conclusively proven whether efficacy is retained when MSCs are administered immediately following cryopreservation; therefore, this is a critical step on the translational path. Differences in MSC characteristics, particularly their immune modulation capabilities, were linked to their source of origin and cryopreservation status and, as such, direct comparison studies are required [19,20,21,22].
We hypothesised that UC-MSCs would modulate inflammation and enhance repair after VILI, in a comparable manner to the “gold standard” BM-MSCs, and that cryopreservation would not alter MSC efficacy.
2. Results:
2.1. Cyclic Stretch-Induced Epithelial Injury In Vitro Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).
Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).
2.2. Pulmonary Epithelial Wound Healing In Vitro Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).
Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).
2.3. Injury Resolution Following In Vivo Ventilation-Induced ARDS Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.
Modulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).
Restoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).
Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.
Modulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).
Restoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).
2.1. Cyclic Stretch-Induced Epithelial Injury In Vitro:
Resolution of In Vitro Stretch Injury: 120 h of mechanical cyclic stretch caused a significant induction of the NFκB transcription factor (p < 0.001) and a significant decrease in the viability of alveolar epithelial cells (p < 0.001) (Figure 1). BM-MSC conditioned medium (CM) significantly attenuated NFκB activation (p < 0.05), as did UC-MSC CM (p < 0.01) (Figure 1A). Both BM-MSC CM and UC-MSC CM significantly reversed the decrease in cell viability (p < 0.05) in comparison to the DF CM control (Figure 1B).
2.2. Pulmonary Epithelial Wound Healing In Vitro:
Wound Healing: Treatment with BM-MSC CM or UC-MSC CM significantly enhanced wound restitution in alveolar epithelial cells following scratch wounding in vitro, when compared to the MEM-α control (p < 0.001) and DF CM control (p < 0.01 and 0.05, respectively) (Figure 2).
2.3. Injury Resolution Following In Vivo Ventilation-Induced ARDS:
Recovery of Lung Function: VILI caused a significant decrement in oxygenation, lung compliance and lung permeability compared to protective ventilation (Figure 3). Both fresh BM-MSCs and UC-MSCs restored arterial oxygenation (p < 0.001) (Figure 3A) when compared to the vehicle (PBS) control group. Importantly, thawed, cryopreserved UC-MSCs also restored arterial oxygenation (p < 0.001), demonstrating that these cells retained efficacy post cryopreservation. The decrement in static lung compliance induced by VILI was restored by BM-MSCs and UC-MSCs (p < 0.001 and 0.01, respectively), while thawed, cryopreserved UC-MSCs were similarly effective (p < 0.01) (Figure 3B). BM-MSCs and UC-MSCs increased the restoration of alveolar barrier permeability, as shown by a reduction in the lung wet:dry weight ratio (p < 0.01 and 0.05, respectively) (Figure 3C) and a reduction in the accumulated total protein in the airspace (p < 0.01) (Figure 3D). The thawed cryopreserved UC-MSCs retained their efficacy and also restored alveolar fluid clearance (p < 0.05) and protein concentrations (p < 0.01) to normal levels.
Modulation of the Inflammatory Response: VILI caused a significant inflammatory response (Figure 4). Fresh BM-MSCs and both fresh and cryopreserved UC-MSCs comparably decreased alveolar cell counts (p < 0.01, 0.05 and 0.01, respectively) (Figure 4A) and alveolar neutrophil counts (p < 0.01) (Figure 4B) when compared to the vehicle (PBS) control group. IL-6 release, induced following VILI, was significantly attenuated by fresh BM-MSCs (p < 0.05) (Figure 4C). While IL-6 concentrations were lower in the BAL of animals that received freshly delivered UC-MSCs, this was not statistically significant (Figure 4C). In contrast, thawed, cryopreserved UC-MSCs reduced alveolar IL-6 concentrations (p < 0.05 (Figure 4C). All MSC treatment groups demonstrated significantly reduced alveolar IL-1β concentrations (p < 0.001, 0.01 and 0.001, respectively) when compared to the control group (Figure 4D).
Restoration of Lung Structure: VILI caused significant alveolar epithelial structural damage. Treatment with BM-MSCs and UC-MSCs, whether fresh or following cryopreservation, significantly enhanced the restoration of lung histologic structure post VILI (p < 0.001), depicted as percentage airspace (Figure 5A) and the amelioration of interstitial and alveolar inflammatory cell infiltration (Figure 5B).
3. Discussion:
This study demonstrates several novel findings and concludes that UC-MSCs are comparably effective to the “gold standard” BM-MSCs for enhancing repair in the injured epithelium and lung and that UC-MSCs retain efficacy when delivered thawed, post cryopreservation. These findings have important implications for the translation and production of MSC therapies.
3.1. The Secretome of UC-MCS and BM-MSCs Comparably Rescues the Injured Lung Epithelium Treatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.
Treatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.
3.2. UC-MSCs and BM-MSCs Comparably Restore Lung Function This study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.
Administration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.
This study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.
Administration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.
3.3. UC-MSCs and BM-MSCs Comparably Modulate the Inflammatory Response The delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.
The administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].
The delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.
The administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].
3.4. UC-MSCs and BM-MSCs Comparably Restore Lung Structure Alveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].
Alveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].
3.1. The Secretome of UC-MCS and BM-MSCs Comparably Rescues the Injured Lung Epithelium:
Treatment with the secretome of either BM-MSCs or UC-MSCs comparably attenuated mechanical stretch injury and enhanced repair following wound injury in vitro. Similar findings were observed in previous studies [8,10] and this further supports the hypothesis that the mechanism of therapeutic action of MSCs involves, in part, the release of paracrine mediators.
3.2. UC-MSCs and BM-MSCs Comparably Restore Lung Function:
This study demonstrated for the first time, that UC-MSCs are comparably efficacious to BM-MSCs, in restoring oxygenation and compliance in a rat model of recovery following VILI. We previously reported this finding for fresh BM-MSCs in VILI [23], but this is the first study to directly compare the efficacy of BM-MSCs to UC-MSCs in this preclinical model. This study also observed that UC-MSCs retained their efficacy to achieve the aforementioned effects after cryopreservation, which is a significant advance for clinical translation in terms of production and delivery. Previous studies reported that cryopreserved BM-MSCs were efficacious in restoring oxygenation and compliance in a rat model of pneumonia [8] and this can be considered alongside the findings of this study to further support the use of cryopreserved MSCs.
Administration of either BM-MSCs or UC-MSC cells was also comparable in the restoration of alveolar membrane integrity, as both treatments were shown to equally restore alveolar fluid clearance and reduce protein concentration in the injured lung. The administration of BM-MSCs was previously shown to lower protein concentrations and fluid retention in the lungs of animals with VILI, but this study is the first to obverse these findings for UC-MSCs in VILI. Furthermore, cryopreservation did not hinder the efficacy of UC-MSCs to restore alveolar membrane integrity following VILI, which is a novel finding in this pre-clinical model of ARDS. One study previously observed that cryopreserved UC-MSCs significantly attenuated protein concentration in the lungs of rats with pneumonia [24]. Overall, these studies further support the use of cryopreserved MSCs in future clinical studies.
3.3. UC-MSCs and BM-MSCs Comparably Modulate the Inflammatory Response:
The delivery of fresh, either BM-MSC or UC-MSC cell doses, significantly modified the inflammatory response to VILI as evidenced by resolved inflammatory cell infiltration in the injured lung, and cryopreservation did not hamper the efficacy of this MSC response. Similar observations were reported in rat models of E.coli-induced lung injury [8,9], but this is the first study to observe these findings in a VILI model.
The administration of MSCs in this animal model revealed a modified BAL cytokine profile. Freshly delivered BM-MSCs significantly relieved the release of IL-6, whereas UC-MSCs did not. Interestingly, the cryopreserved cells did show a significant benefit. MSCs that are harvested on the same day, pooled, cryopreserved and then thawed for delivery, are likely to present as a more homogenous cell dose. Cryopreservation can therefore reduce the variability in MSC efficacy that can be attributed to either cell culture conditions or donor differences. BM-MSCs and UC-MSCs were previously observed to reduce BAL IL-6 in a rat pneumonia model [9]. Finally, fresh BM-MSCs, and either fresh or cryopreserved UC-MSCs, significantly reduced pro-inflammatory IL-1β release in the BAL. Overall, it is clear that neither the cell source nor cryopreservation can hinder the immunomodulatory effects of MSCs in this VILI animal model, and this agrees with previously published reports in other models of ARDS [8,9].
3.4. UC-MSCs and BM-MSCs Comparably Restore Lung Structure:
Alveolar airspace and lung structure was significantly restored by MSC treatment, confirming that the MSCs from either BM or UC sources are equally efficacious, even after cryopreservation, in promoting resolution from injury in VILI. Similar observations in other preclinical models of ARDS were reported [8,24].
4. Materials and Methods:
4.1. Cell Culture A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).
MSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.
Preparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.
A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).
MSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.
Preparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.
4.2. Cyclic Mechanical Cell Stretch As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.
As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.
4.3. Stretch Injury Assessment Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).
Viability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.
Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).
Viability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.
4.4. In Vitro Scratch Wounds A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).
A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).
4.5. Rodent Model of Resolution Post Ventilation-Induced Lung Injury All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.
Induction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].
Experimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.
All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.
Induction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].
Experimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.
4.6. Assessment of Lung Injury and Recovery In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].
Ex Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.
In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].
Ex Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.
4.7. Statistical Analysis The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.
The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.
4.1. Cell Culture:
A549/NF-κB-luciferase Cell Line Culture: NFκB inflammatory signaling is strongly involved in stretch-induced lung inflammation and injury, as well as ARDS development [25,26,27]. A549/NFκB-luciferase cells (Panomics, Fremont, CA, USA) were purchased as cryopreserved 3-passage culture and used at passages 4–10. These cells have an integrated chromosomal luciferase reporter construct that is regulated by NFκB, and are used for examining NFκB transcription factor activity in vitro. Breifly, A549 cells were co-transfected with a NFκB luciferase reporter plasmid and hygromycin-resistant plasmid, and then selected with hygromycin in culture. A TNF-α and luciferase assay was then used to select hygromycin-resistant cell clones. A549/NFκB-luciferase cells were passaged in RPMI-1640 growth medium (Sigma-Aldrich Ireland Ltd., Wicklow, Ireland) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% penicillin G (100 U/mL) and streptomycin (100µg/mL) solution (Sigma-Aldrich), 1% L-glutamine (0.2 mg/mL) (Sigma-Aldrich) and hygromycin (50 μg/mL final) (Roche Life Science, Penzberg, Germany). These cells were maintained in a humidified (95%) tissue culture incubator saturated with a gas mixture containing 5% CO2 and 20% O2 in air at 37 °C. These cells were sub-cultured with 0.025% trypsin/0.05 mM EDTA (GIBCO®, Invitrogen Corporation, Grand Island, NY, USA) and cryopreserved in CryoStor™ cell preservation medium (Sigma-Aldrich).
MSC and Dermal Fibroblast (DF) Isolation, Culture and Expansion: Human BM-MSCs and UC-MSCs were isolated as previously described [9,28] and used at passages 1–3 for all experiments. Briefly, for BM-MSCs, bone marrow aspirates were obtained from healthy donors after ethical consent and were subjected to Ficoll density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK). Mononuclear cells were selected by plastic adherence and cell surface marker expression. For UC-MSCs, umbilical cord tissue was ethically obtained after consent and physically disassociated. The tissue was then subjected to enzymatic breakdown in culture media containing Collagenase 1 (2 mg/mL) (Sigma Aldrich) at 37 °C for less than one hour. The cells were filtered and a single cell suspension was obtained. Cells were selected by plastic adherence and surface marker expression. MSCs were cultured in Alpha Minimum Essential Eagle Medium plus Glutamax (MEM-α) (GIBCO®) supplemented with 10% FCS, penicillin G (100 U/mL) streptomycin (100 μg/mL) and FGF-1 (10 ng/mL) (PeproTech EC Ltd., London, UK). Cells were maintained in 95% humidity, 5% CO2 and 2% O2 (hypoxia) at 37 °C. These cells were sub-cultured with 0.025% trypsin-0.05/mM EDTA and cryopreserved in CryoStor™ cell preservation medium (200 μL per 1 million cells). DFs were used as control cells. DFs were derived from skin punch biopsies (3 mm), secured and cultured in 6-well plates (Sarstedt, Newton, NC, USA) until 80–90% confluent, then expanded and maintained as described above. For animal dosing, cells were reconstituted in 1 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich). Cryopreserved MSCs were stored for up to two months and cell viability after thawing was between 95–97% as determined by trypan blue exclusion.
Preparation of CM from DFs and MSCs: On day 1, the cells (passage 1–3) were seeded in a T175 tissue culture flask (Sarstedt) at 1 × 105 cells/cm2 in complete MEM-α medium (20 mLs). Forty-eight hours later (Day 3), the cells were washed three times in PBS solution before the addition of 20 mLs of serum-free MEM-α medium. The CM was collected 48 h later (Day 5) and then stored at −80 °C for later use. The CM was discarded after two freeze–thaw cycles.
4.2. Cyclic Mechanical Cell Stretch:
As previously described [25], A549/NFκB-luciferase cells were seeded to laminin-coated 6-well Bioflex plates (Flexcell International, Burlington, NC, USA), mounted onto the Flexcell FX-4000T® Tension Plus® baseplate (Flexcell International) and subjected to 22% equibiaxial stretch at a frequency of 0.1 Hz for 120 h. Non-stretched cells were used as control cells. Cells were maintained in their respective treatments for the entire 120 h. Following stretch, the cells were harvested for luciferase and viability assays.
4.3. Stretch Injury Assessment:
Luciferase Assay for NFκB Activity: Cells were harvested and pelleted then mixed with 50 μL of SolarGlow SuperBright (Molecutools, Dublin, Ireland) luciferase assay substrate for 5 min. Luminescence was measured in a VICTOR™ X plate reader (Perkin Elmer, Boston, MA, USA).
Viability Assay: Metabolic/mitochondrial activity was assessed by the thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) assay, as previously described [29]. Briefly, 5% of intact harvested cells were incubated with 100 μL of MTT solution (100 μg/mL final concentration) in complete RPMI medium, in a tissue culture incubator (5% CO2) for 2 h. 50 μL of DMSO was then added to each sample, and samples were kept at room temperature on an orbital mixer for 30 min. Absorbance values were measured at 550 nm.
4.4. In Vitro Scratch Wounds:
A549/NFκB-luciferase cells were seeded at 1 × 105 cells per cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1 mL pipette tip (Sarstedt). The cells were washed with PBS and their respective treatments were added. Wound restitution was assessed over 48 h in scanned images using the edge finding function in image analysis software (GNU Image Manipulation Program).
4.5. Rodent Model of Resolution Post Ventilation-Induced Lung Injury:
All work was approved by the Animal Care in Research Ethics Committee of the National University of Ireland Galway and conducted under license from the Health Products Regulatory Agency Ireland (License B100/4253). Specific-pathogen-free adult male Sprague Dawley rats (Charles River Laboratories, Kent, UK), weighing between 350–450 g, were used in all experiments.
Induction of VILI: As previously described [10], rats were anaesthetized with isoflurane gas and intravenous access was obtained via the tail vein. A laryngoscopy was performed and a 14 G catheter (BD Insyte®, Becton Dickinson Ltd., Oxfordshire, UK) was used to intubate the animal [30] for ventilation using a small animal ventilator (CWE SAR 830 AP; CWE Inc., Ardmore, PA, USA). Anaesthesia was maintained with repeated boli of Alfaxan® (Alfaxadone 0.9% (w/v) and alfadolone acetate 0.3% (w/v); Vétoquinol SA, Lure Cedex, France) and paralysis with cisatracurium besylate 0.5 mg·kg−1 (GlaxoSmithKline, Dublin, Ireland). Following baseline ventilation, static compliance was measured and VILI was induced using the following ventilator settings: FiO2 of 0.3, Pinsp 35 cmH2O, respiratory rate 18 min−1, and PEEP 0 cmH2O. Following development of severe VILI, as evidenced by a 50% decrease in respiratory static compliance, injurious ventilation was discontinued, and the animals were allowed to recover [8].
Experimental Design: Fifteen minutes post induction of VILI, animals were randomized to receive intravenous administrations of 1 × 107 MSCs/kg that were: (i) fresh BM-MSCs; (ii) fresh UC-MSCs; or (iii) thawed, cryopreserved UC-MSCs. Control animals received PBS solution and the extent of inflammation and injury resolution in all groups was measured at 24 h.
4.6. Assessment of Lung Injury and Recovery:
In Vivo Assessment: At 24 h post induction of VILI, animals were re-anaesthetized as described above, intravenous access was obtained via tail vein, and a tracheostomy tube was inserted [10]. Following gaining intra-arterial access, anaesthesia was maintained with Alfaxan®and paralysis with cisatracurium besylate, and mechanical ventilation commenced. Arterial blood pressure, airway pressure, lung static compliance and arterial blood gas analyses were performed as previously described [8].
Ex Vivo Analyses: Following exsanguination under anesthesia, bronchoalveolar lavage (BAL) was performed, and BAL fluid differential leukocyte counts were completed. BAL concentrations of IL-6 and IL-1β, were determined using ELISA (R&D Systems, Oxfordshire, UK) and BAL protein concentrations were also measured (Micro BCA; Pierce, IL, USA) as per the manufacturers guidelines. The left lung was isolated and fixed in 4% paraformaldehyde solution (PFA), sectioned, stained with haematoxylin/eosin and histologic lung damage determined using quantitative stereological techniques [31]. All ex vivo analyses were performed by blinded investigators.
4.7. Statistical Analysis:
The distribution of the data was tested for normality using Kolmogorov–Smirnov tests. Data sets were analysed by a one-way analysis of variance (ANOVA), with a post hoc Student–Newman–Keuls test, for between group comparisons. Data are presented as mean ± standard deviation (SD). A p value of <0.05 was considered statistically significant. The power and sample size of the in vivo study was determined using estimations of variance for 2 key indices of ARDS (oxygenation and compliance) based on previously published data [10,32] and the G*Power 3 program [33]. The study included n = 7 animals per group and the power of the study was >0.80.
5. Conclusions:
The study confirms that cryopreservation has no deleterious effect on the therapeutic efficacy of UC-MSCs in this injury and repair model, and this further supports the use of MSCs for different clinical presentations of ARDS. UC-MSCs represent a more readily available and cost-effective source of MSCs that is suitable for large-scale expansion and industrial production. The integrity and consistency of this cell product can be further maintained with cryopreservation without the loss of therapeutic efficacy, and signifies a huge advantage for the use of MSCs as a therapy for patients with ARDS.
| Background:
Ventilator-induced lung injury (VILI) frequently worsens acute respiratory distress syndrome (ARDS) severity. Human mesenchymal stem/stromal cells (MSCs) offer considerable therapeutic promise, but the key impediments of clinical translation stem from limitations due to cell source and availability, and concerns regarding the loss of efficacy following cryopreservation. These experiments compared the efficacy of umbilical-cord-derived MSCs (UC-MSCs), a readily available and homogenous tissue source, to the previously more widely utilised bone-marrow-derived MSCs (BM-MSCs). We assessed their capacity to limit inflammation, resolve injury and enhance repair in relevant lung mechanical stretch models, and the impact of cryopreservation on therapeutic efficacy.
Methods:
In series 1, confluent alveolar epithelial layers were subjected to cyclic mechanical stretch (22% equibiaxial strain) and wound injury, and the potential of the secretome from BM- and UC-derived MSCs to attenuate epithelial inflammation and cell death, and enhance wound repair was determined. In series 2, anesthetized rats underwent VILI, and later received, in a randomised manner, 1 × 107 MSCs/kg intravenously, that were: (i) fresh BM-MSCs, (ii) fresh UC-MSCs or (iii) cryopreserved UC-MSCs. Control animals received a vehicle (PBS). The extent of the resolution of inflammation and injury, and repair was measured at 24 h.
Results:
Conditioned medium from BM-MSCs and UC-MSCs comparably decreased stretch-induced pulmonary epithelial inflammation and cell death. BM-MSCs and UC-MSCs comparably enhanced wound resolution. In animals subjected to VILI, both fresh BM-MSCs and UC-MSCs enhanced injury resolution and repair, while cryopreserved UC-MSCs comparably retained their efficacy.
Conclusions:
Cryopreserved UC-MSCs can reduce stretch-induced inflammation and cell death, enhance wound resolution, and enhance injury resolution and repair following VILI. Cryopreserved UC-MSCs represent a more abundant, cost-efficient, less variable and equally efficacious source of therapeutic MSC product. | 1. Introduction:
Mesenchymal stem/stromal cells (MSCs) show promise as a therapeutic strategy for sepsis and acute respiratory distress syndrome (ARDS). MSCs were observed to modulate the inflammatory process, improve alveolar epithelial barrier function, attenuate lung injury and reduce the overall mortality in diverse preclinical sepsis and ARDS animal models [1,2,3,4,5,6,7], including our model of E. coli-induced pneumonia [8,9] and repair from ventilator-induced lung injury (VILI) [10]. VILI is an inflammatory process that can result from certain mechanical ventilation strategies, which are necessary to ensure adequate oxygenation but often worsen ARDS. The mechanisms of action of MSCs in VILI are numerous, including the secretion of a range of paracrine mediators and microparticles that can ameliorate the evolution of injury and inflammation, as well as promote tissue repair and recovery [11]. Importantly, MSCs demonstrate utility in human lungs ex vivo [12], and MSCs are well tolerated, showing promising efficacy in a number of phase I/phase II clinical trials [13].
As early phase trials progress, three key issues in relation to the clinical translation of MSCs have been highlighted which are the source of the cell, the protocol of cell expansion, and the mechanism of storage and transport. The industrialization of bone marrow (BM) MSC production for large-scale phase III clinical trials necessitates extensive culture expansion from each bone marrow donation in order to secure suitable doses for systemic delivery [14]. MSCs, which are expanded in cultures, exhibit telomere shortening, which is one factor causative of senescence and an eventual form of cell exhaustion and cessation of proliferation. [15,16]. Other expansion protocol factors can also contribute to phenotypic changes in the cell therapy which could diminish efficacy, reduce cell longevity after engraftment and hamper their regenerative and immunomodulatory properties [16].
The human umbilical cord is a rich source of MSCs and preparing them from this tissue has many obvious advantages when compared to the traditional alternatives. These advantages are particularly clear with regard to the production volumes of the MSCs without introducing senescence. The umbilical cord is also easily procured, as it is a waste tissue where harvesting does not require any sort of invasive procedure as with bone marrow or adipose MSCs. From one donor, the umbilical cord can produce more early-passage MSCs (by one order of magnitude) than a typical bone marrow donation, instead facilitating the manufacturing of large quantities of the lower-passage MSCs to provide the greatest immunomodulatory capacity [17]. Due to the age of their source, all UC-MSCs are essentially the same age, which increases the homogeneity of the final cell product, enhancing reproducibility in the recipient [18].
Finally, the majority of previous studies using MSCs use them as harvested directly from culture, but this approach may only be of utility for chronic diseases and even then is of reduced practicality. It remains to be conclusively proven whether efficacy is retained when MSCs are administered immediately following cryopreservation; therefore, this is a critical step on the translational path. Differences in MSC characteristics, particularly their immune modulation capabilities, were linked to their source of origin and cryopreservation status and, as such, direct comparison studies are required [19,20,21,22].
We hypothesised that UC-MSCs would modulate inflammation and enhance repair after VILI, in a comparable manner to the “gold standard” BM-MSCs, and that cryopreservation would not alter MSC efficacy.
5. Conclusions:
The study confirms that cryopreservation has no deleterious effect on the therapeutic efficacy of UC-MSCs in this injury and repair model, and this further supports the use of MSCs for different clinical presentations of ARDS. UC-MSCs represent a more readily available and cost-effective source of MSCs that is suitable for large-scale expansion and industrial production. The integrity and consistency of this cell product can be further maintained with cryopreservation without the loss of therapeutic efficacy, and signifies a huge advantage for the use of MSCs as a therapy for patients with ARDS. | Background:
Ventilator-induced lung injury (VILI) frequently worsens acute respiratory distress syndrome (ARDS) severity. Human mesenchymal stem/stromal cells (MSCs) offer considerable therapeutic promise, but the key impediments of clinical translation stem from limitations due to cell source and availability, and concerns regarding the loss of efficacy following cryopreservation. These experiments compared the efficacy of umbilical-cord-derived MSCs (UC-MSCs), a readily available and homogenous tissue source, to the previously more widely utilised bone-marrow-derived MSCs (BM-MSCs). We assessed their capacity to limit inflammation, resolve injury and enhance repair in relevant lung mechanical stretch models, and the impact of cryopreservation on therapeutic efficacy.
Methods:
In series 1, confluent alveolar epithelial layers were subjected to cyclic mechanical stretch (22% equibiaxial strain) and wound injury, and the potential of the secretome from BM- and UC-derived MSCs to attenuate epithelial inflammation and cell death, and enhance wound repair was determined. In series 2, anesthetized rats underwent VILI, and later received, in a randomised manner, 1 × 107 MSCs/kg intravenously, that were: (i) fresh BM-MSCs, (ii) fresh UC-MSCs or (iii) cryopreserved UC-MSCs. Control animals received a vehicle (PBS). The extent of the resolution of inflammation and injury, and repair was measured at 24 h.
Results:
Conditioned medium from BM-MSCs and UC-MSCs comparably decreased stretch-induced pulmonary epithelial inflammation and cell death. BM-MSCs and UC-MSCs comparably enhanced wound resolution. In animals subjected to VILI, both fresh BM-MSCs and UC-MSCs enhanced injury resolution and repair, while cryopreserved UC-MSCs comparably retained their efficacy.
Conclusions:
Cryopreserved UC-MSCs can reduce stretch-induced inflammation and cell death, enhance wound resolution, and enhance injury resolution and repair following VILI. Cryopreserved UC-MSCs represent a more abundant, cost-efficient, less variable and equally efficacious source of therapeutic MSC product. | 10,701 | 395 | [
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|||||
Risk behaviors among Italian healthcare students: a cross-sectional study for health promotion of future healthcare workers. | 30990476 | Risk behaviors are frequent among young adults and they are particularly relevant when considering healthcare students. | BACKGROUND | Healthcare students filled an anonymous multiple-choice questionnaire on the occasion of the occupational health visit that preceded their hospital internship. The questionnaire covered socio-demographic characteristics (including student's working status and cohabitation) and risk behaviors. We evaluated the prevalence of risk behaviors and their association with socio-demographic characteristics. | METHODS | The sample consisted of 494 students (65% women): 23.2% were smokers, 7.9% had excessive bodyweight, 35% did not practice any physical activity and 50% reported binge drinking at least once in the last 12 months. We found associations of male sex (30.5%) and being nursing students (29.9%) with smoking habit. The frequency of binge drinking was higher in men (38.4%), working students (53.9%), and among those who lived without family (50%). Physical inactivity was associated with female sex (44.2%) and living without family (57.1%). Finally, the co-presence of 2 risk behaviors or more was higher in men (36.8%), in nursing students (39.6%) and in working students (44.7%). | RESULTS | Our findings regarding the prevalence of risk behaviors and their potential association with socio-demographic factors may be a clue to the definition of targeted strategies aimed at reducing of risk behaviors among healthcare students. | CONCLUSIONS | [
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] | 7809966 | Introduction | Risk behaviors are lifestyle characteristics likely to cause potential harm to the subject, both from a physical and a psychological point of view. They generally include smoking habit, alcohol abuse, physical inactivity, and excessive bodyweight (5). Scientific literature shows that risky habits are usually adopted during adolescence (4). Nevertheless, they could exacerbate and become permanent between 18 and 30 years, when young adults experience a significant amount of stress (22). In fact, during this time, individuals often leave their usual social contexts and confront new academic and working challenges. In addition, there is evidence that unhealthy lifestyles among young adults are strongly linked to disabilities and health problems (12, 28). Therefore, if these behaviors are detected and modified at an early stage, dangerous developments could be avoided (23). In recent years, some academic institutions have tried to address the problem among their students, implementing the concept of “health promoting universities and colleges”. Several documents have been produced, the last being the “Okanagan Charter”, aimed at creating campus culture of well-being and improving the health of the people who live, learn, and work in university campuses, including students (20).
The problem of risk behaviors becomes stringent when considering healthcare students. They – as future health care providers – will play a key role in health promotion and their attitude highly influences not only their health and performance, but also the health status of their patients as well as of the general population. Some studies suggest that the healthy behaviors of healthcare workers influence patients’ attitudes towards preventive counselling and their motivation towards healthy lifestyle choices (9). So, healthcare workers and students who themselves lead healthy lifestyles are more likely to have a positive influence on patients and help them make healthy choices (1, 8, 17).
Furthermore, since these students are in close contact with patients, risk behaviors may be a problem during their hospital internship. In fact, risk behaviors of these students could affect their health, increase occupational injuries and impair the relation with patients (14). For example, binge drinking and alcohol abuse may cause sleep disruptions and alterations in the circadian rhythm in young people, hence increasing the risks of clinical mistakes and work-related injuries in unskilled students. Similarly, overweight and obesity may contribute to musculoskeletal disorders and physical injuries, mainly within nursing students (14). Smoking habit might increase the cancer risk among x-ray technician students, potentially exposed to ionizing radiations. Finally, occupational hazards in healthcare sector (long working hours, shiftwork, stress, limited access to healthy and regular food, sedentary jobs) could themselves influence employees’ health risk behavior.
For these reasons, universities should provide healthcare students with adequate training on the promotion of healthy behaviors. In order to design efficient strategies for the reduction of unhealthy habits, academic institutions should conduct studies to examine the influence of risk behaviors within the specific population of healthcare students (20). Italian occupational physicians started to address this issue during a conference organized in Milan in 2016, attended by the representatives of all the universities of Lombardy, the most populous and richest region in Italy (24).
Our study aims to investigate the prevalence of risk behaviors (smoking, binge drinking, physical inactivity, and excessive bodyweight) in a population of healthcare students attending an Italian university of Northern Italy. Our focus was on the evaluation of the possible associations between unhealthy behaviors and some socio-demographic factors in such a population. We might, in this way, identify specific health promotion programs targeted at the population of healthcare students (6). | Methods | The study analyzed a population of healthcare students, in the Department of medicine and surgery of a University in Northern Italy. The subjects attended the following courses: medicine and surgery, dentistry, nursing, physiotherapy, biomedical laboratory techniques, x-ray techniques, and neuro- and psychomotricity therapies of infancy. Data were collected during the academic year 2015-2016. All students were invited to fill in an anonymous self-administered questionnaire on the occasion of the health examination that precedes the hospital internship. Participation was voluntary. We collected the following socio-demographic characteristics: sex, type of course (medical and dental schools; nursing school; other healthcare schools), working status (non-working student, working student), and cohabitation (with family, without family). The questionnaire also included items on the following risk behaviors: smoking habit (yes, no), number of cigarettes per day, binge drinking in the last year (yes, no), frequency of binge drinking (never in the last 2 months, at least once in the last 2 months), physical activity (active, inactive), and BMI (<25 kg/m2, ≥25 kg/m2). We also considered the number of coexisting risk behaviors (smoking, binge drinking, BMI ≥25 kg/m2 and physical inactivity). In our inquiry, we adopted a definition of binge drinking given by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), namely the consumption within a period of two hours of ≥5 UA (Unit of Alcohol) for men, and of ≥4 UA for women.
Statistical analysis Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.
Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data. | Results | The students who accepted to fill in the questionnaire and included in the survey were 494 (65% women). Students were equally distributed across three degree courses: Medicine/Dentistry (33.4%), Nursing (34%) and Other Health Professions (32.6%). Eight point four percent of them declared to be a working student, 10% were living without family.
As shown in table 1, 23.2% of the students were smokers and 5.5% smoked between 10 and 20 cigarettes a day (no student declared to smoke more than 20 cigarettes per day). Half of the students referred to have practiced binge drinking at least once in the last 12 months, and 14.6% of them declared more than one episode every two months. Thirty-five percent of the individuals did not practice any weekly physical activity. Excessive bodyweight (BMI ≥25 kg/m2) was present in 7.9% of subjects. In particular, 6.6% were overweight and 1.3% were obese. As to the co-existence of risk behaviors, 23.2% of students did not present any of the four risk behaviors (smoking habit, binge drinking, physical inactivity, and BMI ≥25 kg/m2) and 29.9% presented two or more risk behaviors. In detail, 46.9% presented with one risk factor, 21.5% with two risk behaviors, 8.2% with three risk behaviors, and only one student with four risk behaviors.
Prevalence of risk behaviors (n. 494)
Associations between risk behaviors and socio-demographic factors We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.
Associations between smoking and socio-demographic characteristics
* Only smoker students. Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Associations between binge drinking and socio-demographic characteristics
*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.
Chi-square for categorical variables and t-test for age.
Associations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics
Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.
Associations between smoking and socio-demographic characteristics
* Only smoker students. Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Associations between binge drinking and socio-demographic characteristics
*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.
Chi-square for categorical variables and t-test for age.
Associations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics
Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age. | Conclusions | When considering the influence of risk behaviors on young adults, healthcare students represent a sensitive group of individuals. Healthcare students with risk behaviors might put not only their health at risk, but also the health of their patients. This condition is relevant not only during their internship while they attend the university courses, but also in the future perspective of a healthcare employment. Therefore, as health promoting universities, academic institutions should act to minimize the impact of negative behaviors on all their students. By doing so, they could help not only the students, but the community as a whole (20). This goal could be achieved by adopting health promotion programs. Notably, several universities have already implemented programs to improve the wellbeing of their students and of the communities they belong to (11, 19, 26, 27). Such initiatives should be tailored to the population characteristics.
For this reason, by providing information on prevalence of risk behaviors and the potential associations with socio-demographic factors, this study might be an important starting point in the definition of targeted strategies, aimed at reducing risk behaviors. The occupational health physicians working in universities and in other educational institutions, could play an important role in the assessment of health promotion needs. Indeed, the preventive and periodic health examinations are not only convenient opportunities for medical counselling, but also for collecting important data on population characteristics. This survey examined the health and wellbeing behaviors of its own healthcare student population, and highlighted some behaviors that are similar to expectations, and others that are not. This information will enable to tailor programs and interventions aimed at specific groups of students. For example, in our study population health promotion interventions should be mainly addressed towards nursing students, who seemed to have a higher prevalence of coexisting risk factors (particularly smoking habit). Similarly, specific interventions should also be addressed towards male students and working students, while physical activity should be mainly improved among female students.
The present study also has limitations. First, the design of the study was monocentric, since students were selected from a single university. Therefore, the sample cannot be considered representative of the actual Italian population of healthcare students. Second, the average age of the students was not homogenous across degree courses and statistically significant differences were found among them. Finally, individuals within our population could have minimized the magnitude of their risk behaviors, even if the questionnaire was anonymous. In fact, they could have concealed their unhealthy behaviors on account of their role as healthcare students.
Despite these limitations, our study is the first, to our knowledge, to investigate risk behaviors (including binge drinking) in a broad population of healthcare students, analyzing differences across different healthcare courses.
In conclusion, university attendance is a transitional period offering good opportunities for acquisition of healthy lifestyles. Occupational Health Services in the universities could be a reference point for all the initiatives of health promotion addressed to the student population, i.e. the tomorrow’s workforce.
No potential conflict of interest relevant to this article was reported by the authors | [
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"Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.",
"We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.\nAssociations between smoking and socio-demographic characteristics\n* Only smoker students. Data are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.\nAssociations between binge drinking and socio-demographic characteristics\n*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.\nChi-square for categorical variables and t-test for age.\nAssociations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics\nData are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.",
"The prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.",
"Fifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).\nOther Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).",
"Our study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).\nWithin our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.",
"When examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.\nRegarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students."
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"Conclusions"
] | [
"Risk behaviors are lifestyle characteristics likely to cause potential harm to the subject, both from a physical and a psychological point of view. They generally include smoking habit, alcohol abuse, physical inactivity, and excessive bodyweight (5). Scientific literature shows that risky habits are usually adopted during adolescence (4). Nevertheless, they could exacerbate and become permanent between 18 and 30 years, when young adults experience a significant amount of stress (22). In fact, during this time, individuals often leave their usual social contexts and confront new academic and working challenges. In addition, there is evidence that unhealthy lifestyles among young adults are strongly linked to disabilities and health problems (12, 28). Therefore, if these behaviors are detected and modified at an early stage, dangerous developments could be avoided (23). In recent years, some academic institutions have tried to address the problem among their students, implementing the concept of “health promoting universities and colleges”. Several documents have been produced, the last being the “Okanagan Charter”, aimed at creating campus culture of well-being and improving the health of the people who live, learn, and work in university campuses, including students (20).\nThe problem of risk behaviors becomes stringent when considering healthcare students. They – as future health care providers – will play a key role in health promotion and their attitude highly influences not only their health and performance, but also the health status of their patients as well as of the general population. Some studies suggest that the healthy behaviors of healthcare workers influence patients’ attitudes towards preventive counselling and their motivation towards healthy lifestyle choices (9). So, healthcare workers and students who themselves lead healthy lifestyles are more likely to have a positive influence on patients and help them make healthy choices (1, 8, 17).\nFurthermore, since these students are in close contact with patients, risk behaviors may be a problem during their hospital internship. In fact, risk behaviors of these students could affect their health, increase occupational injuries and impair the relation with patients (14). For example, binge drinking and alcohol abuse may cause sleep disruptions and alterations in the circadian rhythm in young people, hence increasing the risks of clinical mistakes and work-related injuries in unskilled students. Similarly, overweight and obesity may contribute to musculoskeletal disorders and physical injuries, mainly within nursing students (14). Smoking habit might increase the cancer risk among x-ray technician students, potentially exposed to ionizing radiations. Finally, occupational hazards in healthcare sector (long working hours, shiftwork, stress, limited access to healthy and regular food, sedentary jobs) could themselves influence employees’ health risk behavior.\nFor these reasons, universities should provide healthcare students with adequate training on the promotion of healthy behaviors. In order to design efficient strategies for the reduction of unhealthy habits, academic institutions should conduct studies to examine the influence of risk behaviors within the specific population of healthcare students (20). Italian occupational physicians started to address this issue during a conference organized in Milan in 2016, attended by the representatives of all the universities of Lombardy, the most populous and richest region in Italy (24).\nOur study aims to investigate the prevalence of risk behaviors (smoking, binge drinking, physical inactivity, and excessive bodyweight) in a population of healthcare students attending an Italian university of Northern Italy. Our focus was on the evaluation of the possible associations between unhealthy behaviors and some socio-demographic factors in such a population. We might, in this way, identify specific health promotion programs targeted at the population of healthcare students (6).",
"The study analyzed a population of healthcare students, in the Department of medicine and surgery of a University in Northern Italy. The subjects attended the following courses: medicine and surgery, dentistry, nursing, physiotherapy, biomedical laboratory techniques, x-ray techniques, and neuro- and psychomotricity therapies of infancy. Data were collected during the academic year 2015-2016. All students were invited to fill in an anonymous self-administered questionnaire on the occasion of the health examination that precedes the hospital internship. Participation was voluntary. We collected the following socio-demographic characteristics: sex, type of course (medical and dental schools; nursing school; other healthcare schools), working status (non-working student, working student), and cohabitation (with family, without family). The questionnaire also included items on the following risk behaviors: smoking habit (yes, no), number of cigarettes per day, binge drinking in the last year (yes, no), frequency of binge drinking (never in the last 2 months, at least once in the last 2 months), physical activity (active, inactive), and BMI (<25 kg/m2, ≥25 kg/m2). We also considered the number of coexisting risk behaviors (smoking, binge drinking, BMI ≥25 kg/m2 and physical inactivity). In our inquiry, we adopted a definition of binge drinking given by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), namely the consumption within a period of two hours of ≥5 UA (Unit of Alcohol) for men, and of ≥4 UA for women.\n Statistical analysis Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.\nAge was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.",
"Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.",
"The students who accepted to fill in the questionnaire and included in the survey were 494 (65% women). Students were equally distributed across three degree courses: Medicine/Dentistry (33.4%), Nursing (34%) and Other Health Professions (32.6%). Eight point four percent of them declared to be a working student, 10% were living without family.\nAs shown in table 1, 23.2% of the students were smokers and 5.5% smoked between 10 and 20 cigarettes a day (no student declared to smoke more than 20 cigarettes per day). Half of the students referred to have practiced binge drinking at least once in the last 12 months, and 14.6% of them declared more than one episode every two months. Thirty-five percent of the individuals did not practice any weekly physical activity. Excessive bodyweight (BMI ≥25 kg/m2) was present in 7.9% of subjects. In particular, 6.6% were overweight and 1.3% were obese. As to the co-existence of risk behaviors, 23.2% of students did not present any of the four risk behaviors (smoking habit, binge drinking, physical inactivity, and BMI ≥25 kg/m2) and 29.9% presented two or more risk behaviors. In detail, 46.9% presented with one risk factor, 21.5% with two risk behaviors, 8.2% with three risk behaviors, and only one student with four risk behaviors.\nPrevalence of risk behaviors (n. 494)\n Associations between risk behaviors and socio-demographic factors We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.\nAssociations between smoking and socio-demographic characteristics\n* Only smoker students. Data are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.\nAssociations between binge drinking and socio-demographic characteristics\n*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.\nChi-square for categorical variables and t-test for age.\nAssociations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics\nData are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.\nWe analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.\nAssociations between smoking and socio-demographic characteristics\n* Only smoker students. Data are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.\nAssociations between binge drinking and socio-demographic characteristics\n*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.\nChi-square for categorical variables and t-test for age.\nAssociations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics\nData are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.",
"We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.\nAssociations between smoking and socio-demographic characteristics\n* Only smoker students. Data are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.\nAssociations between binge drinking and socio-demographic characteristics\n*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.\nChi-square for categorical variables and t-test for age.\nAssociations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics\nData are shown as n (%) or mean±standard deviation.\nChi-square or Fisher’s exact test for categorical variables and t-test for age.",
"We discuss the data from our survey in the light of similar data recorded by ISTAT (Italian National Statistical Institute) among young adults in recent years (13) and of the results from surveys conducted on a population of Italian university students and of healthcare students (6, 21).\n Smoking habit The prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.\nThe prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.\n Binge drinking Fifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).\nOther Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).\nFifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).\nOther Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).\n Physical inactivity and excessive bodyweight Our study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).\nWithin our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.\nOur study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).\nWithin our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.\n Associations of coexisting risk behaviors When examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.\nRegarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students.\nWhen examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.\nRegarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students.",
"The prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.",
"Fifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).\nOther Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).",
"Our study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).\nWithin our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.",
"When examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.\nRegarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students.",
"When considering the influence of risk behaviors on young adults, healthcare students represent a sensitive group of individuals. Healthcare students with risk behaviors might put not only their health at risk, but also the health of their patients. This condition is relevant not only during their internship while they attend the university courses, but also in the future perspective of a healthcare employment. Therefore, as health promoting universities, academic institutions should act to minimize the impact of negative behaviors on all their students. By doing so, they could help not only the students, but the community as a whole (20). This goal could be achieved by adopting health promotion programs. Notably, several universities have already implemented programs to improve the wellbeing of their students and of the communities they belong to (11, 19, 26, 27). Such initiatives should be tailored to the population characteristics.\nFor this reason, by providing information on prevalence of risk behaviors and the potential associations with socio-demographic factors, this study might be an important starting point in the definition of targeted strategies, aimed at reducing risk behaviors. The occupational health physicians working in universities and in other educational institutions, could play an important role in the assessment of health promotion needs. Indeed, the preventive and periodic health examinations are not only convenient opportunities for medical counselling, but also for collecting important data on population characteristics. This survey examined the health and wellbeing behaviors of its own healthcare student population, and highlighted some behaviors that are similar to expectations, and others that are not. This information will enable to tailor programs and interventions aimed at specific groups of students. For example, in our study population health promotion interventions should be mainly addressed towards nursing students, who seemed to have a higher prevalence of coexisting risk factors (particularly smoking habit). Similarly, specific interventions should also be addressed towards male students and working students, while physical activity should be mainly improved among female students.\nThe present study also has limitations. First, the design of the study was monocentric, since students were selected from a single university. Therefore, the sample cannot be considered representative of the actual Italian population of healthcare students. Second, the average age of the students was not homogenous across degree courses and statistically significant differences were found among them. Finally, individuals within our population could have minimized the magnitude of their risk behaviors, even if the questionnaire was anonymous. In fact, they could have concealed their unhealthy behaviors on account of their role as healthcare students.\nDespite these limitations, our study is the first, to our knowledge, to investigate risk behaviors (including binge drinking) in a broad population of healthcare students, analyzing differences across different healthcare courses.\nIn conclusion, university attendance is a transitional period offering good opportunities for acquisition of healthy lifestyles. Occupational Health Services in the universities could be a reference point for all the initiatives of health promotion addressed to the student population, i.e. the tomorrow’s workforce.\nNo potential conflict of interest relevant to this article was reported by the authors"
] | [
"intro",
"methods",
null,
"results",
null,
"discussion",
null,
null,
null,
null,
"conclusions"
] | [
"risk behaviors",
"healthcare students",
"smoking",
"binge drinking",
"physical inactivity"
] | Introduction:
Risk behaviors are lifestyle characteristics likely to cause potential harm to the subject, both from a physical and a psychological point of view. They generally include smoking habit, alcohol abuse, physical inactivity, and excessive bodyweight (5). Scientific literature shows that risky habits are usually adopted during adolescence (4). Nevertheless, they could exacerbate and become permanent between 18 and 30 years, when young adults experience a significant amount of stress (22). In fact, during this time, individuals often leave their usual social contexts and confront new academic and working challenges. In addition, there is evidence that unhealthy lifestyles among young adults are strongly linked to disabilities and health problems (12, 28). Therefore, if these behaviors are detected and modified at an early stage, dangerous developments could be avoided (23). In recent years, some academic institutions have tried to address the problem among their students, implementing the concept of “health promoting universities and colleges”. Several documents have been produced, the last being the “Okanagan Charter”, aimed at creating campus culture of well-being and improving the health of the people who live, learn, and work in university campuses, including students (20).
The problem of risk behaviors becomes stringent when considering healthcare students. They – as future health care providers – will play a key role in health promotion and their attitude highly influences not only their health and performance, but also the health status of their patients as well as of the general population. Some studies suggest that the healthy behaviors of healthcare workers influence patients’ attitudes towards preventive counselling and their motivation towards healthy lifestyle choices (9). So, healthcare workers and students who themselves lead healthy lifestyles are more likely to have a positive influence on patients and help them make healthy choices (1, 8, 17).
Furthermore, since these students are in close contact with patients, risk behaviors may be a problem during their hospital internship. In fact, risk behaviors of these students could affect their health, increase occupational injuries and impair the relation with patients (14). For example, binge drinking and alcohol abuse may cause sleep disruptions and alterations in the circadian rhythm in young people, hence increasing the risks of clinical mistakes and work-related injuries in unskilled students. Similarly, overweight and obesity may contribute to musculoskeletal disorders and physical injuries, mainly within nursing students (14). Smoking habit might increase the cancer risk among x-ray technician students, potentially exposed to ionizing radiations. Finally, occupational hazards in healthcare sector (long working hours, shiftwork, stress, limited access to healthy and regular food, sedentary jobs) could themselves influence employees’ health risk behavior.
For these reasons, universities should provide healthcare students with adequate training on the promotion of healthy behaviors. In order to design efficient strategies for the reduction of unhealthy habits, academic institutions should conduct studies to examine the influence of risk behaviors within the specific population of healthcare students (20). Italian occupational physicians started to address this issue during a conference organized in Milan in 2016, attended by the representatives of all the universities of Lombardy, the most populous and richest region in Italy (24).
Our study aims to investigate the prevalence of risk behaviors (smoking, binge drinking, physical inactivity, and excessive bodyweight) in a population of healthcare students attending an Italian university of Northern Italy. Our focus was on the evaluation of the possible associations between unhealthy behaviors and some socio-demographic factors in such a population. We might, in this way, identify specific health promotion programs targeted at the population of healthcare students (6).
Methods:
The study analyzed a population of healthcare students, in the Department of medicine and surgery of a University in Northern Italy. The subjects attended the following courses: medicine and surgery, dentistry, nursing, physiotherapy, biomedical laboratory techniques, x-ray techniques, and neuro- and psychomotricity therapies of infancy. Data were collected during the academic year 2015-2016. All students were invited to fill in an anonymous self-administered questionnaire on the occasion of the health examination that precedes the hospital internship. Participation was voluntary. We collected the following socio-demographic characteristics: sex, type of course (medical and dental schools; nursing school; other healthcare schools), working status (non-working student, working student), and cohabitation (with family, without family). The questionnaire also included items on the following risk behaviors: smoking habit (yes, no), number of cigarettes per day, binge drinking in the last year (yes, no), frequency of binge drinking (never in the last 2 months, at least once in the last 2 months), physical activity (active, inactive), and BMI (<25 kg/m2, ≥25 kg/m2). We also considered the number of coexisting risk behaviors (smoking, binge drinking, BMI ≥25 kg/m2 and physical inactivity). In our inquiry, we adopted a definition of binge drinking given by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), namely the consumption within a period of two hours of ≥5 UA (Unit of Alcohol) for men, and of ≥4 UA for women.
Statistical analysis Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.
Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.
Statistical analysis:
Age was presented as a mean with standard deviation (SD), while dichotomous variables were presented as numbers and proportions. For statistical analysis, several tests were used. Student t-test was used to compare means. The association between socio-demographic characteristics and risk factors was evaluated by Pearson’s chi-square test or Fisher’s exact test (when the expected frequencies were less than or equal to 5). To assess the impact of missing data we performed sensitivity analysis including only questionnaires reporting all the data.
Results:
The students who accepted to fill in the questionnaire and included in the survey were 494 (65% women). Students were equally distributed across three degree courses: Medicine/Dentistry (33.4%), Nursing (34%) and Other Health Professions (32.6%). Eight point four percent of them declared to be a working student, 10% were living without family.
As shown in table 1, 23.2% of the students were smokers and 5.5% smoked between 10 and 20 cigarettes a day (no student declared to smoke more than 20 cigarettes per day). Half of the students referred to have practiced binge drinking at least once in the last 12 months, and 14.6% of them declared more than one episode every two months. Thirty-five percent of the individuals did not practice any weekly physical activity. Excessive bodyweight (BMI ≥25 kg/m2) was present in 7.9% of subjects. In particular, 6.6% were overweight and 1.3% were obese. As to the co-existence of risk behaviors, 23.2% of students did not present any of the four risk behaviors (smoking habit, binge drinking, physical inactivity, and BMI ≥25 kg/m2) and 29.9% presented two or more risk behaviors. In detail, 46.9% presented with one risk factor, 21.5% with two risk behaviors, 8.2% with three risk behaviors, and only one student with four risk behaviors.
Prevalence of risk behaviors (n. 494)
Associations between risk behaviors and socio-demographic factors We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.
Associations between smoking and socio-demographic characteristics
* Only smoker students. Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Associations between binge drinking and socio-demographic characteristics
*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.
Chi-square for categorical variables and t-test for age.
Associations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics
Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.
Associations between smoking and socio-demographic characteristics
* Only smoker students. Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Associations between binge drinking and socio-demographic characteristics
*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.
Chi-square for categorical variables and t-test for age.
Associations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics
Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Associations between risk behaviors and socio-demographic factors:
We analyzed the associations between risks behaviors and socio-demographic characteristics. We found statistically significant associations between smoking habit and sex and type of course (nursing students had the highest smoking habit) (table 2). We found an association of the number of cigarettes smoked per day with occupational status: 50% of working students smoked more than 10 cigarettes per day, while only 20.2% of non-working students smoked more than 10 cigarettes per day (table 2). Binge drinking, was associated with sex (male) (table 3). Among those who practiced binge drinking at least once every 2 months, 38.4% were men, 53.9% were working students, and 50% lived without family (table 3). No associations with socio-demographic characteristics was evident for BMI (table 4). We found significant associations of physical inactivity with sex and cohabitation status, 44.2% of women and 57.1% of subjects living without family were physically inactive (table 4). The number of coexisting risk behaviors was associated with sex, type of course, and occupational status. Among men 36.8% had two or more coexisting risk behaviors. Similarly, two or more risk behaviors were presented in 39.6% of nursing students and 44.7% of working students (table 4). The reliability of our results was confirmed in the sensitivity analysis. No differences in these results were observed considering only the subjects with all data reported in the questionnaire.
Associations between smoking and socio-demographic characteristics
* Only smoker students. Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Associations between binge drinking and socio-demographic characteristics
*Only Binge drinking. Data are shown as n (%) or mean±standard deviation.
Chi-square for categorical variables and t-test for age.
Associations between overweight, physical inactivity, coexisting risk factors and socio-demographic characteristics
Data are shown as n (%) or mean±standard deviation.
Chi-square or Fisher’s exact test for categorical variables and t-test for age.
Discussion:
We discuss the data from our survey in the light of similar data recorded by ISTAT (Italian National Statistical Institute) among young adults in recent years (13) and of the results from surveys conducted on a population of Italian university students and of healthcare students (6, 21).
Smoking habit The prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.
The prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.
Binge drinking Fifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).
Other Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).
Fifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).
Other Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).
Physical inactivity and excessive bodyweight Our study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).
Within our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.
Our study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).
Within our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.
Associations of coexisting risk behaviors When examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.
Regarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students.
When examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.
Regarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students.
Smoking habit:
The prevalence of smokers was 23.2%, consistent with data regarding smoking habit among Italian young adults (19% between 18 and 19 years of age, and 23.9% between 20 and 24 years) (13). This prevalence was lower than among Italian university students in other surveys (38.5%) (6). When analyzing papers on healthcare students we found that smoking prevalence ranged from 19.2% to 45% (10, 18, 25). It is worth noting that the prevalence of smoking was higher among Italians than foreign students (2, 16). The association between smoking habit and sex (male) and student course (nursing students) was observed in other researchers (3, 6, 7, 13). In our study, 76.1% of students smoked less than 10 cigarettes a day. This result was consistent with data of the Italian young adult population (6, 13). Overall, our research showed an association between total number of cigarettes smoked per day and working status of the student.
Binge drinking:
Fifty percent of the students practiced binge drinking at least once within the last 12 months. This figure was higher than in the Italian young adult population (17% between 18 and 24 years old) (13). It could be explained by the different definition of binge drinking adopted by ISTAT, i.e. the consumption of ≥6 UA, within a single occasion, irrespective of sex. In contrast, we followed the definition given by the NIAAA, i.e. the consumption within a period of two hours of ≥5 UA for men, and of ≥4 UA for women. We also confirmed the association between binge drinking and sex, as evidenced in other surveys (13). Finally, 70.4% of binge drinkers declared less than one episode every two months (i.e. < 6 times per year).
Other Italian surveys did not seem to have specifically investigated binge drinking but only alcohol drinking frequency. For example, a recent study conducted in 2017 at the Second University of Naples on under- and post-graduate healthcare students evidenced that 76.1% of subjects drink alcohol regularly (85% of medical students, 77.4% of medical residents, and 63% of other healthcare students). This behavior proved to be higher among men, smokers, younger subjects, and students with lower BMI (15). In another survey on university students, the prevalence of subjects who declared to drink weekly alcoholic beverages was 42.2% (21).
Physical inactivity and excessive bodyweight:
Our study revealed that 35% of students were physically inactive (i.e. they never or almost never practice physical activity). Our subjects proved to be less active than Italian young adults (13) and to be more physically inactive than other Italian university students (6). Our study confirmed the association between physical inactivity and sex (female) and cohabitation status (living without family) (6, 13).
Within our sample, 7.9% of subjects had excessive bodyweight. Data regarding Italian young adults (18-24 years old) derived from ISTAT surveys, show 17.4% of subjects with excessive bodyweight (13). Our prevalence was also lower than among Italian university students (11.2% of excessive bodyweight) (6). Interestingly, we could not find any correlation between excessive bodyweight and the considered socio-demographic factors. In spite of a higher prevalence of physical inactivity, our population showed a lower BMI than general population. This figure could be explained by more attention paid to diet and healthy food within our population of healthcare students.
Associations of coexisting risk behaviors:
When examining the coexistence of smoking habit, binge drinking, physical inactivity, and excessive bodyweight, 48.9% of the students had one risk behavior, 21.5% two risk behaviors, and 8.4% three or four risk behaviors; the remaining 23.2% declared no risk behaviors. These results were at odds with ISTAT data regarding Italian young adults, according to which 32.2% of subjects presented with no risk factor, 39.1% with one risk factor, 22.3% with two risk behaviors, and 6.4% with three or four risk behaviors (13). The difference between these results could be explained by several factors. ISTAT examined a broader age group (14-44 years old) and considered in addition to the four factors reported in our study, the influence of high daily consumption of alcohol. Our study, in addition, adopted a more stringent definition of binge drinking as compared with the one used by ISTAT.
Regarding the associations with total number of coexisting risk behaviors, this study confirmed a documented correlation with sex (male) (6) and showed for the first time an association with degree course (nursing students) and occupational status (working students). On the contrary, it did not show any association with subjects’ age. No other studies examined the coexistence of risk behaviors within Italian university students and healthcare students.
Conclusions:
When considering the influence of risk behaviors on young adults, healthcare students represent a sensitive group of individuals. Healthcare students with risk behaviors might put not only their health at risk, but also the health of their patients. This condition is relevant not only during their internship while they attend the university courses, but also in the future perspective of a healthcare employment. Therefore, as health promoting universities, academic institutions should act to minimize the impact of negative behaviors on all their students. By doing so, they could help not only the students, but the community as a whole (20). This goal could be achieved by adopting health promotion programs. Notably, several universities have already implemented programs to improve the wellbeing of their students and of the communities they belong to (11, 19, 26, 27). Such initiatives should be tailored to the population characteristics.
For this reason, by providing information on prevalence of risk behaviors and the potential associations with socio-demographic factors, this study might be an important starting point in the definition of targeted strategies, aimed at reducing risk behaviors. The occupational health physicians working in universities and in other educational institutions, could play an important role in the assessment of health promotion needs. Indeed, the preventive and periodic health examinations are not only convenient opportunities for medical counselling, but also for collecting important data on population characteristics. This survey examined the health and wellbeing behaviors of its own healthcare student population, and highlighted some behaviors that are similar to expectations, and others that are not. This information will enable to tailor programs and interventions aimed at specific groups of students. For example, in our study population health promotion interventions should be mainly addressed towards nursing students, who seemed to have a higher prevalence of coexisting risk factors (particularly smoking habit). Similarly, specific interventions should also be addressed towards male students and working students, while physical activity should be mainly improved among female students.
The present study also has limitations. First, the design of the study was monocentric, since students were selected from a single university. Therefore, the sample cannot be considered representative of the actual Italian population of healthcare students. Second, the average age of the students was not homogenous across degree courses and statistically significant differences were found among them. Finally, individuals within our population could have minimized the magnitude of their risk behaviors, even if the questionnaire was anonymous. In fact, they could have concealed their unhealthy behaviors on account of their role as healthcare students.
Despite these limitations, our study is the first, to our knowledge, to investigate risk behaviors (including binge drinking) in a broad population of healthcare students, analyzing differences across different healthcare courses.
In conclusion, university attendance is a transitional period offering good opportunities for acquisition of healthy lifestyles. Occupational Health Services in the universities could be a reference point for all the initiatives of health promotion addressed to the student population, i.e. the tomorrow’s workforce.
No potential conflict of interest relevant to this article was reported by the authors
| Background:
Risk behaviors are frequent among young adults and they are particularly relevant when considering healthcare students.
Methods:
Healthcare students filled an anonymous multiple-choice questionnaire on the occasion of the occupational health visit that preceded their hospital internship. The questionnaire covered socio-demographic characteristics (including student's working status and cohabitation) and risk behaviors. We evaluated the prevalence of risk behaviors and their association with socio-demographic characteristics.
Results:
The sample consisted of 494 students (65% women): 23.2% were smokers, 7.9% had excessive bodyweight, 35% did not practice any physical activity and 50% reported binge drinking at least once in the last 12 months. We found associations of male sex (30.5%) and being nursing students (29.9%) with smoking habit. The frequency of binge drinking was higher in men (38.4%), working students (53.9%), and among those who lived without family (50%). Physical inactivity was associated with female sex (44.2%) and living without family (57.1%). Finally, the co-presence of 2 risk behaviors or more was higher in men (36.8%), in nursing students (39.6%) and in working students (44.7%).
Conclusions:
Our findings regarding the prevalence of risk behaviors and their potential association with socio-demographic factors may be a clue to the definition of targeted strategies aimed at reducing of risk behaviors among healthcare students. | Introduction:
Risk behaviors are lifestyle characteristics likely to cause potential harm to the subject, both from a physical and a psychological point of view. They generally include smoking habit, alcohol abuse, physical inactivity, and excessive bodyweight (5). Scientific literature shows that risky habits are usually adopted during adolescence (4). Nevertheless, they could exacerbate and become permanent between 18 and 30 years, when young adults experience a significant amount of stress (22). In fact, during this time, individuals often leave their usual social contexts and confront new academic and working challenges. In addition, there is evidence that unhealthy lifestyles among young adults are strongly linked to disabilities and health problems (12, 28). Therefore, if these behaviors are detected and modified at an early stage, dangerous developments could be avoided (23). In recent years, some academic institutions have tried to address the problem among their students, implementing the concept of “health promoting universities and colleges”. Several documents have been produced, the last being the “Okanagan Charter”, aimed at creating campus culture of well-being and improving the health of the people who live, learn, and work in university campuses, including students (20).
The problem of risk behaviors becomes stringent when considering healthcare students. They – as future health care providers – will play a key role in health promotion and their attitude highly influences not only their health and performance, but also the health status of their patients as well as of the general population. Some studies suggest that the healthy behaviors of healthcare workers influence patients’ attitudes towards preventive counselling and their motivation towards healthy lifestyle choices (9). So, healthcare workers and students who themselves lead healthy lifestyles are more likely to have a positive influence on patients and help them make healthy choices (1, 8, 17).
Furthermore, since these students are in close contact with patients, risk behaviors may be a problem during their hospital internship. In fact, risk behaviors of these students could affect their health, increase occupational injuries and impair the relation with patients (14). For example, binge drinking and alcohol abuse may cause sleep disruptions and alterations in the circadian rhythm in young people, hence increasing the risks of clinical mistakes and work-related injuries in unskilled students. Similarly, overweight and obesity may contribute to musculoskeletal disorders and physical injuries, mainly within nursing students (14). Smoking habit might increase the cancer risk among x-ray technician students, potentially exposed to ionizing radiations. Finally, occupational hazards in healthcare sector (long working hours, shiftwork, stress, limited access to healthy and regular food, sedentary jobs) could themselves influence employees’ health risk behavior.
For these reasons, universities should provide healthcare students with adequate training on the promotion of healthy behaviors. In order to design efficient strategies for the reduction of unhealthy habits, academic institutions should conduct studies to examine the influence of risk behaviors within the specific population of healthcare students (20). Italian occupational physicians started to address this issue during a conference organized in Milan in 2016, attended by the representatives of all the universities of Lombardy, the most populous and richest region in Italy (24).
Our study aims to investigate the prevalence of risk behaviors (smoking, binge drinking, physical inactivity, and excessive bodyweight) in a population of healthcare students attending an Italian university of Northern Italy. Our focus was on the evaluation of the possible associations between unhealthy behaviors and some socio-demographic factors in such a population. We might, in this way, identify specific health promotion programs targeted at the population of healthcare students (6).
Conclusions:
When considering the influence of risk behaviors on young adults, healthcare students represent a sensitive group of individuals. Healthcare students with risk behaviors might put not only their health at risk, but also the health of their patients. This condition is relevant not only during their internship while they attend the university courses, but also in the future perspective of a healthcare employment. Therefore, as health promoting universities, academic institutions should act to minimize the impact of negative behaviors on all their students. By doing so, they could help not only the students, but the community as a whole (20). This goal could be achieved by adopting health promotion programs. Notably, several universities have already implemented programs to improve the wellbeing of their students and of the communities they belong to (11, 19, 26, 27). Such initiatives should be tailored to the population characteristics.
For this reason, by providing information on prevalence of risk behaviors and the potential associations with socio-demographic factors, this study might be an important starting point in the definition of targeted strategies, aimed at reducing risk behaviors. The occupational health physicians working in universities and in other educational institutions, could play an important role in the assessment of health promotion needs. Indeed, the preventive and periodic health examinations are not only convenient opportunities for medical counselling, but also for collecting important data on population characteristics. This survey examined the health and wellbeing behaviors of its own healthcare student population, and highlighted some behaviors that are similar to expectations, and others that are not. This information will enable to tailor programs and interventions aimed at specific groups of students. For example, in our study population health promotion interventions should be mainly addressed towards nursing students, who seemed to have a higher prevalence of coexisting risk factors (particularly smoking habit). Similarly, specific interventions should also be addressed towards male students and working students, while physical activity should be mainly improved among female students.
The present study also has limitations. First, the design of the study was monocentric, since students were selected from a single university. Therefore, the sample cannot be considered representative of the actual Italian population of healthcare students. Second, the average age of the students was not homogenous across degree courses and statistically significant differences were found among them. Finally, individuals within our population could have minimized the magnitude of their risk behaviors, even if the questionnaire was anonymous. In fact, they could have concealed their unhealthy behaviors on account of their role as healthcare students.
Despite these limitations, our study is the first, to our knowledge, to investigate risk behaviors (including binge drinking) in a broad population of healthcare students, analyzing differences across different healthcare courses.
In conclusion, university attendance is a transitional period offering good opportunities for acquisition of healthy lifestyles. Occupational Health Services in the universities could be a reference point for all the initiatives of health promotion addressed to the student population, i.e. the tomorrow’s workforce.
No potential conflict of interest relevant to this article was reported by the authors | Background:
Risk behaviors are frequent among young adults and they are particularly relevant when considering healthcare students.
Methods:
Healthcare students filled an anonymous multiple-choice questionnaire on the occasion of the occupational health visit that preceded their hospital internship. The questionnaire covered socio-demographic characteristics (including student's working status and cohabitation) and risk behaviors. We evaluated the prevalence of risk behaviors and their association with socio-demographic characteristics.
Results:
The sample consisted of 494 students (65% women): 23.2% were smokers, 7.9% had excessive bodyweight, 35% did not practice any physical activity and 50% reported binge drinking at least once in the last 12 months. We found associations of male sex (30.5%) and being nursing students (29.9%) with smoking habit. The frequency of binge drinking was higher in men (38.4%), working students (53.9%), and among those who lived without family (50%). Physical inactivity was associated with female sex (44.2%) and living without family (57.1%). Finally, the co-presence of 2 risk behaviors or more was higher in men (36.8%), in nursing students (39.6%) and in working students (44.7%).
Conclusions:
Our findings regarding the prevalence of risk behaviors and their potential association with socio-demographic factors may be a clue to the definition of targeted strategies aimed at reducing of risk behaviors among healthcare students. | 6,360 | 285 | [
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] | [CONTENT] risk behaviors | healthcare students | smoking | binge drinking | physical inactivity [SUMMARY] | [CONTENT] risk behaviors | healthcare students | smoking | binge drinking | physical inactivity [SUMMARY] | [CONTENT] risk behaviors | healthcare students | smoking | binge drinking | physical inactivity [SUMMARY] | [CONTENT] risk behaviors | healthcare students | smoking | binge drinking | physical inactivity [SUMMARY] | [CONTENT] risk behaviors | healthcare students | smoking | binge drinking | physical inactivity [SUMMARY] | [CONTENT] risk behaviors | healthcare students | smoking | binge drinking | physical inactivity [SUMMARY] | [CONTENT] Alcohol Drinking | Cross-Sectional Studies | Female | Health Personnel | Health Promotion | Humans | Male | Prevalence | Risk-Taking | Students | Universities | Young Adult [SUMMARY] | [CONTENT] Alcohol Drinking | Cross-Sectional Studies | Female | Health Personnel | Health Promotion | Humans | Male | Prevalence | Risk-Taking | Students | Universities | Young Adult [SUMMARY] | [CONTENT] Alcohol Drinking | Cross-Sectional Studies | Female | Health Personnel | Health Promotion | Humans | Male | Prevalence | Risk-Taking | Students | Universities | Young Adult [SUMMARY] | [CONTENT] Alcohol Drinking | Cross-Sectional Studies | Female | Health Personnel | Health Promotion | Humans | Male | Prevalence | Risk-Taking | Students | Universities | Young Adult [SUMMARY] | [CONTENT] Alcohol Drinking | Cross-Sectional Studies | Female | Health Personnel | Health Promotion | Humans | Male | Prevalence | Risk-Taking | Students | Universities | Young Adult [SUMMARY] | [CONTENT] Alcohol Drinking | Cross-Sectional Studies | Female | Health Personnel | Health Promotion | Humans | Male | Prevalence | Risk-Taking | Students | Universities | Young Adult [SUMMARY] | [CONTENT] student risk behaviors | students risk behaviors | risk behaviors lifestyle | behaviors health risk | unhealthy habits academic [SUMMARY] | [CONTENT] student risk behaviors | students risk behaviors | risk behaviors lifestyle | behaviors health risk | unhealthy habits academic [SUMMARY] | [CONTENT] student risk behaviors | students risk behaviors | risk behaviors lifestyle | behaviors health risk | unhealthy habits academic [SUMMARY] | [CONTENT] student risk behaviors | students risk behaviors | risk behaviors lifestyle | behaviors health risk | unhealthy habits academic [SUMMARY] | [CONTENT] student risk behaviors | students risk behaviors | risk behaviors lifestyle | behaviors health risk | unhealthy habits academic [SUMMARY] | [CONTENT] student risk behaviors | students risk behaviors | risk behaviors lifestyle | behaviors health risk | unhealthy habits academic [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | drinking | binge | binge drinking | italian | healthcare | data [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | drinking | binge | binge drinking | italian | healthcare | data [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | drinking | binge | binge drinking | italian | healthcare | data [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | drinking | binge | binge drinking | italian | healthcare | data [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | drinking | binge | binge drinking | italian | healthcare | data [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | drinking | binge | binge drinking | italian | healthcare | data [SUMMARY] | [CONTENT] health | students | behaviors | patients | healthy | risk | healthcare | risk behaviors | injuries | problem [SUMMARY] | [CONTENT] test | analysis | following | statistical analysis | m2 | 25 kg | 25 kg m2 | kg | kg m2 | statistical [SUMMARY] | [CONTENT] table | associations | behaviors | risk | students | risk behaviors | test | socio demographic characteristics | demographic characteristics | shown [SUMMARY] | [CONTENT] health | students | behaviors | population | risk | healthcare | health promotion | promotion | universities | risk behaviors [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | test | italian | binge | drinking | 13 | binge drinking [SUMMARY] | [CONTENT] students | risk | behaviors | risk behaviors | test | italian | binge | drinking | 13 | binge drinking [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] Healthcare ||| ||| [SUMMARY] | [CONTENT] 494 | 65% | 23.2% | 7.9% | 35% | 50% | the last 12 months ||| 30.5% | 29.9% ||| 38.4% | 53.9% | 50% ||| 44.2% | 57.1% ||| 2 | 36.8% | 39.6% | 44.7% [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] ||| Healthcare ||| ||| ||| ||| 494 | 65% | 23.2% | 7.9% | 35% | 50% | the last 12 months ||| 30.5% | 29.9% ||| 38.4% | 53.9% | 50% ||| 44.2% | 57.1% ||| 2 | 36.8% | 39.6% | 44.7% ||| [SUMMARY] | [CONTENT] ||| Healthcare ||| ||| ||| ||| 494 | 65% | 23.2% | 7.9% | 35% | 50% | the last 12 months ||| 30.5% | 29.9% ||| 38.4% | 53.9% | 50% ||| 44.2% | 57.1% ||| 2 | 36.8% | 39.6% | 44.7% ||| [SUMMARY] |
||||||
Accuracy of suprascapular notch cross-sectional area by MRI in the diagnosis of suprascapular nerve entrapment syndrome: a retrospective pilot study. | 35700981 | Previous studies have demonstrated that morphological changes in the suprascapular notch are closely associated with suprascapular nerve entrapment syndrome (SNES). Thus, we hypothesized that the suprascapular notch cross-sectional area (SSNCSA) could be a good diagnostic parameter to assess SNES. | BACKGROUND | We acquired suprascapular notch data from 10 patients with SNES and 10 healthy individuals who had undergone shoulder magnetic resonance imaging (S-MRI) and had no evidence of SNES. T2-weighted coronal magnetic resonance images were acquired from the shoulder. We analyzed the SSNCSA at the shoulder on S-MRI using our image-analysis program (INFINITT PACS). The SSNCSA was measured as the suprascapular notch, which was the most affected site in coronal S-MRI images. | METHODS | The mean SSNCSA was 64.50 ± 8.93 mm2 in the control group and 44.94 ± 10.40 mm2 in the SNES group. Patients with SNES had significantly lower SSNCSA (P < 0.01) than those in the control group. Receiver operating curve analysis showed that the best cut-off of the SSNCSA was 57.49 mm2, with 80.0% sensitivity, 80.0% specificity, and an area under the curve of 0.92 (95% CI [0.79, 1.00]). | RESULTS | The SSNCSA was found to have acceptable diagnostic properties for detecting SNES. We hope that these results will help diagnose SNES objectively. | CONCLUSIONS | [
"Humans",
"Scapula",
"Pilot Projects",
"Retrospective Studies",
"Nerve Compression Syndromes",
"Magnetic Resonance Imaging"
] | 9726457 | Introduction | Suprascapular nerve entrapment syndrome (SNES) is a peripheral neuropathy caused by compression of the suprascapular nerve. SNES is uncommon in patients with shoulder dysfunction. However, SNES has clinical implications because it innervates approximately 60–70% of the shoulder joint and often leads to pain over the lateral and posterior aspects of the shoulder as well as weakness of the infraspinatus or supraspinatus muscles due to suprascapular nerve innervation [1–6]. Trauma or traction injury due to repetitive overhead activities or massive rotator cuff tears occurs in SNES [7–10]. SNES should be differentiated from disorders of the cervical part of the spinal cord, damage to the brachial plexus, cervical discopathy, or diseases of the shoulder joint, such as damage to the rotator cuff or degeneration of the shoulder [11–13]. Thus, an exact diagnosis is important for managing SNES.
The diagnosis of SNES is typically based on physical examinations and interview [14,15]. Other additional examinations for the diagnosis of SNES include imaging modalities (ultrasonography, X-ray, and computed tomography), electromyography (EMG), and assessment of conduction velocity from the neck nerve point to the supraspinatus muscles [10,16–19]. Although EMG is the gold standard for the diagnosis of SNES, shoulder magnetic resonance imaging (S-MRI) is also useful for the analysis of pathologic abnormalities of the suprascapular notch [2]. Podgórski et al. [20] reported that there are many anatomical variations in the suprascapular notch region, and the shapes of the suprascapular notch are highly diverse. In addition, the suprascapular nerve is most commonly compressed at the suprascapular notch [2]. However, few studies have investigated how morphological changes in the suprascapular notch affect the SNES. Moreover, no studies have examined the clinical optimal cut-off point of the suprascapular notch cross-sectional area (SSNCSA to diagnose SNES).
Therefore, to assess the relationship between SNES and the suprascapular notch, we developed a new morphological diagnostic parameter called SSNCSA. The SSNCSA has not yet been analyzed for its correlation with SNES. We hypothesized that the SSNCSA is an important morphological parameter in the diagnosis of SNES. | null | null | Results | Demographic characteristics were not significantly different between the groups. The average SSNCSA was 64.50 ± 8.93 mm2 in the control group and 44.94 ± 10.40 mm2 in the SNES group (Table 1). Patients with SNES had significantly lower SSNCSA scores (P < 0.01) than those in the control group (Table 1). The ROC analysis showed that the best cut-off value of the SSNCSA was 57.49 mm2, with 80.0% sensitivity, 80.0% specificity, and the AUC of SSNCSA was 0.92 (95% CI [0.79, 1.00]) (Table 2, Fig. 2). | null | null | [
"Participants",
"Imaging parameters",
"Image analysis",
"Statistical analysis"
] | [
"This original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.\nThe SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.\nThe SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).",
"S-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.",
"SSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).",
"We compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data."
] | [
null,
null,
null,
null
] | [
"Introduction",
"Materials and Methods",
"Participants",
"Imaging parameters",
"Image analysis",
"Statistical analysis",
"Results",
"Discussion"
] | [
"Suprascapular nerve entrapment syndrome (SNES) is a peripheral neuropathy caused by compression of the suprascapular nerve. SNES is uncommon in patients with shoulder dysfunction. However, SNES has clinical implications because it innervates approximately 60–70% of the shoulder joint and often leads to pain over the lateral and posterior aspects of the shoulder as well as weakness of the infraspinatus or supraspinatus muscles due to suprascapular nerve innervation [1–6]. Trauma or traction injury due to repetitive overhead activities or massive rotator cuff tears occurs in SNES [7–10]. SNES should be differentiated from disorders of the cervical part of the spinal cord, damage to the brachial plexus, cervical discopathy, or diseases of the shoulder joint, such as damage to the rotator cuff or degeneration of the shoulder [11–13]. Thus, an exact diagnosis is important for managing SNES.\nThe diagnosis of SNES is typically based on physical examinations and interview [14,15]. Other additional examinations for the diagnosis of SNES include imaging modalities (ultrasonography, X-ray, and computed tomography), electromyography (EMG), and assessment of conduction velocity from the neck nerve point to the supraspinatus muscles [10,16–19]. Although EMG is the gold standard for the diagnosis of SNES, shoulder magnetic resonance imaging (S-MRI) is also useful for the analysis of pathologic abnormalities of the suprascapular notch [2]. Podgórski et al. [20] reported that there are many anatomical variations in the suprascapular notch region, and the shapes of the suprascapular notch are highly diverse. In addition, the suprascapular nerve is most commonly compressed at the suprascapular notch [2]. However, few studies have investigated how morphological changes in the suprascapular notch affect the SNES. Moreover, no studies have examined the clinical optimal cut-off point of the suprascapular notch cross-sectional area (SSNCSA to diagnose SNES).\nTherefore, to assess the relationship between SNES and the suprascapular notch, we developed a new morphological diagnostic parameter called SSNCSA. The SSNCSA has not yet been analyzed for its correlation with SNES. We hypothesized that the SSNCSA is an important morphological parameter in the diagnosis of SNES.",
"Participants This original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.\nThe SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.\nThe SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).\nThis original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.\nThe SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.\nThe SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).\nImaging parameters S-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.\nS-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.\nImage analysis SSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).\nSSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).\nStatistical analysis We compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data.\nWe compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data.",
"This original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.\nThe SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.\nThe SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).",
"S-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.",
"SSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).",
"We compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data.",
"Demographic characteristics were not significantly different between the groups. The average SSNCSA was 64.50 ± 8.93 mm2 in the control group and 44.94 ± 10.40 mm2 in the SNES group (Table 1). Patients with SNES had significantly lower SSNCSA scores (P < 0.01) than those in the control group (Table 1). The ROC analysis showed that the best cut-off value of the SSNCSA was 57.49 mm2, with 80.0% sensitivity, 80.0% specificity, and the AUC of SSNCSA was 0.92 (95% CI [0.79, 1.00]) (Table 2, Fig. 2).",
"This pilot study aimed to determine the clinical implications of SSNCSA in SNES. The present study showed that SSNCSA of 57.49 mm2 had 80.0% sensitivity and 80.0% specificity for predicting SNES. This result demonstrates that the SSNCSA could be a meaningful predictor of SNES.\nSNES is a neuropathic condition in which the suprascapular nerve is compressed along the pathway. Traumatic injuries, such as clavicular fractures, scapular fractures, proximal humerus fractures, and dislocation of the acromioclavicular joint or shoulder, are common causes of suprascapular nerve damage [4,12,13,21]. Most importantly, suprascapular nerve compression most commonly occurs at the suprascapular notch, and its symptoms and signs are caused by nerve compression based on morphological changes in the suprascapular notch [2,22,23]. Structures around the suprascapular nerve can be compressed and injured by various mechanical factors. Direct compression in the suprascapular notch region (e.g., labral cyst, ganglion cyst, tumor) [11], continuous nerve irritation after rotator cuff injuries, or inflammation of the shoulder can all lead to SNES [3,24].\nSNES diagnosis is based on patient history and physical examinations while ruling out other similar pathologies, including cervical radiculopathy, cervical discopathy, and various rotator cuff injuries. Imaging studies can also be used for diagnosis [14,15]. Ultrasound, X-ray, computed tomography, and MRI provide information on the suprascapular nerve and surrounding structures, helping diagnose SNES [15,25,26]. Nerve conduction velocity and EMG are the gold standards for SNES diagnosis [14]. However, nerve conduction velocity and EMG cannot be performed in all patients and the efficacy of EMG is low. Although EMG can confirm nerve conduction problems or muscle weakness, it is not useful for the differential diagnosis of various shoulder diseases. Moreover, negative EMG results cannot rule out SNES when clinical signs and symptoms are highly suspicious of SNES [14]. Therefore, MRI is more commonly performed in shoulder injury patients than EMG, and the diagnostic criteria of MRI can be a useful tool to diagnose SNES.\nEven though it has been reported that SNES is more likely to occur in patients with a V-shaped or narrow suprascapular notch, a significant correlation between the suprascapular notch type and SNES has not been confirmed. Ürgüden et al. [9] reported that Rangachery types 4 and 5 of the SSN may increase the risk of suprascapular nerve injury during rotator cuff tear operations. However, no clinical studies have been conducted to prove this theory. Polguj et al. [17] reported that the size of the suprascapular notch is a major risk factor for SNES [22]; however, there is no study to analyze suprascapular notch objectively. In other words, the narrowed suprascapular notch is considered a major morphological parameter of SNES. Therefore, we believe that analyzing the cross-sectional area of the suprascapular notch is the most important factor in the diagnosis of SNES, and we designed the present study to prove the correlation between SSNCSA and SNES.\nAs the bone margin of the suprascapular notch has a wavy or curved contour and multiple signal intensities within the narrowed site, it is difficult to measure. Thus, the length or thickness of the suprascapular notch is not an appropriate measurement for diagnosing SNES. Instead, the cross-sectional area of the suprascapular notch may predict SNES effectively because the SSNCSA, which measures the whole cross-sectional area of the suprascapular notch represents the space of the SSN limited by surrounding structures. This study demonstrated that SSNCSA is a good morphological measurement diagnostic tool for SNES.\nThis study has some limitations. First, SNES has multiple causes such as rotator cuff tears, trauma, and repetitive overhead activities. Additionally, the structures around the suprascapular nerve, such as the supraspinatus muscle, infraspinatus muscles, suprascapular ligament, suprascapular notch, and spinoglenoid notches, were not considered. However, we focused only on the suprascapular notch, where the suprascapular nerve is most commonly compressed. In future studies, we will investigate other anatomical structures that affect SNES, especially the spinoglenoid notch, where the suprascapular nerve is also commonly compressed. Second, there might be some errors in the measurements of SSNCSA on S-MRI. Although we attempted to analyze this morphologic measurement method in the best plane that presents the suprascapular notch in the coronal image section, the coronal images we measured in the section image could be inhomogeneous because of differences in the cutting level or angle in the S-MRI as a result of individual anatomical differences and technical errors. Third, there are several alternative imaging diagnostic tools to evaluate SNES, such as ultrasound examination or computed tomography; however, this study analyzed only the measurement of the SSNCSA on S-MRI. Fourth, functional instability was not analyzed because it is a subjective finding that may vary from one interpretation to another. The goal of this study was to provide objective morphological indicators. Fifth, only a small number of patients were enrolled in the study. We enrolled all patients diagnosed with SNES at our hospital; there were only 10 SNES patients. Although this pilot study investigated a small number of patients, it is valuable because it provides diagnostic criteria using S-MRI, especially SSNCSA. Sixth, since this was a retrospective study, patients who underwent S-MRI and had no structural abnormalities were enrolled in the control group. The control group might have experienced shoulder pain and may not represent the normal population. In a future study, healthy individuals should be recruited in the control group and scanned for S-MRI prospectively, and a more accurate SSNCSA of normal people can be obtained.\nDespite several limitations, we present the diagnostic criteria for SNES using S-MRI for the first time, especially using SSNCSA. In addition, the present study showed that the SSNCSA can be an objective and useful diagnostic tool for SNES.\nWe concluded that these data strengthen the finding that SSNCSA plays a significant role in determining SNES."
] | [
"intro",
"materials|methods",
null,
null,
null,
null,
"results",
"discussion"
] | [
"Anatomy",
"Diagnosis",
"Magnetic resonance imaging",
"Nerve compression syndromes",
"Shoulder",
"Syndrome"
] | Introduction:
Suprascapular nerve entrapment syndrome (SNES) is a peripheral neuropathy caused by compression of the suprascapular nerve. SNES is uncommon in patients with shoulder dysfunction. However, SNES has clinical implications because it innervates approximately 60–70% of the shoulder joint and often leads to pain over the lateral and posterior aspects of the shoulder as well as weakness of the infraspinatus or supraspinatus muscles due to suprascapular nerve innervation [1–6]. Trauma or traction injury due to repetitive overhead activities or massive rotator cuff tears occurs in SNES [7–10]. SNES should be differentiated from disorders of the cervical part of the spinal cord, damage to the brachial plexus, cervical discopathy, or diseases of the shoulder joint, such as damage to the rotator cuff or degeneration of the shoulder [11–13]. Thus, an exact diagnosis is important for managing SNES.
The diagnosis of SNES is typically based on physical examinations and interview [14,15]. Other additional examinations for the diagnosis of SNES include imaging modalities (ultrasonography, X-ray, and computed tomography), electromyography (EMG), and assessment of conduction velocity from the neck nerve point to the supraspinatus muscles [10,16–19]. Although EMG is the gold standard for the diagnosis of SNES, shoulder magnetic resonance imaging (S-MRI) is also useful for the analysis of pathologic abnormalities of the suprascapular notch [2]. Podgórski et al. [20] reported that there are many anatomical variations in the suprascapular notch region, and the shapes of the suprascapular notch are highly diverse. In addition, the suprascapular nerve is most commonly compressed at the suprascapular notch [2]. However, few studies have investigated how morphological changes in the suprascapular notch affect the SNES. Moreover, no studies have examined the clinical optimal cut-off point of the suprascapular notch cross-sectional area (SSNCSA to diagnose SNES).
Therefore, to assess the relationship between SNES and the suprascapular notch, we developed a new morphological diagnostic parameter called SSNCSA. The SSNCSA has not yet been analyzed for its correlation with SNES. We hypothesized that the SSNCSA is an important morphological parameter in the diagnosis of SNES.
Materials and Methods:
Participants This original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.
The SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.
The SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).
This original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.
The SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.
The SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).
Imaging parameters S-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.
S-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.
Image analysis SSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).
SSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).
Statistical analysis We compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data.
We compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data.
Participants:
This original research protocol was approved by The Catholic Kwandong University Institutional Review Board (IRB no. IS21RISI0021). We reviewed electronic medical records of patients who had visited the shoulder orthopedic clinic with SNES from November 2015 to December 2020 and who had taken S-MRI within six months of the visit.
The SNES group included patients diagnosed with SNES by attending physicians according to their history, physical examination, and imaging modality. In addition, the final diagnosis was confirmed using EMG. Exclusion criteria were as follows: (1) history of scapular fracture, (2) history of shoulder surgery, and (3) no available S-MRI. Patients who underwent S-MRI and had no structural abnormalities were included in the control group.
The SNES group comprised 10 patients. There were 8 (80.0%) men and 2 (20.0%) women, with an average age of 43.90 ± 15.57 years (range, 18–60 years) (Table 1). To compare SSNCSA between individuals with and without SNES, we enrolled a control group consisting of individuals who wanted to undergo S-MRI for accurate diagnosis. The control group included patients with shoulder pain who wanted to undergo S-MRI. Moreover, patients in the control group did not show any abnormal findings on S-MRI. The control group comprised 10 individuals (6 men and 4 women) with a mean age of 42.70 ± 13.28 years (range, 20–73 years).
Imaging parameters:
S-MRI was performed on a 3.0T MR unit (MAGNETOM Skyra; Siemens Medical Solutions, Germany) and 3T Ingina (Philips Medical Systems, The Netherlands) scanners, and T2-weighted coronal plane turbo spin-echo images were acquired from all enrolled patients. The following S-MRI sequences were used: slice plane axial, field of view of 160 × 160 cm, repetition time of 619.0 milliseconds, echo time 13.0 milliseconds, flip angle 35 degrees, slice thickness 3.00 mm, matrix size 512 × 307 pixels, number of signals averaged = 2, scan time 4 min 32 s, and 3 > echo train length.
Image analysis:
SSNCSA measurements were done by a board-certified pain specialist with 15 years of experience who was blinded to the shoulder state. We obtained coronal T2-weighted turbo spin echoS-MRI images that presented the best visualization of the suprascapular nerve. We measured SSNCSA on S-MRI using our image-analysis program (INFINITT PACS, ver. 3.0; INFINITT Healthcare, Seoul, Korea) (Fig. 1).
Statistical analysis:
We compared the SSNCSA between the SNES and normal individuals using independent t-tests. Statistical significance was set at P < 0.05. Receiver operating curve (ROC) curve analysis was used to present the diagnostic values of SSNCSA in the diagnosis of SNES, and the diagnostic values included the cut-off points, area under the curve (AUC), specificity, and sensitivity. SPSS (version 22.0; IBM Inc., USA) was used to analyze the collected data.
Results:
Demographic characteristics were not significantly different between the groups. The average SSNCSA was 64.50 ± 8.93 mm2 in the control group and 44.94 ± 10.40 mm2 in the SNES group (Table 1). Patients with SNES had significantly lower SSNCSA scores (P < 0.01) than those in the control group (Table 1). The ROC analysis showed that the best cut-off value of the SSNCSA was 57.49 mm2, with 80.0% sensitivity, 80.0% specificity, and the AUC of SSNCSA was 0.92 (95% CI [0.79, 1.00]) (Table 2, Fig. 2).
Discussion:
This pilot study aimed to determine the clinical implications of SSNCSA in SNES. The present study showed that SSNCSA of 57.49 mm2 had 80.0% sensitivity and 80.0% specificity for predicting SNES. This result demonstrates that the SSNCSA could be a meaningful predictor of SNES.
SNES is a neuropathic condition in which the suprascapular nerve is compressed along the pathway. Traumatic injuries, such as clavicular fractures, scapular fractures, proximal humerus fractures, and dislocation of the acromioclavicular joint or shoulder, are common causes of suprascapular nerve damage [4,12,13,21]. Most importantly, suprascapular nerve compression most commonly occurs at the suprascapular notch, and its symptoms and signs are caused by nerve compression based on morphological changes in the suprascapular notch [2,22,23]. Structures around the suprascapular nerve can be compressed and injured by various mechanical factors. Direct compression in the suprascapular notch region (e.g., labral cyst, ganglion cyst, tumor) [11], continuous nerve irritation after rotator cuff injuries, or inflammation of the shoulder can all lead to SNES [3,24].
SNES diagnosis is based on patient history and physical examinations while ruling out other similar pathologies, including cervical radiculopathy, cervical discopathy, and various rotator cuff injuries. Imaging studies can also be used for diagnosis [14,15]. Ultrasound, X-ray, computed tomography, and MRI provide information on the suprascapular nerve and surrounding structures, helping diagnose SNES [15,25,26]. Nerve conduction velocity and EMG are the gold standards for SNES diagnosis [14]. However, nerve conduction velocity and EMG cannot be performed in all patients and the efficacy of EMG is low. Although EMG can confirm nerve conduction problems or muscle weakness, it is not useful for the differential diagnosis of various shoulder diseases. Moreover, negative EMG results cannot rule out SNES when clinical signs and symptoms are highly suspicious of SNES [14]. Therefore, MRI is more commonly performed in shoulder injury patients than EMG, and the diagnostic criteria of MRI can be a useful tool to diagnose SNES.
Even though it has been reported that SNES is more likely to occur in patients with a V-shaped or narrow suprascapular notch, a significant correlation between the suprascapular notch type and SNES has not been confirmed. Ürgüden et al. [9] reported that Rangachery types 4 and 5 of the SSN may increase the risk of suprascapular nerve injury during rotator cuff tear operations. However, no clinical studies have been conducted to prove this theory. Polguj et al. [17] reported that the size of the suprascapular notch is a major risk factor for SNES [22]; however, there is no study to analyze suprascapular notch objectively. In other words, the narrowed suprascapular notch is considered a major morphological parameter of SNES. Therefore, we believe that analyzing the cross-sectional area of the suprascapular notch is the most important factor in the diagnosis of SNES, and we designed the present study to prove the correlation between SSNCSA and SNES.
As the bone margin of the suprascapular notch has a wavy or curved contour and multiple signal intensities within the narrowed site, it is difficult to measure. Thus, the length or thickness of the suprascapular notch is not an appropriate measurement for diagnosing SNES. Instead, the cross-sectional area of the suprascapular notch may predict SNES effectively because the SSNCSA, which measures the whole cross-sectional area of the suprascapular notch represents the space of the SSN limited by surrounding structures. This study demonstrated that SSNCSA is a good morphological measurement diagnostic tool for SNES.
This study has some limitations. First, SNES has multiple causes such as rotator cuff tears, trauma, and repetitive overhead activities. Additionally, the structures around the suprascapular nerve, such as the supraspinatus muscle, infraspinatus muscles, suprascapular ligament, suprascapular notch, and spinoglenoid notches, were not considered. However, we focused only on the suprascapular notch, where the suprascapular nerve is most commonly compressed. In future studies, we will investigate other anatomical structures that affect SNES, especially the spinoglenoid notch, where the suprascapular nerve is also commonly compressed. Second, there might be some errors in the measurements of SSNCSA on S-MRI. Although we attempted to analyze this morphologic measurement method in the best plane that presents the suprascapular notch in the coronal image section, the coronal images we measured in the section image could be inhomogeneous because of differences in the cutting level or angle in the S-MRI as a result of individual anatomical differences and technical errors. Third, there are several alternative imaging diagnostic tools to evaluate SNES, such as ultrasound examination or computed tomography; however, this study analyzed only the measurement of the SSNCSA on S-MRI. Fourth, functional instability was not analyzed because it is a subjective finding that may vary from one interpretation to another. The goal of this study was to provide objective morphological indicators. Fifth, only a small number of patients were enrolled in the study. We enrolled all patients diagnosed with SNES at our hospital; there were only 10 SNES patients. Although this pilot study investigated a small number of patients, it is valuable because it provides diagnostic criteria using S-MRI, especially SSNCSA. Sixth, since this was a retrospective study, patients who underwent S-MRI and had no structural abnormalities were enrolled in the control group. The control group might have experienced shoulder pain and may not represent the normal population. In a future study, healthy individuals should be recruited in the control group and scanned for S-MRI prospectively, and a more accurate SSNCSA of normal people can be obtained.
Despite several limitations, we present the diagnostic criteria for SNES using S-MRI for the first time, especially using SSNCSA. In addition, the present study showed that the SSNCSA can be an objective and useful diagnostic tool for SNES.
We concluded that these data strengthen the finding that SSNCSA plays a significant role in determining SNES.
| Background:
Previous studies have demonstrated that morphological changes in the suprascapular notch are closely associated with suprascapular nerve entrapment syndrome (SNES). Thus, we hypothesized that the suprascapular notch cross-sectional area (SSNCSA) could be a good diagnostic parameter to assess SNES.
Methods:
We acquired suprascapular notch data from 10 patients with SNES and 10 healthy individuals who had undergone shoulder magnetic resonance imaging (S-MRI) and had no evidence of SNES. T2-weighted coronal magnetic resonance images were acquired from the shoulder. We analyzed the SSNCSA at the shoulder on S-MRI using our image-analysis program (INFINITT PACS). The SSNCSA was measured as the suprascapular notch, which was the most affected site in coronal S-MRI images.
Results:
The mean SSNCSA was 64.50 ± 8.93 mm2 in the control group and 44.94 ± 10.40 mm2 in the SNES group. Patients with SNES had significantly lower SSNCSA (P < 0.01) than those in the control group. Receiver operating curve analysis showed that the best cut-off of the SSNCSA was 57.49 mm2, with 80.0% sensitivity, 80.0% specificity, and an area under the curve of 0.92 (95% CI [0.79, 1.00]).
Conclusions:
The SSNCSA was found to have acceptable diagnostic properties for detecting SNES. We hope that these results will help diagnose SNES objectively. | null | null | 3,408 | 264 | [
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|||
Brazilian propolis mitigates impaired glucose and lipid metabolism in experimental periodontitis in mice. | 27576340 | Periodontitis has been implicated as a risk factor for metabolic disorders associated with insulin resistance. Recently, we have demonstrated that orally administered Porphyromonas gingivalis, a representative periodontopathic bacterium, induces endotoxemia via reduced gut barrier function coupled with changes in gut microbiota composition, resulting in systemic inflammation and insulin resistance. Propolis, a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants, can positively affect metabolic disorders in various experimental models. In this study, we thus aimed to clarify the effect of propolis on impaired glucose and lipid metabolism induced by P. gingivalis administration. | BACKGROUND | Eight-week-old male C57BL/6 mice were orally administered P. gingivalis strain W83, propolis ethanol extract powder with P. gingivalis, or vehicle. We then analyzed the expression profile of glucose and lipid metabolism-related genes in the liver and adipose tissues. Serum endotoxin levels were also evaluated by a limulus amebocyte lysate test. In addition, we performed histological analysis of the liver and quantified alveolar bone loss by measuring the root surface area on the lower first molar. | METHODS | Oral administration of P. gingivalis induced downregulation of genes that improve insulin sensitivity in adipose tissue (C1qtnf9, Irs1, and Sirt1), but upregulation of genes associated with lipid droplet formation and gluconeogenesis (Plin2, Acox, and G6pc). However, concomitant administration of propolis abrogated these adverse effects of P. gingivalis. Consistent with gene expression, histological analysis showed that administered propolis suppressed hepatic steatosis induced by P. gingivalis. Furthermore, propolis inhibited the elevation of serum endotoxin levels induced by P. gingivalis administration. Contrary to the systemic effects, propolis had no beneficial effect on alveolar bone loss. | RESULTS | These results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases. | CONCLUSION | [
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] | 5006533 | Background | Periodontal diseases are mainly chronic infectious diseases resulting from responses to a complex dental plaque microbiome containing various periodontopathic bacteria species. Epidemiological studies suggest that periodontitis is a risk factor for various systemic diseases, such as type 2 diabetes [1, 2], atherosclerotic vascular diseases [3, 4], and non-alcoholic fatty liver disease [5]. Among the various periodontopathic bacteria, considerable research has been focused on the role of Porphyromonas gingivalis as a possible mechanism linking periodontal and other human diseases, due to its unique pathogenicity [6] and its association with various diseases. Although the possible significance of common susceptibility cannot be discounted, there are several hypothetical causal mechanisms linking periodontal disease and systemic diseases. First, bacteria from dental plaque invade gingival tissue through ulcerated sulcular epithelial linings of periodontal pockets and then disseminate into systemic circulation. Second, various proinflammatory cytokines produced in inflamed periodontal tissue, which can also enter systemic circulation, are delivered to various tissues and organs and thereby induce an inflammatory response [7].
Interestingly, links between the diseases related to periodontitis and dysbiosis of the gut microbiota are becoming more evident [8, 9]. Recently, we revealed that oral administration of P. gingivalis altered gut microbiota and elicited endotoxemia, thereby inducing systemic inflammation and insulin resistance [10, 11].
Propolis is a plant product collected by honeybees as a resinous mixture from various plants that is mixed with beeswax and other bee secretions. Although the chemical composition of propolis depends on its location of origin, it basically contains beneficial substances, such as phenolic acids, flavonoids, and vitamins [12, 13]. However, the details of the main ingredients have not yet been disclosed.
Propolis has been used as a folk medicine since ancient times due to its anti-inflammatory [14, 15], anti-microbial [16, 17], anti-oxidant [12], and anti-tumour properties [18, 19]. In addition, previous studies have shown the beneficial effect of propolis on diabetes mellitus. Fuliang et al. revealed that administering propolis improved blood glucose levels and modulates glucose and blood lipid metabolism in experimental rat models of diabetes [20]. Kitamura et al. demonstrated that the propolis extract restored glucose intolerance and insulin resistance, as well as blood glucose and plasma cholesterol levels using ob/ob mice [21]. Furthermore, because of its effectiveness on enterobacteria [22], propolis is expected to replace probiotics as a novel regulator of gut microbiota.
In the present study, we evaluated whether systemic inflammation and insulin resistance induced by periodontopathic bacteria through intestinal reactions could be suppressed by administering propolis. | null | null | Results | Effect of P. gingivalis administration and/or propolis on the body weight After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the altered gene expression in the liver by P. gingivalis administration Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Administering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Administering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Effect of propolis on the altered gene expression in the adipose tissue by P. gingivalis administration Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the serum endotoxin levels in P. gingivalis-administered mice Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the alveolar bone resorption in P. gingivalis-administered mice As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice) | Conclusion | Although further studies are needed to clarify the underlying mechanism by which propolis suppresses P. gingivalis-induced metabolic disturbance, our results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases. | [
"Mice",
"Bacterial cultures",
"Oral administration",
"Analysis of gene expression in the liver and adipose tissues",
"Quantification of alveolar bone loss",
"Endotoxin assay",
"Histological analysis of the liver tissue",
"Statistical analysis",
"Effect of P. gingivalis administration and/or propolis on the body weight",
"Effect of propolis on the altered gene expression in the liver by P. gingivalis administration",
"Effect of propolis on the altered gene expression in the adipose tissue by P. gingivalis administration",
"Effect of propolis on the serum endotoxin levels in P. gingivalis-administered mice",
"Effect of propolis on the alveolar bone resorption in P. gingivalis-administered mice"
] | [
"Eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.",
"P. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.",
"The murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).\nDuring the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.",
"Total RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.",
"The amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.",
"Endotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.",
"The liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.",
"Prior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.",
"After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)",
"Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nAdministering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)\nHistological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)",
"Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)",
"Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)",
"As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)\nQuantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Mice",
"Bacterial cultures",
"Oral administration",
"Analysis of gene expression in the liver and adipose tissues",
"Quantification of alveolar bone loss",
"Endotoxin assay",
"Histological analysis of the liver tissue",
"Statistical analysis",
"Results",
"Effect of P. gingivalis administration and/or propolis on the body weight",
"Effect of propolis on the altered gene expression in the liver by P. gingivalis administration",
"Effect of propolis on the altered gene expression in the adipose tissue by P. gingivalis administration",
"Effect of propolis on the serum endotoxin levels in P. gingivalis-administered mice",
"Effect of propolis on the alveolar bone resorption in P. gingivalis-administered mice",
"Discussion",
"Conclusion"
] | [
"Periodontal diseases are mainly chronic infectious diseases resulting from responses to a complex dental plaque microbiome containing various periodontopathic bacteria species. Epidemiological studies suggest that periodontitis is a risk factor for various systemic diseases, such as type 2 diabetes [1, 2], atherosclerotic vascular diseases [3, 4], and non-alcoholic fatty liver disease [5]. Among the various periodontopathic bacteria, considerable research has been focused on the role of Porphyromonas gingivalis as a possible mechanism linking periodontal and other human diseases, due to its unique pathogenicity [6] and its association with various diseases. Although the possible significance of common susceptibility cannot be discounted, there are several hypothetical causal mechanisms linking periodontal disease and systemic diseases. First, bacteria from dental plaque invade gingival tissue through ulcerated sulcular epithelial linings of periodontal pockets and then disseminate into systemic circulation. Second, various proinflammatory cytokines produced in inflamed periodontal tissue, which can also enter systemic circulation, are delivered to various tissues and organs and thereby induce an inflammatory response [7].\nInterestingly, links between the diseases related to periodontitis and dysbiosis of the gut microbiota are becoming more evident [8, 9]. Recently, we revealed that oral administration of P. gingivalis altered gut microbiota and elicited endotoxemia, thereby inducing systemic inflammation and insulin resistance [10, 11].\nPropolis is a plant product collected by honeybees as a resinous mixture from various plants that is mixed with beeswax and other bee secretions. Although the chemical composition of propolis depends on its location of origin, it basically contains beneficial substances, such as phenolic acids, flavonoids, and vitamins [12, 13]. However, the details of the main ingredients have not yet been disclosed.\nPropolis has been used as a folk medicine since ancient times due to its anti-inflammatory [14, 15], anti-microbial [16, 17], anti-oxidant [12], and anti-tumour properties [18, 19]. In addition, previous studies have shown the beneficial effect of propolis on diabetes mellitus. Fuliang et al. revealed that administering propolis improved blood glucose levels and modulates glucose and blood lipid metabolism in experimental rat models of diabetes [20]. Kitamura et al. demonstrated that the propolis extract restored glucose intolerance and insulin resistance, as well as blood glucose and plasma cholesterol levels using ob/ob mice [21]. Furthermore, because of its effectiveness on enterobacteria [22], propolis is expected to replace probiotics as a novel regulator of gut microbiota.\nIn the present study, we evaluated whether systemic inflammation and insulin resistance induced by periodontopathic bacteria through intestinal reactions could be suppressed by administering propolis.",
" Mice Eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.\nEight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.\n Bacterial cultures P. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.\nP. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.\n Oral administration The murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).\nDuring the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.\nThe murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).\nDuring the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.\n Analysis of gene expression in the liver and adipose tissues Total RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.\nTotal RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.\n Quantification of alveolar bone loss The amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.\nThe amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.\n Endotoxin assay Endotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.\nEndotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.\n Histological analysis of the liver tissue The liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.\nThe liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.\n Statistical analysis Prior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.\nPrior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.",
"Eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.",
"P. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.",
"The murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).\nDuring the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.",
"Total RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.",
"The amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.",
"Endotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.",
"The liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.",
"Prior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.",
" Effect of P. gingivalis administration and/or propolis on the body weight After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nAfter treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\n Effect of propolis on the altered gene expression in the liver by P. gingivalis administration Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nAdministering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)\nHistological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)\nOral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nAdministering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)\nHistological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)\n Effect of propolis on the altered gene expression in the adipose tissue by P. gingivalis administration Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nIl6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\n Effect of propolis on the serum endotoxin levels in P. gingivalis-administered mice Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nOral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\n Effect of propolis on the alveolar bone resorption in P. gingivalis-administered mice As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)\nQuantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)\nAs with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)\nQuantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)",
"After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)",
"Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nAdministering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)\nHistological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b\nP. gingivalis- administered mice, and c\nP. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)",
"Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)",
"Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)\nEffect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)",
"As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)\nQuantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)",
"Periodontal disease is a chronic inflammatory disease likely resulting from dysbiosis of the oral microbiota. Epidemiological studies indicate its association with an increased risk of various diseases, such as diabetes [1, 2], atherosclerotic vascular diseases [3, 4], and rheumatoid arthritis [5]. The underlying mechanisms linking periodontal disease and these diseases have been considered to be endotoxemia and proinflammatory cytokines derived from gingival lesions [7]. We have shown that repeated oral inoculation of P. gingivalis induced elevation of serum inflammatory markers (serum amyloid A and IL-6) in mice [27]. However, in this mouse model, administered P. gingivalis was not detected in the blood or insulin target tissues and there was little inflammation in the gingival tissues, suggesting that conventional mechanisms for the relationship between periodontitis and systemic diseases may not be applicable, at least in this mouse model. Recently, we have also demonstrated altered gut bacterial composition by repeated oral administration of P. gingivalis as a novel underlying mechanism [10, 11]. Since the gut microbial changes are associated with decreased gut barrier function, as evidenced by reduced expression of the tight junction protein gene (Tjp1) and elevated endotoxin levels in the systemic circulation in our previous studies [10, 11], inflammation of adipose and liver tissue seen in the present study was expected to be from bacteria influx and/or bacterial products into these tissues.\nIn the present study, we have demonstrated that oral administration of P. gingivalis altered gene expression associated with tissue-specific pathological changes relating to metabolic syndromes. In the liver, the expression of Plin2 and Acox1 was significantly upregulated. Plin2 is reported to be strongly associated with lipid droplet formation in the liver [28] and Acox1 is involved in fatty acid oxidation [29]. Both genes play important roles in non-alcoholic fatty liver diseases. P. gingivalis administration also seemed to affect glucose metabolism by increasing the expression of G6pc. G6pc positively regulates gluconeogenesis and can increase blood glucose levels [30].\nAdipose tissue plays an important role in mediating insulin sensitivity through various molecules, including adipocytokines. C1qtnf9 encoding C1q/TNF-related protein (CTRP)9 is a novel and highly conserved paralog of adiponectin and has salutary effects on glucose metabolism and vascular function [31, 32]. Irs1 [33] and Sirt1 contribute to improving insulin sensitivity. SIRT1 not only stimulates a glucose-dependent insulin secretion from pancreatic beta cells, but also directly stimulates insulin signaling pathways in insulin-sensitive organs [34]. Tnf and Il6 are further adipocytokines with strong, natural proinflammatory traits that negatively impact insulin signaling. Angptl4 can also be involved in insulin resistance [35]. Taken together, oral administration of P. gingivalis has harmful effects on insulin sensitivity.\nAdministration of propolis during the experimental period seemed to show no serious adverse effects, as indicated by no change in body weight among the groups. In support of previous studies [21, 36], administering propolis alleviated the detrimental effects on glucose and lipid metabolism induced by oral administration of P. gingivalis. Although we have not analyzed the detailed mechanisms by which propolis suppress these harmful effects, it is possible that administration of propolis could affect gut barrier function, either by alteration of gut microbiota composition or direct elevation of gut barrier function. In support of this idea, our previous studies clearly demonstrate that oral administration of P. gingivalis changes gut microbiota composition, inducing sustained endotoxemia and systemic inflammation [10, 11]. Another previous work shows high-fat diet-induced obesity influences gut microbiota and induces metabolic endotoxemia [37]. This endotoxemia is considered to be a result of reduced gut barrier function. In the present study, we also observed that administering P. gingivalis tended to increase serum endotoxin levels, while administering propolis completely abrogated the effect of P. gingivalis. Although administering propolis had no effect on alveolar bone resorption induced by P. gingivalis indicating no local effects on periodontal tissue, low levels of endotoxin activity in the propolis-administered mice could be mediated by anti-microbial effect on P. gingivalis. In this regard, several studies have demonstrated a significant antimicrobial effect of propolis on periodontopathic bacteria such as P. gingivalis [38–41] and an inhibitory effect on alveolar bone resorption in ligature-induced periodontitis in rats [42, 43]. However, our study demonstrated no suppressive effect of propolis on alveolar bone resorption. The difference between our study and the study by Toker et al. [43] could be due at least in part to the different methods for inducing experimental periodontitis. Alternatively, propolis administration may have induced changes in gut microbiota composition or strengthened the gut barrier function. One previous study showed that propolis upregulated tight junction proteins in Caco-3 cells and that in vivo administration of propolis increased colonic epithelial ZO-1 expression [44]. Furthermore, it has been demonstrated that a gut metabolite of linoleic acid ameliorates inflammation-induced intestinal epithelial barrier impairments, suggesting that gut microbiota composition is crucial for the maintenance of gut barrier function [45]. Thus, it is reasonable to consider the beneficial effects of propolis on gut microbiota.",
"Although further studies are needed to clarify the underlying mechanism by which propolis suppresses P. gingivalis-induced metabolic disturbance, our results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases."
] | [
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] | [
"Periodontitis",
"Propolis",
"\nPorphyromonas gingivalis\n",
"Liver",
"Adipose tissue",
"Endotoxemia"
] | Background:
Periodontal diseases are mainly chronic infectious diseases resulting from responses to a complex dental plaque microbiome containing various periodontopathic bacteria species. Epidemiological studies suggest that periodontitis is a risk factor for various systemic diseases, such as type 2 diabetes [1, 2], atherosclerotic vascular diseases [3, 4], and non-alcoholic fatty liver disease [5]. Among the various periodontopathic bacteria, considerable research has been focused on the role of Porphyromonas gingivalis as a possible mechanism linking periodontal and other human diseases, due to its unique pathogenicity [6] and its association with various diseases. Although the possible significance of common susceptibility cannot be discounted, there are several hypothetical causal mechanisms linking periodontal disease and systemic diseases. First, bacteria from dental plaque invade gingival tissue through ulcerated sulcular epithelial linings of periodontal pockets and then disseminate into systemic circulation. Second, various proinflammatory cytokines produced in inflamed periodontal tissue, which can also enter systemic circulation, are delivered to various tissues and organs and thereby induce an inflammatory response [7].
Interestingly, links between the diseases related to periodontitis and dysbiosis of the gut microbiota are becoming more evident [8, 9]. Recently, we revealed that oral administration of P. gingivalis altered gut microbiota and elicited endotoxemia, thereby inducing systemic inflammation and insulin resistance [10, 11].
Propolis is a plant product collected by honeybees as a resinous mixture from various plants that is mixed with beeswax and other bee secretions. Although the chemical composition of propolis depends on its location of origin, it basically contains beneficial substances, such as phenolic acids, flavonoids, and vitamins [12, 13]. However, the details of the main ingredients have not yet been disclosed.
Propolis has been used as a folk medicine since ancient times due to its anti-inflammatory [14, 15], anti-microbial [16, 17], anti-oxidant [12], and anti-tumour properties [18, 19]. In addition, previous studies have shown the beneficial effect of propolis on diabetes mellitus. Fuliang et al. revealed that administering propolis improved blood glucose levels and modulates glucose and blood lipid metabolism in experimental rat models of diabetes [20]. Kitamura et al. demonstrated that the propolis extract restored glucose intolerance and insulin resistance, as well as blood glucose and plasma cholesterol levels using ob/ob mice [21]. Furthermore, because of its effectiveness on enterobacteria [22], propolis is expected to replace probiotics as a novel regulator of gut microbiota.
In the present study, we evaluated whether systemic inflammation and insulin resistance induced by periodontopathic bacteria through intestinal reactions could be suppressed by administering propolis.
Methods:
Mice Eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.
Eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.
Bacterial cultures P. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.
P. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.
Oral administration The murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).
During the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.
The murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).
During the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.
Analysis of gene expression in the liver and adipose tissues Total RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Total RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Quantification of alveolar bone loss The amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.
The amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.
Endotoxin assay Endotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.
Endotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.
Histological analysis of the liver tissue The liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
The liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Statistical analysis Prior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.
Prior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.
Mice:
Eight-week-old male C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimatized under specific pathogen-free conditions and fed regular chow and sterile water until the commencement of infection at nine weeks of age.
Bacterial cultures:
P. gingivalis (strain W83) was cultured in modified Gifu anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) in an anaerobic jar (Becton Dickinson Microbiology System, Cockeysville, MD, USA) in the presence of an AnaeroPack™ (Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan) for 48 h at 37 °C. Bacterial suspensions were prepared in phosphate-buffered saline (PBS) without Mg2+/Ca2+ using established growth curves and spectrophotometric analysis. The number of colony-forming units (CFUs) was standardized by measuring optical density at 600 nm.
Oral administration:
The murine experimental periodontitis model was developed according to Baker et al. with slight modifications [23]. In the present study, mice did not receive antibiotic pretreatment before P. gingivalis administration since systemic antimicrobial treatment affects the gut microbiota composition and alters metabolism [24]. In addition, we have observed P. gingivalis-induced alveolar bone resorption without antibiotic pretreatment [25]. A total of 109 CFUs of live P. gingivalis suspended in 100 μL PBS with 2 % carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) was given to each mouse via a feeding needle three times a week for 5 weeks. The number of administered bacteria was determined based on the number of P. gingivalis contained in saliva of patients with severe periodontitis [26] and adjusted for body weight. The control group was sham-administered 100 μL PBS with 2 % carboxymethyl cellulose without P. gingivalis. In the propolis group, powdered ethanol extracts of propolis mixed with 2 % carboxymethyl cellulose (200 mg/kg weight) were administered every day during the experimental period in addition to P. gingivalis administration three times a week for 5 weeks. The propolis ethanol extract, including 55 % propolis extract as solid content, standardized to contain minimum 8.0 % artepillin C, was obtained from Yamada Bee Company, Inc. (Okayama, Japan).
During the experimental period, all mice were allowed to eat and drink ad libitum. One day after the final treatment, mice were euthanized with CO2 and their tissues were collected.
Analysis of gene expression in the liver and adipose tissues:
Total RNA from the liver and adipose tissues were extracted using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA). cDNA was synthesized with Transcriptor Universal cDNA Master (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Primers and probes specific for real-time PCR were purchased from Life Technologies Corporation. Reactions were carried out in a final volume of 25 μL in a LightCycler® 96 System (Roche) using TaqMan Gene Expression Assays (Life Technologies Corporation) containing 900 nM each of the forward and reverse primers and a 250 nM probe. The reactions consisted of a 10-min incubation at 95 °C, followed by 45 cycles of a 2-step amplification procedure, consisting of annealing/extension at 60 °C for 1 min and denaturation for 15 s at 95 °C. LightCycler® 96 software (Roche) was used to analyze the standards and carry out the quantification. The relative quantity of each mRNA was normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
Quantification of alveolar bone loss:
The amount of bone loss was assessed from images obtained using a stereomicroscope fitted with a video image marker measurement system (DP2-BSW; Olympus, Tokyo, Japan). We determined the area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar. We measured alveolar bone loss in the mice in a blind manner.
Endotoxin assay:
Endotoxin levels were determined in sera collected at the end of the experimental period using a Limulus amebocyte lysate test (QCL-1000TM, BioWhittaker, Walkersville, MD, USA) according to the manufacturer’s instructions. Serum samples were diluted 1-to-4 for the assay. Optical densities were measured using an ELISA plate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA) at 405 nm.
Histological analysis of the liver tissue:
The liver tissues of three mice from each group were fixed in 10 % formalin. Briefly, samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Statistical analysis:
Prior to beginning the present study, the sample size calculation was performed based on the data from our previous study. With an alpha error of 0.05 and a statistical power of 0.8, the sample size was calculated to require more than seven animals in each group. This was verified by post-hoc analyses. The data were tested for normality using the Kolmogorov-Smirnov test. Because some, but not all, data sets showed a parametric distribution even in a particular gene expression among different treatment groups, all data were assessed by the Kruskal-Wallis test with a post-hoc Dunn’s multiple comparison test using GraphPad Prism® (GraphPad Software, Inc., La Jolla, CA). A probability value of p < 0.05 was considered statistically significant.
Results:
Effect of P. gingivalis administration and/or propolis on the body weight After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the altered gene expression in the liver by P. gingivalis administration Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Administering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Administering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Effect of propolis on the altered gene expression in the adipose tissue by P. gingivalis administration Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the serum endotoxin levels in P. gingivalis-administered mice Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the alveolar bone resorption in P. gingivalis-administered mice As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Effect of P. gingivalis administration and/or propolis on the body weight:
After treatment with P. gingivalis and propolis, there were no differences among the sham-administered, P. gingivalis-administered, or P. gingivalis and propolis-administered groups in body weight (Fig. 1) or food intake.Fig. 1Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or P. gingivalis and propolis on body weight. Body weight changes during experimental period in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are mean ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the altered gene expression in the liver by P. gingivalis administration:
Oral administration of P. gingivalis significantly increased gene expression of Plin2 and Acox, both of which are associated with lipid metabolism in the liver, while these gene expressions were markedly reduced in the propolis-administered mice (Fig. 2a). Furthermore, administration of propolis suppressed G6pc expression, which positively regulates gluconeogenesis, compared to mice administered P. gingivalis alone. Conversely, there was no significant change in the expression of the insulin signaling gene Irs1 among the three groups (Fig. 2b). Although gene expression of the proinflammatory cytokines IL-6 and TNF-α tended to be elevated from the administered P. gingivalis, administration of propolis had no effect on the expression of these genes (Fig. 2c).Fig. 2Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the liver. Expression of genes related to lipid metabolism a, glucose metabolism b, and inflammation c. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), Propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Administering propolis led to decreased Plin2 and Acox expression, which were associated with the mitigation of hepatic steatosis induced by P. gingivalis administration (Fig. 3).Fig. 3Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Histological analysis of the liver tissue. Sections of liver tissue in a Sham-administered mice, b
P. gingivalis- administered mice, and c
P. gingivalis and propolis-administered mice were H-E stained. Representative results are shown (N = 3 in each group)
Effect of propolis on the altered gene expression in the adipose tissue by P. gingivalis administration:
Il6, Tnf, C1qtnf9, and Adipoq are known as adipocytokines that are expressed in adipocytes to modulate glucose and lipid metabolism. The gene expression of the proinflammatory cytokines IL-6 and TNF-α, which suppress insulin signals, tended to be higher in the P. gingivalis-administered mice. Conversely, C1qtnf9, a gene that improves insulin sensitivity, was more frequently downregulated in the P. gingivalis-administered mice. Administration of propolis ameliorated these changes to gene expression induced by P. gingivalis administration (Fig. 4a). Furthermore, the genes that improve insulin sensitivity, Irs1 and Sirt1, were downregulated in P. gingivalis-administered mice. However, propolis administration reversed the effect of P. gingivalis administration on the expression of these genes. In addition, administration of propolis suppressed the P. gingivalis-induced increased expression of Angptl4, a gene that is supposed to increase insulin resistance (Fig. 4b).Fig. 4Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on gene expression in the epididymal adipose tissue. a Relative gene expression of adipocytokine. b Relative expression of genes related to glucose metabolism. Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), P. gingivalis and propolis-administered mice (N = 8). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the serum endotoxin levels in P. gingivalis-administered mice:
Oral administration of P. gingivalis increased serum endotoxin levels, but was not statistically significant. Administration of propolis not only repressed this increase, but also further lowered serum endotoxin levels below those of untreated mice (Fig. 5).Fig. 5Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of oral administration of P. gingivalis or propolis on endotoxemia. Serum endotoxin (LPS) concentration (EU/mL) were evaluated in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. (*p < 0.05, Kruskal-Wallis test with post-hoc Dunn’s multiple comparison test)
Effect of propolis on the alveolar bone resorption in P. gingivalis-administered mice:
As with the previous studies, oral administration of P. gingivalis induced significant alveolar bone resorption. In spite of the beneficial systemic effect of administering propolis, no effect was observed on the P. gingivalis-induced alveolar bone resorption (Fig. 6).Fig. 6Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Quantification of alveolar bone loss. a The area surrounded by the margin of the submaxillary alveolar bone crest and the cement-enamel junction on the lingual side of the first molar was determined using a stereoscopic microscope. The image analysis was carried out in Sham-administered mice (N = 7), P. gingivalis-administered mice (N = 7), and P. gingivalis and propolis-administered mice (N = 8). All data are means ± SD. No significant difference was observed. b Photographs were taken after the removal of soft tissues. The measured area is displayed with a yellow line. Representative results are shown (N = 7 in Sham- and P. gingivalis-administered mice, N = 8 in P. gingivalis and propolis- administered mice)
Discussion:
Periodontal disease is a chronic inflammatory disease likely resulting from dysbiosis of the oral microbiota. Epidemiological studies indicate its association with an increased risk of various diseases, such as diabetes [1, 2], atherosclerotic vascular diseases [3, 4], and rheumatoid arthritis [5]. The underlying mechanisms linking periodontal disease and these diseases have been considered to be endotoxemia and proinflammatory cytokines derived from gingival lesions [7]. We have shown that repeated oral inoculation of P. gingivalis induced elevation of serum inflammatory markers (serum amyloid A and IL-6) in mice [27]. However, in this mouse model, administered P. gingivalis was not detected in the blood or insulin target tissues and there was little inflammation in the gingival tissues, suggesting that conventional mechanisms for the relationship between periodontitis and systemic diseases may not be applicable, at least in this mouse model. Recently, we have also demonstrated altered gut bacterial composition by repeated oral administration of P. gingivalis as a novel underlying mechanism [10, 11]. Since the gut microbial changes are associated with decreased gut barrier function, as evidenced by reduced expression of the tight junction protein gene (Tjp1) and elevated endotoxin levels in the systemic circulation in our previous studies [10, 11], inflammation of adipose and liver tissue seen in the present study was expected to be from bacteria influx and/or bacterial products into these tissues.
In the present study, we have demonstrated that oral administration of P. gingivalis altered gene expression associated with tissue-specific pathological changes relating to metabolic syndromes. In the liver, the expression of Plin2 and Acox1 was significantly upregulated. Plin2 is reported to be strongly associated with lipid droplet formation in the liver [28] and Acox1 is involved in fatty acid oxidation [29]. Both genes play important roles in non-alcoholic fatty liver diseases. P. gingivalis administration also seemed to affect glucose metabolism by increasing the expression of G6pc. G6pc positively regulates gluconeogenesis and can increase blood glucose levels [30].
Adipose tissue plays an important role in mediating insulin sensitivity through various molecules, including adipocytokines. C1qtnf9 encoding C1q/TNF-related protein (CTRP)9 is a novel and highly conserved paralog of adiponectin and has salutary effects on glucose metabolism and vascular function [31, 32]. Irs1 [33] and Sirt1 contribute to improving insulin sensitivity. SIRT1 not only stimulates a glucose-dependent insulin secretion from pancreatic beta cells, but also directly stimulates insulin signaling pathways in insulin-sensitive organs [34]. Tnf and Il6 are further adipocytokines with strong, natural proinflammatory traits that negatively impact insulin signaling. Angptl4 can also be involved in insulin resistance [35]. Taken together, oral administration of P. gingivalis has harmful effects on insulin sensitivity.
Administration of propolis during the experimental period seemed to show no serious adverse effects, as indicated by no change in body weight among the groups. In support of previous studies [21, 36], administering propolis alleviated the detrimental effects on glucose and lipid metabolism induced by oral administration of P. gingivalis. Although we have not analyzed the detailed mechanisms by which propolis suppress these harmful effects, it is possible that administration of propolis could affect gut barrier function, either by alteration of gut microbiota composition or direct elevation of gut barrier function. In support of this idea, our previous studies clearly demonstrate that oral administration of P. gingivalis changes gut microbiota composition, inducing sustained endotoxemia and systemic inflammation [10, 11]. Another previous work shows high-fat diet-induced obesity influences gut microbiota and induces metabolic endotoxemia [37]. This endotoxemia is considered to be a result of reduced gut barrier function. In the present study, we also observed that administering P. gingivalis tended to increase serum endotoxin levels, while administering propolis completely abrogated the effect of P. gingivalis. Although administering propolis had no effect on alveolar bone resorption induced by P. gingivalis indicating no local effects on periodontal tissue, low levels of endotoxin activity in the propolis-administered mice could be mediated by anti-microbial effect on P. gingivalis. In this regard, several studies have demonstrated a significant antimicrobial effect of propolis on periodontopathic bacteria such as P. gingivalis [38–41] and an inhibitory effect on alveolar bone resorption in ligature-induced periodontitis in rats [42, 43]. However, our study demonstrated no suppressive effect of propolis on alveolar bone resorption. The difference between our study and the study by Toker et al. [43] could be due at least in part to the different methods for inducing experimental periodontitis. Alternatively, propolis administration may have induced changes in gut microbiota composition or strengthened the gut barrier function. One previous study showed that propolis upregulated tight junction proteins in Caco-3 cells and that in vivo administration of propolis increased colonic epithelial ZO-1 expression [44]. Furthermore, it has been demonstrated that a gut metabolite of linoleic acid ameliorates inflammation-induced intestinal epithelial barrier impairments, suggesting that gut microbiota composition is crucial for the maintenance of gut barrier function [45]. Thus, it is reasonable to consider the beneficial effects of propolis on gut microbiota.
Conclusion:
Although further studies are needed to clarify the underlying mechanism by which propolis suppresses P. gingivalis-induced metabolic disturbance, our results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases.
| Background:
Periodontitis has been implicated as a risk factor for metabolic disorders associated with insulin resistance. Recently, we have demonstrated that orally administered Porphyromonas gingivalis, a representative periodontopathic bacterium, induces endotoxemia via reduced gut barrier function coupled with changes in gut microbiota composition, resulting in systemic inflammation and insulin resistance. Propolis, a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants, can positively affect metabolic disorders in various experimental models. In this study, we thus aimed to clarify the effect of propolis on impaired glucose and lipid metabolism induced by P. gingivalis administration.
Methods:
Eight-week-old male C57BL/6 mice were orally administered P. gingivalis strain W83, propolis ethanol extract powder with P. gingivalis, or vehicle. We then analyzed the expression profile of glucose and lipid metabolism-related genes in the liver and adipose tissues. Serum endotoxin levels were also evaluated by a limulus amebocyte lysate test. In addition, we performed histological analysis of the liver and quantified alveolar bone loss by measuring the root surface area on the lower first molar.
Results:
Oral administration of P. gingivalis induced downregulation of genes that improve insulin sensitivity in adipose tissue (C1qtnf9, Irs1, and Sirt1), but upregulation of genes associated with lipid droplet formation and gluconeogenesis (Plin2, Acox, and G6pc). However, concomitant administration of propolis abrogated these adverse effects of P. gingivalis. Consistent with gene expression, histological analysis showed that administered propolis suppressed hepatic steatosis induced by P. gingivalis. Furthermore, propolis inhibited the elevation of serum endotoxin levels induced by P. gingivalis administration. Contrary to the systemic effects, propolis had no beneficial effect on alveolar bone loss.
Conclusions:
These results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases. | Background:
Periodontal diseases are mainly chronic infectious diseases resulting from responses to a complex dental plaque microbiome containing various periodontopathic bacteria species. Epidemiological studies suggest that periodontitis is a risk factor for various systemic diseases, such as type 2 diabetes [1, 2], atherosclerotic vascular diseases [3, 4], and non-alcoholic fatty liver disease [5]. Among the various periodontopathic bacteria, considerable research has been focused on the role of Porphyromonas gingivalis as a possible mechanism linking periodontal and other human diseases, due to its unique pathogenicity [6] and its association with various diseases. Although the possible significance of common susceptibility cannot be discounted, there are several hypothetical causal mechanisms linking periodontal disease and systemic diseases. First, bacteria from dental plaque invade gingival tissue through ulcerated sulcular epithelial linings of periodontal pockets and then disseminate into systemic circulation. Second, various proinflammatory cytokines produced in inflamed periodontal tissue, which can also enter systemic circulation, are delivered to various tissues and organs and thereby induce an inflammatory response [7].
Interestingly, links between the diseases related to periodontitis and dysbiosis of the gut microbiota are becoming more evident [8, 9]. Recently, we revealed that oral administration of P. gingivalis altered gut microbiota and elicited endotoxemia, thereby inducing systemic inflammation and insulin resistance [10, 11].
Propolis is a plant product collected by honeybees as a resinous mixture from various plants that is mixed with beeswax and other bee secretions. Although the chemical composition of propolis depends on its location of origin, it basically contains beneficial substances, such as phenolic acids, flavonoids, and vitamins [12, 13]. However, the details of the main ingredients have not yet been disclosed.
Propolis has been used as a folk medicine since ancient times due to its anti-inflammatory [14, 15], anti-microbial [16, 17], anti-oxidant [12], and anti-tumour properties [18, 19]. In addition, previous studies have shown the beneficial effect of propolis on diabetes mellitus. Fuliang et al. revealed that administering propolis improved blood glucose levels and modulates glucose and blood lipid metabolism in experimental rat models of diabetes [20]. Kitamura et al. demonstrated that the propolis extract restored glucose intolerance and insulin resistance, as well as blood glucose and plasma cholesterol levels using ob/ob mice [21]. Furthermore, because of its effectiveness on enterobacteria [22], propolis is expected to replace probiotics as a novel regulator of gut microbiota.
In the present study, we evaluated whether systemic inflammation and insulin resistance induced by periodontopathic bacteria through intestinal reactions could be suppressed by administering propolis.
Conclusion:
Although further studies are needed to clarify the underlying mechanism by which propolis suppresses P. gingivalis-induced metabolic disturbance, our results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases. | Background:
Periodontitis has been implicated as a risk factor for metabolic disorders associated with insulin resistance. Recently, we have demonstrated that orally administered Porphyromonas gingivalis, a representative periodontopathic bacterium, induces endotoxemia via reduced gut barrier function coupled with changes in gut microbiota composition, resulting in systemic inflammation and insulin resistance. Propolis, a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants, can positively affect metabolic disorders in various experimental models. In this study, we thus aimed to clarify the effect of propolis on impaired glucose and lipid metabolism induced by P. gingivalis administration.
Methods:
Eight-week-old male C57BL/6 mice were orally administered P. gingivalis strain W83, propolis ethanol extract powder with P. gingivalis, or vehicle. We then analyzed the expression profile of glucose and lipid metabolism-related genes in the liver and adipose tissues. Serum endotoxin levels were also evaluated by a limulus amebocyte lysate test. In addition, we performed histological analysis of the liver and quantified alveolar bone loss by measuring the root surface area on the lower first molar.
Results:
Oral administration of P. gingivalis induced downregulation of genes that improve insulin sensitivity in adipose tissue (C1qtnf9, Irs1, and Sirt1), but upregulation of genes associated with lipid droplet formation and gluconeogenesis (Plin2, Acox, and G6pc). However, concomitant administration of propolis abrogated these adverse effects of P. gingivalis. Consistent with gene expression, histological analysis showed that administered propolis suppressed hepatic steatosis induced by P. gingivalis. Furthermore, propolis inhibited the elevation of serum endotoxin levels induced by P. gingivalis administration. Contrary to the systemic effects, propolis had no beneficial effect on alveolar bone loss.
Conclusions:
These results suggest that administration of propolis may be effective in suppressing periodontopathic bacteria-induced metabolic changes that increase the risk of various systemic diseases. | 10,168 | 350 | [
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"gingivalis propolis"
] | [
"disease periodontopathic bacteria",
"periodontal human diseases",
"periodontopathic bacteria induced",
"periodontitis systemic",
"inflammation gingival tissues"
] | null | [CONTENT] Periodontitis | Propolis |
Porphyromonas gingivalis
| Liver | Adipose tissue | Endotoxemia [SUMMARY] | null | [CONTENT] Periodontitis | Propolis |
Porphyromonas gingivalis
| Liver | Adipose tissue | Endotoxemia [SUMMARY] | [CONTENT] Periodontitis | Propolis |
Porphyromonas gingivalis
| Liver | Adipose tissue | Endotoxemia [SUMMARY] | [CONTENT] Periodontitis | Propolis |
Porphyromonas gingivalis
| Liver | Adipose tissue | Endotoxemia [SUMMARY] | [CONTENT] Periodontitis | Propolis |
Porphyromonas gingivalis
| Liver | Adipose tissue | Endotoxemia [SUMMARY] | [CONTENT] Alveolar Bone Loss | Animals | Blood Glucose | Body Weight | Bone Resorption | Brazil | Endotoxemia | Lipid Metabolism | Male | Mice | Mice, Inbred C57BL | Periodontitis | Porphyromonas gingivalis | Propolis | Protective Agents [SUMMARY] | null | [CONTENT] Alveolar Bone Loss | Animals | Blood Glucose | Body Weight | Bone Resorption | Brazil | Endotoxemia | Lipid Metabolism | Male | Mice | Mice, Inbred C57BL | Periodontitis | Porphyromonas gingivalis | Propolis | Protective Agents [SUMMARY] | [CONTENT] Alveolar Bone Loss | Animals | Blood Glucose | Body Weight | Bone Resorption | Brazil | Endotoxemia | Lipid Metabolism | Male | Mice | Mice, Inbred C57BL | Periodontitis | Porphyromonas gingivalis | Propolis | Protective Agents [SUMMARY] | [CONTENT] Alveolar Bone Loss | Animals | Blood Glucose | Body Weight | Bone Resorption | Brazil | Endotoxemia | Lipid Metabolism | Male | Mice | Mice, Inbred C57BL | Periodontitis | Porphyromonas gingivalis | Propolis | Protective Agents [SUMMARY] | [CONTENT] Alveolar Bone Loss | Animals | Blood Glucose | Body Weight | Bone Resorption | Brazil | Endotoxemia | Lipid Metabolism | Male | Mice | Mice, Inbred C57BL | Periodontitis | Porphyromonas gingivalis | Propolis | Protective Agents [SUMMARY] | [CONTENT] disease periodontopathic bacteria | periodontal human diseases | periodontopathic bacteria induced | periodontitis systemic | inflammation gingival tissues [SUMMARY] | null | [CONTENT] disease periodontopathic bacteria | periodontal human diseases | periodontopathic bacteria induced | periodontitis systemic | inflammation gingival tissues [SUMMARY] | [CONTENT] disease periodontopathic bacteria | periodontal human diseases | periodontopathic bacteria induced | periodontitis systemic | inflammation gingival tissues [SUMMARY] | [CONTENT] disease periodontopathic bacteria | periodontal human diseases | periodontopathic bacteria induced | periodontitis systemic | inflammation gingival tissues [SUMMARY] | [CONTENT] disease periodontopathic bacteria | periodontal human diseases | periodontopathic bacteria induced | periodontitis systemic | inflammation gingival tissues [SUMMARY] | [CONTENT] gingivalis | mice | administered | propolis | administered mice | administration | expression | administered mice gingivalis | mice gingivalis | gingivalis propolis [SUMMARY] | null | [CONTENT] gingivalis | mice | administered | propolis | administered mice | administration | expression | administered mice gingivalis | mice gingivalis | gingivalis propolis [SUMMARY] | [CONTENT] gingivalis | mice | administered | propolis | administered mice | administration | expression | administered mice gingivalis | mice gingivalis | gingivalis propolis [SUMMARY] | [CONTENT] gingivalis | mice | administered | propolis | administered mice | administration | expression | administered mice gingivalis | mice gingivalis | gingivalis propolis [SUMMARY] | [CONTENT] gingivalis | mice | administered | propolis | administered mice | administration | expression | administered mice gingivalis | mice gingivalis | gingivalis propolis [SUMMARY] | [CONTENT] diseases | periodontal | propolis | anti | systemic | bacteria | glucose | blood | diabetes | periodontopathic [SUMMARY] | null | [CONTENT] administered mice | administered | gingivalis | mice | propolis | administered mice gingivalis | mice gingivalis | expression | gingivalis propolis | gingivalis administered [SUMMARY] | [CONTENT] induced metabolic | metabolic | induced | changes increase risk systemic | metabolic changes | metabolic changes increase | metabolic changes increase risk | metabolic disturbance | metabolic disturbance results | metabolic disturbance results suggest [SUMMARY] | [CONTENT] gingivalis | administered | mice | administered mice | propolis | mice gingivalis | administered mice gingivalis | test | administration | expression [SUMMARY] | [CONTENT] gingivalis | administered | mice | administered mice | propolis | mice gingivalis | administered mice gingivalis | test | administration | expression [SUMMARY] | [CONTENT] Periodontitis ||| ||| Propolis ||| [SUMMARY] | null | [CONTENT] Irs1 | Acox ||| ||| ||| ||| [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] Periodontitis ||| ||| Propolis ||| ||| Eight-week-old | W83 ||| ||| ||| first ||| Irs1 | Acox ||| ||| ||| ||| ||| [SUMMARY] | [CONTENT] Periodontitis ||| ||| Propolis ||| ||| Eight-week-old | W83 ||| ||| ||| first ||| Irs1 | Acox ||| ||| ||| ||| ||| [SUMMARY] |
|||||
Lifetime expectancy and quality-adjusted life-year in Alzheimer's disease with and without cerebrovascular disease: effects of nursing home replacement and donepezil administration--a retrospective analysis in the Tajiri Project. | 26542372 | We previously demonstrated a positive correlation with nursing home (NH) replacement and donepezil (DNP) administration on lifetime expectancy after the onset of Alzheimer's disease (AD). However, the correlation with quality-adjusted life-year (QALY) remains to be elucidated, along with the additional impact of concomitant cerebrovascular disease (CVD). Based upon our recently reported health state utility values, we retrospectively analyzed the correlation with NH replacement and/or DNP administration on QALY and life expectancy in 'pure' AD (without CVD) and AD with CVD patients. | BACKGROUND | All outpatients at the Tajiri Clinic from 1999-2012 with available medical records and death certificates were included. The entry criteria were a dementia diagnosis (DSM-IV) and diagnoses of pure AD or AD with CVD (NINCDS-ADRDA), medical treatment for more than 3 months, and follow up to less than 1 year before death. The main outcomes were lifetime expectancy (months between the onset of dementia and death) and QALY. | METHODS | We identified 390 subjects, of whom 275 had the diagnosis of dementia that met the entry criteria, including 67 pure AD, 33 AD with CVD, and 110 VaD patients. For the AD patients, 52 had taken DNP and 48 had not received the drug due to treatment prior to the introduction of DNP in 1999 in Japan. For the pure AD group, there were positive correlation between NH and DNP and QALY, as well as lifetime expectancy. As for the AD with CVD group, only a correlation between DNP and lifetime expectancy was noted, with no correlation with QALY. | RESULTS | We found positive correlations between DNP administration and NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such a positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD; thus, indicating the importance of CVD prevention. | CONCLUSIONS | [
"Alzheimer Disease",
"Cerebrovascular Disorders",
"Cholinesterase Inhibitors",
"Comorbidity",
"Donepezil",
"Humans",
"Indans",
"Japan",
"Life Expectancy",
"Nursing Homes",
"Piperidines",
"Quality-Adjusted Life Years",
"Retrospective Studies"
] | 4635582 | Background | There are no curative drugs for Alzheimer’s disease (AD) at present; however, symptomatic drugs, such as cholinesterase inhibitors (ChEIs) can delay progression of the disease. This effect, combined with psychosocial interventions, can increase the quality of life (QOL) [1]. These drugs administration can delay nursing home placement [2] and may reduce mortality for patients living in nursing homes [3] and in the community [4]. Beyond delayed progression and improved QOL, the ultimate outcome of drug treatment should be measured in terms of lifetime expectancy.
The correlations of drug therapy with lifetime expectancy have not been fully investigated with various findings. ChEIs were reported to be able to delay a nursing home replacement, but have no effect on lifetime expectancy [4], or ChEIs were associated with a lower risk of death and myocardial infarction [5]. Using the database of all outpatients with available medical records and death certificates at the Tajiri Clinic from 1999–2012, we identified 100 patients that were diagnosed with AD; 52 had taken DNP and 48 patients had not received the drug due to treatment prior to the introduction of donepezil in 1999 in Japan. We previously reported a positive impact of nursing home (NH) placement and DNP administration on lifetime expectancy after the onset of AD [6].
However, simple prolongation of the lifetime expectancy, with possible decreased QOL, remains a problem. Instead, “healthy life expectancy” has emerged as an important issue. In this respect, the quality-adjusted life-year (QALY) and health state utility values (HSUVs) are major QOL scales that are used in the analyses of health economics of diseases [7]. In Japan, the most common dementia disease is AD with cerebrovascular disease (CVD), followed by ‘pure’ AD (without CVD) [8, 9]. With respect to the need to reconsider QALY in the context of activities of daily living (ADL) levels in dementia, we previously calculated the HSUVs under the conditions of AD and AD with CVD: from previous reports and EQ-5D [10–12], we estimated that the HSUVs of pure AD and AD with CVD for ADL level A (independent walking and eating), B (some problems with walking but sitting without assistance), and C (confined to bed) [13] were 0.61 and 0.58, 0.53 and 0.28, and 0.19 and 0.05, respectively [14].
Using the same database, we re-analyzed the correlation between NH replacement and/or DNP administration and QALY and lifetime expectancy in AD without CVD and AD with CVD. We hypothesized that 1) the drug would exhibit a positive correlation with QALY as well as lifetime expectancy in both AD without CVD and AD with CVD patients, and 2) that NH residency would also exhibit a positive correlation. We analyzed DNP alone, because this drug has been used since 1999 in Japan, whereas other drugs, such as galantamine, have only been used since 2011. The combined effect of DNP and NH residency was also analyzed. Despite the retrospective design, this was a long-term study of the possible effect of donepezil on QALY, as well as the life expectancy of patients with AD without CVD and AD with CVD. | null | null | null | null | Conclusions | Although this report has the limitation as all retrospective analyses: i.e., the lack of randomization, we found a positive correlation between DNP administration as well as NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD, thus indicating the importance of CVD prevention. | [
"Background",
"Methods",
"Dementia diagnosis",
"Analyses",
"Results",
"Demographics",
"Effects of NH replacement and DNP administration",
"Correlations between the periods of DNP use",
"Discussion",
"Summary of the results",
"Positive correlation between DNP and/or NH replacement and lifetime expectancy and QALY in pure AD",
"Concomitant CVD negated the positive correlation with QALY in AD with CVD, but the correlation between DNP and lifetime expectancy remained",
"Socio-economic effects"
] | [
"There are no curative drugs for Alzheimer’s disease (AD) at present; however, symptomatic drugs, such as cholinesterase inhibitors (ChEIs) can delay progression of the disease. This effect, combined with psychosocial interventions, can increase the quality of life (QOL) [1]. These drugs administration can delay nursing home placement [2] and may reduce mortality for patients living in nursing homes [3] and in the community [4]. Beyond delayed progression and improved QOL, the ultimate outcome of drug treatment should be measured in terms of lifetime expectancy.\nThe correlations of drug therapy with lifetime expectancy have not been fully investigated with various findings. ChEIs were reported to be able to delay a nursing home replacement, but have no effect on lifetime expectancy [4], or ChEIs were associated with a lower risk of death and myocardial infarction [5]. Using the database of all outpatients with available medical records and death certificates at the Tajiri Clinic from 1999–2012, we identified 100 patients that were diagnosed with AD; 52 had taken DNP and 48 patients had not received the drug due to treatment prior to the introduction of donepezil in 1999 in Japan. We previously reported a positive impact of nursing home (NH) placement and DNP administration on lifetime expectancy after the onset of AD [6].\nHowever, simple prolongation of the lifetime expectancy, with possible decreased QOL, remains a problem. Instead, “healthy life expectancy” has emerged as an important issue. In this respect, the quality-adjusted life-year (QALY) and health state utility values (HSUVs) are major QOL scales that are used in the analyses of health economics of diseases [7]. In Japan, the most common dementia disease is AD with cerebrovascular disease (CVD), followed by ‘pure’ AD (without CVD) [8, 9]. With respect to the need to reconsider QALY in the context of activities of daily living (ADL) levels in dementia, we previously calculated the HSUVs under the conditions of AD and AD with CVD: from previous reports and EQ-5D [10–12], we estimated that the HSUVs of pure AD and AD with CVD for ADL level A (independent walking and eating), B (some problems with walking but sitting without assistance), and C (confined to bed) [13] were 0.61 and 0.58, 0.53 and 0.28, and 0.19 and 0.05, respectively [14].\nUsing the same database, we re-analyzed the correlation between NH replacement and/or DNP administration and QALY and lifetime expectancy in AD without CVD and AD with CVD. We hypothesized that 1) the drug would exhibit a positive correlation with QALY as well as lifetime expectancy in both AD without CVD and AD with CVD patients, and 2) that NH residency would also exhibit a positive correlation. We analyzed DNP alone, because this drug has been used since 1999 in Japan, whereas other drugs, such as galantamine, have only been used since 2011. The combined effect of DNP and NH residency was also analyzed. Despite the retrospective design, this was a long-term study of the possible effect of donepezil on QALY, as well as the life expectancy of patients with AD without CVD and AD with CVD.",
" Dementia diagnosis Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nAD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.\nAD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nDiagnoses of other dementing diseases were described in the previous report [6].\nWritten informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.\nDiagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nAD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.\nAD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nDiagnoses of other dementing diseases were described in the previous report [6].\nWritten informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.\n Analyses The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.\nThe main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.",
"Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nAD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.\nAD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nDiagnoses of other dementing diseases were described in the previous report [6].\nWritten informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.",
"The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.",
" Demographics As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.\nAs described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.\n Effects of NH replacement and DNP administration Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD\nn = 25\nn = 27\nn = 6\nn = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD\nn = 9\nn = 14\nn = 8\nn = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\nDNP donepezil, NH nursing home\nEffects of NH replacement and DNP administration on lifetime expectancy and QALY\nTwo-way ANOVAs with the covariance of age and gender were performed\n*p < 0.05, **p < 0.01, ***p < 0.001\n\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\n\nDNP donepezil, NH nursing home\nOn top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.\nTable 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD\nn = 25\nn = 27\nn = 6\nn = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD\nn = 9\nn = 14\nn = 8\nn = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\nDNP donepezil, NH nursing home\nEffects of NH replacement and DNP administration on lifetime expectancy and QALY\nTwo-way ANOVAs with the covariance of age and gender were performed\n*p < 0.05, **p < 0.01, ***p < 0.001\n\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\n\nDNP donepezil, NH nursing home\nOn top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.\n Correlations between the periods of DNP use Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group\nPeriod of DNP use and QALY for each AD group\nSpearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group\nPeriod of DNP use and QALY for each AD group",
"As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.",
"Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD\nn = 25\nn = 27\nn = 6\nn = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD\nn = 9\nn = 14\nn = 8\nn = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\nDNP donepezil, NH nursing home\nEffects of NH replacement and DNP administration on lifetime expectancy and QALY\nTwo-way ANOVAs with the covariance of age and gender were performed\n*p < 0.05, **p < 0.01, ***p < 0.001\n\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\n\nDNP donepezil, NH nursing home\nOn top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.",
"Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group\nPeriod of DNP use and QALY for each AD group",
" Summary of the results Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.\nOur previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.\n Positive correlation between DNP and/or NH replacement and lifetime expectancy and QALY in pure AD We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.\nDNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].\nWe previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.\nDNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].\n Concomitant CVD negated the positive correlation with QALY in AD with CVD, but the correlation between DNP and lifetime expectancy remained However, concomitant CVD negated such positive effects. Each factor is discussed in turn.\nThe improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.\nIt is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.\nHowever, concomitant CVD negated such positive effects. Each factor is discussed in turn.\nThe improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.\nIt is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.\n Socio-economic effects We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.\nFigure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].\nWe calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.\nFigure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].",
"Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.",
"We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.\nDNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].",
"However, concomitant CVD negated such positive effects. Each factor is discussed in turn.\nThe improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.\nIt is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.",
"We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.\nFigure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26]."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Dementia diagnosis",
"Analyses",
"Results",
"Demographics",
"Effects of NH replacement and DNP administration",
"Correlations between the periods of DNP use",
"Discussion",
"Summary of the results",
"Positive correlation between DNP and/or NH replacement and lifetime expectancy and QALY in pure AD",
"Concomitant CVD negated the positive correlation with QALY in AD with CVD, but the correlation between DNP and lifetime expectancy remained",
"Socio-economic effects",
"Conclusions"
] | [
"There are no curative drugs for Alzheimer’s disease (AD) at present; however, symptomatic drugs, such as cholinesterase inhibitors (ChEIs) can delay progression of the disease. This effect, combined with psychosocial interventions, can increase the quality of life (QOL) [1]. These drugs administration can delay nursing home placement [2] and may reduce mortality for patients living in nursing homes [3] and in the community [4]. Beyond delayed progression and improved QOL, the ultimate outcome of drug treatment should be measured in terms of lifetime expectancy.\nThe correlations of drug therapy with lifetime expectancy have not been fully investigated with various findings. ChEIs were reported to be able to delay a nursing home replacement, but have no effect on lifetime expectancy [4], or ChEIs were associated with a lower risk of death and myocardial infarction [5]. Using the database of all outpatients with available medical records and death certificates at the Tajiri Clinic from 1999–2012, we identified 100 patients that were diagnosed with AD; 52 had taken DNP and 48 patients had not received the drug due to treatment prior to the introduction of donepezil in 1999 in Japan. We previously reported a positive impact of nursing home (NH) placement and DNP administration on lifetime expectancy after the onset of AD [6].\nHowever, simple prolongation of the lifetime expectancy, with possible decreased QOL, remains a problem. Instead, “healthy life expectancy” has emerged as an important issue. In this respect, the quality-adjusted life-year (QALY) and health state utility values (HSUVs) are major QOL scales that are used in the analyses of health economics of diseases [7]. In Japan, the most common dementia disease is AD with cerebrovascular disease (CVD), followed by ‘pure’ AD (without CVD) [8, 9]. With respect to the need to reconsider QALY in the context of activities of daily living (ADL) levels in dementia, we previously calculated the HSUVs under the conditions of AD and AD with CVD: from previous reports and EQ-5D [10–12], we estimated that the HSUVs of pure AD and AD with CVD for ADL level A (independent walking and eating), B (some problems with walking but sitting without assistance), and C (confined to bed) [13] were 0.61 and 0.58, 0.53 and 0.28, and 0.19 and 0.05, respectively [14].\nUsing the same database, we re-analyzed the correlation between NH replacement and/or DNP administration and QALY and lifetime expectancy in AD without CVD and AD with CVD. We hypothesized that 1) the drug would exhibit a positive correlation with QALY as well as lifetime expectancy in both AD without CVD and AD with CVD patients, and 2) that NH residency would also exhibit a positive correlation. We analyzed DNP alone, because this drug has been used since 1999 in Japan, whereas other drugs, such as galantamine, have only been used since 2011. The combined effect of DNP and NH residency was also analyzed. Despite the retrospective design, this was a long-term study of the possible effect of donepezil on QALY, as well as the life expectancy of patients with AD without CVD and AD with CVD.",
" Dementia diagnosis Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nAD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.\nAD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nDiagnoses of other dementing diseases were described in the previous report [6].\nWritten informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.\nDiagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nAD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.\nAD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nDiagnoses of other dementing diseases were described in the previous report [6].\nWritten informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.\n Analyses The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.\nThe main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.",
"Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nAD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.\nAD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.\nDiagnoses of other dementing diseases were described in the previous report [6].\nWritten informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.",
"The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.",
" Demographics As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.\nAs described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.\n Effects of NH replacement and DNP administration Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD\nn = 25\nn = 27\nn = 6\nn = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD\nn = 9\nn = 14\nn = 8\nn = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\nDNP donepezil, NH nursing home\nEffects of NH replacement and DNP administration on lifetime expectancy and QALY\nTwo-way ANOVAs with the covariance of age and gender were performed\n*p < 0.05, **p < 0.01, ***p < 0.001\n\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\n\nDNP donepezil, NH nursing home\nOn top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.\nTable 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD\nn = 25\nn = 27\nn = 6\nn = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD\nn = 9\nn = 14\nn = 8\nn = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\nDNP donepezil, NH nursing home\nEffects of NH replacement and DNP administration on lifetime expectancy and QALY\nTwo-way ANOVAs with the covariance of age and gender were performed\n*p < 0.05, **p < 0.01, ***p < 0.001\n\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\n\nDNP donepezil, NH nursing home\nOn top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.\n Correlations between the periods of DNP use Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group\nPeriod of DNP use and QALY for each AD group\nSpearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group\nPeriod of DNP use and QALY for each AD group",
"As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.",
"Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD\nn = 25\nn = 27\nn = 6\nn = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD\nn = 9\nn = 14\nn = 8\nn = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\nDNP donepezil, NH nursing home\nEffects of NH replacement and DNP administration on lifetime expectancy and QALY\nTwo-way ANOVAs with the covariance of age and gender were performed\n*p < 0.05, **p < 0.01, ***p < 0.001\n\nAD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year\n\nDNP donepezil, NH nursing home\nOn top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.",
"Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group\nPeriod of DNP use and QALY for each AD group",
" Summary of the results Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.\nOur previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.\n Positive correlation between DNP and/or NH replacement and lifetime expectancy and QALY in pure AD We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.\nDNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].\nWe previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.\nDNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].\n Concomitant CVD negated the positive correlation with QALY in AD with CVD, but the correlation between DNP and lifetime expectancy remained However, concomitant CVD negated such positive effects. Each factor is discussed in turn.\nThe improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.\nIt is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.\nHowever, concomitant CVD negated such positive effects. Each factor is discussed in turn.\nThe improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.\nIt is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.\n Socio-economic effects We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.\nFigure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].\nWe calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.\nFigure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].",
"Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.",
"We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.\nDNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].",
"However, concomitant CVD negated such positive effects. Each factor is discussed in turn.\nThe improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.\nIt is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.",
"We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.\nFigure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan\nThe “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].",
"Although this report has the limitation as all retrospective analyses: i.e., the lack of randomization, we found a positive correlation between DNP administration as well as NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD, thus indicating the importance of CVD prevention."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
"conclusion"
] | [
"Quality-adjusted life-year",
"Cerebrovascular disease",
"Alzheimer’s disease",
"Donepezil"
] | Background:
There are no curative drugs for Alzheimer’s disease (AD) at present; however, symptomatic drugs, such as cholinesterase inhibitors (ChEIs) can delay progression of the disease. This effect, combined with psychosocial interventions, can increase the quality of life (QOL) [1]. These drugs administration can delay nursing home placement [2] and may reduce mortality for patients living in nursing homes [3] and in the community [4]. Beyond delayed progression and improved QOL, the ultimate outcome of drug treatment should be measured in terms of lifetime expectancy.
The correlations of drug therapy with lifetime expectancy have not been fully investigated with various findings. ChEIs were reported to be able to delay a nursing home replacement, but have no effect on lifetime expectancy [4], or ChEIs were associated with a lower risk of death and myocardial infarction [5]. Using the database of all outpatients with available medical records and death certificates at the Tajiri Clinic from 1999–2012, we identified 100 patients that were diagnosed with AD; 52 had taken DNP and 48 patients had not received the drug due to treatment prior to the introduction of donepezil in 1999 in Japan. We previously reported a positive impact of nursing home (NH) placement and DNP administration on lifetime expectancy after the onset of AD [6].
However, simple prolongation of the lifetime expectancy, with possible decreased QOL, remains a problem. Instead, “healthy life expectancy” has emerged as an important issue. In this respect, the quality-adjusted life-year (QALY) and health state utility values (HSUVs) are major QOL scales that are used in the analyses of health economics of diseases [7]. In Japan, the most common dementia disease is AD with cerebrovascular disease (CVD), followed by ‘pure’ AD (without CVD) [8, 9]. With respect to the need to reconsider QALY in the context of activities of daily living (ADL) levels in dementia, we previously calculated the HSUVs under the conditions of AD and AD with CVD: from previous reports and EQ-5D [10–12], we estimated that the HSUVs of pure AD and AD with CVD for ADL level A (independent walking and eating), B (some problems with walking but sitting without assistance), and C (confined to bed) [13] were 0.61 and 0.58, 0.53 and 0.28, and 0.19 and 0.05, respectively [14].
Using the same database, we re-analyzed the correlation between NH replacement and/or DNP administration and QALY and lifetime expectancy in AD without CVD and AD with CVD. We hypothesized that 1) the drug would exhibit a positive correlation with QALY as well as lifetime expectancy in both AD without CVD and AD with CVD patients, and 2) that NH residency would also exhibit a positive correlation. We analyzed DNP alone, because this drug has been used since 1999 in Japan, whereas other drugs, such as galantamine, have only been used since 2011. The combined effect of DNP and NH residency was also analyzed. Despite the retrospective design, this was a long-term study of the possible effect of donepezil on QALY, as well as the life expectancy of patients with AD without CVD and AD with CVD.
Methods:
Dementia diagnosis Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.
AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.
AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.
Diagnoses of other dementing diseases were described in the previous report [6].
Written informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.
Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.
AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.
AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.
Diagnoses of other dementing diseases were described in the previous report [6].
Written informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.
Analyses The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.
The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.
Dementia diagnosis:
Diagnoses of the following diseases were determined during a meeting of two neurologists, a psychiatrist, and a physician.AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.
AD without CVD was diagnosed in patients who met the NINCDS-ADRDA criteria for probable AD [15] and had no CVD on MRI. On MRI, low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and high signal intensity surrounding the low signal intensity areas on FLAIR images were considered to indicate CVD.
AD with CVD was diagnosed according to the NINCDS-ADRDA criteria for probable AD and on evidence for the presence of CVD on MRI; however, CVD lesions were judged to be concomitant with AD and not responsible for cognitive deterioration.
Diagnoses of other dementing diseases were described in the previous report [6].
Written informed consent was obtained from each patient and from the family of those with dementia at entry according to the Declaration of Helsinki (BMJ 1991; 302: 1194). The study was approved by the ethical committee of Tohoku University Graduate School of Medicine, as well as those of the Osaki-Tajiri SKIP Center.
Analyses:
The main outcomes were QALY and lifetime expectancy (i.e., the number of months between the onset of dementia and death). The onset of dementia was confirmed by extensive hearing of medical histories from their families. Two-way ANOVA with the covariance of age and sex, included the effects of NH replacement and DNP administration for the AD and AD with CVD groups. Spearman’s correlations were used to examine the relationship between the DNP use periods and QALY as well as the lifetime expectancy in both groups. Data were available for all 100 participants. The sample size was sufficiently powered to analyze the group effect at a significance level of p < 0.05. The error protection was = 0.05 and power was 0.8, thus the effected size was 15 for each group.
Results:
Demographics As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.
As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.
Effects of NH replacement and DNP administration Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD
n = 25
n = 27
n = 6
n = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD
n = 9
n = 14
n = 8
n = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001
AD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year
DNP donepezil, NH nursing home
Effects of NH replacement and DNP administration on lifetime expectancy and QALY
Two-way ANOVAs with the covariance of age and gender were performed
*p < 0.05, **p < 0.01, ***p < 0.001
AD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year
DNP donepezil, NH nursing home
On top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.
Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD
n = 25
n = 27
n = 6
n = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD
n = 9
n = 14
n = 8
n = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001
AD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year
DNP donepezil, NH nursing home
Effects of NH replacement and DNP administration on lifetime expectancy and QALY
Two-way ANOVAs with the covariance of age and gender were performed
*p < 0.05, **p < 0.01, ***p < 0.001
AD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year
DNP donepezil, NH nursing home
On top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.
Correlations between the periods of DNP use Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group
Period of DNP use and QALY for each AD group
Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group
Period of DNP use and QALY for each AD group
Demographics:
As described previously, of the 100 patients with AD and AD with CVD (both of which being diseases that can be treated with DNP), 52 received DNP and 48 patients did not receive the drug due to treatment prior to the introduction of DNP in 1999 in Japan.
Effects of NH replacement and DNP administration:
Table 1 presents the effects of NH replacement and DNP administration on lifetime expectancy and QALY. For the pure AD group, there were positive SNH and DNP effects on QALY as well as lifetime expectancy. As for the AD with CVD group, there were no NH effects, only the DNP effect on life expectancy with no effect on QALY.Table 1Effects of NH replacement and DNP administration on lifetime expectancy and QALYown homesNH replacementF-valuescovarianceno DNPDNPno DNPDNPNHDNPinteractagesexAD
n = 25
n = 27
n = 6
n = 7lifetime expectancy56.3 (44.6)84.7 (36.3)122.5 (59.7)156.4 (37.8)20.442***4.704*0.0020.8732.482QALY26.4 (17.8)47.0 (20.5)53.1 (18.2)75.7 (18.5)15.123***11.859***0.0000.8543.738AD with CVD
n = 9
n = 14
n = 8
n = 4lifetime expectancy49.9 (41.6)77.6 (32.9)57.5 (44.6)119.0 (46.2)2.2077.861**1.0340.2650.364QALY30.4 (21.3)41.9 (16.1)30.8 (27.1)54.4 (29.4)0.743.6080.650.0772.162Two-way ANOVAs with the covariance of age and gender were performed*p < 0.05, **p < 0.01, ***p < 0.001
AD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year
DNP donepezil, NH nursing home
Effects of NH replacement and DNP administration on lifetime expectancy and QALY
Two-way ANOVAs with the covariance of age and gender were performed
*p < 0.05, **p < 0.01, ***p < 0.001
AD Alzheimer's disease, CVD cerebrovascular diseases, QALY quality-adusted life-year
DNP donepezil, NH nursing home
On top of the previous analysis (DNP vs Non-DNP) [6], AD with/without CVD were herein analyzed; thus the significant p-values would be better less than 0.05/2 = 0.025. The correlation between DNP and lifetime expectancy in the AD without CVD group (F = 4.704) in Table 1 may be influenced by the familywise error.
Correlations between the periods of DNP use:
Spearman’s correlation analyses revealed that all relationships between the period of DNP use and QALY, as well as lifetime expectancy were significantly positive (biologically meaningful) in the AD and AD with CVD patients. Figure 1 presents the period of DNP use and QALY for each AD group.Fig. 1Period of DNP use and QALY for each AD group
Period of DNP use and QALY for each AD group
Discussion:
Summary of the results Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.
Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.
Positive correlation between DNP and/or NH replacement and lifetime expectancy and QALY in pure AD We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.
DNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].
We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.
DNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].
Concomitant CVD negated the positive correlation with QALY in AD with CVD, but the correlation between DNP and lifetime expectancy remained However, concomitant CVD negated such positive effects. Each factor is discussed in turn.
The improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.
It is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.
However, concomitant CVD negated such positive effects. Each factor is discussed in turn.
The improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.
It is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.
Socio-economic effects We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.
Figure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan
The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan
The “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].
We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.
Figure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan
The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan
The “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].
Summary of the results:
Our previous study reported the positive effects of DNP administration and NH replacement on lifetime expectancy after the onset of AD; however, simple prolongation of lifetime expectancy with possible decreased QOL remains a problem. Instead, “healthy life expectancy,” i.e., QALY, remains an important issue. Herein, we report the effects on QALY. No covariance effects of age and sex may be due to all patients were old and male to female ratio was not so different between the groups.
Positive correlation between DNP and/or NH replacement and lifetime expectancy and QALY in pure AD:
We previously discussed that while the possible correlation between DNP and mortality remains uncertain, there is also uncertainty regarding its effect on QALY. DNP may have a negative effect on aspiration pneumonia due to the side effect of nausea. An increased gastro-esophageal reflex may also induce pneumonia. This suggests that the effect of DNP on lifetime expectancy or QALY was not purely pharmacological.
DNP administration can improve psychomotor speed or attention function, associated with the frontal lobe [16, 17]. It is consistent with the higher mortality in older adults with lower perceptual speed [18]. Their activities of daily living (ADL) are also stimulated. In a study of the long-term effects of DNP and the use of community-based home help service, the drug was reported to maintain higher self-supported levels of instrumental ADL [19]. Rehabilitation also exhibits a long-term effect in decreasing mortality, and particularly improves motor disability and ADL [20], and prevents aspiration pneumonia [21].
Concomitant CVD negated the positive correlation with QALY in AD with CVD, but the correlation between DNP and lifetime expectancy remained:
However, concomitant CVD negated such positive effects. Each factor is discussed in turn.
The improvement of psychomotor speed or attention function after DNP administration could be poorer in the AD with CVD than in the pure AD patients. By definition, no concomitant CVDs were located in the “strategic” areas, such as the thalamus in the AD with CVD patients; however, the effect of white matter changes on the cholinergic network can be considered. Indeed, the CHIPS scores in these patients were higher than the pure AD patients (data not shown). Thus the effect of DNP on psychomotor speed or attention function is not “full-blown,” and the possible lack of a “synergistic” effect with psychosocial intervention performed in the NH cannot lead to any improvement of QALY.
It is noteworthy that the effect of DNP remained with respect to lifetime expectancy. There were no remarkable differences in the incidence of pneumonia and/or respiratory failure as the cause of death between the AD and AD with CVD groups, and it is only possible to speculate the reason for the effect of DNP on lifetime expectancy in the absence of an effect on QALY. Taking into consideration nausea as a side effect of DNP, together with increased gastro-esophageal reflux, both may induce pneumonia; the effect of DNP on lifetime expectancy was not considered as being purely pharmacological. However, increasing the level of acetylcholine to approach a healthy level in the brain might influence the “energy level” of whole body and thus the biological lifetime would be prolonged. Another possibility is that when the QALY is evaluated based on the HSUV, which is mainly based on ADLs, meaningful but statistically small changes of QALY might be underestimated.
Socio-economic effects:
We calculated QALY of AD and AD with CVD, based on the HSUV for AD by taking ADL into consideration. There were no previous reports that estimated the HSUVs according to the AD severity with ADL extent, or considered complications, including CVD, but only domestic studies on items of AD [22], long-term care [23], and the extent of ADL [24] were found but there were no reports that evaluated each of these issues together. We estimated the HSUV value based on the ADL. We suggest that the analysis of healthcare economics of dementia in Japan could be improved through consideration of the extent of ADL, in addition to the severity of AD.
Figure 2 presents our hypothesis of the concept of the dementia state. In Western countries, few mild AD patients also exhibit CVD and low levels of physical ADL. However, in Japan, most mild AD patients could exhibit CVD and low levels of physical ADL. This state is called ‘boke' in Japanese.Fig. 2The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan
The models of the relationship between Alzheimer’s disease, cerebrovascular disease, and low level of physical ADL in Europe and the United States and Japan
The “Orange Plan”, a 5-year plan for the promotion of measures against dementia in Japan, suggests that people with dementia want to live in their own home as long as possible. However, people with dementia or mild cognitive impairment (MCI) face difficulties in maintaining their daily lives. Maintaining a good level of drug compliance remains an essential part in preserving their lives at home. Also, the findings suggest that QALY in dementia is affected by CVD; thus, indicating the importance of CVD prevention. Indeed, the most common dementing disease in Japan is AD with CVD. Comprehensive measures for stroke and dementia prevention (primary, secondary, and tertiary) in the community are necessary to maintain the QOL in older adults [25, 26].
Conclusions:
Although this report has the limitation as all retrospective analyses: i.e., the lack of randomization, we found a positive correlation between DNP administration as well as NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD, thus indicating the importance of CVD prevention.
| Background:
We previously demonstrated a positive correlation with nursing home (NH) replacement and donepezil (DNP) administration on lifetime expectancy after the onset of Alzheimer's disease (AD). However, the correlation with quality-adjusted life-year (QALY) remains to be elucidated, along with the additional impact of concomitant cerebrovascular disease (CVD). Based upon our recently reported health state utility values, we retrospectively analyzed the correlation with NH replacement and/or DNP administration on QALY and life expectancy in 'pure' AD (without CVD) and AD with CVD patients.
Methods:
All outpatients at the Tajiri Clinic from 1999-2012 with available medical records and death certificates were included. The entry criteria were a dementia diagnosis (DSM-IV) and diagnoses of pure AD or AD with CVD (NINCDS-ADRDA), medical treatment for more than 3 months, and follow up to less than 1 year before death. The main outcomes were lifetime expectancy (months between the onset of dementia and death) and QALY.
Results:
We identified 390 subjects, of whom 275 had the diagnosis of dementia that met the entry criteria, including 67 pure AD, 33 AD with CVD, and 110 VaD patients. For the AD patients, 52 had taken DNP and 48 had not received the drug due to treatment prior to the introduction of DNP in 1999 in Japan. For the pure AD group, there were positive correlation between NH and DNP and QALY, as well as lifetime expectancy. As for the AD with CVD group, only a correlation between DNP and lifetime expectancy was noted, with no correlation with QALY.
Conclusions:
We found positive correlations between DNP administration and NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such a positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD; thus, indicating the importance of CVD prevention. | Background:
There are no curative drugs for Alzheimer’s disease (AD) at present; however, symptomatic drugs, such as cholinesterase inhibitors (ChEIs) can delay progression of the disease. This effect, combined with psychosocial interventions, can increase the quality of life (QOL) [1]. These drugs administration can delay nursing home placement [2] and may reduce mortality for patients living in nursing homes [3] and in the community [4]. Beyond delayed progression and improved QOL, the ultimate outcome of drug treatment should be measured in terms of lifetime expectancy.
The correlations of drug therapy with lifetime expectancy have not been fully investigated with various findings. ChEIs were reported to be able to delay a nursing home replacement, but have no effect on lifetime expectancy [4], or ChEIs were associated with a lower risk of death and myocardial infarction [5]. Using the database of all outpatients with available medical records and death certificates at the Tajiri Clinic from 1999–2012, we identified 100 patients that were diagnosed with AD; 52 had taken DNP and 48 patients had not received the drug due to treatment prior to the introduction of donepezil in 1999 in Japan. We previously reported a positive impact of nursing home (NH) placement and DNP administration on lifetime expectancy after the onset of AD [6].
However, simple prolongation of the lifetime expectancy, with possible decreased QOL, remains a problem. Instead, “healthy life expectancy” has emerged as an important issue. In this respect, the quality-adjusted life-year (QALY) and health state utility values (HSUVs) are major QOL scales that are used in the analyses of health economics of diseases [7]. In Japan, the most common dementia disease is AD with cerebrovascular disease (CVD), followed by ‘pure’ AD (without CVD) [8, 9]. With respect to the need to reconsider QALY in the context of activities of daily living (ADL) levels in dementia, we previously calculated the HSUVs under the conditions of AD and AD with CVD: from previous reports and EQ-5D [10–12], we estimated that the HSUVs of pure AD and AD with CVD for ADL level A (independent walking and eating), B (some problems with walking but sitting without assistance), and C (confined to bed) [13] were 0.61 and 0.58, 0.53 and 0.28, and 0.19 and 0.05, respectively [14].
Using the same database, we re-analyzed the correlation between NH replacement and/or DNP administration and QALY and lifetime expectancy in AD without CVD and AD with CVD. We hypothesized that 1) the drug would exhibit a positive correlation with QALY as well as lifetime expectancy in both AD without CVD and AD with CVD patients, and 2) that NH residency would also exhibit a positive correlation. We analyzed DNP alone, because this drug has been used since 1999 in Japan, whereas other drugs, such as galantamine, have only been used since 2011. The combined effect of DNP and NH residency was also analyzed. Despite the retrospective design, this was a long-term study of the possible effect of donepezil on QALY, as well as the life expectancy of patients with AD without CVD and AD with CVD.
Conclusions:
Although this report has the limitation as all retrospective analyses: i.e., the lack of randomization, we found a positive correlation between DNP administration as well as NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD, thus indicating the importance of CVD prevention. | Background:
We previously demonstrated a positive correlation with nursing home (NH) replacement and donepezil (DNP) administration on lifetime expectancy after the onset of Alzheimer's disease (AD). However, the correlation with quality-adjusted life-year (QALY) remains to be elucidated, along with the additional impact of concomitant cerebrovascular disease (CVD). Based upon our recently reported health state utility values, we retrospectively analyzed the correlation with NH replacement and/or DNP administration on QALY and life expectancy in 'pure' AD (without CVD) and AD with CVD patients.
Methods:
All outpatients at the Tajiri Clinic from 1999-2012 with available medical records and death certificates were included. The entry criteria were a dementia diagnosis (DSM-IV) and diagnoses of pure AD or AD with CVD (NINCDS-ADRDA), medical treatment for more than 3 months, and follow up to less than 1 year before death. The main outcomes were lifetime expectancy (months between the onset of dementia and death) and QALY.
Results:
We identified 390 subjects, of whom 275 had the diagnosis of dementia that met the entry criteria, including 67 pure AD, 33 AD with CVD, and 110 VaD patients. For the AD patients, 52 had taken DNP and 48 had not received the drug due to treatment prior to the introduction of DNP in 1999 in Japan. For the pure AD group, there were positive correlation between NH and DNP and QALY, as well as lifetime expectancy. As for the AD with CVD group, only a correlation between DNP and lifetime expectancy was noted, with no correlation with QALY.
Conclusions:
We found positive correlations between DNP administration and NH replacement and lifetime expectancy and QALY after the onset of AD. However, concomitant CVD negated such a positive correlation with QALY. The findings suggest that QALY in AD is affected by CVD; thus, indicating the importance of CVD prevention. | 6,845 | 372 | [
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] | null | null | [CONTENT] Quality-adjusted life-year | Cerebrovascular disease | Alzheimer’s disease | Donepezil [SUMMARY] | null | null | [CONTENT] Quality-adjusted life-year | Cerebrovascular disease | Alzheimer’s disease | Donepezil [SUMMARY] | [CONTENT] Quality-adjusted life-year | Cerebrovascular disease | Alzheimer’s disease | Donepezil [SUMMARY] | [CONTENT] Quality-adjusted life-year | Cerebrovascular disease | Alzheimer’s disease | Donepezil [SUMMARY] | [CONTENT] Alzheimer Disease | Cerebrovascular Disorders | Cholinesterase Inhibitors | Comorbidity | Donepezil | Humans | Indans | Japan | Life Expectancy | Nursing Homes | Piperidines | Quality-Adjusted Life Years | Retrospective Studies [SUMMARY] | null | null | [CONTENT] Alzheimer Disease | Cerebrovascular Disorders | Cholinesterase Inhibitors | Comorbidity | Donepezil | Humans | Indans | Japan | Life Expectancy | Nursing Homes | Piperidines | Quality-Adjusted Life Years | Retrospective Studies [SUMMARY] | [CONTENT] Alzheimer Disease | Cerebrovascular Disorders | Cholinesterase Inhibitors | Comorbidity | Donepezil | Humans | Indans | Japan | Life Expectancy | Nursing Homes | Piperidines | Quality-Adjusted Life Years | Retrospective Studies [SUMMARY] | [CONTENT] Alzheimer Disease | Cerebrovascular Disorders | Cholinesterase Inhibitors | Comorbidity | Donepezil | Humans | Indans | Japan | Life Expectancy | Nursing Homes | Piperidines | Quality-Adjusted Life Years | Retrospective Studies [SUMMARY] | [CONTENT] alzheimer disease ad | dementia want live | life expectancy patients | expectancy correlations drug | drug therapy lifetime [SUMMARY] | null | null | [CONTENT] alzheimer disease ad | dementia want live | life expectancy patients | expectancy correlations drug | drug therapy lifetime [SUMMARY] | [CONTENT] alzheimer disease ad | dementia want live | life expectancy patients | expectancy correlations drug | drug therapy lifetime [SUMMARY] | [CONTENT] alzheimer disease ad | dementia want live | life expectancy patients | expectancy correlations drug | drug therapy lifetime [SUMMARY] | [CONTENT] ad | cvd | dnp | qaly | expectancy | lifetime | ad cvd | lifetime expectancy | effect | patients [SUMMARY] | null | null | [CONTENT] ad | cvd | dnp | qaly | expectancy | lifetime | ad cvd | lifetime expectancy | effect | patients [SUMMARY] | [CONTENT] ad | cvd | dnp | qaly | expectancy | lifetime | ad cvd | lifetime expectancy | effect | patients [SUMMARY] | [CONTENT] ad | cvd | dnp | qaly | expectancy | lifetime | ad cvd | lifetime expectancy | effect | patients [SUMMARY] | [CONTENT] ad | drugs | cvd | ad cvd | expectancy | nursing | ad cvd ad cvd | ad cvd ad | cheis | delay [SUMMARY] | null | null | [CONTENT] positive correlation | qaly | cvd | correlation | positive | qaly findings suggest | correlation dnp administration nh | randomization found positive correlation | randomization found positive | ad affected [SUMMARY] | [CONTENT] ad | dnp | cvd | qaly | expectancy | effect | lifetime | lifetime expectancy | adl | ad cvd [SUMMARY] | [CONTENT] ad | dnp | cvd | qaly | expectancy | effect | lifetime | lifetime expectancy | adl | ad cvd [SUMMARY] | [CONTENT] NH | DNP ||| QALY ||| NH | DNP | QALY | ' AD | AD | CVD [SUMMARY] | null | null | [CONTENT] DNP | NH | QALY ||| QALY ||| QALY | AD | CVD [SUMMARY] | [CONTENT] NH | DNP ||| QALY ||| NH | DNP | QALY | ' AD | AD | CVD ||| the Tajiri Clinic | 1999-2012 ||| AD or AD | CVD | NINCDS-ADRDA | more than 3 months | less than 1 year ||| months | QALY ||| ||| 390 | 275 | 67 | AD | 33 AD | CVD | 110 ||| 52 | DNP | 48 | DNP | 1999 | Japan ||| NH | DNP | QALY ||| CVD | DNP | QALY ||| DNP | NH | QALY ||| QALY ||| QALY | AD | CVD [SUMMARY] | [CONTENT] NH | DNP ||| QALY ||| NH | DNP | QALY | ' AD | AD | CVD ||| the Tajiri Clinic | 1999-2012 ||| AD or AD | CVD | NINCDS-ADRDA | more than 3 months | less than 1 year ||| months | QALY ||| ||| 390 | 275 | 67 | AD | 33 AD | CVD | 110 ||| 52 | DNP | 48 | DNP | 1999 | Japan ||| NH | DNP | QALY ||| CVD | DNP | QALY ||| DNP | NH | QALY ||| QALY ||| QALY | AD | CVD [SUMMARY] |
||||
High incidence of postoperative silent venous thromboembolism in ulcerative colitis: a retrospective observational study. | 34011335 | The incidence of postoperative venous thromboembolism (VTE) is high in patients with inflammatory bowel disease. We aimed to analyze the incidence and predictive factors of postoperative VTE in patients with ulcerative colitis. | BACKGROUND | Patients with ulcerative colitis who underwent colon and rectum surgery during 2010-2018 were included. We retrospectively investigated the incidence of postoperative VTE. | METHODS | A total of 140 colorectal surgery cases were included. Postoperative VTE was detected in 24 (17.1 %). Portal-mesenteric venous thrombosis was the most frequent VTE (18 cases; 75 %); of these, 15 patients underwent total proctocolectomy (TPC) with ileal pouch-anal anastomosis (IPAA). In univariate analysis, VTE occurred more frequently in patients with neoplasia than in those refractory to medications (27.2 % vs. 12.5 %; p < 0.031). TPC with IPAA was more often associated with VTE development (28 %) than total colectomy (10.5 %) or proctectomy (5.9 %). On logistic regression analysis, TPC with IPAA, total colectomy, long operation time (> 4 h), and high serum D-dimer level (> 5.3 µg/mL) on the day following surgery were identified as predictive risk factors. | RESULTS | Postoperative VTE occurred frequently and asymptomatically, especially after TPC with IPAA. Serum D-dimer level on the day after surgery may be a useful predictor of VTE. | CONCLUSIONS | [
"Colitis, Ulcerative",
"Humans",
"Incidence",
"Postoperative Complications",
"Proctocolectomy, Restorative",
"Retrospective Studies",
"Venous Thromboembolism"
] | 8132420 | Background | Venous thromboembolism (VTE) is a well-known complication after colorectal surgery that leads to morbidity and mortality in hospitalized as well as discharged patients. In particular, patients with ulcerative colitis (UC) show a high incidence of VTE postoperatively. Many previous studies conducted in Western countries found that the risk of postoperative VTE in patients with UC was 2–3-fold higher than in patients with colorectal cancer, with VTE rates of 4.1–5.8 % [1, 2].
Current guidelines from the American Society of Colon and Rectal Surgeons recommend pharmacological prophylaxis with low-dose unfractionated heparin or low-molecular-weight heparin in patients with moderate and high risk for VTE following abdominal surgery. Patients with inflammatory bowel disease (IBD) are defined as having a high risk for VTE [3].
Despite many reports on postoperative VTE and guidelines that recommend pharmacological prophylaxis, it is not sufficiently administered in clinical practice. Possible reasons include the risk of postoperative bleeding from the remnant rectum with acute flare, epidural hematoma, and mainly low recognition of postoperative VTE by surgeons.
We suspected that more cases of postoperative VTE occurred in patients with UC in our clinical setting than described in the findings of the previous reports. As the data in the previous studies were extracted from large nationwide databases, they were considered reliable because there is less likelihood of bias in such large datasets. However, VTE with poor symptoms such as slight fever elevation or abnormal blood test data only may not have been included in these data. We therefore conducted a retrospective study of postoperative VTE in patients with UC in an IBD-specialized facility in Japan and reviewed all computed tomography (CT) scans that were performed postoperatively. The aim of the study was to clarify the incidence of postoperative VTE including portal–mesenteric venous thrombosis (PMVT) and identify predictive factors for VTE. | null | null | Results | We included a total of 110 patients with UC in this study. The number of surgical cases was 143. The surgeries comprised 64 cases of TPC with ileal pouch-anal anastomosis (IPAA) as a two-stage procedure, 40 cases of TC with ileostomy, 34 cases of proctectomy with IPAA, 4 cases of partial colectomy, and one case of TC with ileorectal anastomosis. Thirty-three of the 34 patient who underwent proctectomy with IPAA had previously undergone TC with ileostomy as a three-stage procedure (Fig. 1).
Of the remaining 140 cases, postoperative VTE was detected in 24 cases (17.1 %) at 7 (5.2–9.7) days after surgery. Specifically, PMVT occurred in 18 cases (12.6 %), PE in 4 cases (2.8 %), femoral venous thrombus in 2 cases (1.4 %), subclavian vein thrombus in 1 case (0.7 %), and jugular vein thrombus in 1 case (0.7 %). Among these cases, two patients had multiple VTEs; one had PE + DVT and the other had PE + PMVT. PE was detected after TPC with IPAA in three cases and after TC in one case. Two patients with subclavian vein thrombus and jugular vein thrombus had central venous catheters after TC.
PMVT occurred most frequently, and 15 of these cases occurred after TPC with IPAA (Fig. 2). All 17 cases of liver portal vein thrombosis were detected in the right hepatic lobe, and 5 of these cases showed mesenteric venous thrombosis simultaneously. With respect to mesenteric venous thrombosis, five cases were in the inferior mesenteric vein, two cases were in the superior mesenteric vein, and one case was in the splenic vein. Patient backgrounds with VTE were not significantly different with those without VTE, except for the reason for surgery (Table 1).
Fig. 2Numbers of VTE cases according to surgical procedures. The correlations between surgical procedures and thrombosis sites were determined. PMVT frequently occurred after TPC with IPAA. PE and DVT occurred more frequently in patients who underwent TC with acute flare-ups of UC. DVT included central vein and femoral vein thrombus. TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, PMV portal–mesenteric venous thrombosis
Numbers of VTE cases according to surgical procedures. The correlations between surgical procedures and thrombosis sites were determined. PMVT frequently occurred after TPC with IPAA. PE and DVT occurred more frequently in patients who underwent TC with acute flare-ups of UC. DVT included central vein and femoral vein thrombus. TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, PMV portal–mesenteric venous thrombosis
Table 1Patient characteristics according to venous thromboembolismOverallVTE +VTE −p valueN = 140 N = 24 N = 116Gender, n (%)Male97 (69.3)15 (62.5)82 (70.6)0.428Female43 (31.7)9 (37.5)34 (29.3)Age, median (range)54 (41.2–63)48 (38.2–57.5)55.5 (42–64.7)0.158BMI, median (range)21 (18.7–24.1)21.8 (18.4–24)20.5 (18.7–24.1)0.81ASA-PS ≧ 3, n (%)8 (5.7)1 (4.2)7 (6.0)0.719Current smoking, n (%)13 (9.3)2 (8.3)11 (9.48)0.86Preoperative therapy, n (%)Steroid59 (42.1)13 (54.2)46 (39.6)0.19Immunosuppressive agents48 (34.2)9 (37.5)39 (33.6)0.72Biological agents20 (14.2)3 (12.5)17 (14.7)0.78Comorbidity, n (%)HT20 (14.2)5 (20.8)15 (8.6)0.31HL11 (7.9)1 (4.2)10 (8.6)0.46DM15 (10.7)015 (12.9)0.073Indication, n (%)Cancer/ dysplasia44 (31.4)12 (50)32 (27.5)Refractory to medication96 (68.5)12 (50)84 (87.5)0.0313*VTE venous thromboembolism, BMI body mass index, ASA-PS American Society of Anesthesiology-physical status, HT hypertension, HL hyperlipidemia, DM diabetes mellitus*P < 0.05
Patient characteristics according to venous thromboembolism
VTE venous thromboembolism, BMI body mass index, ASA-PS American Society of Anesthesiology-physical status, HT hypertension, HL hyperlipidemia, DM diabetes mellitus
*P < 0.05
Contrast-enhanced CT after surgery was conducted in 91 cases (65 %). The most frequent reasons for CT examinations were suspicion of pelvic abscess with fever elevation and white blood cell elevations, or outlet obstruction at the stoma site with abdominal pain and ileus. In the 24 cases with VTE, the reasons for CT examinations were AST/ALT elevation in 13 cases, white blood cell elevation in 10 cases, fever elevation in 6 cases, D-dimer elevation in 5 cases, and dyspnea in one case. Complications other than VTE were ileus in 6 cases (25 %) and pelvic abscess in 3 cases (4.2 %) (Table 2).
Table 2Peri-operative results according to venous thromboembolismOverallVTE +VTE −p valueN = 140 N = 24 N = 116ProcedureTPC IPAA, n (%)64 (45.7)18(75)46 (39.7)TC, n (%)38 (27.1)4 (16.7)34 (29.3)Proctectomy + IPAA, n (%)34 (24.2)2 (8.3)32 (27.6)Other, n (%)4 (2.8)04 (3.4)0.0144*IPAA96 (68.6)20 (83.3)76 (65.5)0.087Operative time, min.(range)291 (203–383)340 (255–476)277 (181–355)0.006*Blood loss, ml (range)300 (155–522)326 (230–528)275 (12–520)0.1775ComplicationsIleus, n (%)28 (20)6 (25)22 (19)0.57Anastomosis leakage, n (%)9 (6.4)1 (4.2)8 (6.9)0.629Pelvic abscess, n (%)24 (17)3 (12.5)21 (18)0.497Emergency operation36 (27.6)5 (20.8)31 (26.7)0.5478Prophylaxis with anticoagulant, n (%)44 (31.4)10 (41.7)34 (29.3 %)0.2956D-dimer, µg/ml (range)POD1 D-dimer,3.1 (2.2–5.8)5.6 (2.7–8.9)3 (2.2–4.7)0.0218*POD3 D-dimer4.4 (3.1–8.25)7 (3.5–10.6)4.1 (3.1–7.7)0.083Peak D-dimer8.85 (5.9–13)10.1 (7.4–15.3)8.7 (5.7–12)0.072VTE venous thromboembolism, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, POD post-operative day*P < 0.05
Peri-operative results according to venous thromboembolism
VTE venous thromboembolism, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, POD post-operative day
*P < 0.05
The rate of postoperative VTE prophylaxis with anticoagulant was 31.4 % in all patients, 41.7 % in the VTE (+) group, and 29.3 % in the VTE (−) group, respectively. Limited to the TPC + IPAA group, that was no significant difference in postoperative VTE prophylaxis between patients with VTE (9 cases; 50 %) and patients without VTE (19 cases; 41.3 %). With regard to surgical procedures, medical prophylaxis was performed in 28 cases (43.7 %) in the TPC + IPAA group, 6 cases (16.2 %) in the TC group, and 10 cases (29.4 %) in the proctectomy + IPAA group.
On univariate analysis, VTE occurred more frequently in patients with neoplasia (cancer or high-grade dysplasia) than in those refractory to medications (27.2 % vs. 12.5 %; p < 0.031). There were no significant differences in age, body mass index, American Society of Anesthesiology-physical status (ASA-PS) score ≥ 3, current smoking, preoperative therapy, and comorbidity between the VTE (+) group and VTE (−) group (Table 1). The perioperative results showed significant differences in the rates of VTE according to surgical procedures. Patients who underwent TPC with IPAA were more likely to develop VTE (28 %) than those who underwent TC (10.5 %) or proctectomy (5.9 %). Operation time in the VTE (+) group was significantly longer than that in the VTE (−) group (340 vs. 291 min, p = 0.006). Laboratory data indicated that serum D-dimer levels on the day following surgery were higher in the VTE (+) group (5.6 µg/mL vs. 3.1 µg/mL, p = 0.00218). D-dimer levels at day 3 after surgery were also higher in the VTE (+) group, but the difference was not significant (Table 2).
On logistic regression analysis, we identified TPC with IPAA, TC, long operation time (> 4 h), and serum D-dimer level > 5.3 mg/dL on the day following surgery as predictive risk factors for postoperative VTE in patients with UC (Table 3).
Table 3Predictive risk factors in logistic regression analysisOR95 % CI
p valueProcedureProctectomy + IPAAReference−−TC45.881.44–28630.0304*TPC + IPAA17.121.675–433.70.015*IndicationCancer/dysplasia0.450.06–2.890.405Operative time(> 4 h)28.841.23–12600.0358*D-dimer at Day1(> 5.3 µg/dl )3.691.02–14.780.0452*OR odds ratio, CI confidence interval, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy*P < 0.05
Predictive risk factors in logistic regression analysis
OR odds ratio, CI confidence interval, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy
*P < 0.05 | Conclusions | We found a high frequency of postoperative VTE, especially PMVT, in our retrospective dataset, particularly in one-quarter of patient who underwent TPC with IPAA. In most cases, the symptoms were mild, but may have the potential to become symptomatic with growth. It is important to recognize these findings, and to conduct strict anticoagulant therapy. | [
"Background",
"Data collection and variables",
"Outcome",
"Statistical analysis",
"Ethical considerations"
] | [
"Venous thromboembolism (VTE) is a well-known complication after colorectal surgery that leads to morbidity and mortality in hospitalized as well as discharged patients. In particular, patients with ulcerative colitis (UC) show a high incidence of VTE postoperatively. Many previous studies conducted in Western countries found that the risk of postoperative VTE in patients with UC was 2–3-fold higher than in patients with colorectal cancer, with VTE rates of 4.1–5.8 % [1, 2].\nCurrent guidelines from the American Society of Colon and Rectal Surgeons recommend pharmacological prophylaxis with low-dose unfractionated heparin or low-molecular-weight heparin in patients with moderate and high risk for VTE following abdominal surgery. Patients with inflammatory bowel disease (IBD) are defined as having a high risk for VTE [3].\nDespite many reports on postoperative VTE and guidelines that recommend pharmacological prophylaxis, it is not sufficiently administered in clinical practice. Possible reasons include the risk of postoperative bleeding from the remnant rectum with acute flare, epidural hematoma, and mainly low recognition of postoperative VTE by surgeons.\nWe suspected that more cases of postoperative VTE occurred in patients with UC in our clinical setting than described in the findings of the previous reports. As the data in the previous studies were extracted from large nationwide databases, they were considered reliable because there is less likelihood of bias in such large datasets. However, VTE with poor symptoms such as slight fever elevation or abnormal blood test data only may not have been included in these data. We therefore conducted a retrospective study of postoperative VTE in patients with UC in an IBD-specialized facility in Japan and reviewed all computed tomography (CT) scans that were performed postoperatively. The aim of the study was to clarify the incidence of postoperative VTE including portal–mesenteric venous thrombosis (PMVT) and identify predictive factors for VTE.",
"We performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.\n\nFig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nSchema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nThe preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.\nAll patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.",
"The primary outcome was incidence of in-hospital VTE after abdominopelvic surgery. The secondary outcome was predictive risk factors for VTE.\nIn this study, VTE included pulmonary embolism (PE), deep vein thrombosis (DVT), and PMVT. DVT included thrombosis at the vena cava, internal jugular vein, subclavian vein, and femoral vein. The lower and upper limbs were not evaluated in the study.\nStatistical analysis Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.\nAnalyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.\nEthical considerations \nThis study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.\n\nThis study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.",
"Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.",
"\nThis study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet."
] | [
null,
null,
null,
null,
null
] | [
"Background",
"Materials and methods",
"Data collection and variables",
"Outcome",
"Statistical analysis",
"Ethical considerations",
"Results",
"Discussion",
"Conclusions"
] | [
"Venous thromboembolism (VTE) is a well-known complication after colorectal surgery that leads to morbidity and mortality in hospitalized as well as discharged patients. In particular, patients with ulcerative colitis (UC) show a high incidence of VTE postoperatively. Many previous studies conducted in Western countries found that the risk of postoperative VTE in patients with UC was 2–3-fold higher than in patients with colorectal cancer, with VTE rates of 4.1–5.8 % [1, 2].\nCurrent guidelines from the American Society of Colon and Rectal Surgeons recommend pharmacological prophylaxis with low-dose unfractionated heparin or low-molecular-weight heparin in patients with moderate and high risk for VTE following abdominal surgery. Patients with inflammatory bowel disease (IBD) are defined as having a high risk for VTE [3].\nDespite many reports on postoperative VTE and guidelines that recommend pharmacological prophylaxis, it is not sufficiently administered in clinical practice. Possible reasons include the risk of postoperative bleeding from the remnant rectum with acute flare, epidural hematoma, and mainly low recognition of postoperative VTE by surgeons.\nWe suspected that more cases of postoperative VTE occurred in patients with UC in our clinical setting than described in the findings of the previous reports. As the data in the previous studies were extracted from large nationwide databases, they were considered reliable because there is less likelihood of bias in such large datasets. However, VTE with poor symptoms such as slight fever elevation or abnormal blood test data only may not have been included in these data. We therefore conducted a retrospective study of postoperative VTE in patients with UC in an IBD-specialized facility in Japan and reviewed all computed tomography (CT) scans that were performed postoperatively. The aim of the study was to clarify the incidence of postoperative VTE including portal–mesenteric venous thrombosis (PMVT) and identify predictive factors for VTE.",
"Data collection and variables We performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.\n\nFig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nSchema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nThe preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.\nAll patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.\nWe performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.\n\nFig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nSchema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nThe preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.\nAll patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.",
"We performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.\n\nFig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nSchema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis\nThe preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.\nAll patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.",
"The primary outcome was incidence of in-hospital VTE after abdominopelvic surgery. The secondary outcome was predictive risk factors for VTE.\nIn this study, VTE included pulmonary embolism (PE), deep vein thrombosis (DVT), and PMVT. DVT included thrombosis at the vena cava, internal jugular vein, subclavian vein, and femoral vein. The lower and upper limbs were not evaluated in the study.\nStatistical analysis Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.\nAnalyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.\nEthical considerations \nThis study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.\n\nThis study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.",
"Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.",
"\nThis study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.",
"We included a total of 110 patients with UC in this study. The number of surgical cases was 143. The surgeries comprised 64 cases of TPC with ileal pouch-anal anastomosis (IPAA) as a two-stage procedure, 40 cases of TC with ileostomy, 34 cases of proctectomy with IPAA, 4 cases of partial colectomy, and one case of TC with ileorectal anastomosis. Thirty-three of the 34 patient who underwent proctectomy with IPAA had previously undergone TC with ileostomy as a three-stage procedure (Fig. 1).\nOf the remaining 140 cases, postoperative VTE was detected in 24 cases (17.1 %) at 7 (5.2–9.7) days after surgery. Specifically, PMVT occurred in 18 cases (12.6 %), PE in 4 cases (2.8 %), femoral venous thrombus in 2 cases (1.4 %), subclavian vein thrombus in 1 case (0.7 %), and jugular vein thrombus in 1 case (0.7 %). Among these cases, two patients had multiple VTEs; one had PE + DVT and the other had PE + PMVT. PE was detected after TPC with IPAA in three cases and after TC in one case. Two patients with subclavian vein thrombus and jugular vein thrombus had central venous catheters after TC.\nPMVT occurred most frequently, and 15 of these cases occurred after TPC with IPAA (Fig. 2). All 17 cases of liver portal vein thrombosis were detected in the right hepatic lobe, and 5 of these cases showed mesenteric venous thrombosis simultaneously. With respect to mesenteric venous thrombosis, five cases were in the inferior mesenteric vein, two cases were in the superior mesenteric vein, and one case was in the splenic vein. Patient backgrounds with VTE were not significantly different with those without VTE, except for the reason for surgery (Table 1).\n\nFig. 2Numbers of VTE cases according to surgical procedures. The correlations between surgical procedures and thrombosis sites were determined. PMVT frequently occurred after TPC with IPAA. PE and DVT occurred more frequently in patients who underwent TC with acute flare-ups of UC. DVT included central vein and femoral vein thrombus. TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, PMV portal–mesenteric venous thrombosis\nNumbers of VTE cases according to surgical procedures. The correlations between surgical procedures and thrombosis sites were determined. PMVT frequently occurred after TPC with IPAA. PE and DVT occurred more frequently in patients who underwent TC with acute flare-ups of UC. DVT included central vein and femoral vein thrombus. TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, PMV portal–mesenteric venous thrombosis\n\nTable 1Patient characteristics according to venous thromboembolismOverallVTE +VTE −p valueN = 140 N = 24 N = 116Gender, n (%)Male97 (69.3)15 (62.5)82 (70.6)0.428Female43 (31.7)9 (37.5)34 (29.3)Age, median (range)54 (41.2–63)48 (38.2–57.5)55.5 (42–64.7)0.158BMI, median (range)21 (18.7–24.1)21.8 (18.4–24)20.5 (18.7–24.1)0.81ASA-PS ≧ 3, n (%)8 (5.7)1 (4.2)7 (6.0)0.719Current smoking, n (%)13 (9.3)2 (8.3)11 (9.48)0.86Preoperative therapy, n (%)Steroid59 (42.1)13 (54.2)46 (39.6)0.19Immunosuppressive agents48 (34.2)9 (37.5)39 (33.6)0.72Biological agents20 (14.2)3 (12.5)17 (14.7)0.78Comorbidity, n (%)HT20 (14.2)5 (20.8)15 (8.6)0.31HL11 (7.9)1 (4.2)10 (8.6)0.46DM15 (10.7)015 (12.9)0.073Indication, n (%)Cancer/ dysplasia44 (31.4)12 (50)32 (27.5)Refractory to medication96 (68.5)12 (50)84 (87.5)0.0313*VTE venous thromboembolism, BMI body mass index, ASA-PS American Society of Anesthesiology-physical status, HT hypertension, HL hyperlipidemia, DM diabetes mellitus*P < 0.05\nPatient characteristics according to venous thromboembolism\nVTE venous thromboembolism, BMI body mass index, ASA-PS American Society of Anesthesiology-physical status, HT hypertension, HL hyperlipidemia, DM diabetes mellitus\n*P < 0.05\nContrast-enhanced CT after surgery was conducted in 91 cases (65 %). The most frequent reasons for CT examinations were suspicion of pelvic abscess with fever elevation and white blood cell elevations, or outlet obstruction at the stoma site with abdominal pain and ileus. In the 24 cases with VTE, the reasons for CT examinations were AST/ALT elevation in 13 cases, white blood cell elevation in 10 cases, fever elevation in 6 cases, D-dimer elevation in 5 cases, and dyspnea in one case. Complications other than VTE were ileus in 6 cases (25 %) and pelvic abscess in 3 cases (4.2 %) (Table 2).\n\nTable 2Peri-operative results according to venous thromboembolismOverallVTE +VTE −p valueN = 140 N = 24 N = 116ProcedureTPC IPAA, n (%)64 (45.7)18(75)46 (39.7)TC, n (%)38 (27.1)4 (16.7)34 (29.3)Proctectomy + IPAA, n (%)34 (24.2)2 (8.3)32 (27.6)Other, n (%)4 (2.8)04 (3.4)0.0144*IPAA96 (68.6)20 (83.3)76 (65.5)0.087Operative time, min.(range)291 (203–383)340 (255–476)277 (181–355)0.006*Blood loss, ml (range)300 (155–522)326 (230–528)275 (12–520)0.1775ComplicationsIleus, n (%)28 (20)6 (25)22 (19)0.57Anastomosis leakage, n (%)9 (6.4)1 (4.2)8 (6.9)0.629Pelvic abscess, n (%)24 (17)3 (12.5)21 (18)0.497Emergency operation36 (27.6)5 (20.8)31 (26.7)0.5478Prophylaxis with anticoagulant, n (%)44 (31.4)10 (41.7)34 (29.3 %)0.2956D-dimer, µg/ml (range)POD1 D-dimer,3.1 (2.2–5.8)5.6 (2.7–8.9)3 (2.2–4.7)0.0218*POD3 D-dimer4.4 (3.1–8.25)7 (3.5–10.6)4.1 (3.1–7.7)0.083Peak D-dimer8.85 (5.9–13)10.1 (7.4–15.3)8.7 (5.7–12)0.072VTE venous thromboembolism, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, POD post-operative day*P < 0.05\nPeri-operative results according to venous thromboembolism\nVTE venous thromboembolism, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, POD post-operative day\n*P < 0.05\nThe rate of postoperative VTE prophylaxis with anticoagulant was 31.4 % in all patients, 41.7 % in the VTE (+) group, and 29.3 % in the VTE (−) group, respectively. Limited to the TPC + IPAA group, that was no significant difference in postoperative VTE prophylaxis between patients with VTE (9 cases; 50 %) and patients without VTE (19 cases; 41.3 %). With regard to surgical procedures, medical prophylaxis was performed in 28 cases (43.7 %) in the TPC + IPAA group, 6 cases (16.2 %) in the TC group, and 10 cases (29.4 %) in the proctectomy + IPAA group.\nOn univariate analysis, VTE occurred more frequently in patients with neoplasia (cancer or high-grade dysplasia) than in those refractory to medications (27.2 % vs. 12.5 %; p < 0.031). There were no significant differences in age, body mass index, American Society of Anesthesiology-physical status (ASA-PS) score ≥ 3, current smoking, preoperative therapy, and comorbidity between the VTE (+) group and VTE (−) group (Table 1). The perioperative results showed significant differences in the rates of VTE according to surgical procedures. Patients who underwent TPC with IPAA were more likely to develop VTE (28 %) than those who underwent TC (10.5 %) or proctectomy (5.9 %). Operation time in the VTE (+) group was significantly longer than that in the VTE (−) group (340 vs. 291 min, p = 0.006). Laboratory data indicated that serum D-dimer levels on the day following surgery were higher in the VTE (+) group (5.6 µg/mL vs. 3.1 µg/mL, p = 0.00218). D-dimer levels at day 3 after surgery were also higher in the VTE (+) group, but the difference was not significant (Table 2).\nOn logistic regression analysis, we identified TPC with IPAA, TC, long operation time (> 4 h), and serum D-dimer level > 5.3 mg/dL on the day following surgery as predictive risk factors for postoperative VTE in patients with UC (Table 3).\n\nTable 3Predictive risk factors in logistic regression analysisOR95 % CI\np valueProcedureProctectomy + IPAAReference−−TC45.881.44–28630.0304*TPC + IPAA17.121.675–433.70.015*IndicationCancer/dysplasia0.450.06–2.890.405Operative time(> 4 h)28.841.23–12600.0358*D-dimer at Day1(> 5.3 µg/dl )3.691.02–14.780.0452*OR odds ratio, CI confidence interval, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy*P < 0.05\nPredictive risk factors in logistic regression analysis\nOR odds ratio, CI confidence interval, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy\n*P < 0.05",
"In this study, postoperative VTE in patients with UC was detected in 24 cases (17.1 %) at a median of 7 days after surgery. PMVT was the most common type of VTE (18 cases; 12.8 %), and occurred most frequently after TPC with IPAA (23.4 %). Three predictive risk factors were detected in the logistic regression analysis (Table 3), namely elevated D-dimer on the day after surgery, surgical procedure (TPC with IPAA, TC), and long operation time (> 4 h).\nIBD is a well-known independent risk factor for VTE, and patients with UC have a higher risk of VTE than those with Crohn’s disease [2, 4]. In patients with IBD, the risk of developing DVT and PE was 1.98- and 1.80-fold higher compared with other individuals, respectively, and the rates of postoperative VTE were 2.5–3.8 %, which was higher than the 2.4 % observed among patients with colorectal cancer [5–9]. The incidence rate of VTE in the present study (17.1 %) was higher than those in the previous reports. More than two-thirds of the cases had PMVT. When limited to DVT and PE, there were 7 cases (4.9 %), which was close to the frequencies reported in the previous studies.\nCT examination after surgery was conducted for 91 cases (65 %), mostly because of abnormal laboratory data or fever elevation to rule out abscess, ileus, or VTE. In the 24 cases with VTE, only 5 cases were suspected of having VTE before CT examination, and the other 19 cases were unexpected. Frequent postoperative CT examinations for patients with mild symptoms made it possible to detect many VTE cases. Easy performance of CT may be related to the Universal Insurance System covering all people in Japan.\nPMVT was most frequently detected after TPC with IPAA in the present study (23.4 %). There are few previous studies on PMVT. The reported rate of PMVT after colorectal surgery was 2.9–4.9 % [10, 11, 12]. IBD is a frequent cause of PMVT after abdominal surgery, with a reported rate of 8.3 % [13]. As a surgical procedure, restorative TPC with IPAA for patients with UC was identified as an independent predictor of PMVT, and the incidence rate was high at 5.8–10 % [14, 11, 15, 16]. In the procedure for ileal pouch construction and anal anastomosis, there were several factors that can promote thrombus formation in addition to the nature of IBD. These factors comprised long operation time, pelvic surgery in the lithotomy position, manipulation of mesenteric vessels, and traction of the superior mesentery vessels related to IPAA reconstruction. A dehydrated state caused by the covering ileostomy was also related to thrombosis, and occurred for all patients treated at our institution.\nD-dimer elevation (> 5.3 mg/dL) on the day after surgery was identified as a predictive risk factor for postoperative VTE in this study, with sensitivity of 55 % and specificity of 78.5 %. D-dimer is not a specific marker for thrombus formation. Disease flares, bleeding, surgical invasion, and septic condition are also related to D-dimer elevation, even when the patients do not have VTE. Actually, 5 patients in the VTE (−) group had very high D-dimer levels (> 10 µg/mL), and all of them underwent emergency TC for bleeding or perforation with severe bowel inflammation. Therefore, D-dimer elevation is not a strong predictor of VTE, but can be easily used for suspicion of thrombus formation. It may be more useful for patients undergoing TPC or proctectomy whose preoperative condition is relatively stable compared with patients undergoing TC.\nThis study had several limitations. The study was designed retrospectively at a single institution, and the sample number was small. Variables that could be collected from the database were limited. Pharmacological prophylaxis was not administered to all patients, and the administration period was not defined. CT examination was conducted in all patients preoperatively, but not in all patients postoperatively, and the lower limbs were not examined for venous thrombus formation. Data for lower limb examinations using ultrasound imaging were not collected. Finally, the percentage of postoperative VTE prophylaxis with anticoagulant was only 31.4 %. This percentage is too small, given that the risks of VTE in patients with UC are well known. The current guideline states that strict administration of postoperative pharmacological prophylaxis is necessary, except for cases with a risk of bleeding from the remnant rectum [3].",
"We found a high frequency of postoperative VTE, especially PMVT, in our retrospective dataset, particularly in one-quarter of patient who underwent TPC with IPAA. In most cases, the symptoms were mild, but may have the potential to become symptomatic with growth. It is important to recognize these findings, and to conduct strict anticoagulant therapy."
] | [
null,
"materials|methods",
null,
null,
null,
null,
"results",
"discussion",
"conclusion"
] | [
"Ulcerative colitis",
"Prophylaxis",
"D-dimer",
"Ileal pouch",
"Portal–mesenteric venous thrombosis"
] | Background:
Venous thromboembolism (VTE) is a well-known complication after colorectal surgery that leads to morbidity and mortality in hospitalized as well as discharged patients. In particular, patients with ulcerative colitis (UC) show a high incidence of VTE postoperatively. Many previous studies conducted in Western countries found that the risk of postoperative VTE in patients with UC was 2–3-fold higher than in patients with colorectal cancer, with VTE rates of 4.1–5.8 % [1, 2].
Current guidelines from the American Society of Colon and Rectal Surgeons recommend pharmacological prophylaxis with low-dose unfractionated heparin or low-molecular-weight heparin in patients with moderate and high risk for VTE following abdominal surgery. Patients with inflammatory bowel disease (IBD) are defined as having a high risk for VTE [3].
Despite many reports on postoperative VTE and guidelines that recommend pharmacological prophylaxis, it is not sufficiently administered in clinical practice. Possible reasons include the risk of postoperative bleeding from the remnant rectum with acute flare, epidural hematoma, and mainly low recognition of postoperative VTE by surgeons.
We suspected that more cases of postoperative VTE occurred in patients with UC in our clinical setting than described in the findings of the previous reports. As the data in the previous studies were extracted from large nationwide databases, they were considered reliable because there is less likelihood of bias in such large datasets. However, VTE with poor symptoms such as slight fever elevation or abnormal blood test data only may not have been included in these data. We therefore conducted a retrospective study of postoperative VTE in patients with UC in an IBD-specialized facility in Japan and reviewed all computed tomography (CT) scans that were performed postoperatively. The aim of the study was to clarify the incidence of postoperative VTE including portal–mesenteric venous thrombosis (PMVT) and identify predictive factors for VTE.
Materials and methods:
Data collection and variables We performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.
Fig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis
Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis
The preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.
All patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.
We performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.
Fig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis
Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis
The preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.
All patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.
Data collection and variables:
We performed this retrospective observational study using a prospectively maintained database of patients with UC who underwent abdominopelvic bowel resection at Hiroshima University Hospital between January 2010 and December 2018. In total, we included 143 surgical cases among 110 patients. All patients were Asian Japanese. Restorative proctocolectomy and proctectomy were conducted with rectal mucosectomy and hand-sewn ileal pouch–anal anastomosis (IPAA) with a J pouch. Thirty-three patients with total colectomy (TC) underwent proctectomy with IPAA as a second surgery. When VTE was detected after TC, proctectomy was conducted after the thrombus had disappeared. Three patients who underwent TC were excluded because they showed preoperative VTE (Fig. 1). We reviewed the patients’ electronic medical charts to obtain information regarding patient characteristics, surgical procedures, and postoperative data. Emergency surgery was defined as unplanned surgery on admission.
Fig. 1Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis
Schema of the enrolled patients and surgical procedures. We enrolled a total of 110 patients, including 143 surgical cases. After total colectomy, 33 patients underwent proctocolectomy with IPAA. Thirty-three of 34 patients who underwent proctectomy with IPAA had previously undergone total colectomy. Three cases with total colectomy were excluded from the analysis because they had preoperative venous thromboembolism. IPAA ileal pouch–anal anastomosis
The preoperative state of venous thrombosis was assessed using contrast-enhanced CT, with an image range from chest to pelvis, just prior to emergency surgery and within 4 weeks prior to elective surgery. Presence of postoperative VTE was assessed by contrast-enhanced CT examination from chest to pelvis as well as supplementary ultrasound examination. Preoperative therapy included use of steroid just prior to surgery, immunosuppressive agents within 1 week before surgery, and biological agents within 6 weeks prior to surgery.
All patients underwent early mobilization on the day following surgery and received mechanical VTE prophylaxis with compression stockings and an intermittent pneumatic compression device from the time of surgery until the start of walking. As pharmacological prophylaxis, low-molecular-weight heparin or unfractionated heparin was administered for 7 days after surgery. In our institution, we introduced pharmacological prophylaxis in 2015, except for cases with a risk of bleeding. Preoperative pharmacological prophylaxis was not administered to any patients.
Outcome:
The primary outcome was incidence of in-hospital VTE after abdominopelvic surgery. The secondary outcome was predictive risk factors for VTE.
In this study, VTE included pulmonary embolism (PE), deep vein thrombosis (DVT), and PMVT. DVT included thrombosis at the vena cava, internal jugular vein, subclavian vein, and femoral vein. The lower and upper limbs were not evaluated in the study.
Statistical analysis Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.
Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.
Ethical considerations
This study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.
This study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.
Statistical analysis:
Analyses were conducted using JMP version 11 (SAS Institute Japan Ltd., Tokyo, Japan). Statistics are reported as median with interquartile range (IQR) for continuous variables and frequency with percentage for categorical variables. Normally distributed continuous variables were analyzed using the t-test, and nonnormally distributed continuous variables were analyzed with the nonparametric Wilcoxon’s rank-sum test. Fisher’s exact test was performed for categorical variables. Multivariable logistic regression analysis was conducted to identify predictive risk factors for surgical recurrence at the anastomosis site. Each analysis was performed using a two-sided 5 % significance level and 95 % confidence interval.
Ethical considerations:
This study was approved by the Hiroshima University Institutional Review Board (E-1636). Participants received information about the conduct of the research, including the purpose of the study, and were given the opportunity to decline participation in the study. Because the opt-out method can be used without informed consent from the patients, the information was published on the internet.
Results:
We included a total of 110 patients with UC in this study. The number of surgical cases was 143. The surgeries comprised 64 cases of TPC with ileal pouch-anal anastomosis (IPAA) as a two-stage procedure, 40 cases of TC with ileostomy, 34 cases of proctectomy with IPAA, 4 cases of partial colectomy, and one case of TC with ileorectal anastomosis. Thirty-three of the 34 patient who underwent proctectomy with IPAA had previously undergone TC with ileostomy as a three-stage procedure (Fig. 1).
Of the remaining 140 cases, postoperative VTE was detected in 24 cases (17.1 %) at 7 (5.2–9.7) days after surgery. Specifically, PMVT occurred in 18 cases (12.6 %), PE in 4 cases (2.8 %), femoral venous thrombus in 2 cases (1.4 %), subclavian vein thrombus in 1 case (0.7 %), and jugular vein thrombus in 1 case (0.7 %). Among these cases, two patients had multiple VTEs; one had PE + DVT and the other had PE + PMVT. PE was detected after TPC with IPAA in three cases and after TC in one case. Two patients with subclavian vein thrombus and jugular vein thrombus had central venous catheters after TC.
PMVT occurred most frequently, and 15 of these cases occurred after TPC with IPAA (Fig. 2). All 17 cases of liver portal vein thrombosis were detected in the right hepatic lobe, and 5 of these cases showed mesenteric venous thrombosis simultaneously. With respect to mesenteric venous thrombosis, five cases were in the inferior mesenteric vein, two cases were in the superior mesenteric vein, and one case was in the splenic vein. Patient backgrounds with VTE were not significantly different with those without VTE, except for the reason for surgery (Table 1).
Fig. 2Numbers of VTE cases according to surgical procedures. The correlations between surgical procedures and thrombosis sites were determined. PMVT frequently occurred after TPC with IPAA. PE and DVT occurred more frequently in patients who underwent TC with acute flare-ups of UC. DVT included central vein and femoral vein thrombus. TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, PMV portal–mesenteric venous thrombosis
Numbers of VTE cases according to surgical procedures. The correlations between surgical procedures and thrombosis sites were determined. PMVT frequently occurred after TPC with IPAA. PE and DVT occurred more frequently in patients who underwent TC with acute flare-ups of UC. DVT included central vein and femoral vein thrombus. TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, PMV portal–mesenteric venous thrombosis
Table 1Patient characteristics according to venous thromboembolismOverallVTE +VTE −p valueN = 140 N = 24 N = 116Gender, n (%)Male97 (69.3)15 (62.5)82 (70.6)0.428Female43 (31.7)9 (37.5)34 (29.3)Age, median (range)54 (41.2–63)48 (38.2–57.5)55.5 (42–64.7)0.158BMI, median (range)21 (18.7–24.1)21.8 (18.4–24)20.5 (18.7–24.1)0.81ASA-PS ≧ 3, n (%)8 (5.7)1 (4.2)7 (6.0)0.719Current smoking, n (%)13 (9.3)2 (8.3)11 (9.48)0.86Preoperative therapy, n (%)Steroid59 (42.1)13 (54.2)46 (39.6)0.19Immunosuppressive agents48 (34.2)9 (37.5)39 (33.6)0.72Biological agents20 (14.2)3 (12.5)17 (14.7)0.78Comorbidity, n (%)HT20 (14.2)5 (20.8)15 (8.6)0.31HL11 (7.9)1 (4.2)10 (8.6)0.46DM15 (10.7)015 (12.9)0.073Indication, n (%)Cancer/ dysplasia44 (31.4)12 (50)32 (27.5)Refractory to medication96 (68.5)12 (50)84 (87.5)0.0313*VTE venous thromboembolism, BMI body mass index, ASA-PS American Society of Anesthesiology-physical status, HT hypertension, HL hyperlipidemia, DM diabetes mellitus*P < 0.05
Patient characteristics according to venous thromboembolism
VTE venous thromboembolism, BMI body mass index, ASA-PS American Society of Anesthesiology-physical status, HT hypertension, HL hyperlipidemia, DM diabetes mellitus
*P < 0.05
Contrast-enhanced CT after surgery was conducted in 91 cases (65 %). The most frequent reasons for CT examinations were suspicion of pelvic abscess with fever elevation and white blood cell elevations, or outlet obstruction at the stoma site with abdominal pain and ileus. In the 24 cases with VTE, the reasons for CT examinations were AST/ALT elevation in 13 cases, white blood cell elevation in 10 cases, fever elevation in 6 cases, D-dimer elevation in 5 cases, and dyspnea in one case. Complications other than VTE were ileus in 6 cases (25 %) and pelvic abscess in 3 cases (4.2 %) (Table 2).
Table 2Peri-operative results according to venous thromboembolismOverallVTE +VTE −p valueN = 140 N = 24 N = 116ProcedureTPC IPAA, n (%)64 (45.7)18(75)46 (39.7)TC, n (%)38 (27.1)4 (16.7)34 (29.3)Proctectomy + IPAA, n (%)34 (24.2)2 (8.3)32 (27.6)Other, n (%)4 (2.8)04 (3.4)0.0144*IPAA96 (68.6)20 (83.3)76 (65.5)0.087Operative time, min.(range)291 (203–383)340 (255–476)277 (181–355)0.006*Blood loss, ml (range)300 (155–522)326 (230–528)275 (12–520)0.1775ComplicationsIleus, n (%)28 (20)6 (25)22 (19)0.57Anastomosis leakage, n (%)9 (6.4)1 (4.2)8 (6.9)0.629Pelvic abscess, n (%)24 (17)3 (12.5)21 (18)0.497Emergency operation36 (27.6)5 (20.8)31 (26.7)0.5478Prophylaxis with anticoagulant, n (%)44 (31.4)10 (41.7)34 (29.3 %)0.2956D-dimer, µg/ml (range)POD1 D-dimer,3.1 (2.2–5.8)5.6 (2.7–8.9)3 (2.2–4.7)0.0218*POD3 D-dimer4.4 (3.1–8.25)7 (3.5–10.6)4.1 (3.1–7.7)0.083Peak D-dimer8.85 (5.9–13)10.1 (7.4–15.3)8.7 (5.7–12)0.072VTE venous thromboembolism, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, POD post-operative day*P < 0.05
Peri-operative results according to venous thromboembolism
VTE venous thromboembolism, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy, POD post-operative day
*P < 0.05
The rate of postoperative VTE prophylaxis with anticoagulant was 31.4 % in all patients, 41.7 % in the VTE (+) group, and 29.3 % in the VTE (−) group, respectively. Limited to the TPC + IPAA group, that was no significant difference in postoperative VTE prophylaxis between patients with VTE (9 cases; 50 %) and patients without VTE (19 cases; 41.3 %). With regard to surgical procedures, medical prophylaxis was performed in 28 cases (43.7 %) in the TPC + IPAA group, 6 cases (16.2 %) in the TC group, and 10 cases (29.4 %) in the proctectomy + IPAA group.
On univariate analysis, VTE occurred more frequently in patients with neoplasia (cancer or high-grade dysplasia) than in those refractory to medications (27.2 % vs. 12.5 %; p < 0.031). There were no significant differences in age, body mass index, American Society of Anesthesiology-physical status (ASA-PS) score ≥ 3, current smoking, preoperative therapy, and comorbidity between the VTE (+) group and VTE (−) group (Table 1). The perioperative results showed significant differences in the rates of VTE according to surgical procedures. Patients who underwent TPC with IPAA were more likely to develop VTE (28 %) than those who underwent TC (10.5 %) or proctectomy (5.9 %). Operation time in the VTE (+) group was significantly longer than that in the VTE (−) group (340 vs. 291 min, p = 0.006). Laboratory data indicated that serum D-dimer levels on the day following surgery were higher in the VTE (+) group (5.6 µg/mL vs. 3.1 µg/mL, p = 0.00218). D-dimer levels at day 3 after surgery were also higher in the VTE (+) group, but the difference was not significant (Table 2).
On logistic regression analysis, we identified TPC with IPAA, TC, long operation time (> 4 h), and serum D-dimer level > 5.3 mg/dL on the day following surgery as predictive risk factors for postoperative VTE in patients with UC (Table 3).
Table 3Predictive risk factors in logistic regression analysisOR95 % CI
p valueProcedureProctectomy + IPAAReference−−TC45.881.44–28630.0304*TPC + IPAA17.121.675–433.70.015*IndicationCancer/dysplasia0.450.06–2.890.405Operative time(> 4 h)28.841.23–12600.0358*D-dimer at Day1(> 5.3 µg/dl )3.691.02–14.780.0452*OR odds ratio, CI confidence interval, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy*P < 0.05
Predictive risk factors in logistic regression analysis
OR odds ratio, CI confidence interval, TPC total proctocolectomy, IPAA ileal pouch–anal anastomosis, TC total colectomy
*P < 0.05
Discussion:
In this study, postoperative VTE in patients with UC was detected in 24 cases (17.1 %) at a median of 7 days after surgery. PMVT was the most common type of VTE (18 cases; 12.8 %), and occurred most frequently after TPC with IPAA (23.4 %). Three predictive risk factors were detected in the logistic regression analysis (Table 3), namely elevated D-dimer on the day after surgery, surgical procedure (TPC with IPAA, TC), and long operation time (> 4 h).
IBD is a well-known independent risk factor for VTE, and patients with UC have a higher risk of VTE than those with Crohn’s disease [2, 4]. In patients with IBD, the risk of developing DVT and PE was 1.98- and 1.80-fold higher compared with other individuals, respectively, and the rates of postoperative VTE were 2.5–3.8 %, which was higher than the 2.4 % observed among patients with colorectal cancer [5–9]. The incidence rate of VTE in the present study (17.1 %) was higher than those in the previous reports. More than two-thirds of the cases had PMVT. When limited to DVT and PE, there were 7 cases (4.9 %), which was close to the frequencies reported in the previous studies.
CT examination after surgery was conducted for 91 cases (65 %), mostly because of abnormal laboratory data or fever elevation to rule out abscess, ileus, or VTE. In the 24 cases with VTE, only 5 cases were suspected of having VTE before CT examination, and the other 19 cases were unexpected. Frequent postoperative CT examinations for patients with mild symptoms made it possible to detect many VTE cases. Easy performance of CT may be related to the Universal Insurance System covering all people in Japan.
PMVT was most frequently detected after TPC with IPAA in the present study (23.4 %). There are few previous studies on PMVT. The reported rate of PMVT after colorectal surgery was 2.9–4.9 % [10, 11, 12]. IBD is a frequent cause of PMVT after abdominal surgery, with a reported rate of 8.3 % [13]. As a surgical procedure, restorative TPC with IPAA for patients with UC was identified as an independent predictor of PMVT, and the incidence rate was high at 5.8–10 % [14, 11, 15, 16]. In the procedure for ileal pouch construction and anal anastomosis, there were several factors that can promote thrombus formation in addition to the nature of IBD. These factors comprised long operation time, pelvic surgery in the lithotomy position, manipulation of mesenteric vessels, and traction of the superior mesentery vessels related to IPAA reconstruction. A dehydrated state caused by the covering ileostomy was also related to thrombosis, and occurred for all patients treated at our institution.
D-dimer elevation (> 5.3 mg/dL) on the day after surgery was identified as a predictive risk factor for postoperative VTE in this study, with sensitivity of 55 % and specificity of 78.5 %. D-dimer is not a specific marker for thrombus formation. Disease flares, bleeding, surgical invasion, and septic condition are also related to D-dimer elevation, even when the patients do not have VTE. Actually, 5 patients in the VTE (−) group had very high D-dimer levels (> 10 µg/mL), and all of them underwent emergency TC for bleeding or perforation with severe bowel inflammation. Therefore, D-dimer elevation is not a strong predictor of VTE, but can be easily used for suspicion of thrombus formation. It may be more useful for patients undergoing TPC or proctectomy whose preoperative condition is relatively stable compared with patients undergoing TC.
This study had several limitations. The study was designed retrospectively at a single institution, and the sample number was small. Variables that could be collected from the database were limited. Pharmacological prophylaxis was not administered to all patients, and the administration period was not defined. CT examination was conducted in all patients preoperatively, but not in all patients postoperatively, and the lower limbs were not examined for venous thrombus formation. Data for lower limb examinations using ultrasound imaging were not collected. Finally, the percentage of postoperative VTE prophylaxis with anticoagulant was only 31.4 %. This percentage is too small, given that the risks of VTE in patients with UC are well known. The current guideline states that strict administration of postoperative pharmacological prophylaxis is necessary, except for cases with a risk of bleeding from the remnant rectum [3].
Conclusions:
We found a high frequency of postoperative VTE, especially PMVT, in our retrospective dataset, particularly in one-quarter of patient who underwent TPC with IPAA. In most cases, the symptoms were mild, but may have the potential to become symptomatic with growth. It is important to recognize these findings, and to conduct strict anticoagulant therapy.
| Background:
The incidence of postoperative venous thromboembolism (VTE) is high in patients with inflammatory bowel disease. We aimed to analyze the incidence and predictive factors of postoperative VTE in patients with ulcerative colitis.
Methods:
Patients with ulcerative colitis who underwent colon and rectum surgery during 2010-2018 were included. We retrospectively investigated the incidence of postoperative VTE.
Results:
A total of 140 colorectal surgery cases were included. Postoperative VTE was detected in 24 (17.1 %). Portal-mesenteric venous thrombosis was the most frequent VTE (18 cases; 75 %); of these, 15 patients underwent total proctocolectomy (TPC) with ileal pouch-anal anastomosis (IPAA). In univariate analysis, VTE occurred more frequently in patients with neoplasia than in those refractory to medications (27.2 % vs. 12.5 %; p < 0.031). TPC with IPAA was more often associated with VTE development (28 %) than total colectomy (10.5 %) or proctectomy (5.9 %). On logistic regression analysis, TPC with IPAA, total colectomy, long operation time (> 4 h), and high serum D-dimer level (> 5.3 µg/mL) on the day following surgery were identified as predictive risk factors.
Conclusions:
Postoperative VTE occurred frequently and asymptomatically, especially after TPC with IPAA. Serum D-dimer level on the day after surgery may be a useful predictor of VTE. | Background:
Venous thromboembolism (VTE) is a well-known complication after colorectal surgery that leads to morbidity and mortality in hospitalized as well as discharged patients. In particular, patients with ulcerative colitis (UC) show a high incidence of VTE postoperatively. Many previous studies conducted in Western countries found that the risk of postoperative VTE in patients with UC was 2–3-fold higher than in patients with colorectal cancer, with VTE rates of 4.1–5.8 % [1, 2].
Current guidelines from the American Society of Colon and Rectal Surgeons recommend pharmacological prophylaxis with low-dose unfractionated heparin or low-molecular-weight heparin in patients with moderate and high risk for VTE following abdominal surgery. Patients with inflammatory bowel disease (IBD) are defined as having a high risk for VTE [3].
Despite many reports on postoperative VTE and guidelines that recommend pharmacological prophylaxis, it is not sufficiently administered in clinical practice. Possible reasons include the risk of postoperative bleeding from the remnant rectum with acute flare, epidural hematoma, and mainly low recognition of postoperative VTE by surgeons.
We suspected that more cases of postoperative VTE occurred in patients with UC in our clinical setting than described in the findings of the previous reports. As the data in the previous studies were extracted from large nationwide databases, they were considered reliable because there is less likelihood of bias in such large datasets. However, VTE with poor symptoms such as slight fever elevation or abnormal blood test data only may not have been included in these data. We therefore conducted a retrospective study of postoperative VTE in patients with UC in an IBD-specialized facility in Japan and reviewed all computed tomography (CT) scans that were performed postoperatively. The aim of the study was to clarify the incidence of postoperative VTE including portal–mesenteric venous thrombosis (PMVT) and identify predictive factors for VTE.
Conclusions:
We found a high frequency of postoperative VTE, especially PMVT, in our retrospective dataset, particularly in one-quarter of patient who underwent TPC with IPAA. In most cases, the symptoms were mild, but may have the potential to become symptomatic with growth. It is important to recognize these findings, and to conduct strict anticoagulant therapy. | Background:
The incidence of postoperative venous thromboembolism (VTE) is high in patients with inflammatory bowel disease. We aimed to analyze the incidence and predictive factors of postoperative VTE in patients with ulcerative colitis.
Methods:
Patients with ulcerative colitis who underwent colon and rectum surgery during 2010-2018 were included. We retrospectively investigated the incidence of postoperative VTE.
Results:
A total of 140 colorectal surgery cases were included. Postoperative VTE was detected in 24 (17.1 %). Portal-mesenteric venous thrombosis was the most frequent VTE (18 cases; 75 %); of these, 15 patients underwent total proctocolectomy (TPC) with ileal pouch-anal anastomosis (IPAA). In univariate analysis, VTE occurred more frequently in patients with neoplasia than in those refractory to medications (27.2 % vs. 12.5 %; p < 0.031). TPC with IPAA was more often associated with VTE development (28 %) than total colectomy (10.5 %) or proctectomy (5.9 %). On logistic regression analysis, TPC with IPAA, total colectomy, long operation time (> 4 h), and high serum D-dimer level (> 5.3 µg/mL) on the day following surgery were identified as predictive risk factors.
Conclusions:
Postoperative VTE occurred frequently and asymptomatically, especially after TPC with IPAA. Serum D-dimer level on the day after surgery may be a useful predictor of VTE. | 5,232 | 286 | [
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] | null | [CONTENT] Ulcerative colitis | Prophylaxis | D-dimer | Ileal pouch | Portal–mesenteric venous thrombosis [SUMMARY] | null | [CONTENT] Ulcerative colitis | Prophylaxis | D-dimer | Ileal pouch | Portal–mesenteric venous thrombosis [SUMMARY] | [CONTENT] Ulcerative colitis | Prophylaxis | D-dimer | Ileal pouch | Portal–mesenteric venous thrombosis [SUMMARY] | [CONTENT] Ulcerative colitis | Prophylaxis | D-dimer | Ileal pouch | Portal–mesenteric venous thrombosis [SUMMARY] | [CONTENT] Ulcerative colitis | Prophylaxis | D-dimer | Ileal pouch | Portal–mesenteric venous thrombosis [SUMMARY] | [CONTENT] Colitis, Ulcerative | Humans | Incidence | Postoperative Complications | Proctocolectomy, Restorative | Retrospective Studies | Venous Thromboembolism [SUMMARY] | null | [CONTENT] Colitis, Ulcerative | Humans | Incidence | Postoperative Complications | Proctocolectomy, Restorative | Retrospective Studies | Venous Thromboembolism [SUMMARY] | [CONTENT] Colitis, Ulcerative | Humans | Incidence | Postoperative Complications | Proctocolectomy, Restorative | Retrospective Studies | Venous Thromboembolism [SUMMARY] | [CONTENT] Colitis, Ulcerative | Humans | Incidence | Postoperative Complications | Proctocolectomy, Restorative | Retrospective Studies | Venous Thromboembolism [SUMMARY] | [CONTENT] Colitis, Ulcerative | Humans | Incidence | Postoperative Complications | Proctocolectomy, Restorative | Retrospective Studies | Venous Thromboembolism [SUMMARY] | [CONTENT] patients inflammatory bowel | preoperative venous thromboembolism | postoperative pharmacological prophylaxis | risk bleeding preoperative | heparin patients moderate [SUMMARY] | null | [CONTENT] patients inflammatory bowel | preoperative venous thromboembolism | postoperative pharmacological prophylaxis | risk bleeding preoperative | heparin patients moderate [SUMMARY] | [CONTENT] patients inflammatory bowel | preoperative venous thromboembolism | postoperative pharmacological prophylaxis | risk bleeding preoperative | heparin patients moderate [SUMMARY] | [CONTENT] patients inflammatory bowel | preoperative venous thromboembolism | postoperative pharmacological prophylaxis | risk bleeding preoperative | heparin patients moderate [SUMMARY] | [CONTENT] patients inflammatory bowel | preoperative venous thromboembolism | postoperative pharmacological prophylaxis | risk bleeding preoperative | heparin patients moderate [SUMMARY] | [CONTENT] patients | vte | cases | ipaa | surgery | total | underwent | surgical | tc | colectomy [SUMMARY] | null | [CONTENT] patients | vte | cases | ipaa | surgery | total | underwent | surgical | tc | colectomy [SUMMARY] | [CONTENT] patients | vte | cases | ipaa | surgery | total | underwent | surgical | tc | colectomy [SUMMARY] | [CONTENT] patients | vte | cases | ipaa | surgery | total | underwent | surgical | tc | colectomy [SUMMARY] | [CONTENT] patients | vte | cases | ipaa | surgery | total | underwent | surgical | tc | colectomy [SUMMARY] | [CONTENT] vte | patients | postoperative | postoperative vte | previous | uc | low | large | high risk vte | recommend pharmacological prophylaxis [SUMMARY] | null | [CONTENT] cases | vte | tpc | ipaa | tc | group | vein | total | venous | 24 [SUMMARY] | [CONTENT] symptoms mild | especially pmvt retrospective | especially | retrospective dataset particularly quarter | retrospective dataset particularly | retrospective dataset | tpc ipaa cases symptoms | anticoagulant therapy | found high | found high frequency [SUMMARY] | [CONTENT] patients | vte | cases | total | ipaa | surgery | variables | study | underwent | colectomy [SUMMARY] | [CONTENT] patients | vte | cases | total | ipaa | surgery | variables | study | underwent | colectomy [SUMMARY] | [CONTENT] ||| [SUMMARY] | null | [CONTENT] 140 ||| 24 | 17.1 % ||| VTE | 18 | 75 % | 15 | IPAA ||| neoplasia | 27.2 % | 12.5 % | 0.031 ||| IPAA | 28 % | 10.5 % | 5.9 % ||| TPC | IPAA | 4 | 5.3 | the day [SUMMARY] | [CONTENT] TPC | IPAA ||| Serum D-dimer | the day | VTE [SUMMARY] | [CONTENT] ||| ||| 2010-2018 ||| ||| 140 ||| 24 | 17.1 % ||| VTE | 18 | 75 % | 15 | IPAA ||| neoplasia | 27.2 % | 12.5 % | 0.031 ||| IPAA | 28 % | 10.5 % | 5.9 % ||| TPC | IPAA | 4 | 5.3 | the day ||| TPC | IPAA ||| Serum D-dimer | the day | VTE [SUMMARY] | [CONTENT] ||| ||| 2010-2018 ||| ||| 140 ||| 24 | 17.1 % ||| VTE | 18 | 75 % | 15 | IPAA ||| neoplasia | 27.2 % | 12.5 % | 0.031 ||| IPAA | 28 % | 10.5 % | 5.9 % ||| TPC | IPAA | 4 | 5.3 | the day ||| TPC | IPAA ||| Serum D-dimer | the day | VTE [SUMMARY] |
|||||
The effect of amiloride in decreasing albuminuria in patients with diabetic kidney diseases: a prospective, crossover, open-label study. | 33657976 | Diabetic kidney diseases (DKD) were the leading cause of End-stage renal diseases worldwide. Albuminuria was a target for treatment in DKD and decreasing albuminuria was particularly important for improving its prognosis. However, there is still a lack of specific treatment for DKD. | BACKGROUND | We conducted a prospective, crossover, open-label study to investigate the effect of amiloride in patients with DKD. Safety and efficacy were assessed by monitoring urine protein creatinine ratio(uPCR), urinary albumin creatinine ratio (uACR), blood pressure, weight, serum sodium, serum potassium, cholesterol, triglyceride, uric acid, serum soluble urokinase-type plasminogen activator receptor (suPAR) and urinary suPAR. Ten subjects were enrolled in the trial. | METHODS | In this prospective, crossover, open-label design, amiloride could induce a significant decrease of uACR in DKD. The decrease of serum and urinary suPAR in the amiloride/hydrochlorothiazide (HCTZ) group was also significant compared with those patients using HCTZ as the control group. Correlation analysis showed that the levels of urinary suPAR were positively associated with uPCR and uACR. No significant difference in blood pressure, weight, serum sodium, serum potassium, cholesterol, triglyceride, uric acid was seen between the amiloride/HCTZ group and the control group. | RESULTS | In summary, among patients with DKD, amiloride could decrease albuminuria without severe side effects, which was accompanied by the significant decline of urinary suPAR. | CONCLUSION | [
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] | 7935116 | Introduction | Diabetic kidney diseases (DKD) were the leading cause of end-stage renal diseases and renal replacement therapy worldwide [1]. Recently the prevalence of DKD had been increasing dramatically in China and chronic kidney disease (CKD) related to diabetes had become more common than CKD related to glomerulonephritis [2]. The clinicopathological presentation of DKD was characterized by progressive albuminuria, hyperfiltration, associative glomerular injury, and tubulointerstitial fibrosis [3]. Moreover, persistent albuminuria could predict early deterioration of renal function [4] and was an independent risk factor of cardiovascular disease [5] and a target for the treatment of DKD [6]. Therefore, decrease albuminuria in DKD is particularly essential for improving its prognosis and reduce complications.
Albuminuria in DKD result from the damage of the glomerular filtration barrier, which leads to the excessive filtration of plasma protein and other macromolecular substances and exceeds the reabsorption ability of renal tubules. The podocyte plays a key role in the glomerular filtration barrier and its disruption results in a dramatic loss of function leading to albuminuria. The major biological functions of podocytes are to limit the passage of albumin from the circulation into the urine and to maintain overall glomerular integrity [7]. Thus, exploration of the drugs which target the improvement of the damaged podocyte would be conducive to improve albuminuria and delay the progression in DKD.
Recent studies have found that the increased expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes could induce podocyte injury and albuminuria [8–12]. uPAR is required for activation of β3 integrin signaling within the kidney podocyte, which would lead to accelerated podocyte foot process dynamics and dysregulate the shape and function of the glomerular filtration barrier [11]. Increasing soluble urokinase-type plasminogen activator receptor (suPAR) levels in mice would induce podocyte abnormalities while reducing suPAR levels and blocking suPAR actions could improve kidney morphology [13].
Amiloride, a pyrazine compound inhibiting sodium reabsorption through sodium channels in renal epithelial cells, has been used as a diuretic in clinical practice. Our previous study had found that amiloride inhibited podocyte uPAR induction and reduced proteinuria in the 5/6 nephrectomy rat model and the LPS mouse model of transient proteinuria [14]. Moreover, amiloride could inhibit urine uPAR activity which attenuated plasminogen activation in vivo [15]. These results suggest that amiloride may have shown a potential effect on reducing albuminuria. Since there was no specific immunosuppressive therapy for DKD, we performed this prospective, crossover, open-label study to investigate the safety and effectiveness of amiloride in decreasing albuminuria in DKD. | null | null | Results | Comparison of the pretherapeutic baseline characteristics between patients received amiloride/HCTZ and HCTZ Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.
Comparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.
Comparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Amiloride’s effect in decreasing uACR and suPAR After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).
Comparison of outcomes between amiloride and HCTZ over the course of the study.
uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).
Comparison of outcomes between amiloride and HCTZ over the course of the study.
uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Association between urinary suPAR and uPCR/uACR Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.
(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.
Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.
(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.
The safety of amiloride during the treatment periods Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).
Comparison of side effect between amiloride and HCTZ over the course of the study.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure.
Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).
Comparison of side effect between amiloride and HCTZ over the course of the study.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure. | Conclusions | Amiloride could induce a significant decrease of albuminuria, which was accompanied by the significant decline of urinary suPAR. No serious side effects were found in DKD patients using amiloride during treatment periods. In summary, amiloride might be an efficient and safe treatment strategy for decreasing albuminuria in DKD. | [
"Subjects and data collection",
"Study design and outcome",
"Detection of serum and urinary suPAR",
"Statistical analyses",
"Comparison of the pretherapeutic baseline characteristics between patients received amiloride/HCTZ and HCTZ",
"Amiloride’s effect in decreasing uACR and suPAR",
"Association between urinary suPAR and uPCR/uACR",
"The safety of amiloride during the treatment periods"
] | [
"Ten patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.\nThe clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.",
"The study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.\nFlow chart of the patients enrolled in the study.",
"Serum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).\nBlank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.",
"All data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.",
"Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.\nComparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.",
"After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).\nComparison of outcomes between amiloride and HCTZ over the course of the study.\nuPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.",
"Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.\n(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.",
"Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).\nComparison of side effect between amiloride and HCTZ over the course of the study.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure."
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Materials and methods",
"Subjects and data collection",
"Study design and outcome",
"Detection of serum and urinary suPAR",
"Statistical analyses",
"Results",
"Comparison of the pretherapeutic baseline characteristics between patients received amiloride/HCTZ and HCTZ",
"Amiloride’s effect in decreasing uACR and suPAR",
"Association between urinary suPAR and uPCR/uACR",
"The safety of amiloride during the treatment periods",
"Discussion",
"Conclusions",
"Supplementary Material"
] | [
"Diabetic kidney diseases (DKD) were the leading cause of end-stage renal diseases and renal replacement therapy worldwide [1]. Recently the prevalence of DKD had been increasing dramatically in China and chronic kidney disease (CKD) related to diabetes had become more common than CKD related to glomerulonephritis [2]. The clinicopathological presentation of DKD was characterized by progressive albuminuria, hyperfiltration, associative glomerular injury, and tubulointerstitial fibrosis [3]. Moreover, persistent albuminuria could predict early deterioration of renal function [4] and was an independent risk factor of cardiovascular disease [5] and a target for the treatment of DKD [6]. Therefore, decrease albuminuria in DKD is particularly essential for improving its prognosis and reduce complications.\nAlbuminuria in DKD result from the damage of the glomerular filtration barrier, which leads to the excessive filtration of plasma protein and other macromolecular substances and exceeds the reabsorption ability of renal tubules. The podocyte plays a key role in the glomerular filtration barrier and its disruption results in a dramatic loss of function leading to albuminuria. The major biological functions of podocytes are to limit the passage of albumin from the circulation into the urine and to maintain overall glomerular integrity [7]. Thus, exploration of the drugs which target the improvement of the damaged podocyte would be conducive to improve albuminuria and delay the progression in DKD.\nRecent studies have found that the increased expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes could induce podocyte injury and albuminuria [8–12]. uPAR is required for activation of β3 integrin signaling within the kidney podocyte, which would lead to accelerated podocyte foot process dynamics and dysregulate the shape and function of the glomerular filtration barrier [11]. Increasing soluble urokinase-type plasminogen activator receptor (suPAR) levels in mice would induce podocyte abnormalities while reducing suPAR levels and blocking suPAR actions could improve kidney morphology [13].\nAmiloride, a pyrazine compound inhibiting sodium reabsorption through sodium channels in renal epithelial cells, has been used as a diuretic in clinical practice. Our previous study had found that amiloride inhibited podocyte uPAR induction and reduced proteinuria in the 5/6 nephrectomy rat model and the LPS mouse model of transient proteinuria [14]. Moreover, amiloride could inhibit urine uPAR activity which attenuated plasminogen activation in vivo [15]. These results suggest that amiloride may have shown a potential effect on reducing albuminuria. Since there was no specific immunosuppressive therapy for DKD, we performed this prospective, crossover, open-label study to investigate the safety and effectiveness of amiloride in decreasing albuminuria in DKD.",
"Subjects and data collection Ten patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.\nThe clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.\nTen patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.\nThe clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.\nStudy design and outcome The study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.\nFlow chart of the patients enrolled in the study.\nThe study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.\nFlow chart of the patients enrolled in the study.\nDetection of serum and urinary suPAR Serum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).\nBlank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.\nSerum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).\nBlank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.\nStatistical analyses All data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.\nAll data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.",
"Ten patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.\nThe clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.",
"The study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.\nFlow chart of the patients enrolled in the study.",
"Serum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).\nBlank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.",
"All data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.",
"Comparison of the pretherapeutic baseline characteristics between patients received amiloride/HCTZ and HCTZ Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.\nComparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.\nAmong ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.\nComparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.\nAmiloride’s effect in decreasing uACR and suPAR After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).\nComparison of outcomes between amiloride and HCTZ over the course of the study.\nuPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.\nAfter the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).\nComparison of outcomes between amiloride and HCTZ over the course of the study.\nuPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.\nAssociation between urinary suPAR and uPCR/uACR Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.\n(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.\nCorrelation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.\n(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.\nThe safety of amiloride during the treatment periods Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).\nComparison of side effect between amiloride and HCTZ over the course of the study.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure.\nAmong ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).\nComparison of side effect between amiloride and HCTZ over the course of the study.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure.",
"Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.\nComparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.",
"After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).\nComparison of outcomes between amiloride and HCTZ over the course of the study.\nuPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.",
"Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.\n(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.",
"Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).\nComparison of side effect between amiloride and HCTZ over the course of the study.\nSBP: Systolic blood pressure; DBP: Diastolic blood pressure.",
"DKD is the most common cause of CKD and ESRD worldwide and develops in approximately 40% of type 2 diabetic patients [18]. DKD is a common microvascular complication of diabetes mellitus (DM) and most of the risk for all-cause and cardiovascular disease mortality in DM patients is related to the presence of DKD [19].\nProgressively larger amounts of albuminuria are the most common manifestation in DKD and then classically followed by a relentless decline in kidney function, renal impairment, and ultimately ESRD. Albuminuria is a well-known factor that links podocyte damage to tubulointerstitial injury, which triggers tubulointerstitial inflammation and fibrogenesis, and accelerates the decline of renal function [20]. Therefore, reducing albuminuria in DKD is particularly important to delay its progression and improve outcomes.\nThe impairment of the glomerular filtration barrier is an important pathophysiological basis for albuminuria in DKD. The glomerular filtration barrier consists of endothelial cells, the glomerular basement membrane (GBM), and podocytes. Podocytes have a prominent role in maintaining the integrity of the glomerular filter. Podocytes injury would lead to the effacement of foot processes and the detachment or apoptosis of podocytes [21]. The loss of integrity of the podocyte seal results in the diffusion and convection of proteins from the circulation into the urinary space and finally leads to the occurrence of albuminuria [22]. Moreover, several pathogenic mechanisms involved in podocyte injury would beget ultrastructural changes in podocytes and albuminuria. Previous researches had found that mutation of the gene encoding the podocyte-expressed protein nephrin would result in congenital albuminuria [23]. Disorder of cross-talk between podocytes and the other cells involved in the glomerular filtration barrier might also lead to albuminuria. Several studies [19,24–26] had found that the podocyte lesion is the primary causes for glomerular diseases characterized by massive proteinuria, such as minimal change nephropathy (MCD), focal glomerulosclerosis (FSGS), membranous nephropathy (MN), lupus nephritis (LN), DKD and is related to disease progression.\nPrevious findings [11] had indicated that increasing expression of uPAR in podocytes would result in the podocytes lesion and the occurrence of albuminuria. uPAR is a multidomain glycoprotein tethered to the cell membrane with a glycosylphosphotidylinositol (GPI) anchor and was originally identified as a saturable binding site for urokinase on the cell surface. uPAR is required for activation of β3 integrin within the kidney podocyte, which is one of the main proteins that anchors podocytes to the underlying GBM. Increased β3 integrin activation within podocytes would cause accelerated podocyte foot process dynamics, which dysregulates the shape and function of the glomerular filtration barrier. Wei [11] et al. had found that lipopolysaccharide (LPS) injection would induce the higher expression of uPAR in podocytes. Mice lacking uPAR were protected from LPS-mediated proteinuria but develop the disease after expression of a constitutively active β3 integrin. Knockdown of uPAR in podocytes would decrease the number of migrating podocytes and promote motility, which showed a physiological role for uPAR signaling in the regulation of kidney permeability. Moreover, Zhao [27] et al. had demonstrated that urinary suPAR was specifically elevated in primary FSGS and was related to disease severity. The elevated urinary suPAR could activate β3 integrin on human podocytes. Therefore, it indicated that restraining the expression of uPAR in podocytes may be a potential therapeutic target for the clinical management of albuminuria in DKD.\nAmiloride is a Na channel blocker and inhibits the Na+/H+ and Na+/Ca2+ antiporters, which have been widely used as a diuretic. Previous studies had suggested that amiloride could inhibit the expression of uPAR in tumor-infiltrating lymphocytes [28] and colon cancer cells [29,30]. Our former works had indicated that amiloride was able to inhibit the expression of uPAR and thereby reduced the mobility of podocytes, which provided a new therapeutic paradigm for the clinical management of albuminuria. Therefore, the effect of amiloride in decreasing albuminuria in DKD was explored in this prospective, crossover, open-label study.\nIn this crossover design, amiloride induced a significant decrease of uACR in DKD. Additionally, the decrease of serum and urinary suPAR in the amiloride/HCTZ group was significant compared with the HCTZ group. Correlation analysis had also shown that the levels of urinary suPAR were positively associated with uPCR and uACR. Based on the above results, we concluded that amiloride was able to reduce albuminuria in DKD and may be associated with the decreased levels of urinary suPAR, which was an indicator for disease severity [27]. Recent researches had revealed that urinary suPAR was significantly increased in patients with massive proteinuria and decreased accompanying with the remission of proteinuria. Mette Staehr [28] et al. had demonstrated that urinary uPA protein and activity were increased in rats treated with puromycin aminonucleoside. And amiloride could inhibit urinary uPA activity, which attenuated plasminogen activation and urine protease activity. Our studies had also found that amiloride could reduce urinary and serum suPAR, which may be a relevant target for amiloride in reducing albuminuria.\nAs Na channel blocker, amiloride would inhibit the exchange of Na+–K+ and Na+–H+ in distal tubules and collecting ducts of the kidney, thus increasing the excretion of Na+ and water and reducing the excretion of K+ and H+. Therefore, hyperkalemia and hyponatremia were the most common side effect of amiloride. In this study, significant hyperkalemia occurred in two patients and no patients experienced hyponatremia. And the serum potassium in both patients could return to normal after hypokalemia treatment. Additionally, there was no significant influence in fasting glucose, cholesterol, triglyceride, unic acid with amiloride/HCTZ when compared with HCTZ. Furthermore, amiloride would substantially reduce the SBP and volume status by blocking both ENaC and urokinase [31,32]. However, we had not found significant reductions in weight or SBP with amiloride/HCTZ compared with HCTZ. Our inability to detect significant effects on blood pressure and weight may be associated with the small sample size in this study. Unruh [33] et al. had also demonstrated the safety of amiloride in patients with type 2 diabetes, normal renal function, and proteinuria, which provided important research evidence for amiloride’s application in decreasing albuminuria in DKD.\nThe strength of this study mainly discussed the effect and safety of amiloride in decreasing albuminuria in DKD patients, which would provide a new application for the traditional diuretic. However, our study still has some limitations. First of all, we only enrolled small sizes sample from a single-center, and long-term renal prognosis had not been explored in DKD patients. Furthermore, the follow-up period should be extended in order to investigate the long-term effect of amiloride in DKD.",
"Amiloride could induce a significant decrease of albuminuria, which was accompanied by the significant decline of urinary suPAR. No serious side effects were found in DKD patients using amiloride during treatment periods. In summary, amiloride might be an efficient and safe treatment strategy for decreasing albuminuria in DKD.",
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"Diabetic kidney disease",
"amiloride",
"albuminuria",
"soluble urokinase-type plasminogen activator receptor"
] | Introduction:
Diabetic kidney diseases (DKD) were the leading cause of end-stage renal diseases and renal replacement therapy worldwide [1]. Recently the prevalence of DKD had been increasing dramatically in China and chronic kidney disease (CKD) related to diabetes had become more common than CKD related to glomerulonephritis [2]. The clinicopathological presentation of DKD was characterized by progressive albuminuria, hyperfiltration, associative glomerular injury, and tubulointerstitial fibrosis [3]. Moreover, persistent albuminuria could predict early deterioration of renal function [4] and was an independent risk factor of cardiovascular disease [5] and a target for the treatment of DKD [6]. Therefore, decrease albuminuria in DKD is particularly essential for improving its prognosis and reduce complications.
Albuminuria in DKD result from the damage of the glomerular filtration barrier, which leads to the excessive filtration of plasma protein and other macromolecular substances and exceeds the reabsorption ability of renal tubules. The podocyte plays a key role in the glomerular filtration barrier and its disruption results in a dramatic loss of function leading to albuminuria. The major biological functions of podocytes are to limit the passage of albumin from the circulation into the urine and to maintain overall glomerular integrity [7]. Thus, exploration of the drugs which target the improvement of the damaged podocyte would be conducive to improve albuminuria and delay the progression in DKD.
Recent studies have found that the increased expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes could induce podocyte injury and albuminuria [8–12]. uPAR is required for activation of β3 integrin signaling within the kidney podocyte, which would lead to accelerated podocyte foot process dynamics and dysregulate the shape and function of the glomerular filtration barrier [11]. Increasing soluble urokinase-type plasminogen activator receptor (suPAR) levels in mice would induce podocyte abnormalities while reducing suPAR levels and blocking suPAR actions could improve kidney morphology [13].
Amiloride, a pyrazine compound inhibiting sodium reabsorption through sodium channels in renal epithelial cells, has been used as a diuretic in clinical practice. Our previous study had found that amiloride inhibited podocyte uPAR induction and reduced proteinuria in the 5/6 nephrectomy rat model and the LPS mouse model of transient proteinuria [14]. Moreover, amiloride could inhibit urine uPAR activity which attenuated plasminogen activation in vivo [15]. These results suggest that amiloride may have shown a potential effect on reducing albuminuria. Since there was no specific immunosuppressive therapy for DKD, we performed this prospective, crossover, open-label study to investigate the safety and effectiveness of amiloride in decreasing albuminuria in DKD.
Materials and methods:
Subjects and data collection Ten patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.
The clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.
Ten patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.
The clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.
Study design and outcome The study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.
Flow chart of the patients enrolled in the study.
The study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.
Flow chart of the patients enrolled in the study.
Detection of serum and urinary suPAR Serum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).
Blank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.
Serum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).
Blank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.
Statistical analyses All data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.
All data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.
Subjects and data collection:
Ten patients who were clinically or pathological diagnosed with DKD in Guangdong Provincial People’s Hospital from May 2018 to March 2019 and fulfilled the inclusion criteria were enrolled in this prospective, crossover, open-label study. DKD is clinical diagnosed in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of >300 mg/24h, or a urine albumin-to-creatinine ratio (uACR) of >300 mg/gCr, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease. Moreover, DKD could also be pathologically diagnosed with morphological changes such as mesangial expansion and thickening of the glomerular and tubular basement membranes, as well as typical glomerulosclerosis with nodular mesangial lesions (Kimmelstiel–Wilson lesions). Among ten DKD patients enrolled, five patients were clinically diagnosed while five patients met pathological diagnostic criteria [16,17]. The inclusion criteria of this study were as follows: patients were over 14 years old; patients with good treatment compliance; urine protein creatinine ratio (uPCR) ≥500 mg/g Cr; patients signed written informed consent. Those who met the following criteria were excluded: eGFR ≤30 mL/min/1.73m2, previous intolerance or allergy to hydrochlorothiazide (HCTZ), patients with gout attack history within half a year, patients with active infectious diseases, patients with severe cardiopulmonary disease and central nervous system dysfunction, patients with a history of malignancy, patients’ life expectancy was less than 1 year, patients were pregnant, lactating or without contraceptive measures, patients who participated in other clinical trials within 3 months before enrollment, patients who were using immunosuppressive agents or glucocorticoids within 12 weeks before enrollment, patients failed to sign written informed consent or were unwilling to comply with the research protocol approved by the researcher. The procedures of this study were performed in accordance with medical ethics and the Helsinki Declaration and approved by the Ethics Committee of Guangdong Provincial People’s Hospital (No. GDREC2017318H). The registered clinical trial number was NCT03170336.
The clinical parameters were derived from electronic medical records and the hospital’s computerized database manually, including age, gender, weight, blood pressure, eGFR, serum creatine, uPCR, urinary albumin creatinine ratio (uACR), serum albumin, cholesterol, triglyceride, fasting glucose, uric acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR, and urinary suPAR. The serum and urine specimens of the enrolled patients were collected before and after the treatment period and stored at −80° celsius. Serum suPAR and urinary suPAR were measured by the enzyme-linked immunosorbent assay (ELISA) method.
Study design and outcome:
The study had a prospective, open-label, single-center, crossover design. In order to avoid hyperkalemia caused by amiloride, we used compound preparation of amiloride in this study, which contained 2.5 milligrams of amiloride and 25 milligrams of hydrochlorothiazide (HCTZ). Patients were orally administered with compound preparation of amiloride in doses of 5 mg of amiloride and 50 mg of HCTZ daily for 12 weeks treatment periods or with HCTZ in doses of 50 mg daily for 12 weeks treatment periods. Following more than 4-week washout periods, participants crossed over to the other treatment for 12 weeks. Outcomes were assessed at the end of each 12 weeks active treatment period (Figure 1). The randomization was performed centrally and patients were allocated to the different treatment sequences using random numbers. Guangdong Provincial People’s Hospital was responsible for coordinating and managing the investigational drug inventory, storage, distribution, and record-keeping for this clinical research study. This crossover study examined the amiloride’s effect in decreasing albuminuria in DKD patients as the primary outcome. The secondary outcome was the changes in blood pressure, weight, serum sodium, serum potassium, fasting glucose, serum uric acid, cholesterol, triglycerides before and after treatment.
Flow chart of the patients enrolled in the study.
Detection of serum and urinary suPAR:
Serum and urinary suPAR were detected using ELISA (JingMei, Hangzhou, China).
Blank wells and testing sample wells were set separately and 50 µL standard was added to the standard well. 40 μL sample dilution and 10 µL testing sample were added to the testing sample well. 100 µL HRP-Conjugate reagent were added to each well, except blank well. After closing the plate with the closure plate membrane, the plate was incubated for 60 min at 37 °C. The 20-fold wash solution was diluted into one-fold with distilled water and reserved. The closure plate membrane was uncovered and the liquid was discarded. Washing buffer was added to every well for 30 s and then were drained with 5 times repeat. 450 µL Chromogen Solution A and 50 µL Chromogen Solution B were added to each well, then evade the light preservation for 15 min at 37 °C. After that, 50 µL stop solutions were added to each well. The color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of suPAR in samples is then determined by comparing the OD of the samples to the standard curve.
Statistical analyses:
All data were analyzed using SPSS statistical software for Windows, version 23.0 (SPSS, Inc., Chicago, IL, USA). The measurement data accorded with normal distribution were expressed as mean ± SD and differences between the two groups were compared using Student’s t-test. The non-normally distributed data were expressed as medians (25th, 75th percentiles) and differences between two groups were compared using nonparametric Mann-Whitney U test. Categorical variables were compared using the χ2 test or Fisher exact test. Analysis of variance for two-period crossover design was performed to evaluate the therapeutic outcome and side effects. Correlation analysis was used to explore the correlation between variables. Two-tailed tests were used for all comparisons and a p < 0.05 was considered to be statistically significant.
Results:
Comparison of the pretherapeutic baseline characteristics between patients received amiloride/HCTZ and HCTZ Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.
Comparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.
Comparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Amiloride’s effect in decreasing uACR and suPAR After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).
Comparison of outcomes between amiloride and HCTZ over the course of the study.
uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).
Comparison of outcomes between amiloride and HCTZ over the course of the study.
uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Association between urinary suPAR and uPCR/uACR Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.
(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.
Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.
(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.
The safety of amiloride during the treatment periods Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).
Comparison of side effect between amiloride and HCTZ over the course of the study.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure.
Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).
Comparison of side effect between amiloride and HCTZ over the course of the study.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure.
Comparison of the pretherapeutic baseline characteristics between patients received amiloride/HCTZ and HCTZ:
Among ten DKD patients enrolled, five patients received amiloride/HCTZ, and after more than 4-week washout periods they were orally administrated with HCTZ. Another five patients were orally administrated with HCTZ and after more than 4-week washout periods they received amiloride/HCTZ. Mean age (±SD) was 57.1 ± 12.7 and 5 (50%) were men. Table 1 shows the participant baseline characteristics between two groups (Amiloride/HCTZ vs HCTZ) before treatment. There was no difference in pretherapeutic weight, blood pressure, eGFR, serum creatine, uPCR, uACR, serum albumin, cholesterol, triglyceride, fasting glucose, unic acid, hemoglobin, platelet, serum sodium, serum potassium, serum suPAR and urinary suPAR between patients received amiloride/HCTZ and HCTZ.
Comparison of the baseline characteristics between patients received amiloride/HCTZ and HCTZ before treatment.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure; eGFR: estimated glomerular filtration rate; uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Amiloride’s effect in decreasing uACR and suPAR:
After the treatment period of 12 weeks, uACR, serum, and urinary suPAR decreased significantly in patients who received amiloride/HCTZ compared with those administrated with HCTZ. The decline in uACR over the 12 weeks after treatment was 605.0 (172.0, 1395.5) mg/gCr in the amiloride/HCTZ group and −353.0(−1090.3, 502.3) mg/gCr in the HCTZ group, with a resulting treatment difference between two groups (F = 8.279, p = 0.021). However, the decline in uPCR over the 12 weeks after treatment was 1385.5 (518.5, 3218.0) mg/gCr in the amiloride/HCTZ group and −390.0(−1402.5, 1185.8) mg/gCr in the HCTZ group, without a significant difference between groups (F = 5.131, p = 0.053). Moreover, the levels of serum suPAR decreased by 7.1 (−2.7, 14.9) ng/mL from the baseline while the decrease of urinary suPAR was 5.2 (1.8, 49.7) in the amiloride/HCTZ group, with a significant difference compared with the HCTZ groups (serum suPAR: F = 32.313, p < 0.001; urinary suPAR: F = 6.188, p = 0.038) (Table 2).
Comparison of outcomes between amiloride and HCTZ over the course of the study.
uPCR: urine protein creatinine ratio; uACR: urinary albumin creatinine ratio; suPAR: soluble urokinase-type plasminogen activator receptor.
Association between urinary suPAR and uPCR/uACR:
Correlation analysis showed that the baseline urinary suPAR was positively associated with uPCR (r = 0.521, p = 0.018) (Figure 2(A)). Additionally, the baseline urinary suPAR was also positively associated with uACR (r = 0.514, p = 0.020) (Figure 2(B)). However, neither uPCR nor uACR was found to have a significant association with serum suPAR (Supplementary Figure 1(A,B)). We defined that the effect of amiloride in decreasing uACR in DKD may be associated with the decline of urinary suPAR.
(A, B) The correlation between urinary protein/albumin creatinine ratio and urinary suPAR.
The safety of amiloride during the treatment periods:
Among ten DKD patients enrolled, one patient in the amiloride/HCTZ group and one patient in the HCTZ group experienced hyperkalemia during the treatment periods. Serum K+ in the amiloride/HCTZ group increased by 0.1 (−0.1, 0.9) mmol/L while 0.0 (−0.3, 0.2) mmol/L in the HCTZ group. Treatment effect on serum K+ in the amiloride/HCTZ group had no significant difference compared with those in the HCTZ group. Additionally, outcomes including changes in blood pressure, weight, serum Na+, serum uric acid, cholesterol, triglycerides before and after treatment were compared between the amiloride/HCTZ and HCTZ group. Analysis of variance for the two-period crossover design showed that there was no significant difference in side effects between groups (Table 3).
Comparison of side effect between amiloride and HCTZ over the course of the study.
SBP: Systolic blood pressure; DBP: Diastolic blood pressure.
Discussion:
DKD is the most common cause of CKD and ESRD worldwide and develops in approximately 40% of type 2 diabetic patients [18]. DKD is a common microvascular complication of diabetes mellitus (DM) and most of the risk for all-cause and cardiovascular disease mortality in DM patients is related to the presence of DKD [19].
Progressively larger amounts of albuminuria are the most common manifestation in DKD and then classically followed by a relentless decline in kidney function, renal impairment, and ultimately ESRD. Albuminuria is a well-known factor that links podocyte damage to tubulointerstitial injury, which triggers tubulointerstitial inflammation and fibrogenesis, and accelerates the decline of renal function [20]. Therefore, reducing albuminuria in DKD is particularly important to delay its progression and improve outcomes.
The impairment of the glomerular filtration barrier is an important pathophysiological basis for albuminuria in DKD. The glomerular filtration barrier consists of endothelial cells, the glomerular basement membrane (GBM), and podocytes. Podocytes have a prominent role in maintaining the integrity of the glomerular filter. Podocytes injury would lead to the effacement of foot processes and the detachment or apoptosis of podocytes [21]. The loss of integrity of the podocyte seal results in the diffusion and convection of proteins from the circulation into the urinary space and finally leads to the occurrence of albuminuria [22]. Moreover, several pathogenic mechanisms involved in podocyte injury would beget ultrastructural changes in podocytes and albuminuria. Previous researches had found that mutation of the gene encoding the podocyte-expressed protein nephrin would result in congenital albuminuria [23]. Disorder of cross-talk between podocytes and the other cells involved in the glomerular filtration barrier might also lead to albuminuria. Several studies [19,24–26] had found that the podocyte lesion is the primary causes for glomerular diseases characterized by massive proteinuria, such as minimal change nephropathy (MCD), focal glomerulosclerosis (FSGS), membranous nephropathy (MN), lupus nephritis (LN), DKD and is related to disease progression.
Previous findings [11] had indicated that increasing expression of uPAR in podocytes would result in the podocytes lesion and the occurrence of albuminuria. uPAR is a multidomain glycoprotein tethered to the cell membrane with a glycosylphosphotidylinositol (GPI) anchor and was originally identified as a saturable binding site for urokinase on the cell surface. uPAR is required for activation of β3 integrin within the kidney podocyte, which is one of the main proteins that anchors podocytes to the underlying GBM. Increased β3 integrin activation within podocytes would cause accelerated podocyte foot process dynamics, which dysregulates the shape and function of the glomerular filtration barrier. Wei [11] et al. had found that lipopolysaccharide (LPS) injection would induce the higher expression of uPAR in podocytes. Mice lacking uPAR were protected from LPS-mediated proteinuria but develop the disease after expression of a constitutively active β3 integrin. Knockdown of uPAR in podocytes would decrease the number of migrating podocytes and promote motility, which showed a physiological role for uPAR signaling in the regulation of kidney permeability. Moreover, Zhao [27] et al. had demonstrated that urinary suPAR was specifically elevated in primary FSGS and was related to disease severity. The elevated urinary suPAR could activate β3 integrin on human podocytes. Therefore, it indicated that restraining the expression of uPAR in podocytes may be a potential therapeutic target for the clinical management of albuminuria in DKD.
Amiloride is a Na channel blocker and inhibits the Na+/H+ and Na+/Ca2+ antiporters, which have been widely used as a diuretic. Previous studies had suggested that amiloride could inhibit the expression of uPAR in tumor-infiltrating lymphocytes [28] and colon cancer cells [29,30]. Our former works had indicated that amiloride was able to inhibit the expression of uPAR and thereby reduced the mobility of podocytes, which provided a new therapeutic paradigm for the clinical management of albuminuria. Therefore, the effect of amiloride in decreasing albuminuria in DKD was explored in this prospective, crossover, open-label study.
In this crossover design, amiloride induced a significant decrease of uACR in DKD. Additionally, the decrease of serum and urinary suPAR in the amiloride/HCTZ group was significant compared with the HCTZ group. Correlation analysis had also shown that the levels of urinary suPAR were positively associated with uPCR and uACR. Based on the above results, we concluded that amiloride was able to reduce albuminuria in DKD and may be associated with the decreased levels of urinary suPAR, which was an indicator for disease severity [27]. Recent researches had revealed that urinary suPAR was significantly increased in patients with massive proteinuria and decreased accompanying with the remission of proteinuria. Mette Staehr [28] et al. had demonstrated that urinary uPA protein and activity were increased in rats treated with puromycin aminonucleoside. And amiloride could inhibit urinary uPA activity, which attenuated plasminogen activation and urine protease activity. Our studies had also found that amiloride could reduce urinary and serum suPAR, which may be a relevant target for amiloride in reducing albuminuria.
As Na channel blocker, amiloride would inhibit the exchange of Na+–K+ and Na+–H+ in distal tubules and collecting ducts of the kidney, thus increasing the excretion of Na+ and water and reducing the excretion of K+ and H+. Therefore, hyperkalemia and hyponatremia were the most common side effect of amiloride. In this study, significant hyperkalemia occurred in two patients and no patients experienced hyponatremia. And the serum potassium in both patients could return to normal after hypokalemia treatment. Additionally, there was no significant influence in fasting glucose, cholesterol, triglyceride, unic acid with amiloride/HCTZ when compared with HCTZ. Furthermore, amiloride would substantially reduce the SBP and volume status by blocking both ENaC and urokinase [31,32]. However, we had not found significant reductions in weight or SBP with amiloride/HCTZ compared with HCTZ. Our inability to detect significant effects on blood pressure and weight may be associated with the small sample size in this study. Unruh [33] et al. had also demonstrated the safety of amiloride in patients with type 2 diabetes, normal renal function, and proteinuria, which provided important research evidence for amiloride’s application in decreasing albuminuria in DKD.
The strength of this study mainly discussed the effect and safety of amiloride in decreasing albuminuria in DKD patients, which would provide a new application for the traditional diuretic. However, our study still has some limitations. First of all, we only enrolled small sizes sample from a single-center, and long-term renal prognosis had not been explored in DKD patients. Furthermore, the follow-up period should be extended in order to investigate the long-term effect of amiloride in DKD.
Conclusions:
Amiloride could induce a significant decrease of albuminuria, which was accompanied by the significant decline of urinary suPAR. No serious side effects were found in DKD patients using amiloride during treatment periods. In summary, amiloride might be an efficient and safe treatment strategy for decreasing albuminuria in DKD.
Supplementary Material:
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| Background:
Diabetic kidney diseases (DKD) were the leading cause of End-stage renal diseases worldwide. Albuminuria was a target for treatment in DKD and decreasing albuminuria was particularly important for improving its prognosis. However, there is still a lack of specific treatment for DKD.
Methods:
We conducted a prospective, crossover, open-label study to investigate the effect of amiloride in patients with DKD. Safety and efficacy were assessed by monitoring urine protein creatinine ratio(uPCR), urinary albumin creatinine ratio (uACR), blood pressure, weight, serum sodium, serum potassium, cholesterol, triglyceride, uric acid, serum soluble urokinase-type plasminogen activator receptor (suPAR) and urinary suPAR. Ten subjects were enrolled in the trial.
Results:
In this prospective, crossover, open-label design, amiloride could induce a significant decrease of uACR in DKD. The decrease of serum and urinary suPAR in the amiloride/hydrochlorothiazide (HCTZ) group was also significant compared with those patients using HCTZ as the control group. Correlation analysis showed that the levels of urinary suPAR were positively associated with uPCR and uACR. No significant difference in blood pressure, weight, serum sodium, serum potassium, cholesterol, triglyceride, uric acid was seen between the amiloride/HCTZ group and the control group.
Conclusions:
In summary, among patients with DKD, amiloride could decrease albuminuria without severe side effects, which was accompanied by the significant decline of urinary suPAR. | Introduction:
Diabetic kidney diseases (DKD) were the leading cause of end-stage renal diseases and renal replacement therapy worldwide [1]. Recently the prevalence of DKD had been increasing dramatically in China and chronic kidney disease (CKD) related to diabetes had become more common than CKD related to glomerulonephritis [2]. The clinicopathological presentation of DKD was characterized by progressive albuminuria, hyperfiltration, associative glomerular injury, and tubulointerstitial fibrosis [3]. Moreover, persistent albuminuria could predict early deterioration of renal function [4] and was an independent risk factor of cardiovascular disease [5] and a target for the treatment of DKD [6]. Therefore, decrease albuminuria in DKD is particularly essential for improving its prognosis and reduce complications.
Albuminuria in DKD result from the damage of the glomerular filtration barrier, which leads to the excessive filtration of plasma protein and other macromolecular substances and exceeds the reabsorption ability of renal tubules. The podocyte plays a key role in the glomerular filtration barrier and its disruption results in a dramatic loss of function leading to albuminuria. The major biological functions of podocytes are to limit the passage of albumin from the circulation into the urine and to maintain overall glomerular integrity [7]. Thus, exploration of the drugs which target the improvement of the damaged podocyte would be conducive to improve albuminuria and delay the progression in DKD.
Recent studies have found that the increased expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes could induce podocyte injury and albuminuria [8–12]. uPAR is required for activation of β3 integrin signaling within the kidney podocyte, which would lead to accelerated podocyte foot process dynamics and dysregulate the shape and function of the glomerular filtration barrier [11]. Increasing soluble urokinase-type plasminogen activator receptor (suPAR) levels in mice would induce podocyte abnormalities while reducing suPAR levels and blocking suPAR actions could improve kidney morphology [13].
Amiloride, a pyrazine compound inhibiting sodium reabsorption through sodium channels in renal epithelial cells, has been used as a diuretic in clinical practice. Our previous study had found that amiloride inhibited podocyte uPAR induction and reduced proteinuria in the 5/6 nephrectomy rat model and the LPS mouse model of transient proteinuria [14]. Moreover, amiloride could inhibit urine uPAR activity which attenuated plasminogen activation in vivo [15]. These results suggest that amiloride may have shown a potential effect on reducing albuminuria. Since there was no specific immunosuppressive therapy for DKD, we performed this prospective, crossover, open-label study to investigate the safety and effectiveness of amiloride in decreasing albuminuria in DKD.
Conclusions:
Amiloride could induce a significant decrease of albuminuria, which was accompanied by the significant decline of urinary suPAR. No serious side effects were found in DKD patients using amiloride during treatment periods. In summary, amiloride might be an efficient and safe treatment strategy for decreasing albuminuria in DKD. | Background:
Diabetic kidney diseases (DKD) were the leading cause of End-stage renal diseases worldwide. Albuminuria was a target for treatment in DKD and decreasing albuminuria was particularly important for improving its prognosis. However, there is still a lack of specific treatment for DKD.
Methods:
We conducted a prospective, crossover, open-label study to investigate the effect of amiloride in patients with DKD. Safety and efficacy were assessed by monitoring urine protein creatinine ratio(uPCR), urinary albumin creatinine ratio (uACR), blood pressure, weight, serum sodium, serum potassium, cholesterol, triglyceride, uric acid, serum soluble urokinase-type plasminogen activator receptor (suPAR) and urinary suPAR. Ten subjects were enrolled in the trial.
Results:
In this prospective, crossover, open-label design, amiloride could induce a significant decrease of uACR in DKD. The decrease of serum and urinary suPAR in the amiloride/hydrochlorothiazide (HCTZ) group was also significant compared with those patients using HCTZ as the control group. Correlation analysis showed that the levels of urinary suPAR were positively associated with uPCR and uACR. No significant difference in blood pressure, weight, serum sodium, serum potassium, cholesterol, triglyceride, uric acid was seen between the amiloride/HCTZ group and the control group.
Conclusions:
In summary, among patients with DKD, amiloride could decrease albuminuria without severe side effects, which was accompanied by the significant decline of urinary suPAR. | 7,769 | 280 | [
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] | null | [CONTENT] Diabetic kidney disease | amiloride | albuminuria | soluble urokinase-type plasminogen activator receptor [SUMMARY] | null | [CONTENT] Diabetic kidney disease | amiloride | albuminuria | soluble urokinase-type plasminogen activator receptor [SUMMARY] | [CONTENT] Diabetic kidney disease | amiloride | albuminuria | soluble urokinase-type plasminogen activator receptor [SUMMARY] | [CONTENT] Diabetic kidney disease | amiloride | albuminuria | soluble urokinase-type plasminogen activator receptor [SUMMARY] | [CONTENT] Diabetic kidney disease | amiloride | albuminuria | soluble urokinase-type plasminogen activator receptor [SUMMARY] | [CONTENT] Aged | Albuminuria | Amiloride | Creatinine | Cross-Over Studies | Diabetic Nephropathies | Drug Combinations | Female | Humans | Hydrochlorothiazide | Male | Middle Aged | Prospective Studies | Receptors, Urokinase Plasminogen Activator | Treatment Outcome [SUMMARY] | null | [CONTENT] Aged | Albuminuria | Amiloride | Creatinine | Cross-Over Studies | Diabetic Nephropathies | Drug Combinations | Female | Humans | Hydrochlorothiazide | Male | Middle Aged | Prospective Studies | Receptors, Urokinase Plasminogen Activator | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Albuminuria | Amiloride | Creatinine | Cross-Over Studies | Diabetic Nephropathies | Drug Combinations | Female | Humans | Hydrochlorothiazide | Male | Middle Aged | Prospective Studies | Receptors, Urokinase Plasminogen Activator | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Albuminuria | Amiloride | Creatinine | Cross-Over Studies | Diabetic Nephropathies | Drug Combinations | Female | Humans | Hydrochlorothiazide | Male | Middle Aged | Prospective Studies | Receptors, Urokinase Plasminogen Activator | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Albuminuria | Amiloride | Creatinine | Cross-Over Studies | Diabetic Nephropathies | Drug Combinations | Female | Humans | Hydrochlorothiazide | Male | Middle Aged | Prospective Studies | Receptors, Urokinase Plasminogen Activator | Treatment Outcome [SUMMARY] | [CONTENT] diabetic kidney | introduction diabetic kidney | dkd decrease albuminuria | decreasing albuminuria dkd | albuminuria dkd glomerular [SUMMARY] | null | [CONTENT] diabetic kidney | introduction diabetic kidney | dkd decrease albuminuria | decreasing albuminuria dkd | albuminuria dkd glomerular [SUMMARY] | [CONTENT] diabetic kidney | introduction diabetic kidney | dkd decrease albuminuria | decreasing albuminuria dkd | albuminuria dkd glomerular [SUMMARY] | [CONTENT] diabetic kidney | introduction diabetic kidney | dkd decrease albuminuria | decreasing albuminuria dkd | albuminuria dkd glomerular [SUMMARY] | [CONTENT] diabetic kidney | introduction diabetic kidney | dkd decrease albuminuria | decreasing albuminuria dkd | albuminuria dkd glomerular [SUMMARY] | [CONTENT] hctz | amiloride | patients | serum | supar | urinary | treatment | amiloride hctz | dkd | urinary supar [SUMMARY] | null | [CONTENT] hctz | amiloride | patients | serum | supar | urinary | treatment | amiloride hctz | dkd | urinary supar [SUMMARY] | [CONTENT] hctz | amiloride | patients | serum | supar | urinary | treatment | amiloride hctz | dkd | urinary supar [SUMMARY] | [CONTENT] hctz | amiloride | patients | serum | supar | urinary | treatment | amiloride hctz | dkd | urinary supar [SUMMARY] | [CONTENT] hctz | amiloride | patients | serum | supar | urinary | treatment | amiloride hctz | dkd | urinary supar [SUMMARY] | [CONTENT] podocyte | albuminuria | dkd | renal | upar | kidney | glomerular | filtration | glomerular filtration barrier | function [SUMMARY] | null | [CONTENT] hctz | amiloride hctz | group | hctz group | amiloride | supar | serum | urinary | uacr | received [SUMMARY] | [CONTENT] amiloride | albuminuria | significant | summary amiloride efficient safe | safe treatment | significant decline urinary | significant decline | effects found | effects found dkd | effects found dkd patients [SUMMARY] | [CONTENT] hctz | amiloride | patients | supar | amiloride hctz | serum | urinary | group | hctz group | treatment [SUMMARY] | [CONTENT] hctz | amiloride | patients | supar | amiloride hctz | serum | urinary | group | hctz group | treatment [SUMMARY] | [CONTENT] ||| Albuminuria | DKD ||| DKD [SUMMARY] | null | [CONTENT] DKD ||| HCTZ | HCTZ ||| ||| [SUMMARY] | [CONTENT] DKD [SUMMARY] | [CONTENT] ||| Albuminuria | DKD ||| DKD ||| DKD ||| ||| Ten ||| ||| DKD ||| HCTZ | HCTZ ||| ||| ||| DKD [SUMMARY] | [CONTENT] ||| Albuminuria | DKD ||| DKD ||| DKD ||| ||| Ten ||| ||| DKD ||| HCTZ | HCTZ ||| ||| ||| DKD [SUMMARY] |
|||||
Standardization in laboratory medicine: Two years' experience from category 1 EQA programs in Spain. | 30591811 | Standardization is the ability to obtain interchangeable results leading to same medical interpretation. External quality assessment (EQA) is the main support of the on-going harmonization initiatives. Aim of study was to evaluate results obtained from two years category 1 EQA program experience in Spain and determine the impact of applying this type of EQA program on the analytical standardization. | INTRODUCTION | According to the analytical method, traceability and instrument different groups were established which results were evaluated by calculating mean, coefficient of variation and percent of deviation to the reference value. Analytical performance specifications used to the results' evaluation were derived from biological variation for bias and from the inter-laboratory coefficients of variation found in a previous pilot study. | MATERIALS AND METHODS | Only creatinine measured by enzymatic methods gave excellent results, although few laboratories used this method. Creatine kinase and GGT gave good precision and bias in all, but one instrument studied. For the remaining analytes (ALT, ALP, AST, bilirubin, calcium, chloride, glucose, magnesium, potassium, sodium, total protein and urate) some improvement is still necessary to achieve satisfactory standardization in our setting. | RESULTS | The two years of category 1 EQA program experience in Spain have manifested a lack of standardization of 17 most frequent biochemistry tests used in our laboratories. The impact of the information obtained on the lack of standardization is to recommend abandoning methods such as ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium. | CONCLUSIONS | [
"Clinical Laboratory Techniques",
"Creatine Kinase",
"Creatinine",
"Humans",
"Quality Assurance, Health Care",
"Spain",
"gamma-Glutamyltransferase"
] | 6294154 | Introduction | The main objective of clinical laboratory is to provide clear, reliable and useful information for clinical decision-making. Current healthcare systems imply performing laboratory tests in different locations, so standardization among laboratories become one of the cornerstones of the quality patient‘s care. Standardization can be defined as the ability to obtain interchangeable results (within certain analytical quality uncertainty) in order to achieve the same medical decision, regardless of the analytical procedure (method, traceability and instrument), measurement units and reference intervals.
The standardization should be based on six basic pillars, which include in vitro diagnostic companies, reference materials, reference methods, reference laboratories, medical laboratories and external quality assessment (EQA) organizations (1). Recently, Greaves noted that EQA is not just a pillar but the central support for on-going harmonization (2). Discordance in results between laboratories and methods should become a practice no longer accepted.
It is widely accepted that the best strategy to organize an EQA scheme is to use fresh frozen commutable control samples with values assigned by reference laboratories using reference methods, which can be found on www.harmonization.net (3, 4).
Spanish Society of Laboratory Medicine (SEQCML) is a non-profit scientific organization that has been providing EQA schemes in Spain since 1980 by using stabilized control materials. Since 2013 a category 1 program has been organized for basic biochemistry analytes. According to Miller et al. this kind of program distributes commutable control materials with reference-measurement procedure (RMP) assigned values and replicate samples in surveys are tested (3). Accuracy of individual laboratories is assessed by comparison with the RMP, while reproducibility is checked both intra- and inter-laboratory, and standardization is assessed by comparison of measurement procedure calibration traceability with RMP. Two initial surveys were performed in 2013 and 2014, as preliminary experiences and regular annual surveys have been organized since 2015. For a proper assessment of bias, having adequate information of measurement’s traceability is therefore a crucial point (5, 6).
Another important aspect to consider is the analytical performance specification (APS) or acceptability limits selected for the evaluation of the derived results. When APS are based on biological variation (BV), it is highly recommended to use the gradual classification of APS according to its strictness: optimal, desirable and minimal (7). It should be noted that the APS grade could be selected according to the limitations of the current state of the art, being defined as the performance achieved by about 80% of laboratories. According to this criterion, in this study the minimal BV-based APS grade was selected for electrolytes evaluation, while desirable BV APS were chosen for enzymes and substrates.
In this regard, a performance worse than the minimum APS should alert the laboratory that its results could be at risk and clinical decision-making might be detrimentally affected. Likewise, a performance reaching the minimal grade suggest that further improvement may be beneficial for patients (8, 9).
The aim of this work is to evaluate the results obtained from two years category 1 EQA program, 2015 and 2016 surveys, performed in our country and to assess the impact of applying this kind of EQA program over the analytical standardization. Evaluation is based on the inter-laboratory imprecision and the bias of the peer group means compared with the reference method values. | null | null | Results | All results exceeding the mean ± 3 standard deviation of each group were rejected as outliers. The number of rejected participant laboratories was 5 for the 2015 survey and 10 for the 2016 survey. Moreover, 30 results for lactate dehydrogenase (LD) which were 100% higher than the others due to the different substrate (pyruvate instead of lactate) were also excluded from the study. Results for bias are presented in Figures 1-17. Results for the inter-laboratory imprecision of each peer group for electrolytes, enzymes and substrates are presented in Tables 3-5 and compared with the APS for inter-laboratory imprecision (APSIL) from the pilot 2014 survey (16). An overview of the standardization achieved in our setting, according to the bias and the imprecision calculated for instruments, is presented in Table 6. | Conclusions | The two years of category 1 EQA program experience in our country have manifested a lack of standardization of the 17 more frequent general biochemistry tests used in our laboratories. The application of this kind of EQA program allows estimating measurement procedure-traceability-instrument bias in a way that can be expanded to what happens with real patient samples. The impact of the information obtained by category 1 EQA program on the lack of standardization is: to recommend abandoning methods such as for ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, pyruvate-lactate for LD, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium. | [] | [] | [] | [
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"The main objective of clinical laboratory is to provide clear, reliable and useful information for clinical decision-making. Current healthcare systems imply performing laboratory tests in different locations, so standardization among laboratories become one of the cornerstones of the quality patient‘s care. Standardization can be defined as the ability to obtain interchangeable results (within certain analytical quality uncertainty) in order to achieve the same medical decision, regardless of the analytical procedure (method, traceability and instrument), measurement units and reference intervals.\nThe standardization should be based on six basic pillars, which include in vitro diagnostic companies, reference materials, reference methods, reference laboratories, medical laboratories and external quality assessment (EQA) organizations (1). Recently, Greaves noted that EQA is not just a pillar but the central support for on-going harmonization (2). Discordance in results between laboratories and methods should become a practice no longer accepted.\nIt is widely accepted that the best strategy to organize an EQA scheme is to use fresh frozen commutable control samples with values assigned by reference laboratories using reference methods, which can be found on www.harmonization.net (3, 4).\nSpanish Society of Laboratory Medicine (SEQCML) is a non-profit scientific organization that has been providing EQA schemes in Spain since 1980 by using stabilized control materials. Since 2013 a category 1 program has been organized for basic biochemistry analytes. According to Miller et al. this kind of program distributes commutable control materials with reference-measurement procedure (RMP) assigned values and replicate samples in surveys are tested (3). Accuracy of individual laboratories is assessed by comparison with the RMP, while reproducibility is checked both intra- and inter-laboratory, and standardization is assessed by comparison of measurement procedure calibration traceability with RMP. Two initial surveys were performed in 2013 and 2014, as preliminary experiences and regular annual surveys have been organized since 2015. For a proper assessment of bias, having adequate information of measurement’s traceability is therefore a crucial point (5, 6).\nAnother important aspect to consider is the analytical performance specification (APS) or acceptability limits selected for the evaluation of the derived results. When APS are based on biological variation (BV), it is highly recommended to use the gradual classification of APS according to its strictness: optimal, desirable and minimal (7). It should be noted that the APS grade could be selected according to the limitations of the current state of the art, being defined as the performance achieved by about 80% of laboratories. According to this criterion, in this study the minimal BV-based APS grade was selected for electrolytes evaluation, while desirable BV APS were chosen for enzymes and substrates.\nIn this regard, a performance worse than the minimum APS should alert the laboratory that its results could be at risk and clinical decision-making might be detrimentally affected. Likewise, a performance reaching the minimal grade suggest that further improvement may be beneficial for patients (8, 9).\nThe aim of this work is to evaluate the results obtained from two years category 1 EQA program, 2015 and 2016 surveys, performed in our country and to assess the impact of applying this kind of EQA program over the analytical standardization. Evaluation is based on the inter-laboratory imprecision and the bias of the peer group means compared with the reference method values.",
"Commutable control materials were purchased from MCA laboratory (Queen Beatrix Hospital, Winterswijk, The Netherlands) by means of the Stichting Kwaliteitsbewaking Medische Laboratorium Diagnostiek (SKML). According to Cobbaert et al. controls had been prepared from fresh anonymized left-over sera of routine laboratory with exclusion of lipemic, icteric, positive hepatitis B surface antigen (HBsAG), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) samples, and stored frozen at – 84 ºC in aliquots. Pathological concentration ranges were created by adequately mixing pools and by spiking with minerals, recombinant human enzymes and human albumin (10). Commutability had been verified by SKML, as explained by Baadenhuijsen et al. and Jansen et al. (11, 12). Throughout the years commutability has been monitored by including a native, single donation spy-sample (10, 12).\nSix vials of fresh frozen human serum pools at different concentrations were distributed once per year in a single express shipment at – 80 ºC and delivered within 24 hours to laboratories all over Spain. Different lots at different concentrations were provided for each of the two surveys. Participant laboratories were requested to maintain samples at – 20 ºC until analysis, which had to be performed within the following 14 days. Each vial had to be analysed in duplicate, one vial per day, for 6 consecutive days whenever possible. Results were registered on the SEQCML-EQA website, in order to be either individually and globally evaluated.\nA preliminary 2013 survey was carried out in 19 laboratories and was addressed to ascertain whether the logistics of managing a non-stabilized set of control materials was operative in our country. No incidents were observed with temperature maintenance during the time between deliveries of control materials from the provider to the laboratory analysis.\nAnother point of interest of this preliminary survey was to explore whether laboratories could adequately inform about their analytical traceability to standards. Important difficulties were perceived that impelled holding a meeting between EQAs organization and providers, claiming for clear and complete information on calibrators’ traceability.\nIn 2014 first survey was performed, as part of a pilot European study (INPUTs) (Italy, The Netherlands, Portugal, Spain and The United Kingdom), with a total of 20 laboratories participants and whose results has been already published (12, 13). Only about 45% of participants were able to correctly inform about its traceability, so results are not shown in this study. This survey was then considered as a pilot to identify the problems that could impact on the EQA participation and further interpretation of results. For both surveys as well as for those performed in 2015 and 2016, same sample management protocol was applied.\nThe 2015 and 2016 surveys were exclusively run in Spain and included 17 analytes. The number of registered participants was 93 and 105, respectively. The target values of distributed control materials were assigned by the reference methods and laboratories (Table 1).\nResults were categorized by measurement procedure, traceability and instrument. The description of standard materials used by participants for calibration traceability is shown in Table 2. Participant laboratories using the same combination of these three elements were considered as a peer group. The peer groups and the number of laboratories included for each analyte are shown in Figures 1-17.\nCalcium. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Numbers in brackets mean the number of participant laboratories. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. NM-BAPTA: calcium specific amino-polycarboxylic acid.\nChloride. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. ISE - ion selective electrode. Numbers in brackets indicate the laboratories participating for each instrument.\nMagnesium. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value . Lim Bias (m): acceptability limit for bias based on BV, minimum grade. Xil - Xilidil blue. Numbers in brackets indicate the laboratories participating for each instrument.\nPotassium. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. ISE - ion selective electrode. Numbers in brackets indicate the laboratories participating for each instrument.\nSodium. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. ISE - ion selective electrode. Numbers in brackets indicate the laboratories participating for each instrument.\nAlkaline phosphatase. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. AMP - 2-amino-2-methyl-1-propanol. Numbers in brackets indicate the laboratories participating for each instrument.\nAmylase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. G3 - malto trioside. G7 - malto-heptaoside. Numbers in brackets indicate the laboratories participating for each instrument.\nALT. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. Numbers in brackets indicate the laboratories participating for each instrument.\nAST. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. P5P -pyridoxal-5-phosphate. Numbers in brackets indicate the laboratories participating for each instrument.\nCreatine kinase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. NAC - N-acetyl-cysteine. Numbers in brackets indicate the laboratories participating for each instrument.\nGamma glutamyl transferase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. All groups use substrate: γ glutamyl-3carboxy-4nitroanilide > 4mmol/L. The exception is: Siemens Dimension, Vista that uses substrate < 4mmol/L. Numbers in brackets indicate the laboratories participating for each instrument.\nLactate dehydrogenase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. NMG - N-methyl-D-glucamine. DEA - diethanolamine. TRIS -hydroxymethyl-aminomethane. Numbers in brackets indicate the laboratories participating for each instrument.\nBilirubin. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. DPD - 3,5-dicholorophenyl-diazonium- tetrafluoroborate. Numbers in brackets indicate the laboratories participating for each instrument.\nCreatinine. Percentage deviation (Dev%) of peer group mean from the reference value. Methods in figure appearing according the following order: enzymatic, compensated and non-compensated. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. Numbers in brackets indicate the laboratories participating for each instrument.\nGlucose. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. GOD - glucose oxidase. HK - hexokinase. Numbers in brackets indicate the laboratories participating for each instrument.\nTotal protein. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. B - biuret. Numbers in brackets indicate the laboratories participating for each instrument.\nUrate. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. POD – peroxidase. Numbers in brackets indicate the laboratories participating for each instrument.\nCompared to 2015, a new instrument was incorporated in 2016 survey (Bio-systems BA 400), with only 6 participating laboratories. The overall evaluation of the 2015 survey was published on the SEQCML website and was presented at the 2016 EQALM annual meeting (13, 15). Only groups formed by 5 or more final laboratories were considered in this study.\nInter-laboratory imprecision was calculated by averaging the coefficient of variation (CV) obtained from the six controls distributed on the 2016 and 2015 surveys and compared with the best (Dutch) inter-laboratory CV derived from the 2014 pilot study, which used similar six commutable control materials (16).\nBias was calculated by the percent difference between the peer group mean (same measurement procedure, traceability and instrument) and the reference value. The analytical performance specification to apply for bias evaluation was based on the BV data collected on the online 2014 database, which had been elaborated as detailed by Ricós et al., applying the minimum level of requirement for electrolytes and the desirable level for substrates and enzymes (17-19).\nThe results of this study were examined with the particular focus on the most common analytical procedures used in Spain and its repercussion on non-comparable results, detected throughout participation on level 1 EQA schemes.\nStandardization is defined by the attainment of inter-laboratory imprecision within the predefined APS and peer group bias (% mean deviation to the reference value) below the allowed bias derived from BV.",
"All results exceeding the mean ± 3 standard deviation of each group were rejected as outliers. The number of rejected participant laboratories was 5 for the 2015 survey and 10 for the 2016 survey. Moreover, 30 results for lactate dehydrogenase (LD) which were 100% higher than the others due to the different substrate (pyruvate instead of lactate) were also excluded from the study. Results for bias are presented in Figures 1-17. Results for the inter-laboratory imprecision of each peer group for electrolytes, enzymes and substrates are presented in Tables 3-5 and compared with the APS for inter-laboratory imprecision (APSIL) from the pilot 2014 survey (16). An overview of the standardization achieved in our setting, according to the bias and the imprecision calculated for instruments, is presented in Table 6.",
"The percentage of laboratories excluded was higher in 2016 than in 2015 due to better knowledge of the traceability-instrument, so groups were more specific in 2016. This cannot be considered a disadvantage. The results in this study are discussed form the light of their impact on the aims proposed. These are: positive, negative and needed to be dialogued with providers.\nMain positive impacts, which imply an adequate standardization not needing for further improvements, apply to potassium and creatine kinase (CK). Potassium shows inter-laboratory imprecision and bias (Figure 4) within the allowable limits for almost all peer groups. For the remaining electrolytes good inter-laboratory imprecision can also be seen, well in agreement with the 2014 survey (performed in collaboration with other European countries) where all participant laboratories and manufacturers fulfilled the APS for total analytical error at the minimum performance level (20). Creatine kinase show good inter-laboratory imprecision and bias (Figure 10), except for the new group enrolled in the 2016 survey (BA400). So it may be expected a well standardized measurements soon. Negative impacts may be due to several reasons. The aqueous matrix of SRM 915 and 918 used for calcium and sodium, respectively (Figures 1 and 5), produces low results. Lack of commutability of calibration traceability materials was described to be a crucial factor to assure standardization in medical laboratories by Panteghini and Ambruster (21, 22).\nInstrument dependent problems can be seen in this study for alkaline phosphatase (ALP) with low results for Roche users (Figure 6), whereas all participants use same method and traceability; this event causes an important lack of standardization in our country because it is the greatest group. Same results had been seen by Braga et al., and Aloisio et al. who observed discrepancies among Abbott Architect users related to an “experimental” calibration factor provided by the manufacturer (23, 24). Non-standardized ALP results could have a great impact in some clinical scenarios such as hypophosphatemia diagnosis, so an improvement in the results’ traceability becomes a crucial objective (25). Method dependent troubles are seen in four cases.\nFirstly, amylase, were all groups using malto-heptaoside (G7) substrate, as well as the malto-trioside (G3) of Abbott Architect show harmonized results. The remaining G3 groups have unacceptable negative bias (Figure 7). This lack of standardization affects one third of the participants of this study, thus producing a considerable impact on the healthcare in our country. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) testing show unacceptable inter-laboratory imprecision and bias (low results) (Figures 8 and 9) for laboratories that did not add pyridoxal-5-phosphate (P5P) in its measurement procedure. Infusino et al. and Jansen et al. reported that when reagent is supplemented with P5P the ratio of preformed holoenzyme to apoenzyme differs among specimens (12, 26). Gamma glutamyl transferase (GGT), were all groups using substrate of γ-glutamyl-3carboxy-4nitroanilide > 4mmol/L have good precision and bias; however, the Siemens Dimension Vista group that uses a different concentration of substrate (< 4 mmol/L) produces unacceptable high results (Figure 11). Lastly, creatinine shows good inter-laboratory CV. However, only enzymatic methods have good bias at the entire concentration range studied, whereas most of the Jaffe based measurements produce unacceptable high results at low-normal concentrations (≤ 50 mmol/L) and some of them show inconsistent bias along the two surveys evaluated (Figure 14). Part of the 2015 results had been previously published and is in accordance with the 2016 survey, as well as with Jassam et al. that observed as Abbott compensated and Jaffe methods were most affected by glucose interferences, resulting in either under- or over- estimation of GFR and may also lead to errors in the classification of chronically kidney disease (20, 27, 28). Likewise, data reported by Panteghini showed an 18 μmol/L positive bias derived from the Jaffe-based method on a Beckman AU 2710 instrument (29). These results are especially relevant for paediatric population. Our results evidences that for consecutive years the Jaffe method produces false high results at low-normal concentration values, in all the instruments used in our country. Consequently, creatinine is not standardized in our setting and considering the clinical implications associated, Jaffe method should be abandoned. Dialogue with providers is of upmost necessity in several cases. The main negative issue is the lack of adequate information about the calibration traceability of the measurement procedure; this circumstance was observed to affect the 55% of participating laboratories in 2015. In order to address and minimize this issue, the SEQCML- Analytical Quality Commission promoted regular and specific meetings with providers and holding educational communications and workshops in national laboratory congresses (5, 6). This effort seems to have been worthy, observing a decrease in the percentage of wrong-coding traceability from 55% to 20% in 2016.\nSome in vitro diagnostic medical device providers reported their methods for ALT and AST as “IFCC traceable” when no P5P was added; this created a high incidence of wrong codifications by laboratory workers that was solved and recorded by SEQCML after informing of this circumstance to providers and users.\nLactate dehydrogenase measurements gave good inter-laboratory CV in the 2015 survey but not in 2016; the reason for this remains unknown and should be discussed with providers. Bias showed an interesting improvement, resulting in satisfactory results for all users of the lactate to pyruvate based measurement in the 2016 survey (Figure 12).\nOur findings for bilirubin, chloride, glucose, magnesium (irregular inter-laboratory CV and bias), as well as total protein and urate (good inter-laboratory imprecision, but irregular bias) led us to the opinion that a dialogue with providers would be necessary for improving standardization in our country.\nA limitation of this study would be the reduced number of participants in certain groups, due to the fact that this program is still poorly known by many Spanish laboratories. Consequently, one symposium, various workshops in the national congress and specific meetings were organized in 2017, a book has been written in 2018 and other educational activities are planned for the future to overcome this limitation.\nAnother drawback might be that there is a single exercise per year; this could be not enough to guarantee the trueness for the rest of the year. Because the economic difficulty to make more distributions of these controls materials along the year, laboratories in Spain could use our regular EQA schemes (stabilized materials, peer group evaluation, one sample per month) to verify if their analytical performance is maintained along the year.",
"The two years of category 1 EQA program experience in our country have manifested a lack of standardization of the 17 more frequent general biochemistry tests used in our laboratories. The application of this kind of EQA program allows estimating measurement procedure-traceability-instrument bias in a way that can be expanded to what happens with real patient samples. The impact of the information obtained by category 1 EQA program on the lack of standardization is: to recommend abandoning methods such as for ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, pyruvate-lactate for LD, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium."
] | [
"intro",
"materials|methods",
"results",
"discussion",
"conclusions"
] | [
"standardization",
"external quality assessment",
"traceability",
"bias"
] | Introduction:
The main objective of clinical laboratory is to provide clear, reliable and useful information for clinical decision-making. Current healthcare systems imply performing laboratory tests in different locations, so standardization among laboratories become one of the cornerstones of the quality patient‘s care. Standardization can be defined as the ability to obtain interchangeable results (within certain analytical quality uncertainty) in order to achieve the same medical decision, regardless of the analytical procedure (method, traceability and instrument), measurement units and reference intervals.
The standardization should be based on six basic pillars, which include in vitro diagnostic companies, reference materials, reference methods, reference laboratories, medical laboratories and external quality assessment (EQA) organizations (1). Recently, Greaves noted that EQA is not just a pillar but the central support for on-going harmonization (2). Discordance in results between laboratories and methods should become a practice no longer accepted.
It is widely accepted that the best strategy to organize an EQA scheme is to use fresh frozen commutable control samples with values assigned by reference laboratories using reference methods, which can be found on www.harmonization.net (3, 4).
Spanish Society of Laboratory Medicine (SEQCML) is a non-profit scientific organization that has been providing EQA schemes in Spain since 1980 by using stabilized control materials. Since 2013 a category 1 program has been organized for basic biochemistry analytes. According to Miller et al. this kind of program distributes commutable control materials with reference-measurement procedure (RMP) assigned values and replicate samples in surveys are tested (3). Accuracy of individual laboratories is assessed by comparison with the RMP, while reproducibility is checked both intra- and inter-laboratory, and standardization is assessed by comparison of measurement procedure calibration traceability with RMP. Two initial surveys were performed in 2013 and 2014, as preliminary experiences and regular annual surveys have been organized since 2015. For a proper assessment of bias, having adequate information of measurement’s traceability is therefore a crucial point (5, 6).
Another important aspect to consider is the analytical performance specification (APS) or acceptability limits selected for the evaluation of the derived results. When APS are based on biological variation (BV), it is highly recommended to use the gradual classification of APS according to its strictness: optimal, desirable and minimal (7). It should be noted that the APS grade could be selected according to the limitations of the current state of the art, being defined as the performance achieved by about 80% of laboratories. According to this criterion, in this study the minimal BV-based APS grade was selected for electrolytes evaluation, while desirable BV APS were chosen for enzymes and substrates.
In this regard, a performance worse than the minimum APS should alert the laboratory that its results could be at risk and clinical decision-making might be detrimentally affected. Likewise, a performance reaching the minimal grade suggest that further improvement may be beneficial for patients (8, 9).
The aim of this work is to evaluate the results obtained from two years category 1 EQA program, 2015 and 2016 surveys, performed in our country and to assess the impact of applying this kind of EQA program over the analytical standardization. Evaluation is based on the inter-laboratory imprecision and the bias of the peer group means compared with the reference method values.
Materials and methods:
Commutable control materials were purchased from MCA laboratory (Queen Beatrix Hospital, Winterswijk, The Netherlands) by means of the Stichting Kwaliteitsbewaking Medische Laboratorium Diagnostiek (SKML). According to Cobbaert et al. controls had been prepared from fresh anonymized left-over sera of routine laboratory with exclusion of lipemic, icteric, positive hepatitis B surface antigen (HBsAG), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) samples, and stored frozen at – 84 ºC in aliquots. Pathological concentration ranges were created by adequately mixing pools and by spiking with minerals, recombinant human enzymes and human albumin (10). Commutability had been verified by SKML, as explained by Baadenhuijsen et al. and Jansen et al. (11, 12). Throughout the years commutability has been monitored by including a native, single donation spy-sample (10, 12).
Six vials of fresh frozen human serum pools at different concentrations were distributed once per year in a single express shipment at – 80 ºC and delivered within 24 hours to laboratories all over Spain. Different lots at different concentrations were provided for each of the two surveys. Participant laboratories were requested to maintain samples at – 20 ºC until analysis, which had to be performed within the following 14 days. Each vial had to be analysed in duplicate, one vial per day, for 6 consecutive days whenever possible. Results were registered on the SEQCML-EQA website, in order to be either individually and globally evaluated.
A preliminary 2013 survey was carried out in 19 laboratories and was addressed to ascertain whether the logistics of managing a non-stabilized set of control materials was operative in our country. No incidents were observed with temperature maintenance during the time between deliveries of control materials from the provider to the laboratory analysis.
Another point of interest of this preliminary survey was to explore whether laboratories could adequately inform about their analytical traceability to standards. Important difficulties were perceived that impelled holding a meeting between EQAs organization and providers, claiming for clear and complete information on calibrators’ traceability.
In 2014 first survey was performed, as part of a pilot European study (INPUTs) (Italy, The Netherlands, Portugal, Spain and The United Kingdom), with a total of 20 laboratories participants and whose results has been already published (12, 13). Only about 45% of participants were able to correctly inform about its traceability, so results are not shown in this study. This survey was then considered as a pilot to identify the problems that could impact on the EQA participation and further interpretation of results. For both surveys as well as for those performed in 2015 and 2016, same sample management protocol was applied.
The 2015 and 2016 surveys were exclusively run in Spain and included 17 analytes. The number of registered participants was 93 and 105, respectively. The target values of distributed control materials were assigned by the reference methods and laboratories (Table 1).
Results were categorized by measurement procedure, traceability and instrument. The description of standard materials used by participants for calibration traceability is shown in Table 2. Participant laboratories using the same combination of these three elements were considered as a peer group. The peer groups and the number of laboratories included for each analyte are shown in Figures 1-17.
Calcium. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Numbers in brackets mean the number of participant laboratories. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. NM-BAPTA: calcium specific amino-polycarboxylic acid.
Chloride. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. ISE - ion selective electrode. Numbers in brackets indicate the laboratories participating for each instrument.
Magnesium. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value . Lim Bias (m): acceptability limit for bias based on BV, minimum grade. Xil - Xilidil blue. Numbers in brackets indicate the laboratories participating for each instrument.
Potassium. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. ISE - ion selective electrode. Numbers in brackets indicate the laboratories participating for each instrument.
Sodium. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (m): acceptability limit for bias based on BV, minimum grade. ISE - ion selective electrode. Numbers in brackets indicate the laboratories participating for each instrument.
Alkaline phosphatase. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. AMP - 2-amino-2-methyl-1-propanol. Numbers in brackets indicate the laboratories participating for each instrument.
Amylase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. G3 - malto trioside. G7 - malto-heptaoside. Numbers in brackets indicate the laboratories participating for each instrument.
ALT. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. Numbers in brackets indicate the laboratories participating for each instrument.
AST. Percentage deviation (Dev%) of peer group means from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. P5P -pyridoxal-5-phosphate. Numbers in brackets indicate the laboratories participating for each instrument.
Creatine kinase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. NAC - N-acetyl-cysteine. Numbers in brackets indicate the laboratories participating for each instrument.
Gamma glutamyl transferase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. All groups use substrate: γ glutamyl-3carboxy-4nitroanilide > 4mmol/L. The exception is: Siemens Dimension, Vista that uses substrate < 4mmol/L. Numbers in brackets indicate the laboratories participating for each instrument.
Lactate dehydrogenase. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. NMG - N-methyl-D-glucamine. DEA - diethanolamine. TRIS -hydroxymethyl-aminomethane. Numbers in brackets indicate the laboratories participating for each instrument.
Bilirubin. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. DPD - 3,5-dicholorophenyl-diazonium- tetrafluoroborate. Numbers in brackets indicate the laboratories participating for each instrument.
Creatinine. Percentage deviation (Dev%) of peer group mean from the reference value. Methods in figure appearing according the following order: enzymatic, compensated and non-compensated. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. Numbers in brackets indicate the laboratories participating for each instrument.
Glucose. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. GOD - glucose oxidase. HK - hexokinase. Numbers in brackets indicate the laboratories participating for each instrument.
Total protein. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. B - biuret. Numbers in brackets indicate the laboratories participating for each instrument.
Urate. Percentage deviation (Dev%) of peer group mean from the reference value. X axis shows reference values of the six control materials. Y axis shows percent deviation of peer group mean versus the reference value. Lim Bias (d): acceptability limit for bias based on BV, desirable grade. POD – peroxidase. Numbers in brackets indicate the laboratories participating for each instrument.
Compared to 2015, a new instrument was incorporated in 2016 survey (Bio-systems BA 400), with only 6 participating laboratories. The overall evaluation of the 2015 survey was published on the SEQCML website and was presented at the 2016 EQALM annual meeting (13, 15). Only groups formed by 5 or more final laboratories were considered in this study.
Inter-laboratory imprecision was calculated by averaging the coefficient of variation (CV) obtained from the six controls distributed on the 2016 and 2015 surveys and compared with the best (Dutch) inter-laboratory CV derived from the 2014 pilot study, which used similar six commutable control materials (16).
Bias was calculated by the percent difference between the peer group mean (same measurement procedure, traceability and instrument) and the reference value. The analytical performance specification to apply for bias evaluation was based on the BV data collected on the online 2014 database, which had been elaborated as detailed by Ricós et al., applying the minimum level of requirement for electrolytes and the desirable level for substrates and enzymes (17-19).
The results of this study were examined with the particular focus on the most common analytical procedures used in Spain and its repercussion on non-comparable results, detected throughout participation on level 1 EQA schemes.
Standardization is defined by the attainment of inter-laboratory imprecision within the predefined APS and peer group bias (% mean deviation to the reference value) below the allowed bias derived from BV.
Results:
All results exceeding the mean ± 3 standard deviation of each group were rejected as outliers. The number of rejected participant laboratories was 5 for the 2015 survey and 10 for the 2016 survey. Moreover, 30 results for lactate dehydrogenase (LD) which were 100% higher than the others due to the different substrate (pyruvate instead of lactate) were also excluded from the study. Results for bias are presented in Figures 1-17. Results for the inter-laboratory imprecision of each peer group for electrolytes, enzymes and substrates are presented in Tables 3-5 and compared with the APS for inter-laboratory imprecision (APSIL) from the pilot 2014 survey (16). An overview of the standardization achieved in our setting, according to the bias and the imprecision calculated for instruments, is presented in Table 6.
Discussion:
The percentage of laboratories excluded was higher in 2016 than in 2015 due to better knowledge of the traceability-instrument, so groups were more specific in 2016. This cannot be considered a disadvantage. The results in this study are discussed form the light of their impact on the aims proposed. These are: positive, negative and needed to be dialogued with providers.
Main positive impacts, which imply an adequate standardization not needing for further improvements, apply to potassium and creatine kinase (CK). Potassium shows inter-laboratory imprecision and bias (Figure 4) within the allowable limits for almost all peer groups. For the remaining electrolytes good inter-laboratory imprecision can also be seen, well in agreement with the 2014 survey (performed in collaboration with other European countries) where all participant laboratories and manufacturers fulfilled the APS for total analytical error at the minimum performance level (20). Creatine kinase show good inter-laboratory imprecision and bias (Figure 10), except for the new group enrolled in the 2016 survey (BA400). So it may be expected a well standardized measurements soon. Negative impacts may be due to several reasons. The aqueous matrix of SRM 915 and 918 used for calcium and sodium, respectively (Figures 1 and 5), produces low results. Lack of commutability of calibration traceability materials was described to be a crucial factor to assure standardization in medical laboratories by Panteghini and Ambruster (21, 22).
Instrument dependent problems can be seen in this study for alkaline phosphatase (ALP) with low results for Roche users (Figure 6), whereas all participants use same method and traceability; this event causes an important lack of standardization in our country because it is the greatest group. Same results had been seen by Braga et al., and Aloisio et al. who observed discrepancies among Abbott Architect users related to an “experimental” calibration factor provided by the manufacturer (23, 24). Non-standardized ALP results could have a great impact in some clinical scenarios such as hypophosphatemia diagnosis, so an improvement in the results’ traceability becomes a crucial objective (25). Method dependent troubles are seen in four cases.
Firstly, amylase, were all groups using malto-heptaoside (G7) substrate, as well as the malto-trioside (G3) of Abbott Architect show harmonized results. The remaining G3 groups have unacceptable negative bias (Figure 7). This lack of standardization affects one third of the participants of this study, thus producing a considerable impact on the healthcare in our country. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) testing show unacceptable inter-laboratory imprecision and bias (low results) (Figures 8 and 9) for laboratories that did not add pyridoxal-5-phosphate (P5P) in its measurement procedure. Infusino et al. and Jansen et al. reported that when reagent is supplemented with P5P the ratio of preformed holoenzyme to apoenzyme differs among specimens (12, 26). Gamma glutamyl transferase (GGT), were all groups using substrate of γ-glutamyl-3carboxy-4nitroanilide > 4mmol/L have good precision and bias; however, the Siemens Dimension Vista group that uses a different concentration of substrate (< 4 mmol/L) produces unacceptable high results (Figure 11). Lastly, creatinine shows good inter-laboratory CV. However, only enzymatic methods have good bias at the entire concentration range studied, whereas most of the Jaffe based measurements produce unacceptable high results at low-normal concentrations (≤ 50 mmol/L) and some of them show inconsistent bias along the two surveys evaluated (Figure 14). Part of the 2015 results had been previously published and is in accordance with the 2016 survey, as well as with Jassam et al. that observed as Abbott compensated and Jaffe methods were most affected by glucose interferences, resulting in either under- or over- estimation of GFR and may also lead to errors in the classification of chronically kidney disease (20, 27, 28). Likewise, data reported by Panteghini showed an 18 μmol/L positive bias derived from the Jaffe-based method on a Beckman AU 2710 instrument (29). These results are especially relevant for paediatric population. Our results evidences that for consecutive years the Jaffe method produces false high results at low-normal concentration values, in all the instruments used in our country. Consequently, creatinine is not standardized in our setting and considering the clinical implications associated, Jaffe method should be abandoned. Dialogue with providers is of upmost necessity in several cases. The main negative issue is the lack of adequate information about the calibration traceability of the measurement procedure; this circumstance was observed to affect the 55% of participating laboratories in 2015. In order to address and minimize this issue, the SEQCML- Analytical Quality Commission promoted regular and specific meetings with providers and holding educational communications and workshops in national laboratory congresses (5, 6). This effort seems to have been worthy, observing a decrease in the percentage of wrong-coding traceability from 55% to 20% in 2016.
Some in vitro diagnostic medical device providers reported their methods for ALT and AST as “IFCC traceable” when no P5P was added; this created a high incidence of wrong codifications by laboratory workers that was solved and recorded by SEQCML after informing of this circumstance to providers and users.
Lactate dehydrogenase measurements gave good inter-laboratory CV in the 2015 survey but not in 2016; the reason for this remains unknown and should be discussed with providers. Bias showed an interesting improvement, resulting in satisfactory results for all users of the lactate to pyruvate based measurement in the 2016 survey (Figure 12).
Our findings for bilirubin, chloride, glucose, magnesium (irregular inter-laboratory CV and bias), as well as total protein and urate (good inter-laboratory imprecision, but irregular bias) led us to the opinion that a dialogue with providers would be necessary for improving standardization in our country.
A limitation of this study would be the reduced number of participants in certain groups, due to the fact that this program is still poorly known by many Spanish laboratories. Consequently, one symposium, various workshops in the national congress and specific meetings were organized in 2017, a book has been written in 2018 and other educational activities are planned for the future to overcome this limitation.
Another drawback might be that there is a single exercise per year; this could be not enough to guarantee the trueness for the rest of the year. Because the economic difficulty to make more distributions of these controls materials along the year, laboratories in Spain could use our regular EQA schemes (stabilized materials, peer group evaluation, one sample per month) to verify if their analytical performance is maintained along the year.
Conclusions:
The two years of category 1 EQA program experience in our country have manifested a lack of standardization of the 17 more frequent general biochemistry tests used in our laboratories. The application of this kind of EQA program allows estimating measurement procedure-traceability-instrument bias in a way that can be expanded to what happens with real patient samples. The impact of the information obtained by category 1 EQA program on the lack of standardization is: to recommend abandoning methods such as for ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, pyruvate-lactate for LD, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium.
| Background:
Standardization is the ability to obtain interchangeable results leading to same medical interpretation. External quality assessment (EQA) is the main support of the on-going harmonization initiatives. Aim of study was to evaluate results obtained from two years category 1 EQA program experience in Spain and determine the impact of applying this type of EQA program on the analytical standardization.
Methods:
According to the analytical method, traceability and instrument different groups were established which results were evaluated by calculating mean, coefficient of variation and percent of deviation to the reference value. Analytical performance specifications used to the results' evaluation were derived from biological variation for bias and from the inter-laboratory coefficients of variation found in a previous pilot study.
Results:
Only creatinine measured by enzymatic methods gave excellent results, although few laboratories used this method. Creatine kinase and GGT gave good precision and bias in all, but one instrument studied. For the remaining analytes (ALT, ALP, AST, bilirubin, calcium, chloride, glucose, magnesium, potassium, sodium, total protein and urate) some improvement is still necessary to achieve satisfactory standardization in our setting.
Conclusions:
The two years of category 1 EQA program experience in Spain have manifested a lack of standardization of 17 most frequent biochemistry tests used in our laboratories. The impact of the information obtained on the lack of standardization is to recommend abandoning methods such as ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium. | Introduction:
The main objective of clinical laboratory is to provide clear, reliable and useful information for clinical decision-making. Current healthcare systems imply performing laboratory tests in different locations, so standardization among laboratories become one of the cornerstones of the quality patient‘s care. Standardization can be defined as the ability to obtain interchangeable results (within certain analytical quality uncertainty) in order to achieve the same medical decision, regardless of the analytical procedure (method, traceability and instrument), measurement units and reference intervals.
The standardization should be based on six basic pillars, which include in vitro diagnostic companies, reference materials, reference methods, reference laboratories, medical laboratories and external quality assessment (EQA) organizations (1). Recently, Greaves noted that EQA is not just a pillar but the central support for on-going harmonization (2). Discordance in results between laboratories and methods should become a practice no longer accepted.
It is widely accepted that the best strategy to organize an EQA scheme is to use fresh frozen commutable control samples with values assigned by reference laboratories using reference methods, which can be found on www.harmonization.net (3, 4).
Spanish Society of Laboratory Medicine (SEQCML) is a non-profit scientific organization that has been providing EQA schemes in Spain since 1980 by using stabilized control materials. Since 2013 a category 1 program has been organized for basic biochemistry analytes. According to Miller et al. this kind of program distributes commutable control materials with reference-measurement procedure (RMP) assigned values and replicate samples in surveys are tested (3). Accuracy of individual laboratories is assessed by comparison with the RMP, while reproducibility is checked both intra- and inter-laboratory, and standardization is assessed by comparison of measurement procedure calibration traceability with RMP. Two initial surveys were performed in 2013 and 2014, as preliminary experiences and regular annual surveys have been organized since 2015. For a proper assessment of bias, having adequate information of measurement’s traceability is therefore a crucial point (5, 6).
Another important aspect to consider is the analytical performance specification (APS) or acceptability limits selected for the evaluation of the derived results. When APS are based on biological variation (BV), it is highly recommended to use the gradual classification of APS according to its strictness: optimal, desirable and minimal (7). It should be noted that the APS grade could be selected according to the limitations of the current state of the art, being defined as the performance achieved by about 80% of laboratories. According to this criterion, in this study the minimal BV-based APS grade was selected for electrolytes evaluation, while desirable BV APS were chosen for enzymes and substrates.
In this regard, a performance worse than the minimum APS should alert the laboratory that its results could be at risk and clinical decision-making might be detrimentally affected. Likewise, a performance reaching the minimal grade suggest that further improvement may be beneficial for patients (8, 9).
The aim of this work is to evaluate the results obtained from two years category 1 EQA program, 2015 and 2016 surveys, performed in our country and to assess the impact of applying this kind of EQA program over the analytical standardization. Evaluation is based on the inter-laboratory imprecision and the bias of the peer group means compared with the reference method values.
Conclusions:
The two years of category 1 EQA program experience in our country have manifested a lack of standardization of the 17 more frequent general biochemistry tests used in our laboratories. The application of this kind of EQA program allows estimating measurement procedure-traceability-instrument bias in a way that can be expanded to what happens with real patient samples. The impact of the information obtained by category 1 EQA program on the lack of standardization is: to recommend abandoning methods such as for ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, pyruvate-lactate for LD, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium. | Background:
Standardization is the ability to obtain interchangeable results leading to same medical interpretation. External quality assessment (EQA) is the main support of the on-going harmonization initiatives. Aim of study was to evaluate results obtained from two years category 1 EQA program experience in Spain and determine the impact of applying this type of EQA program on the analytical standardization.
Methods:
According to the analytical method, traceability and instrument different groups were established which results were evaluated by calculating mean, coefficient of variation and percent of deviation to the reference value. Analytical performance specifications used to the results' evaluation were derived from biological variation for bias and from the inter-laboratory coefficients of variation found in a previous pilot study.
Results:
Only creatinine measured by enzymatic methods gave excellent results, although few laboratories used this method. Creatine kinase and GGT gave good precision and bias in all, but one instrument studied. For the remaining analytes (ALT, ALP, AST, bilirubin, calcium, chloride, glucose, magnesium, potassium, sodium, total protein and urate) some improvement is still necessary to achieve satisfactory standardization in our setting.
Conclusions:
The two years of category 1 EQA program experience in Spain have manifested a lack of standardization of 17 most frequent biochemistry tests used in our laboratories. The impact of the information obtained on the lack of standardization is to recommend abandoning methods such as ALT, AST without exogenous pyridoxal phosphate, Jaffe method for creatinine, and do not use non-commutable calibrators, such as aqueous solutions for calcium and sodium. | 4,478 | 300 | [] | 5 | [
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] | null | [CONTENT] standardization | external quality assessment | traceability | bias [SUMMARY] | null | [CONTENT] standardization | external quality assessment | traceability | bias [SUMMARY] | [CONTENT] standardization | external quality assessment | traceability | bias [SUMMARY] | [CONTENT] standardization | external quality assessment | traceability | bias [SUMMARY] | [CONTENT] standardization | external quality assessment | traceability | bias [SUMMARY] | [CONTENT] Clinical Laboratory Techniques | Creatine Kinase | Creatinine | Humans | Quality Assurance, Health Care | Spain | gamma-Glutamyltransferase [SUMMARY] | null | [CONTENT] Clinical Laboratory Techniques | Creatine Kinase | Creatinine | Humans | Quality Assurance, Health Care | Spain | gamma-Glutamyltransferase [SUMMARY] | [CONTENT] Clinical Laboratory Techniques | Creatine Kinase | Creatinine | Humans | Quality Assurance, Health Care | Spain | gamma-Glutamyltransferase [SUMMARY] | [CONTENT] Clinical Laboratory Techniques | Creatine Kinase | Creatinine | Humans | Quality Assurance, Health Care | Spain | gamma-Glutamyltransferase [SUMMARY] | [CONTENT] Clinical Laboratory Techniques | Creatine Kinase | Creatinine | Humans | Quality Assurance, Health Care | Spain | gamma-Glutamyltransferase [SUMMARY] | [CONTENT] patient care standardization | analytical standardization | assure standardization medical | standardization laboratories cornerstones | laboratory standardization assessed [SUMMARY] | null | [CONTENT] patient care standardization | analytical standardization | assure standardization medical | standardization laboratories cornerstones | laboratory standardization assessed [SUMMARY] | [CONTENT] patient care standardization | analytical standardization | assure standardization medical | standardization laboratories cornerstones | laboratory standardization assessed [SUMMARY] | [CONTENT] patient care standardization | analytical standardization | assure standardization medical | standardization laboratories cornerstones | laboratory standardization assessed [SUMMARY] | [CONTENT] patient care standardization | analytical standardization | assure standardization medical | standardization laboratories cornerstones | laboratory standardization assessed [SUMMARY] | [CONTENT] reference | bias | group | laboratories | peer | peer group | shows | value | deviation | reference value [SUMMARY] | null | [CONTENT] reference | bias | group | laboratories | peer | peer group | shows | value | deviation | reference value [SUMMARY] | [CONTENT] reference | bias | group | laboratories | peer | peer group | shows | value | deviation | reference value [SUMMARY] | [CONTENT] reference | bias | group | laboratories | peer | peer group | shows | value | deviation | reference value [SUMMARY] | [CONTENT] reference | bias | group | laboratories | peer | peer group | shows | value | deviation | reference value [SUMMARY] | [CONTENT] reference | aps | laboratory | eqa | laboratories | minimal | selected | decision | rmp | results [SUMMARY] | null | [CONTENT] presented | results | survey | rejected | imprecision | inter | laboratory | inter laboratory imprecision | inter laboratory | lactate [SUMMARY] | [CONTENT] eqa program | program | eqa | category eqa program | lack standardization | lack | category eqa | category | frequent | aqueous solutions [SUMMARY] | [CONTENT] results | reference | bias | laboratory | laboratories | group | eqa | program | survey | eqa program [SUMMARY] | [CONTENT] results | reference | bias | laboratory | laboratories | group | eqa | program | survey | eqa program [SUMMARY] | [CONTENT] ||| EQA ||| two years | 1 | EQA | Spain | EQA [SUMMARY] | null | [CONTENT] ||| Creatine | one ||| ALT | ALP | AST | glucose, magnesium [SUMMARY] | [CONTENT] The two years | 1 | EQA | Spain | 17 ||| ALT | Jaffe [SUMMARY] | [CONTENT] ||| EQA ||| two years | 1 | EQA | Spain | EQA ||| ||| ||| ||| Creatine | one ||| ALT | ALP | AST | glucose, magnesium ||| The two years | 1 | EQA | Spain | 17 ||| ALT | Jaffe [SUMMARY] | [CONTENT] ||| EQA ||| two years | 1 | EQA | Spain | EQA ||| ||| ||| ||| Creatine | one ||| ALT | ALP | AST | glucose, magnesium ||| The two years | 1 | EQA | Spain | 17 ||| ALT | Jaffe [SUMMARY] |
|||||
Economic burden of chronic bronchitis in the United States: a retrospective case-control study. | 21311695 | Chronic bronchitis (CB) is often misdiagnosed or diagnosed at a later stage of chronic obstructive pulmonary disease (COPD). We examined how this later diagnosis may impact health care costs and utilization during the 12 months prior to and 24 months post initial CB diagnosis. | BACKGROUND | This retrospective case-control analysis used claims data from a large US database from July 1, 2003 through June 30, 2007. Patients with CB aged 40 years and older were propensity matched (N = 11,674) to patients without evidence of COPD or asthma by demographics, CB diagnosis quarter/year, and comorbidities. Group differences were assessed using Student's t-test and Pearson chi-square test statistics. | METHODS | Six months prediagnosis, CB patients had higher frequencies of any hospitalization (9.6%, 6.7%; P < 0.05), emergency department/urgent care visits (13.3%, 6.7%; P < 0.05), and prescriptions (97.3%, 94.1%; P < 0.05). Six months postdiagnosis, CB patients had 5.6 times more hospitalizations (P < 0.05) and 3.1 times more emergency department/urgent care visits (P < 0.05) compared with controls. Mean total costs (US$) for CB patients 12 months prediagnosis were significantly higher than controls (months 12-7: $4212, $3826; P < 0.05; months 6-1: $5289, $4285; P < 0.05). CB patients had higher mean total costs ($8919; P < 0.05) 6 months postdiagnosis. Costs remained $2429 higher for CB patients 19-24 months postdiagnosis (P < 0.05). | RESULTS | Health care costs and utilization among CB patients are increased both prior to diagnosis and during the 2 years postdiagnosis. This study suggests that not accurately diagnosing CB early has a substantial impact on health care costs, and that the economic burden for CB patients remains elevated even after adjustment for comorbidities associated with COPD. | CONCLUSION | [
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] | 3034282 | Introduction | Chronic bronchitis (CB), an inflammatory condition that affects the central bronchi, is one of two main lung diseases by which patients with chronic obstructive pulmonary disease (COPD) are characterized. Excessive mucus secretion differentiates it from the second, emphysema, which is characterized by permanent enlargement of lung airways and destruction of the walls of the alveoli, making breathing difficult.1 Other symptoms of CB related to lung inflammation and heavy mucus production include cough, production of sputum, and dyspnea.1 In 2008, more than 9.8 million Americans reported having a CB diagnosis.2 While many patients with COPD may suffer from both conditions, the courses of the diseases and response to treatment are frequently different; separate studies of outcomes between the two could assist in optimizing care for these patients.
Estimated annual expenditures for CB treatment total US$11.7 billion, with hospitalizations accounting for US$6 billion of the total costs.3 There are limited studies that examine the costs associated with CB, but research has shown that hospitalizations for acute CB exacerbations accounted for 46%–90% of the costs associated with treatment.4 The diminished quality of life experienced by COPD patients is well documented and suggests that earlier detection, treatment, and reduction of exacerbations may have a substantial economic and psychosocial impact on patients.5–13
The chronic cough and sputum production associated with CB often predates the development of airflow limitation.14 The GOLD (Global Initiative for Chronic Obstructive Lung Disease) guidelines for diagnosis and treatment of COPD recommend early identification of patients with symptoms of CB in order to begin intervention at the earliest stage of COPD.14 Unfortunately, CB symptoms may be dismissed by patients as “smoker’s cough”, or physicians may misdiagnose CB as acute bronchitis or asthma.15,16 Once CB is diagnosed and treatment has commenced, many patients have progressed into more severe COPD stages, resulting in higher consumption of health care resources and increased complexity in clinical management.
Prior research demonstrated higher utilization rates of health care resources for COPD patients when compared with a control population during the 12 months preceding initial COPD diagnosis.17 Utilization trends indicated an increasing use of resources in terms of medical services and pharmacy prescriptions up to the COPD diagnosis, particularly in the final month before diagnosis.17 In addition, total costs for COPD patients have been shown to be higher up to 2 years prior to diagnosis.18 To determine if these trends were apparent for the subset of COPD patients diagnosed with CB, we examined health care utilization and costs for 12 months prior to CB diagnosis and 24 months post diagnosis. | Analysis | Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.
Means and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC). | Results | There were initially 182,169 patients who had a COPD diagnosis at any time; after selection criteria were applied, 11,937 patients met criteria for the CB cohort. A 10% random sample of possible control subjects with 3 or more years of continuous enrollment was selected, of whom 282,078 met the control cohort selection criteria. These patients were randomly reduced to a pool of 65,654 control patients. Propensity score matching was used to match CB patients to control patients on a one-to-one basis, resulting in two cohorts of 11,674 each.
Prior to matching, the two cohorts exhibited significant differences in all matching characteristics with the exception of two of the index date quarter variables (Table 1). After matching, the CB patient cohort and control patient cohort exhibited a significant difference only in the percentage of each population that was male (CB = 41.4%, control = 43.0%; P = 0.0142). The cohorts were similar with respect to comorbidities, geographic region, mean age, and quarter in which the index date occurred.
After matching, the mean age of patients in the CB cohort was 61.5 years; for the controls, 61.8 years. Patients were predominantly from the Northeast (37%) and female (57.8%). The most prevalent comorbidities in each cohort were: uncomplicated hypertension (48.5%), diabetes (19.3%), ischemic heart disease (19.3%), arrhythmia (10.5%), and hypothyroidism (10.5%), shown in Table 1. Among the CB cohort, 22.8% had a history of asthma in addition to their CB diagnosis.
There was a decrease in the number of patients with continuous enrollment beginning 13 months after the index date. Approximately 23% of the CB patients were lost to follow-up during months 13–18, and another 25% in the 19–24 month period after the index date. For the final 6-month observation period (months 19–24), there were 6128 CB patients and 6203 control patients.
As shown in Table 2 and Figure 1, from 1 year and up to 6 months prior to the CB diagnosis there were no differences between the cohorts with respect to the percentage of patients having any IP visit; however, the CB cohort had a higher percentage having any ED/UC visit (9.5%, 6.4%; P < 0.05), and any pharmacy use (93.4%, 91.5%; P < 0.05), while having a lower percentage with any OP visit (90.5%, 92.9%; P < 0.05). In the next 6 months, which was the 6 months immediately prior to diagnosis, patients in the CB cohort, compared with the control cohort, had a higher percentage having any IP visit (9.6%, 6.7%; P < 0.05), and again, higher percentages for any ED/UC visit (13.3%, 6.7%; P < 0.05), and any pharmacy prescription (97.3%, 94.1%; P < 0.05). Percentages of patients having any OP visit were identical for the two cohorts during the 6 months immediately prior to CB diagnosis.
During the first 6 months postindex (Figure 1), patients in the CB cohort had 5.6 times more IP visits (30.4%, 5.4%; P < 0.05) and 3.1 times more ED/UC visits (20.7%, 6.6%; P < 0.05). To a lesser degree, OP visits and pharmacy use were also higher for the CB cohort. Utilization rates remained higher for the CB cohort during all four of the 6-month periods postindex, but beginning in the second 6-month postindex period, utilization rates in the CB cohort declined from the highs reached in the first postindex period and remained fairly stable for the remaining observation periods. The control cohort’s utilization rates declined slightly in each of the four postindex periods. Mean number of events were higher for the CB cohort in all time periods, pre- and postindex, compared with the control cohort except for mean OP visits in the 6-month period immediately preceding the CB diagnosis (Table 3, Figure 2).
Mean total costs (US$) for the CB cohort during both preindex periods were significantly higher than for the control cohort (months 12–7: $4212, $3826; P < 0.05; months 6–1: $5289, $4285; P < 0.05). Component costs were also higher, with the exception that there was no difference in medical services costs during the initial 6 months prior to diagnosis (Table 4). CB patients had higher mean total costs ($8919; P < 0.05) in the 6 months postdiagnosis. Costs for both groups then decreased (Figure 3) but for CB patients remained $2429 higher than for control patients 19–24 months postdiagnosis (P < 0.05). Median costs were all significantly higher for the CB cohort (Table 5). | Conclusion | To our knowledge, this is the first study to use a large enough population sample to facilitate propensity score matching based on comorbidities. Prior studies have not controlled for comorbidity by matching,30 creating potential for residual confounding and other biases in case-control comparisons. Our method allowed for a cleaner assessment of the true impact of CB. The disease burden is greatest during the 6-month period following CB diagnosis, but is also high in periods preceding the diagnosis. In patients diagnosed with chronic bronchitis, compared with patients without evidence of COPD or asthma, health care utilization and costs continue to remain elevated 2 years after diagnosis. This study suggests that not accurately diagnosing CB early may have a substantial impact on health care costs, resulting in a higher economic burden for CB patients than would otherwise be present. | [
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" Study design and data source This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.\nThis case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.\n Sample CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.\nPatients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.\nCB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.\nPatients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.\n Measures Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.\nUtilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.\n Analysis Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.\nMeans and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).\nDescriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.\nMeans and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).",
"This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.",
"CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.\nPatients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.",
"Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.",
"To our knowledge, this is the first study to use a large enough population sample to facilitate propensity score matching based on comorbidities. Prior studies have not controlled for comorbidity by matching,30 creating potential for residual confounding and other biases in case-control comparisons. Our method allowed for a cleaner assessment of the true impact of CB. The disease burden is greatest during the 6-month period following CB diagnosis, but is also high in periods preceding the diagnosis. In patients diagnosed with chronic bronchitis, compared with patients without evidence of COPD or asthma, health care utilization and costs continue to remain elevated 2 years after diagnosis. This study suggests that not accurately diagnosing CB early may have a substantial impact on health care costs, resulting in a higher economic burden for CB patients than would otherwise be present."
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"Chronic bronchitis (CB), an inflammatory condition that affects the central bronchi, is one of two main lung diseases by which patients with chronic obstructive pulmonary disease (COPD) are characterized. Excessive mucus secretion differentiates it from the second, emphysema, which is characterized by permanent enlargement of lung airways and destruction of the walls of the alveoli, making breathing difficult.1 Other symptoms of CB related to lung inflammation and heavy mucus production include cough, production of sputum, and dyspnea.1 In 2008, more than 9.8 million Americans reported having a CB diagnosis.2 While many patients with COPD may suffer from both conditions, the courses of the diseases and response to treatment are frequently different; separate studies of outcomes between the two could assist in optimizing care for these patients.\nEstimated annual expenditures for CB treatment total US$11.7 billion, with hospitalizations accounting for US$6 billion of the total costs.3 There are limited studies that examine the costs associated with CB, but research has shown that hospitalizations for acute CB exacerbations accounted for 46%–90% of the costs associated with treatment.4 The diminished quality of life experienced by COPD patients is well documented and suggests that earlier detection, treatment, and reduction of exacerbations may have a substantial economic and psychosocial impact on patients.5–13\nThe chronic cough and sputum production associated with CB often predates the development of airflow limitation.14 The GOLD (Global Initiative for Chronic Obstructive Lung Disease) guidelines for diagnosis and treatment of COPD recommend early identification of patients with symptoms of CB in order to begin intervention at the earliest stage of COPD.14 Unfortunately, CB symptoms may be dismissed by patients as “smoker’s cough”, or physicians may misdiagnose CB as acute bronchitis or asthma.15,16 Once CB is diagnosed and treatment has commenced, many patients have progressed into more severe COPD stages, resulting in higher consumption of health care resources and increased complexity in clinical management.\nPrior research demonstrated higher utilization rates of health care resources for COPD patients when compared with a control population during the 12 months preceding initial COPD diagnosis.17 Utilization trends indicated an increasing use of resources in terms of medical services and pharmacy prescriptions up to the COPD diagnosis, particularly in the final month before diagnosis.17 In addition, total costs for COPD patients have been shown to be higher up to 2 years prior to diagnosis.18 To determine if these trends were apparent for the subset of COPD patients diagnosed with CB, we examined health care utilization and costs for 12 months prior to CB diagnosis and 24 months post diagnosis.",
" Study design and data source This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.\nThis case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.\n Sample CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.\nPatients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.\nCB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.\nPatients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.\n Measures Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.\nUtilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.\n Analysis Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.\nMeans and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).\nDescriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.\nMeans and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).",
"This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.",
"CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.\nPatients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.",
"Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.",
"Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.\nMeans and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).",
"There were initially 182,169 patients who had a COPD diagnosis at any time; after selection criteria were applied, 11,937 patients met criteria for the CB cohort. A 10% random sample of possible control subjects with 3 or more years of continuous enrollment was selected, of whom 282,078 met the control cohort selection criteria. These patients were randomly reduced to a pool of 65,654 control patients. Propensity score matching was used to match CB patients to control patients on a one-to-one basis, resulting in two cohorts of 11,674 each.\nPrior to matching, the two cohorts exhibited significant differences in all matching characteristics with the exception of two of the index date quarter variables (Table 1). After matching, the CB patient cohort and control patient cohort exhibited a significant difference only in the percentage of each population that was male (CB = 41.4%, control = 43.0%; P = 0.0142). The cohorts were similar with respect to comorbidities, geographic region, mean age, and quarter in which the index date occurred.\nAfter matching, the mean age of patients in the CB cohort was 61.5 years; for the controls, 61.8 years. Patients were predominantly from the Northeast (37%) and female (57.8%). The most prevalent comorbidities in each cohort were: uncomplicated hypertension (48.5%), diabetes (19.3%), ischemic heart disease (19.3%), arrhythmia (10.5%), and hypothyroidism (10.5%), shown in Table 1. Among the CB cohort, 22.8% had a history of asthma in addition to their CB diagnosis.\nThere was a decrease in the number of patients with continuous enrollment beginning 13 months after the index date. Approximately 23% of the CB patients were lost to follow-up during months 13–18, and another 25% in the 19–24 month period after the index date. For the final 6-month observation period (months 19–24), there were 6128 CB patients and 6203 control patients.\nAs shown in Table 2 and Figure 1, from 1 year and up to 6 months prior to the CB diagnosis there were no differences between the cohorts with respect to the percentage of patients having any IP visit; however, the CB cohort had a higher percentage having any ED/UC visit (9.5%, 6.4%; P < 0.05), and any pharmacy use (93.4%, 91.5%; P < 0.05), while having a lower percentage with any OP visit (90.5%, 92.9%; P < 0.05). In the next 6 months, which was the 6 months immediately prior to diagnosis, patients in the CB cohort, compared with the control cohort, had a higher percentage having any IP visit (9.6%, 6.7%; P < 0.05), and again, higher percentages for any ED/UC visit (13.3%, 6.7%; P < 0.05), and any pharmacy prescription (97.3%, 94.1%; P < 0.05). Percentages of patients having any OP visit were identical for the two cohorts during the 6 months immediately prior to CB diagnosis.\nDuring the first 6 months postindex (Figure 1), patients in the CB cohort had 5.6 times more IP visits (30.4%, 5.4%; P < 0.05) and 3.1 times more ED/UC visits (20.7%, 6.6%; P < 0.05). To a lesser degree, OP visits and pharmacy use were also higher for the CB cohort. Utilization rates remained higher for the CB cohort during all four of the 6-month periods postindex, but beginning in the second 6-month postindex period, utilization rates in the CB cohort declined from the highs reached in the first postindex period and remained fairly stable for the remaining observation periods. The control cohort’s utilization rates declined slightly in each of the four postindex periods. Mean number of events were higher for the CB cohort in all time periods, pre- and postindex, compared with the control cohort except for mean OP visits in the 6-month period immediately preceding the CB diagnosis (Table 3, Figure 2).\nMean total costs (US$) for the CB cohort during both preindex periods were significantly higher than for the control cohort (months 12–7: $4212, $3826; P < 0.05; months 6–1: $5289, $4285; P < 0.05). Component costs were also higher, with the exception that there was no difference in medical services costs during the initial 6 months prior to diagnosis (Table 4). CB patients had higher mean total costs ($8919; P < 0.05) in the 6 months postdiagnosis. Costs for both groups then decreased (Figure 3) but for CB patients remained $2429 higher than for control patients 19–24 months postdiagnosis (P < 0.05). Median costs were all significantly higher for the CB cohort (Table 5).",
"This retrospective observational study demonstrated that total health care utilization and costs for CB patients in the US are significantly greater than for a matched control cohort of patients. This was true for both the 12-month period prior to CB diagnosis and for 2 years after diagnosis. Mean utilization and costs in the CB cohort were seen to increase in the 6 months immediately preceding diagnosis, a similar finding to that of Akazawa et al.17 This prediagnosis trend is commensurate with the gradual progression of COPD.\nIn the postindex period, the majority of total health care cost differences between the cohorts were for medical services. Patients in the CB cohort incurred approximately US$2000–$2800 more for medical services across three of the 6-month postindex periods. During the same periods, the CB cohort’s pharmacy costs were US$500–$600 higher than the control cohort. After the initial spike following a diagnosis of chronic bronchitis, medical services costs declined but remained elevated compared with controls, which may be due to increased utilization related to CB events, but also to non-CB related health events.\nPrevalence of various comorbidities has been documented generally in studies of COPD patients, but there is a dearth of information concerning the extent to which comorbid conditions exist in patients with chronic bronchitis. Mapel et al found that in an analysis of a population in which 39% had cardiovascular disease and 13% had diabetes, the presence of comorbid conditions was a better predictor of future costs of COPD patients compared with COPD disease stage as measured by spirometry.23 Similar prevalence rates of comorbidities have been found in other study populations. In a telephone survey of COPD patients, Barr et al found prevalence rates of more than 50% for hypertension and more than 25% for both depression and diabetes.24 A 2005 review of 1090 Canadian patients with COPD levels ranging from asymptomatic to severe identified comorbidity rates of 51% for hypertension, 19% for diabetes, 15% for depression, 13% for peripheral vascular disease, and 10% for asthma.25 Our CB cohort had a higher asthma rate compared with the COPD patients of the Canadian study, 23% versus 10%, respectively; however, in a study of CB patients who were initiating maintenance therapy, Delea et al found an asthma comorbidity rate of 29%, a prevalence rate similar to CB subjects in this analysis.26\nSimilar to a study of COPD patients by Akazawa and colleagues, our study found higher IP, ED/UC, and pharmacy utilization in the 6 months prior to CB diagnosis; higher ED/UC and pharmacy utilization was also observed even earlier, up to 1 year prediagnosis. The utilization differences became pronounced in the 6 months immediately preceding CB diagnosis, reached an apex during the next 6 months that included the index date (diagnosis), and then decreased. However, during the last three of the 6-month periods postindex, differences in mean medical services and pharmacy costs, as well as mean events, remained at levels of greater magnitude than those shown during both 6-month periods prior to CB diagnosis. This suggests that there is strong evidence that the systemic consequences of chronic bronchitis may play a significant role in driving up total health care utilization and costs among patients with chronic bronchitis.\nA strength of our study is that we focused on adults aged 40 years and over, as opposed to an exclusively elderly population. An additional strength is that we examined health care utilization and costs in comparisons of CB and non-CB patients, whereas most of the literature has focused on the broader definition of COPD. A review by Maciewicz et al suggested that the varied aspects of the progression of COPD may be related to the aging process;27 earlier disease identification and treatment initiation may improve patients’ long-term health and economic outcomes, although the total cost savings may be modest due to shorter life expectancy for patients with COPD.28\nThis retrospective study based on claims data is not without limitations. Foremost among these is that the accuracy of diagnoses could not be verified. The results and conclusions of this study are limited to the population studied and the operational definitions of our variables. The analysis is limited to those with CB who utilize health care services and the representation of CB patients in the database we used of our analysis. The geographical representation in this study differed from what has previously been noted for the USA; that is, high prevalence in the South and Midwest.29 In addition, cost estimates may vary when different databases are used. Data were unavailable on both patient smoking behavior and severity of disease among the CB cohort, thus limiting our findings regarding drivers of resource utilization and cost.",
"To our knowledge, this is the first study to use a large enough population sample to facilitate propensity score matching based on comorbidities. Prior studies have not controlled for comorbidity by matching,30 creating potential for residual confounding and other biases in case-control comparisons. Our method allowed for a cleaner assessment of the true impact of CB. The disease burden is greatest during the 6-month period following CB diagnosis, but is also high in periods preceding the diagnosis. In patients diagnosed with chronic bronchitis, compared with patients without evidence of COPD or asthma, health care utilization and costs continue to remain elevated 2 years after diagnosis. This study suggests that not accurately diagnosing CB early may have a substantial impact on health care costs, resulting in a higher economic burden for CB patients than would otherwise be present."
] | [
"intro",
"methods",
"methods",
null,
null,
"methods",
"results",
"discussion",
null
] | [
"chronic bronchitis",
"burden",
"economic",
"chronic obstructive pulmonary disease"
] | Introduction:
Chronic bronchitis (CB), an inflammatory condition that affects the central bronchi, is one of two main lung diseases by which patients with chronic obstructive pulmonary disease (COPD) are characterized. Excessive mucus secretion differentiates it from the second, emphysema, which is characterized by permanent enlargement of lung airways and destruction of the walls of the alveoli, making breathing difficult.1 Other symptoms of CB related to lung inflammation and heavy mucus production include cough, production of sputum, and dyspnea.1 In 2008, more than 9.8 million Americans reported having a CB diagnosis.2 While many patients with COPD may suffer from both conditions, the courses of the diseases and response to treatment are frequently different; separate studies of outcomes between the two could assist in optimizing care for these patients.
Estimated annual expenditures for CB treatment total US$11.7 billion, with hospitalizations accounting for US$6 billion of the total costs.3 There are limited studies that examine the costs associated with CB, but research has shown that hospitalizations for acute CB exacerbations accounted for 46%–90% of the costs associated with treatment.4 The diminished quality of life experienced by COPD patients is well documented and suggests that earlier detection, treatment, and reduction of exacerbations may have a substantial economic and psychosocial impact on patients.5–13
The chronic cough and sputum production associated with CB often predates the development of airflow limitation.14 The GOLD (Global Initiative for Chronic Obstructive Lung Disease) guidelines for diagnosis and treatment of COPD recommend early identification of patients with symptoms of CB in order to begin intervention at the earliest stage of COPD.14 Unfortunately, CB symptoms may be dismissed by patients as “smoker’s cough”, or physicians may misdiagnose CB as acute bronchitis or asthma.15,16 Once CB is diagnosed and treatment has commenced, many patients have progressed into more severe COPD stages, resulting in higher consumption of health care resources and increased complexity in clinical management.
Prior research demonstrated higher utilization rates of health care resources for COPD patients when compared with a control population during the 12 months preceding initial COPD diagnosis.17 Utilization trends indicated an increasing use of resources in terms of medical services and pharmacy prescriptions up to the COPD diagnosis, particularly in the final month before diagnosis.17 In addition, total costs for COPD patients have been shown to be higher up to 2 years prior to diagnosis.18 To determine if these trends were apparent for the subset of COPD patients diagnosed with CB, we examined health care utilization and costs for 12 months prior to CB diagnosis and 24 months post diagnosis.
Data and methods:
Study design and data source This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.
This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.
Sample CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.
Patients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.
CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.
Patients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.
Measures Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.
Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.
Analysis Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.
Means and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).
Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.
Means and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).
Study design and data source:
This case–control analysis used data from the PharMetrics Integrated Database, which contains continuously updated information from enrollment files and facility, professional services, and outpatient pharmacy claims from more than 90 participating US health plans, representing more than 55 million patients. The data include dates of service and International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) diagnosis codes, participating plan payment and billed charge information. The dataset is deidentified and Health Insurance Portability and Accountability Act (HIPAA) compliant.
Sample:
CB and control cohorts were selected using medical claims data for services provided between July 1, 2003 and June 30, 2007. The CB cohort consisted of patients with an initial primary or secondary diagnosis claim of CB (ICD-9-CM codes 491.xx), considered the index event, during the observation period described above. All study patients were required to be aged 40 or older and to have had continuous enrollment in a health plan for at least 12 months prior to the initial diagnosis (index date). Thus the earliest index date was July 1, 2004. In addition to the preindex health plan enrollment period, subjects were also required to remain enrolled in a health plan for a minimum of 1 year, to have at least one prescription claim in both the pre- and postindex observational time periods (to provide evidence of pharmaceutical insurance coverage) and to have no prior claims for any COPD-related outpatient visit.
Patients in the potential control cohort had at least 36 months of continuous enrollment between July 1, 2003 and June 30, 2007. Index dates were randomly assigned to control cohort subjects following the first 12 months of continuous enrollment. Patients in the potential control cohort had no evidence of bronchitis, chronic bronchitis, emphysema, asthma, or unspecified COPD (ICD-9-CM codes 490.xx, 491.xx, 492.xx, 493.xx, 496.xx) during the 12 months prior to the randomly assigned index date, and also had to have at least one medical services claim and one prescription claim pre- and postindex date. Patients with serious lung conditions other than COPD or asthma were excluded from both cohorts.19 The likelihood of a CB diagnosis was calculated by a logistic regression that controlled for demographics (age, sex, and geographic region), quarter and year of index date, and comorbidities (Table 1). CB patients were propensity matched one-to-one to control subjects using the greedy match algorithm, a method that derives matched samples using nearest available pair matching.20,21 CB cases were matched to controls according to age, geographic region, quarter of year of index date, and selected comorbidities to reduce selection bias. Comorbid conditions were identified based on the classifications by Elixhauser et al22 sleep apnea and heart disease (rheumatic heart failure, hypertensive heart disease, ischemic heart disease, and unspecified cardiovascular disease) were also included.
Measures:
Utilization and costs due to all causes in both the pre- and postindex periods were categorized by inpatient hospitalization (IP), emergency department/urgent care (ED/UC), outpatient visits (OP), and pharmacy fills. Utilization and costs occurring on the index date were included in the first 6-month period postindex. We defined costs as the amount paid by the health plan; costs paid by other sources were not included. Costs for IP, OP, and ED/UC were aggregated as medical services costs. Pharmacy costs were kept separate; total costs were the sum of medical services and pharmacy costs. Each category for both cohorts was analyzed for six time periods of 6 months each: two in the preindex period and four during the postindex period.
Analysis:
Descriptive analysis was used for all demographic, patient comorbidity, and outcome variables. Continuous variables were reported as means, standard deviations, and medians. Binary and categorical variables were reported as counts and percentages.
Means and frequencies for utilization and costs were calculated and compared for all outcome categories in both the pre- and postindex periods. Covariate adjustment was incorporated through the use of a propensity score matching technique as previously described. The Student’s t-test and Wilcoxon rank-sum test was used for continuous variables and the Pearson chi-square test for binary use variables. All statistical tests were two-sided with a 0.05 level of significance. Analyses were conducted with SAS software (version 9.1.3 for Windows; SAS Institute, Cary, NC).
Results:
There were initially 182,169 patients who had a COPD diagnosis at any time; after selection criteria were applied, 11,937 patients met criteria for the CB cohort. A 10% random sample of possible control subjects with 3 or more years of continuous enrollment was selected, of whom 282,078 met the control cohort selection criteria. These patients were randomly reduced to a pool of 65,654 control patients. Propensity score matching was used to match CB patients to control patients on a one-to-one basis, resulting in two cohorts of 11,674 each.
Prior to matching, the two cohorts exhibited significant differences in all matching characteristics with the exception of two of the index date quarter variables (Table 1). After matching, the CB patient cohort and control patient cohort exhibited a significant difference only in the percentage of each population that was male (CB = 41.4%, control = 43.0%; P = 0.0142). The cohorts were similar with respect to comorbidities, geographic region, mean age, and quarter in which the index date occurred.
After matching, the mean age of patients in the CB cohort was 61.5 years; for the controls, 61.8 years. Patients were predominantly from the Northeast (37%) and female (57.8%). The most prevalent comorbidities in each cohort were: uncomplicated hypertension (48.5%), diabetes (19.3%), ischemic heart disease (19.3%), arrhythmia (10.5%), and hypothyroidism (10.5%), shown in Table 1. Among the CB cohort, 22.8% had a history of asthma in addition to their CB diagnosis.
There was a decrease in the number of patients with continuous enrollment beginning 13 months after the index date. Approximately 23% of the CB patients were lost to follow-up during months 13–18, and another 25% in the 19–24 month period after the index date. For the final 6-month observation period (months 19–24), there were 6128 CB patients and 6203 control patients.
As shown in Table 2 and Figure 1, from 1 year and up to 6 months prior to the CB diagnosis there were no differences between the cohorts with respect to the percentage of patients having any IP visit; however, the CB cohort had a higher percentage having any ED/UC visit (9.5%, 6.4%; P < 0.05), and any pharmacy use (93.4%, 91.5%; P < 0.05), while having a lower percentage with any OP visit (90.5%, 92.9%; P < 0.05). In the next 6 months, which was the 6 months immediately prior to diagnosis, patients in the CB cohort, compared with the control cohort, had a higher percentage having any IP visit (9.6%, 6.7%; P < 0.05), and again, higher percentages for any ED/UC visit (13.3%, 6.7%; P < 0.05), and any pharmacy prescription (97.3%, 94.1%; P < 0.05). Percentages of patients having any OP visit were identical for the two cohorts during the 6 months immediately prior to CB diagnosis.
During the first 6 months postindex (Figure 1), patients in the CB cohort had 5.6 times more IP visits (30.4%, 5.4%; P < 0.05) and 3.1 times more ED/UC visits (20.7%, 6.6%; P < 0.05). To a lesser degree, OP visits and pharmacy use were also higher for the CB cohort. Utilization rates remained higher for the CB cohort during all four of the 6-month periods postindex, but beginning in the second 6-month postindex period, utilization rates in the CB cohort declined from the highs reached in the first postindex period and remained fairly stable for the remaining observation periods. The control cohort’s utilization rates declined slightly in each of the four postindex periods. Mean number of events were higher for the CB cohort in all time periods, pre- and postindex, compared with the control cohort except for mean OP visits in the 6-month period immediately preceding the CB diagnosis (Table 3, Figure 2).
Mean total costs (US$) for the CB cohort during both preindex periods were significantly higher than for the control cohort (months 12–7: $4212, $3826; P < 0.05; months 6–1: $5289, $4285; P < 0.05). Component costs were also higher, with the exception that there was no difference in medical services costs during the initial 6 months prior to diagnosis (Table 4). CB patients had higher mean total costs ($8919; P < 0.05) in the 6 months postdiagnosis. Costs for both groups then decreased (Figure 3) but for CB patients remained $2429 higher than for control patients 19–24 months postdiagnosis (P < 0.05). Median costs were all significantly higher for the CB cohort (Table 5).
Discussion:
This retrospective observational study demonstrated that total health care utilization and costs for CB patients in the US are significantly greater than for a matched control cohort of patients. This was true for both the 12-month period prior to CB diagnosis and for 2 years after diagnosis. Mean utilization and costs in the CB cohort were seen to increase in the 6 months immediately preceding diagnosis, a similar finding to that of Akazawa et al.17 This prediagnosis trend is commensurate with the gradual progression of COPD.
In the postindex period, the majority of total health care cost differences between the cohorts were for medical services. Patients in the CB cohort incurred approximately US$2000–$2800 more for medical services across three of the 6-month postindex periods. During the same periods, the CB cohort’s pharmacy costs were US$500–$600 higher than the control cohort. After the initial spike following a diagnosis of chronic bronchitis, medical services costs declined but remained elevated compared with controls, which may be due to increased utilization related to CB events, but also to non-CB related health events.
Prevalence of various comorbidities has been documented generally in studies of COPD patients, but there is a dearth of information concerning the extent to which comorbid conditions exist in patients with chronic bronchitis. Mapel et al found that in an analysis of a population in which 39% had cardiovascular disease and 13% had diabetes, the presence of comorbid conditions was a better predictor of future costs of COPD patients compared with COPD disease stage as measured by spirometry.23 Similar prevalence rates of comorbidities have been found in other study populations. In a telephone survey of COPD patients, Barr et al found prevalence rates of more than 50% for hypertension and more than 25% for both depression and diabetes.24 A 2005 review of 1090 Canadian patients with COPD levels ranging from asymptomatic to severe identified comorbidity rates of 51% for hypertension, 19% for diabetes, 15% for depression, 13% for peripheral vascular disease, and 10% for asthma.25 Our CB cohort had a higher asthma rate compared with the COPD patients of the Canadian study, 23% versus 10%, respectively; however, in a study of CB patients who were initiating maintenance therapy, Delea et al found an asthma comorbidity rate of 29%, a prevalence rate similar to CB subjects in this analysis.26
Similar to a study of COPD patients by Akazawa and colleagues, our study found higher IP, ED/UC, and pharmacy utilization in the 6 months prior to CB diagnosis; higher ED/UC and pharmacy utilization was also observed even earlier, up to 1 year prediagnosis. The utilization differences became pronounced in the 6 months immediately preceding CB diagnosis, reached an apex during the next 6 months that included the index date (diagnosis), and then decreased. However, during the last three of the 6-month periods postindex, differences in mean medical services and pharmacy costs, as well as mean events, remained at levels of greater magnitude than those shown during both 6-month periods prior to CB diagnosis. This suggests that there is strong evidence that the systemic consequences of chronic bronchitis may play a significant role in driving up total health care utilization and costs among patients with chronic bronchitis.
A strength of our study is that we focused on adults aged 40 years and over, as opposed to an exclusively elderly population. An additional strength is that we examined health care utilization and costs in comparisons of CB and non-CB patients, whereas most of the literature has focused on the broader definition of COPD. A review by Maciewicz et al suggested that the varied aspects of the progression of COPD may be related to the aging process;27 earlier disease identification and treatment initiation may improve patients’ long-term health and economic outcomes, although the total cost savings may be modest due to shorter life expectancy for patients with COPD.28
This retrospective study based on claims data is not without limitations. Foremost among these is that the accuracy of diagnoses could not be verified. The results and conclusions of this study are limited to the population studied and the operational definitions of our variables. The analysis is limited to those with CB who utilize health care services and the representation of CB patients in the database we used of our analysis. The geographical representation in this study differed from what has previously been noted for the USA; that is, high prevalence in the South and Midwest.29 In addition, cost estimates may vary when different databases are used. Data were unavailable on both patient smoking behavior and severity of disease among the CB cohort, thus limiting our findings regarding drivers of resource utilization and cost.
Conclusion:
To our knowledge, this is the first study to use a large enough population sample to facilitate propensity score matching based on comorbidities. Prior studies have not controlled for comorbidity by matching,30 creating potential for residual confounding and other biases in case-control comparisons. Our method allowed for a cleaner assessment of the true impact of CB. The disease burden is greatest during the 6-month period following CB diagnosis, but is also high in periods preceding the diagnosis. In patients diagnosed with chronic bronchitis, compared with patients without evidence of COPD or asthma, health care utilization and costs continue to remain elevated 2 years after diagnosis. This study suggests that not accurately diagnosing CB early may have a substantial impact on health care costs, resulting in a higher economic burden for CB patients than would otherwise be present.
| Background:
Chronic bronchitis (CB) is often misdiagnosed or diagnosed at a later stage of chronic obstructive pulmonary disease (COPD). We examined how this later diagnosis may impact health care costs and utilization during the 12 months prior to and 24 months post initial CB diagnosis.
Methods:
This retrospective case-control analysis used claims data from a large US database from July 1, 2003 through June 30, 2007. Patients with CB aged 40 years and older were propensity matched (N = 11,674) to patients without evidence of COPD or asthma by demographics, CB diagnosis quarter/year, and comorbidities. Group differences were assessed using Student's t-test and Pearson chi-square test statistics.
Results:
Six months prediagnosis, CB patients had higher frequencies of any hospitalization (9.6%, 6.7%; P < 0.05), emergency department/urgent care visits (13.3%, 6.7%; P < 0.05), and prescriptions (97.3%, 94.1%; P < 0.05). Six months postdiagnosis, CB patients had 5.6 times more hospitalizations (P < 0.05) and 3.1 times more emergency department/urgent care visits (P < 0.05) compared with controls. Mean total costs (US$) for CB patients 12 months prediagnosis were significantly higher than controls (months 12-7: $4212, $3826; P < 0.05; months 6-1: $5289, $4285; P < 0.05). CB patients had higher mean total costs ($8919; P < 0.05) 6 months postdiagnosis. Costs remained $2429 higher for CB patients 19-24 months postdiagnosis (P < 0.05).
Conclusions:
Health care costs and utilization among CB patients are increased both prior to diagnosis and during the 2 years postdiagnosis. This study suggests that not accurately diagnosing CB early has a substantial impact on health care costs, and that the economic burden for CB patients remains elevated even after adjustment for comorbidities associated with COPD. | Introduction:
Chronic bronchitis (CB), an inflammatory condition that affects the central bronchi, is one of two main lung diseases by which patients with chronic obstructive pulmonary disease (COPD) are characterized. Excessive mucus secretion differentiates it from the second, emphysema, which is characterized by permanent enlargement of lung airways and destruction of the walls of the alveoli, making breathing difficult.1 Other symptoms of CB related to lung inflammation and heavy mucus production include cough, production of sputum, and dyspnea.1 In 2008, more than 9.8 million Americans reported having a CB diagnosis.2 While many patients with COPD may suffer from both conditions, the courses of the diseases and response to treatment are frequently different; separate studies of outcomes between the two could assist in optimizing care for these patients.
Estimated annual expenditures for CB treatment total US$11.7 billion, with hospitalizations accounting for US$6 billion of the total costs.3 There are limited studies that examine the costs associated with CB, but research has shown that hospitalizations for acute CB exacerbations accounted for 46%–90% of the costs associated with treatment.4 The diminished quality of life experienced by COPD patients is well documented and suggests that earlier detection, treatment, and reduction of exacerbations may have a substantial economic and psychosocial impact on patients.5–13
The chronic cough and sputum production associated with CB often predates the development of airflow limitation.14 The GOLD (Global Initiative for Chronic Obstructive Lung Disease) guidelines for diagnosis and treatment of COPD recommend early identification of patients with symptoms of CB in order to begin intervention at the earliest stage of COPD.14 Unfortunately, CB symptoms may be dismissed by patients as “smoker’s cough”, or physicians may misdiagnose CB as acute bronchitis or asthma.15,16 Once CB is diagnosed and treatment has commenced, many patients have progressed into more severe COPD stages, resulting in higher consumption of health care resources and increased complexity in clinical management.
Prior research demonstrated higher utilization rates of health care resources for COPD patients when compared with a control population during the 12 months preceding initial COPD diagnosis.17 Utilization trends indicated an increasing use of resources in terms of medical services and pharmacy prescriptions up to the COPD diagnosis, particularly in the final month before diagnosis.17 In addition, total costs for COPD patients have been shown to be higher up to 2 years prior to diagnosis.18 To determine if these trends were apparent for the subset of COPD patients diagnosed with CB, we examined health care utilization and costs for 12 months prior to CB diagnosis and 24 months post diagnosis.
Conclusion:
To our knowledge, this is the first study to use a large enough population sample to facilitate propensity score matching based on comorbidities. Prior studies have not controlled for comorbidity by matching,30 creating potential for residual confounding and other biases in case-control comparisons. Our method allowed for a cleaner assessment of the true impact of CB. The disease burden is greatest during the 6-month period following CB diagnosis, but is also high in periods preceding the diagnosis. In patients diagnosed with chronic bronchitis, compared with patients without evidence of COPD or asthma, health care utilization and costs continue to remain elevated 2 years after diagnosis. This study suggests that not accurately diagnosing CB early may have a substantial impact on health care costs, resulting in a higher economic burden for CB patients than would otherwise be present. | Background:
Chronic bronchitis (CB) is often misdiagnosed or diagnosed at a later stage of chronic obstructive pulmonary disease (COPD). We examined how this later diagnosis may impact health care costs and utilization during the 12 months prior to and 24 months post initial CB diagnosis.
Methods:
This retrospective case-control analysis used claims data from a large US database from July 1, 2003 through June 30, 2007. Patients with CB aged 40 years and older were propensity matched (N = 11,674) to patients without evidence of COPD or asthma by demographics, CB diagnosis quarter/year, and comorbidities. Group differences were assessed using Student's t-test and Pearson chi-square test statistics.
Results:
Six months prediagnosis, CB patients had higher frequencies of any hospitalization (9.6%, 6.7%; P < 0.05), emergency department/urgent care visits (13.3%, 6.7%; P < 0.05), and prescriptions (97.3%, 94.1%; P < 0.05). Six months postdiagnosis, CB patients had 5.6 times more hospitalizations (P < 0.05) and 3.1 times more emergency department/urgent care visits (P < 0.05) compared with controls. Mean total costs (US$) for CB patients 12 months prediagnosis were significantly higher than controls (months 12-7: $4212, $3826; P < 0.05; months 6-1: $5289, $4285; P < 0.05). CB patients had higher mean total costs ($8919; P < 0.05) 6 months postdiagnosis. Costs remained $2429 higher for CB patients 19-24 months postdiagnosis (P < 0.05).
Conclusions:
Health care costs and utilization among CB patients are increased both prior to diagnosis and during the 2 years postdiagnosis. This study suggests that not accurately diagnosing CB early has a substantial impact on health care costs, and that the economic burden for CB patients remains elevated even after adjustment for comorbidities associated with COPD. | 4,960 | 380 | [
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Ambulatory Care | Bronchitis, Chronic | Case-Control Studies | Chi-Square Distribution | Drug Costs | Early Diagnosis | Emergency Medical Services | Female | Health Care Costs | Hospitalization | Humans | Male | Middle Aged | Models, Economic | Propensity Score | Retrospective Studies | Time Factors | Treatment Outcome | United States [SUMMARY] | [CONTENT] Aged | Ambulatory Care | Bronchitis, Chronic | Case-Control Studies | Chi-Square Distribution | Drug Costs | Early Diagnosis | Emergency Medical Services | Female | Health Care Costs | Hospitalization | Humans | Male | Middle Aged | Models, Economic | Propensity Score | Retrospective Studies | Time Factors | Treatment Outcome | United States [SUMMARY] | [CONTENT] Aged | Ambulatory Care | Bronchitis, Chronic | Case-Control Studies | Chi-Square Distribution | Drug Costs | Early Diagnosis | Emergency Medical Services | Female | Health Care Costs | Hospitalization | Humans | Male | Middle Aged | Models, Economic | Propensity Score | Retrospective Studies | Time Factors | Treatment Outcome | United States [SUMMARY] | [CONTENT] Aged | Ambulatory Care | Bronchitis, Chronic | Case-Control Studies | Chi-Square Distribution | Drug Costs | Early Diagnosis | Emergency Medical Services | Female | Health Care Costs | Hospitalization | Humans | Male | Middle Aged | Models, Economic | Propensity Score | Retrospective Studies | Time Factors | Treatment Outcome | United States [SUMMARY] | [CONTENT] Aged | Ambulatory Care | Bronchitis, Chronic | Case-Control Studies | Chi-Square Distribution | Drug Costs | Early Diagnosis | Emergency Medical Services | Female | Health Care Costs | Hospitalization | Humans | Male | Middle Aged | Models, Economic | Propensity Score | Retrospective Studies | Time Factors | Treatment Outcome | United States [SUMMARY] | [CONTENT] copd asthma | consequences chronic bronchitis | chronic bronchitis compared | chronic bronchitis cb | costs copd patients [SUMMARY] | [CONTENT] copd asthma | consequences chronic bronchitis | chronic bronchitis compared | chronic bronchitis cb | costs copd patients [SUMMARY] | [CONTENT] copd asthma | consequences chronic bronchitis | chronic bronchitis compared | chronic bronchitis cb | costs copd patients [SUMMARY] | [CONTENT] copd asthma | consequences chronic bronchitis | chronic bronchitis compared | chronic bronchitis cb | costs copd patients [SUMMARY] | [CONTENT] copd asthma | consequences chronic bronchitis | chronic bronchitis compared | chronic bronchitis cb | costs copd patients [SUMMARY] | [CONTENT] copd asthma | consequences chronic bronchitis | chronic bronchitis compared | chronic bronchitis cb | costs copd patients [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | months | control | copd | health | index [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | months | control | copd | health | index [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | months | control | copd | health | index [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | months | control | copd | health | index [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | months | control | copd | health | index [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | months | control | copd | health | index [SUMMARY] | [CONTENT] cb | copd | patients | treatment | diagnosis | copd patients | resources | associated | cough | symptoms [SUMMARY] | [CONTENT] variables | test | outcome | sas | continuous variables | binary | means | variables reported | reported | continuous [SUMMARY] | [CONTENT] cohort | cb | patients | 05 | cb cohort | higher | months | control | mean | percentage [SUMMARY] | [CONTENT] cb | burden | impact | health care | diagnosis | patients | study | matching | care | greatest [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | copd | months | health | control | postindex [SUMMARY] | [CONTENT] cb | patients | costs | cohort | diagnosis | copd | months | health | control | postindex [SUMMARY] | [CONTENT] ||| the 12 months | 24 months [SUMMARY] | [CONTENT] US | July 1, 2003 through June 30, 2007 ||| CB | 40 years | 11,674 | COPD | quarter/year ||| Student | Pearson chi-square [SUMMARY] | [CONTENT] Six months | CB | 9.6% | 6.7% | P < 0.05 | 13.3% | 6.7% | P < 0.05 | 97.3% | 94.1% | P < 0.05 ||| Six months | CB | 5.6 | 3.1 ||| CB | 12 months | months 12-7 | 3826 | P < 0.05 | months 6-1 | 5289 | 4285 | P < 0.05 ||| CB | 8919 | P < 0.05 | 6 months ||| 2429 | CB | 19-24 months [SUMMARY] | [CONTENT] CB | 2 years ||| CB | COPD [SUMMARY] | [CONTENT] ||| the 12 months | 24 months ||| US | July 1, 2003 through June 30, 2007 ||| CB | 40 years | 11,674 | COPD | quarter/year ||| Student | Pearson chi-square ||| Six months | CB | 9.6% | 6.7% | P < 0.05 | 13.3% | 6.7% | P < 0.05 | 97.3% | 94.1% | P < 0.05 ||| Six months | CB | 5.6 | 3.1 ||| CB | 12 months | months 12-7 | 3826 | P < 0.05 | months 6-1 | 5289 | 4285 | P < 0.05 ||| CB | 8919 | P < 0.05 | 6 months ||| 2429 | CB | 19-24 months ||| CB | 2 years ||| CB | COPD [SUMMARY] | [CONTENT] ||| the 12 months | 24 months ||| US | July 1, 2003 through June 30, 2007 ||| CB | 40 years | 11,674 | COPD | quarter/year ||| Student | Pearson chi-square ||| Six months | CB | 9.6% | 6.7% | P < 0.05 | 13.3% | 6.7% | P < 0.05 | 97.3% | 94.1% | P < 0.05 ||| Six months | CB | 5.6 | 3.1 ||| CB | 12 months | months 12-7 | 3826 | P < 0.05 | months 6-1 | 5289 | 4285 | P < 0.05 ||| CB | 8919 | P < 0.05 | 6 months ||| 2429 | CB | 19-24 months ||| CB | 2 years ||| CB | COPD [SUMMARY] |
||||||
Early Determinants of Length of Hospital Stay: A Case Control Survival Analysis among COVID-19 Patients admitted in a Tertiary Healthcare Facility of East India. | 34704488 | Length of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning. The study aimed to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients. | BACKGROUND | It was a retrospective, record based, unmatched, case control study using hospital records of 334 COVID-19 patients admitted in an East Indian tertiary healthcare facility during May to October 2020. Discharge from the hospital (cases/survivors) was considered as an event while death (control/non-survivors) as right censoring in the case-control survival analysis using cox proportional hazard model. | MATERIALS AND METHODS | Overall, we found the median LOS for the survivors to be 8 days [interquartile range (IQR): 7-10 days] while the same for the non-survivors was 6 days [IQR: 2-11 days]. In the multivariable cox-proportional hazard model; travel distance (>16 km) [adjusted hazard ratio (aHR): 0.69, 95% CI: (0.50-0.95)], mode of transport to the hospital (ambulance) [aHR: 0.62, 95% CI: (0.45-0.85)], breathlessness (yes) [aHR: 0.56, 95% CI: (0.40-0.77)], number of co-morbidities (1-2) [aHR: 0.66, 95% CI: (0.47-0.93)] (≥3) [aHR: 0.16, 95% CI: (0.04-0.65)], COPD/asthma (yes) [ [aHR: 0.11, 95% CI: (0.01-0.79)], DBP (<60/≥90) [aHR: 0.55, 95% CI: (0.35-0.86)] and qSOFA score (≥2) [aHR: 0.33, 95% CI: (0.12-0.92)] were the significant attributes affecting LOS of the COVID-19 patients. | RESULTS | Factors elicited on arrival were found to be significantly associated with LOS. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients. | CONCLUSION | [
"COVID-19",
"Case-Control Studies",
"Humans",
"India",
"Length of Stay",
"Retrospective Studies",
"Risk Factors",
"SARS-CoV-2",
"Survival Analysis",
"Tertiary Healthcare"
] | 8554553 | Introduction | It’s being more than a year since the first case of COVID-19 reported in Wuhan, China.
1
Since then, the pandemic has affected millions of lives around the globe.
2
It has interrupted routine healthcare delivery and challenged healthcare infrastructure of even developed countries.3-8 India reported first ever COVID-19 case in January 2020.
9
Since then, the country has reported over 30 million COVID-19 cases and 4 00 000 deaths associated with the disease.
2
Length of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning.
10
Decreased LOS is reported to be associated with lowered risk of hospital acquired infection, reduction in financial burden for treatment among the patients, higher bed turnover rate of the hospitals (increases bed availability for the other patients) and vice versa can be told for increased LOS.11,12 For COVID-19 the reported associates of LOS are age, gender, nutritional status, presenting symptoms (ie, fever, breathlessness, fatigue, anorexia etc.), co-morbidities (ie, hypertension, heart disease, diabetes etc.), vitals (ie, respiratory rate, blood pressure etc.) laboratory parameters [ie, d-dimer, C-reactive protein (CRP), leucocyte count, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) etc.], radio graphical parameters (ie, chest X-ray, CT scan findings etc.), and medications (ie, tocilizumab, ACEI, ARB, metformin etc.).13-22 Although in terms of healthcare logistic planning the factors that could predict LOS of COVID-19 patients early bears additional importance. Patient characteristics and vital signs at the time of admission are being already linked with the level of care requirement, disease progression and mortality among COVID-19 patients by some prior studies.23-25 Although considering specifically for LOS, no such prior attempts have been made in Indian context.
Early identification of factors influencing LOS of COVID-19 patients may help policymakers to plan healthcare logistics (man, money, and material) for the disease accordingly. On extensive literature search we could only retrieve 2 prior Indian studies which have explored LOS among COVID-19 patients.13,14 Although these 2 studies had their own limitations. The study conducted by Thiruvengadam et al
13
did not report median survival time for the found associated variables. By contrast, Mishra et al
14
analyzed data extracted from media bulletin and elicited very few variables. Moreover, both these studies were conducted in Karnataka (a southern state of India). Therefore, the current retrospective case-control study was planned to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients admitted in a tertiary care hospital of East India. | null | null | Results | The median age of the studied COVID-19 patients was 55 years with interquartile range (IQR) of 40 to 65 years. Overall, we found the median LOS for the survivors to be 8 days [interquartile range (IQR): 7-10 days] while the same for the non-survivors was 6 days [IQR: 2-11 days]. Survivors had more probability of higher LOS compared to non-survivors. In univariate cox-proportional hazard model; higher age, number of co-morbidities and travelling distance to seek healthcare, self-reporting to the healthcare facility, complaint of breathlessness, deranged RR, SpO2, PR, SBP, DBP, GCS, and qSOFA score on admission increased LOS while complaint of sore throat on admission decreased the same. Similarly, among the reported co-morbidities, COPD/asthma was found be associated with slowest recovery followed by CKD, diabetes, hypertension and ischemic heart disease. The median days since symptomatic prior to admission was 6 days with IQR of 4 to 8 days. The duration of symptoms prior to admission was not found to be associated with LOS [HR: 0.85, 95% CI: (0.63-1.16)] (Figure 1 and Table 1).
Kaplan Meier curve showing overall survival of the COVID-19 patients (n = 334).
Univariate Cox Proportional Hazard Model Showing Factors Associated With Length of Hospital Stay Among COVID-19 Patients (n = 334).
Abbreviations: CI: confidence interval; COPD: chronic obstructive pulmonary disease; DBP: diastolic blood pressure; GCS: Glasgow coma scale; HR: hazard ratio; LOS: length of hospital stay (in days); qSOFA: quick sequential organ failure assessment; ref.: reference; SBP: systolic blood pressure.
Due to right censoring in these groups median survival time could not be calculated; HR < 1 indicates higher LOS.
In the multivariable cox-proportional hazard model; travel distance (>16 km) [adjusted hazard ratio (aHR): 0.69, 95% CI: (0.50-0.95)], mode of transport to the hospital (ambulance) [aHR: 0.62, 95% CI: (0.45-0.85)], breathlessness (yes) [aHR: 0.56, 95% CI: (0.40-0.77)], number of co-morbidities (1-2) [aHR: 0.66, 95% CI: (0.47-0.93)] (≥3) [aHR: 0.16, 95% CI: (0.04-0.65)], COPD/asthma (yes) [aHR: 0.11, 95% CI: (0.01-0.79)], DBP (<60/≥90) [aHR: 0.55, 95% CI: (0.35-0.86)] and qSOFA score (≥2) [aHR: 0.33, 95% CI: (0.12-0.92)] were the significant attributes affecting LOS of the COVID-19 patients. Thus, those who resided within 16 km from the health facility, used self-owned/rented vehicle on way to the hospital, did not have breathlessness, COPD/asthma and had no/less number of co-morbidities, normal DBP (between 60 to 89 mmHg) and lower qSOFA score at the time of admission recovered earlier compared to others (Table 2).
Multivariable Cox Proportional Hazard Model Showing Factors Associated with Length of Hospital Stay Among COVID-19 Patients: n = 334.
Abbreviations: aHR, adjusted hazard ratio; CI, confidence interval; COPD, chronic obstructive pulmonary disease; DBP, diastolic blood pressure; GCS, Glasgow coma scale; qSOFA, quick sequential organ failure assessment; ref., reference.
aHR <1 indicates higher LOS.
The multivariable cox-proportional hazard model can be presented as h(t) = h0(t) exp[−0.37 × (travel distance>16)−0.48 × (mode of transport to the hospitalambulance)−0.59 × (breathlessness at admissionyes)–0.42 × (co-morbidity1-2)−1.87 × (co-morbidity≥3)−2.25 × (COPD/asthmayes)−0.60 × (DBP<60/≥90)−1.11 × (qSOFA score≥2)] | Conclusion | Factors elicited on arrival was found to be significantly associated with LOS. Travel distance, mode of transport to the hospital, breathlessness, number of co-morbidities, presence of COPD/ asthma, DBP and qSOFA score which are easily elicitable at the time of admission found to be significantly associated with LOS. Further research is warranted to gain more knowledge on this issue. These future studies should elicit effect of nutritional status and insurance coverage on LOS of COVID-19 patients which we could not do due lack of those data. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients. | [
"Study Setting and Period",
"Sample Size Determination",
"Sampling Technique",
"Data Collection Procedure",
"Statistical Analysis",
"Limitations"
] | [
"It was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.",
"Considering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al\n26\n), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.\n27\n The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.",
"During the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.",
"The variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.",
"The data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.",
"Despite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records."
] | [
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Materials and Methods",
"Study Setting and Period",
"Sample Size Determination",
"Sampling Technique",
"Data Collection Procedure",
"Statistical Analysis",
"Results",
"Discussion",
"Limitations",
"Conclusion"
] | [
"It’s being more than a year since the first case of COVID-19 reported in Wuhan, China.\n1\n Since then, the pandemic has affected millions of lives around the globe.\n2\n It has interrupted routine healthcare delivery and challenged healthcare infrastructure of even developed countries.3-8 India reported first ever COVID-19 case in January 2020.\n9\n Since then, the country has reported over 30 million COVID-19 cases and 4 00 000 deaths associated with the disease.\n2\n\nLength of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning.\n10\n Decreased LOS is reported to be associated with lowered risk of hospital acquired infection, reduction in financial burden for treatment among the patients, higher bed turnover rate of the hospitals (increases bed availability for the other patients) and vice versa can be told for increased LOS.11,12 For COVID-19 the reported associates of LOS are age, gender, nutritional status, presenting symptoms (ie, fever, breathlessness, fatigue, anorexia etc.), co-morbidities (ie, hypertension, heart disease, diabetes etc.), vitals (ie, respiratory rate, blood pressure etc.) laboratory parameters [ie, d-dimer, C-reactive protein (CRP), leucocyte count, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) etc.], radio graphical parameters (ie, chest X-ray, CT scan findings etc.), and medications (ie, tocilizumab, ACEI, ARB, metformin etc.).13-22 Although in terms of healthcare logistic planning the factors that could predict LOS of COVID-19 patients early bears additional importance. Patient characteristics and vital signs at the time of admission are being already linked with the level of care requirement, disease progression and mortality among COVID-19 patients by some prior studies.23-25 Although considering specifically for LOS, no such prior attempts have been made in Indian context.\nEarly identification of factors influencing LOS of COVID-19 patients may help policymakers to plan healthcare logistics (man, money, and material) for the disease accordingly. On extensive literature search we could only retrieve 2 prior Indian studies which have explored LOS among COVID-19 patients.13,14 Although these 2 studies had their own limitations. The study conducted by Thiruvengadam et al\n13\n did not report median survival time for the found associated variables. By contrast, Mishra et al\n14\n analyzed data extracted from media bulletin and elicited very few variables. Moreover, both these studies were conducted in Karnataka (a southern state of India). Therefore, the current retrospective case-control study was planned to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients admitted in a tertiary care hospital of East India.",
"Study Setting and Period It was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.\nIt was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.\nSample Size Determination Considering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al\n26\n), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.\n27\n The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.\nConsidering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al\n26\n), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.\n27\n The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.\nSampling Technique During the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.\nDuring the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.\nData Collection Procedure The variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.\nThe variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.\nStatistical Analysis The data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.\nThe data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.",
"It was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.",
"Considering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al\n26\n), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.\n27\n The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.",
"During the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.",
"The variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.",
"The data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.",
"The median age of the studied COVID-19 patients was 55 years with interquartile range (IQR) of 40 to 65 years. Overall, we found the median LOS for the survivors to be 8 days [interquartile range (IQR): 7-10 days] while the same for the non-survivors was 6 days [IQR: 2-11 days]. Survivors had more probability of higher LOS compared to non-survivors. In univariate cox-proportional hazard model; higher age, number of co-morbidities and travelling distance to seek healthcare, self-reporting to the healthcare facility, complaint of breathlessness, deranged RR, SpO2, PR, SBP, DBP, GCS, and qSOFA score on admission increased LOS while complaint of sore throat on admission decreased the same. Similarly, among the reported co-morbidities, COPD/asthma was found be associated with slowest recovery followed by CKD, diabetes, hypertension and ischemic heart disease. The median days since symptomatic prior to admission was 6 days with IQR of 4 to 8 days. The duration of symptoms prior to admission was not found to be associated with LOS [HR: 0.85, 95% CI: (0.63-1.16)] (Figure 1 and Table 1).\nKaplan Meier curve showing overall survival of the COVID-19 patients (n = 334).\nUnivariate Cox Proportional Hazard Model Showing Factors Associated With Length of Hospital Stay Among COVID-19 Patients (n = 334).\nAbbreviations: CI: confidence interval; COPD: chronic obstructive pulmonary disease; DBP: diastolic blood pressure; GCS: Glasgow coma scale; HR: hazard ratio; LOS: length of hospital stay (in days); qSOFA: quick sequential organ failure assessment; ref.: reference; SBP: systolic blood pressure.\nDue to right censoring in these groups median survival time could not be calculated; HR < 1 indicates higher LOS.\nIn the multivariable cox-proportional hazard model; travel distance (>16 km) [adjusted hazard ratio (aHR): 0.69, 95% CI: (0.50-0.95)], mode of transport to the hospital (ambulance) [aHR: 0.62, 95% CI: (0.45-0.85)], breathlessness (yes) [aHR: 0.56, 95% CI: (0.40-0.77)], number of co-morbidities (1-2) [aHR: 0.66, 95% CI: (0.47-0.93)] (≥3) [aHR: 0.16, 95% CI: (0.04-0.65)], COPD/asthma (yes) [aHR: 0.11, 95% CI: (0.01-0.79)], DBP (<60/≥90) [aHR: 0.55, 95% CI: (0.35-0.86)] and qSOFA score (≥2) [aHR: 0.33, 95% CI: (0.12-0.92)] were the significant attributes affecting LOS of the COVID-19 patients. Thus, those who resided within 16 km from the health facility, used self-owned/rented vehicle on way to the hospital, did not have breathlessness, COPD/asthma and had no/less number of co-morbidities, normal DBP (between 60 to 89 mmHg) and lower qSOFA score at the time of admission recovered earlier compared to others (Table 2).\nMultivariable Cox Proportional Hazard Model Showing Factors Associated with Length of Hospital Stay Among COVID-19 Patients: n = 334.\nAbbreviations: aHR, adjusted hazard ratio; CI, confidence interval; COPD, chronic obstructive pulmonary disease; DBP, diastolic blood pressure; GCS, Glasgow coma scale; qSOFA, quick sequential organ failure assessment; ref., reference.\naHR <1 indicates higher LOS.\nThe multivariable cox-proportional hazard model can be presented as h(t) = h0(t) exp[−0.37 × (travel distance>16)−0.48 × (mode of transport to the hospitalambulance)−0.59 × (breathlessness at admissionyes)–0.42 × (co-morbidity1-2)−1.87 × (co-morbidity≥3)−2.25 × (COPD/asthmayes)−0.60 × (DBP<60/≥90)−1.11 × (qSOFA score≥2)]",
"In this retrospective case-control study we found that higher travelling distance, use of an ambulance as mode of transport, presence of breathlessness at admission, co-morbidities, COPD/asthma, deranged DBP, and higher qSOFA score at admission were associated with greater duration of hospitalization. Demised COVID-19 patients had shorter LOS compared to those who were discharged. Development of a scoring system inculcating found significant attributes of LOS, giving each due weightage as per their adjusted strength of association might be useful in early prediction of LOS of COVID-19 patients. Moreover, linking this scoring system with the logistic management system of the hospital might help in optimal use of the available resources. This might also help in optimizing patient care and alleviate various logistical barriers associated with it (ie, shortage of staff, beds, medicines, equipment’s etc.).\nWe found that patients who required to travel higher distance from their native place to seek healthcare from the present healthcare facility had longer LOS. It might be because our institute is the highest referral institute for most of the healthcare facilities in the state of Bihar. Therefore, usually critically ill patients from the peripheral institutes are used to be referred to our institute. Critically ill COVID-19 patients do usually have a longer LOS, as reported by a systematic review by Rees et al\n26\n Similarly, we found that those who used ambulance as mode of transport to the present healthcare facility had longer LOS. It was previously known that seriously ill and/or patients needing oxygen support during transportation use ambulance as a mode of transport.\n28\n Thus, distance from hospital and mode of transport to the hospital might serve as a reliable proxy indicator of illness severity and LOS.\nIn the current study, those who had breathlessness at the time of admission had longer LOS. This was analogous with the findings of Thiruvengadam et al\n13\n and Liu et al\n18\n but unlike the findings of Wu et al\n17\n who did not find this association. A systematic review and meta-analysis by Jain and Yuan\n29\n reported that dyspnea was the sole symptom which strongly predicted both intensive care unit (ICU) admission and serious disease. The said study also recommended using dyspnea as a measure of risk assessment and clinical management. Thus, breathlessness may be used as a prognostic indicator of LOS as well. With higher number of co-morbidities, LOS increased in the present study. This was identical with the finding of Thiruvengadam et al\n13\n which revealed similar opinion. This may be because with increased in the number of co-morbidities chances of developing a severe form of COVID-19 and mortality also reported to be increased.\n30\n Similarly, we observed that patients with COPD/asthma had slower recovery compared to others. A study in Mexico by Lee et al\n31\n reported that individuals with COPD are at higher risk of hospitalization and death owing to COVID-19. This might be because persons with chronic respiratory diseases like COPD and asthma have prevailing compromised lung function. COVID-19 infection might have further detorieted the lung function among them. This might have compelled them to hospitalize early. Unfortunately, most of them die. Those who survive recover gradually.32,33 Patients with deranged DBP at admission had longer LOS in our study. This was in line with the findings of a study in China by Ran et al\n22\n which reported that with elevation of DBP hazard of mortality among COVID-19 patients also increases. We observed significant association between higher qSOFA score and LOS in the present study. qSOFA score is a previously established prognostic indicator for COVID-19.34-36 An investigation in US by Wilfong et al\n36\n reported that on admission qSOFA score might predict ICU admission and fatality among COVID-19 patients.\nLimitations Despite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records.\nDespite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records.",
"Despite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records.",
"Factors elicited on arrival was found to be significantly associated with LOS. Travel distance, mode of transport to the hospital, breathlessness, number of co-morbidities, presence of COPD/ asthma, DBP and qSOFA score which are easily elicitable at the time of admission found to be significantly associated with LOS. Further research is warranted to gain more knowledge on this issue. These future studies should elicit effect of nutritional status and insurance coverage on LOS of COVID-19 patients which we could not do due lack of those data. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients."
] | [
"intro",
"materials|methods",
null,
null,
null,
null,
null,
"results",
"discussion",
null,
"conclusions"
] | [
"length of stay",
"COVID-19",
"association",
"dyspnea",
"comorbidity"
] | Introduction:
It’s being more than a year since the first case of COVID-19 reported in Wuhan, China.
1
Since then, the pandemic has affected millions of lives around the globe.
2
It has interrupted routine healthcare delivery and challenged healthcare infrastructure of even developed countries.3-8 India reported first ever COVID-19 case in January 2020.
9
Since then, the country has reported over 30 million COVID-19 cases and 4 00 000 deaths associated with the disease.
2
Length of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning.
10
Decreased LOS is reported to be associated with lowered risk of hospital acquired infection, reduction in financial burden for treatment among the patients, higher bed turnover rate of the hospitals (increases bed availability for the other patients) and vice versa can be told for increased LOS.11,12 For COVID-19 the reported associates of LOS are age, gender, nutritional status, presenting symptoms (ie, fever, breathlessness, fatigue, anorexia etc.), co-morbidities (ie, hypertension, heart disease, diabetes etc.), vitals (ie, respiratory rate, blood pressure etc.) laboratory parameters [ie, d-dimer, C-reactive protein (CRP), leucocyte count, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) etc.], radio graphical parameters (ie, chest X-ray, CT scan findings etc.), and medications (ie, tocilizumab, ACEI, ARB, metformin etc.).13-22 Although in terms of healthcare logistic planning the factors that could predict LOS of COVID-19 patients early bears additional importance. Patient characteristics and vital signs at the time of admission are being already linked with the level of care requirement, disease progression and mortality among COVID-19 patients by some prior studies.23-25 Although considering specifically for LOS, no such prior attempts have been made in Indian context.
Early identification of factors influencing LOS of COVID-19 patients may help policymakers to plan healthcare logistics (man, money, and material) for the disease accordingly. On extensive literature search we could only retrieve 2 prior Indian studies which have explored LOS among COVID-19 patients.13,14 Although these 2 studies had their own limitations. The study conducted by Thiruvengadam et al
13
did not report median survival time for the found associated variables. By contrast, Mishra et al
14
analyzed data extracted from media bulletin and elicited very few variables. Moreover, both these studies were conducted in Karnataka (a southern state of India). Therefore, the current retrospective case-control study was planned to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients admitted in a tertiary care hospital of East India.
Materials and Methods:
Study Setting and Period It was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.
It was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.
Sample Size Determination Considering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al
26
), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.
27
The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.
Considering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al
26
), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.
27
The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.
Sampling Technique During the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.
During the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.
Data Collection Procedure The variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.
The variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.
Statistical Analysis The data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.
The data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.
Study Setting and Period:
It was a retrospective, record based, unmatched, case control study using hospital records of COVID-19 patients admitted in an East Indian tertiary healthcare facility situated in Patna city beside holy river Ganges during May to October 2020. Our institute have pioneered COVID-19 care in the region since its emergence with over 400 general and 60 intensive care unit (ICU) beds and over 3000 dedicated healthcare workforces. During July to December, 2020 the institute have also functioned as COVID-19 dedicated tertiary healthcare facility to meet increasing tertiary COVID-19 care needs of the region. Those who were discharged as per medical advice were the cases, while those who died were deemed as controls for the study. Notably those who were admitted in non-COVID-19 wards of the hospital before ultimately being diagnosed as a COVID-19 patient and left against medical advice (LAMA) during the study period were not considered for inclusion.
Sample Size Determination:
Considering 1.75 relative hazard (median LOS in hospital for discharged and died cases were reported to be 14 and 8 days subsequently by Rees et al
26
), equal number of cases and controls, 99% precision and 85% power the minimum sample size for each group was calculated to be 167 using an online sample size calculator.
27
The said online sample size calculator uses following formula for sample size estimation in case of survival analysis: n = (Zα + Zβ)2/(log(RH))2q0q1 where RH = relative hazard, q1 = proportion of subjects that are in group 1 (exposed) and q0 = proportion of subjects that are in group 0 (unexposed); 1-q1.
Sampling Technique:
During the study period in total 2981 COVID-19 patients (tested positive by RTPCR/rapid antigen test) were admitted in the selected healthcare facility. Out of these 297 (9.9%) were LAMA and 234 (7.8%) were admitted in non-COVID-19 wards before being diagnosed as COVID-19 positive. So, these were excluded. Out of the rest, 2450 patients 303 eventually died. Separate line list of these 2147 (87.6%) discharged cases and 303 (12.4%) death cases were prepared. The required number of cases and controls were selected from these line lists using simple random sampling. Microsoft excel 2016 was used to generate random numbers.
Data Collection Procedure:
The variables extracted from the records of the selected COVID-19 patients were age (in completed years), sex (male/female), travel distance (distance of the current healthcare facility from his/her native place) (in kilometers), mode of seeking healthcare (contact tracing/self-reported) and mode of transport to the hospital (self-owned/ rented vehicle/ambulance). Presenting complaints like fever (yes/no), cough (yes/no), breathlessness (yes/no), sore throat (yes/no), diarrhea (yes/no), nausea/ vomiting (yes/no) and fatigue (yes/no) were documented. Presence of co-morbidities like ischemic heart disease (IHD) (yes/no), hypertension (HTN) (yes/no), diabetes (DM) (yes/no), cancer (yes/no), chronic obstructive pulmonary disease (COPD)/asthma (yes/no) and chronic kidney disease (CKD) (yes/no) were elicited. Vitals documented at the time of admission like respiratory rate (RR) (in breaths per minute), temperature (in degree Fahrenheit), oxygen saturation (SpO2) (in percentage), pulse rate (PR) (in beats per minute), systolic blood pressure (SBP) (in mmHg), diastolic blood pressure (DBP) (in mmHg), Glasgow coma scale (GCS) score and quick sequential organ failure assessment (qSOFA) score were also documented for the study.
Statistical Analysis:
The data were first entered in Microsoft excel 2016. Then imported on Statistical Package for Social Sciences (SPSS) (Chicago, USA) (version 22.0) for analysis. We presented categorical variables as frequency (percentage) while median [interquartile range (IQR)] were used to report continuous variables. We considered discharge from the hospital as an event (cases/survivors) while death was deemed as right censoring (control/non-survivors). Overall survival of the study subjects was depicted using Kaplan-Meier curve. To report survival time median [95% confidence interval (CI)] was used. To elicit association between LOS and its associates at first univariate cox proportional hazard regression was done. Attributes which were significant in univariate analysis (P ≤ .05) were entered to multivariable cox proportional hazard model using backward likelihood ratio (LR) method. The multivariable model with highest −2 log likelihood value was finally reported. The strength of association was measured in terms of hazard ratios (HRs) at 95% confidence limits.
Results:
The median age of the studied COVID-19 patients was 55 years with interquartile range (IQR) of 40 to 65 years. Overall, we found the median LOS for the survivors to be 8 days [interquartile range (IQR): 7-10 days] while the same for the non-survivors was 6 days [IQR: 2-11 days]. Survivors had more probability of higher LOS compared to non-survivors. In univariate cox-proportional hazard model; higher age, number of co-morbidities and travelling distance to seek healthcare, self-reporting to the healthcare facility, complaint of breathlessness, deranged RR, SpO2, PR, SBP, DBP, GCS, and qSOFA score on admission increased LOS while complaint of sore throat on admission decreased the same. Similarly, among the reported co-morbidities, COPD/asthma was found be associated with slowest recovery followed by CKD, diabetes, hypertension and ischemic heart disease. The median days since symptomatic prior to admission was 6 days with IQR of 4 to 8 days. The duration of symptoms prior to admission was not found to be associated with LOS [HR: 0.85, 95% CI: (0.63-1.16)] (Figure 1 and Table 1).
Kaplan Meier curve showing overall survival of the COVID-19 patients (n = 334).
Univariate Cox Proportional Hazard Model Showing Factors Associated With Length of Hospital Stay Among COVID-19 Patients (n = 334).
Abbreviations: CI: confidence interval; COPD: chronic obstructive pulmonary disease; DBP: diastolic blood pressure; GCS: Glasgow coma scale; HR: hazard ratio; LOS: length of hospital stay (in days); qSOFA: quick sequential organ failure assessment; ref.: reference; SBP: systolic blood pressure.
Due to right censoring in these groups median survival time could not be calculated; HR < 1 indicates higher LOS.
In the multivariable cox-proportional hazard model; travel distance (>16 km) [adjusted hazard ratio (aHR): 0.69, 95% CI: (0.50-0.95)], mode of transport to the hospital (ambulance) [aHR: 0.62, 95% CI: (0.45-0.85)], breathlessness (yes) [aHR: 0.56, 95% CI: (0.40-0.77)], number of co-morbidities (1-2) [aHR: 0.66, 95% CI: (0.47-0.93)] (≥3) [aHR: 0.16, 95% CI: (0.04-0.65)], COPD/asthma (yes) [aHR: 0.11, 95% CI: (0.01-0.79)], DBP (<60/≥90) [aHR: 0.55, 95% CI: (0.35-0.86)] and qSOFA score (≥2) [aHR: 0.33, 95% CI: (0.12-0.92)] were the significant attributes affecting LOS of the COVID-19 patients. Thus, those who resided within 16 km from the health facility, used self-owned/rented vehicle on way to the hospital, did not have breathlessness, COPD/asthma and had no/less number of co-morbidities, normal DBP (between 60 to 89 mmHg) and lower qSOFA score at the time of admission recovered earlier compared to others (Table 2).
Multivariable Cox Proportional Hazard Model Showing Factors Associated with Length of Hospital Stay Among COVID-19 Patients: n = 334.
Abbreviations: aHR, adjusted hazard ratio; CI, confidence interval; COPD, chronic obstructive pulmonary disease; DBP, diastolic blood pressure; GCS, Glasgow coma scale; qSOFA, quick sequential organ failure assessment; ref., reference.
aHR <1 indicates higher LOS.
The multivariable cox-proportional hazard model can be presented as h(t) = h0(t) exp[−0.37 × (travel distance>16)−0.48 × (mode of transport to the hospitalambulance)−0.59 × (breathlessness at admissionyes)–0.42 × (co-morbidity1-2)−1.87 × (co-morbidity≥3)−2.25 × (COPD/asthmayes)−0.60 × (DBP<60/≥90)−1.11 × (qSOFA score≥2)]
Discussion:
In this retrospective case-control study we found that higher travelling distance, use of an ambulance as mode of transport, presence of breathlessness at admission, co-morbidities, COPD/asthma, deranged DBP, and higher qSOFA score at admission were associated with greater duration of hospitalization. Demised COVID-19 patients had shorter LOS compared to those who were discharged. Development of a scoring system inculcating found significant attributes of LOS, giving each due weightage as per their adjusted strength of association might be useful in early prediction of LOS of COVID-19 patients. Moreover, linking this scoring system with the logistic management system of the hospital might help in optimal use of the available resources. This might also help in optimizing patient care and alleviate various logistical barriers associated with it (ie, shortage of staff, beds, medicines, equipment’s etc.).
We found that patients who required to travel higher distance from their native place to seek healthcare from the present healthcare facility had longer LOS. It might be because our institute is the highest referral institute for most of the healthcare facilities in the state of Bihar. Therefore, usually critically ill patients from the peripheral institutes are used to be referred to our institute. Critically ill COVID-19 patients do usually have a longer LOS, as reported by a systematic review by Rees et al
26
Similarly, we found that those who used ambulance as mode of transport to the present healthcare facility had longer LOS. It was previously known that seriously ill and/or patients needing oxygen support during transportation use ambulance as a mode of transport.
28
Thus, distance from hospital and mode of transport to the hospital might serve as a reliable proxy indicator of illness severity and LOS.
In the current study, those who had breathlessness at the time of admission had longer LOS. This was analogous with the findings of Thiruvengadam et al
13
and Liu et al
18
but unlike the findings of Wu et al
17
who did not find this association. A systematic review and meta-analysis by Jain and Yuan
29
reported that dyspnea was the sole symptom which strongly predicted both intensive care unit (ICU) admission and serious disease. The said study also recommended using dyspnea as a measure of risk assessment and clinical management. Thus, breathlessness may be used as a prognostic indicator of LOS as well. With higher number of co-morbidities, LOS increased in the present study. This was identical with the finding of Thiruvengadam et al
13
which revealed similar opinion. This may be because with increased in the number of co-morbidities chances of developing a severe form of COVID-19 and mortality also reported to be increased.
30
Similarly, we observed that patients with COPD/asthma had slower recovery compared to others. A study in Mexico by Lee et al
31
reported that individuals with COPD are at higher risk of hospitalization and death owing to COVID-19. This might be because persons with chronic respiratory diseases like COPD and asthma have prevailing compromised lung function. COVID-19 infection might have further detorieted the lung function among them. This might have compelled them to hospitalize early. Unfortunately, most of them die. Those who survive recover gradually.32,33 Patients with deranged DBP at admission had longer LOS in our study. This was in line with the findings of a study in China by Ran et al
22
which reported that with elevation of DBP hazard of mortality among COVID-19 patients also increases. We observed significant association between higher qSOFA score and LOS in the present study. qSOFA score is a previously established prognostic indicator for COVID-19.34-36 An investigation in US by Wilfong et al
36
reported that on admission qSOFA score might predict ICU admission and fatality among COVID-19 patients.
Limitations Despite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records.
Despite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records.
Limitations:
Despite adaptation of case-control design, we could not match background characteristics of the cases and controls. It could not be accomplished owing to restricted availability of controls for the study. However, use of simple random sampling might have alleviated chances of bias arising out of non-matching to some extent. There may be various other possible attributes (ie, nutritional status, insurance coverage, etc.) which might influence LOS of COVID patients.20,37 Effect of these factors on LOS could not be elicited due to non-documentation of those data in our hospital records.
Conclusion:
Factors elicited on arrival was found to be significantly associated with LOS. Travel distance, mode of transport to the hospital, breathlessness, number of co-morbidities, presence of COPD/ asthma, DBP and qSOFA score which are easily elicitable at the time of admission found to be significantly associated with LOS. Further research is warranted to gain more knowledge on this issue. These future studies should elicit effect of nutritional status and insurance coverage on LOS of COVID-19 patients which we could not do due lack of those data. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients.
| Background:
Length of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning. The study aimed to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients.
Methods:
It was a retrospective, record based, unmatched, case control study using hospital records of 334 COVID-19 patients admitted in an East Indian tertiary healthcare facility during May to October 2020. Discharge from the hospital (cases/survivors) was considered as an event while death (control/non-survivors) as right censoring in the case-control survival analysis using cox proportional hazard model.
Results:
Overall, we found the median LOS for the survivors to be 8 days [interquartile range (IQR): 7-10 days] while the same for the non-survivors was 6 days [IQR: 2-11 days]. In the multivariable cox-proportional hazard model; travel distance (>16 km) [adjusted hazard ratio (aHR): 0.69, 95% CI: (0.50-0.95)], mode of transport to the hospital (ambulance) [aHR: 0.62, 95% CI: (0.45-0.85)], breathlessness (yes) [aHR: 0.56, 95% CI: (0.40-0.77)], number of co-morbidities (1-2) [aHR: 0.66, 95% CI: (0.47-0.93)] (≥3) [aHR: 0.16, 95% CI: (0.04-0.65)], COPD/asthma (yes) [ [aHR: 0.11, 95% CI: (0.01-0.79)], DBP (<60/≥90) [aHR: 0.55, 95% CI: (0.35-0.86)] and qSOFA score (≥2) [aHR: 0.33, 95% CI: (0.12-0.92)] were the significant attributes affecting LOS of the COVID-19 patients.
Conclusions:
Factors elicited on arrival were found to be significantly associated with LOS. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients. | Introduction:
It’s being more than a year since the first case of COVID-19 reported in Wuhan, China.
1
Since then, the pandemic has affected millions of lives around the globe.
2
It has interrupted routine healthcare delivery and challenged healthcare infrastructure of even developed countries.3-8 India reported first ever COVID-19 case in January 2020.
9
Since then, the country has reported over 30 million COVID-19 cases and 4 00 000 deaths associated with the disease.
2
Length of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning.
10
Decreased LOS is reported to be associated with lowered risk of hospital acquired infection, reduction in financial burden for treatment among the patients, higher bed turnover rate of the hospitals (increases bed availability for the other patients) and vice versa can be told for increased LOS.11,12 For COVID-19 the reported associates of LOS are age, gender, nutritional status, presenting symptoms (ie, fever, breathlessness, fatigue, anorexia etc.), co-morbidities (ie, hypertension, heart disease, diabetes etc.), vitals (ie, respiratory rate, blood pressure etc.) laboratory parameters [ie, d-dimer, C-reactive protein (CRP), leucocyte count, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) etc.], radio graphical parameters (ie, chest X-ray, CT scan findings etc.), and medications (ie, tocilizumab, ACEI, ARB, metformin etc.).13-22 Although in terms of healthcare logistic planning the factors that could predict LOS of COVID-19 patients early bears additional importance. Patient characteristics and vital signs at the time of admission are being already linked with the level of care requirement, disease progression and mortality among COVID-19 patients by some prior studies.23-25 Although considering specifically for LOS, no such prior attempts have been made in Indian context.
Early identification of factors influencing LOS of COVID-19 patients may help policymakers to plan healthcare logistics (man, money, and material) for the disease accordingly. On extensive literature search we could only retrieve 2 prior Indian studies which have explored LOS among COVID-19 patients.13,14 Although these 2 studies had their own limitations. The study conducted by Thiruvengadam et al
13
did not report median survival time for the found associated variables. By contrast, Mishra et al
14
analyzed data extracted from media bulletin and elicited very few variables. Moreover, both these studies were conducted in Karnataka (a southern state of India). Therefore, the current retrospective case-control study was planned to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients admitted in a tertiary care hospital of East India.
Conclusion:
Factors elicited on arrival was found to be significantly associated with LOS. Travel distance, mode of transport to the hospital, breathlessness, number of co-morbidities, presence of COPD/ asthma, DBP and qSOFA score which are easily elicitable at the time of admission found to be significantly associated with LOS. Further research is warranted to gain more knowledge on this issue. These future studies should elicit effect of nutritional status and insurance coverage on LOS of COVID-19 patients which we could not do due lack of those data. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients. | Background:
Length of hospital stay (LOS) for a disease is a vital estimate for healthcare logistics planning. The study aimed to illustrate the effect of factors elicited on arrival on LOS of the COVID-19 patients.
Methods:
It was a retrospective, record based, unmatched, case control study using hospital records of 334 COVID-19 patients admitted in an East Indian tertiary healthcare facility during May to October 2020. Discharge from the hospital (cases/survivors) was considered as an event while death (control/non-survivors) as right censoring in the case-control survival analysis using cox proportional hazard model.
Results:
Overall, we found the median LOS for the survivors to be 8 days [interquartile range (IQR): 7-10 days] while the same for the non-survivors was 6 days [IQR: 2-11 days]. In the multivariable cox-proportional hazard model; travel distance (>16 km) [adjusted hazard ratio (aHR): 0.69, 95% CI: (0.50-0.95)], mode of transport to the hospital (ambulance) [aHR: 0.62, 95% CI: (0.45-0.85)], breathlessness (yes) [aHR: 0.56, 95% CI: (0.40-0.77)], number of co-morbidities (1-2) [aHR: 0.66, 95% CI: (0.47-0.93)] (≥3) [aHR: 0.16, 95% CI: (0.04-0.65)], COPD/asthma (yes) [ [aHR: 0.11, 95% CI: (0.01-0.79)], DBP (<60/≥90) [aHR: 0.55, 95% CI: (0.35-0.86)] and qSOFA score (≥2) [aHR: 0.33, 95% CI: (0.12-0.92)] were the significant attributes affecting LOS of the COVID-19 patients.
Conclusions:
Factors elicited on arrival were found to be significantly associated with LOS. A scoring system inculcating these factors may be developed to predict LOS of the COVID-19 patients. | 5,344 | 406 | [
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] | null | [CONTENT] length of stay | COVID-19 | association | dyspnea | comorbidity [SUMMARY] | null | [CONTENT] length of stay | COVID-19 | association | dyspnea | comorbidity [SUMMARY] | [CONTENT] length of stay | COVID-19 | association | dyspnea | comorbidity [SUMMARY] | [CONTENT] length of stay | COVID-19 | association | dyspnea | comorbidity [SUMMARY] | [CONTENT] length of stay | COVID-19 | association | dyspnea | comorbidity [SUMMARY] | [CONTENT] COVID-19 | Case-Control Studies | Humans | India | Length of Stay | Retrospective Studies | Risk Factors | SARS-CoV-2 | Survival Analysis | Tertiary Healthcare [SUMMARY] | null | [CONTENT] COVID-19 | Case-Control Studies | Humans | India | Length of Stay | Retrospective Studies | Risk Factors | SARS-CoV-2 | Survival Analysis | Tertiary Healthcare [SUMMARY] | [CONTENT] COVID-19 | Case-Control Studies | Humans | India | Length of Stay | Retrospective Studies | Risk Factors | SARS-CoV-2 | Survival Analysis | Tertiary Healthcare [SUMMARY] | [CONTENT] COVID-19 | Case-Control Studies | Humans | India | Length of Stay | Retrospective Studies | Risk Factors | SARS-CoV-2 | Survival Analysis | Tertiary Healthcare [SUMMARY] | [CONTENT] COVID-19 | Case-Control Studies | Humans | India | Length of Stay | Retrospective Studies | Risk Factors | SARS-CoV-2 | Survival Analysis | Tertiary Healthcare [SUMMARY] | [CONTENT] covid 19 infection | mortality covid 19 | hospitalization demised covid | los covid patients | hospital stay covid [SUMMARY] | null | [CONTENT] covid 19 infection | mortality covid 19 | hospitalization demised covid | los covid patients | hospital stay covid [SUMMARY] | [CONTENT] covid 19 infection | mortality covid 19 | hospitalization demised covid | los covid patients | hospital stay covid [SUMMARY] | [CONTENT] covid 19 infection | mortality covid 19 | hospitalization demised covid | los covid patients | hospital stay covid [SUMMARY] | [CONTENT] covid 19 infection | mortality covid 19 | hospitalization demised covid | los covid patients | hospital stay covid [SUMMARY] | [CONTENT] covid | covid 19 | 19 | los | yes | patients | study | hospital | healthcare | covid 19 patients [SUMMARY] | null | [CONTENT] covid | covid 19 | 19 | los | yes | patients | study | hospital | healthcare | covid 19 patients [SUMMARY] | [CONTENT] covid | covid 19 | 19 | los | yes | patients | study | hospital | healthcare | covid 19 patients [SUMMARY] | [CONTENT] covid | covid 19 | 19 | los | yes | patients | study | hospital | healthcare | covid 19 patients [SUMMARY] | [CONTENT] covid | covid 19 | 19 | los | yes | patients | study | hospital | healthcare | covid 19 patients [SUMMARY] | [CONTENT] ie | etc | covid 19 | los | 19 | covid | studies | disease | india | patients [SUMMARY] | null | [CONTENT] ahr | 95 ci | ci | 95 | days | 16 | hazard | hazard model | cox proportional hazard | proportional [SUMMARY] | [CONTENT] significantly | found significantly associated los | found significantly associated | found significantly | significantly associated los | significantly associated | los | associated los | found | los covid 19 patients [SUMMARY] | [CONTENT] yes | 19 | covid 19 | covid | los | patients | study | healthcare | hazard | cases [SUMMARY] | [CONTENT] yes | 19 | covid 19 | covid | los | patients | study | healthcare | hazard | cases [SUMMARY] | [CONTENT] healthcare logistics planning ||| COVID-19 [SUMMARY] | null | [CONTENT] 8 days ||| IQR | 7-10 days | 6 days ||| 2-11 days ||| 16 km | 0.69 | 95% | CI | 0.50-0.95 ||| 0.62 | 95% | CI | 0.45-0.85 ||| 0.56 | 95% | CI | 0.40 | 1-2 ||| 0.66 | 95% | CI | 0.47-0.93 | 0.16 | 95% | CI | 0.04-0.65 ||| 0.11 | 95% | CI | 0.01 | DBP ||| 0.55 | 95% | CI | 0.35-0.86 ||| 0.33 | 95% | CI | 0.12-0.92 | COVID-19 [SUMMARY] | [CONTENT] ||| COVID-19 [SUMMARY] | [CONTENT] healthcare logistics planning ||| COVID-19 ||| 334 | East Indian | May to October 2020 ||| ||| 8 days ||| IQR | 7-10 days | 6 days ||| 2-11 days ||| 16 km | 0.69 | 95% | CI | 0.50-0.95 ||| 0.62 | 95% | CI | 0.45-0.85 ||| 0.56 | 95% | CI | 0.40 | 1-2 ||| 0.66 | 95% | CI | 0.47-0.93 | 0.16 | 95% | CI | 0.04-0.65 ||| 0.11 | 95% | CI | 0.01 | DBP ||| 0.55 | 95% | CI | 0.35-0.86 ||| 0.33 | 95% | CI | 0.12-0.92 | COVID-19 ||| ||| COVID-19 [SUMMARY] | [CONTENT] healthcare logistics planning ||| COVID-19 ||| 334 | East Indian | May to October 2020 ||| ||| 8 days ||| IQR | 7-10 days | 6 days ||| 2-11 days ||| 16 km | 0.69 | 95% | CI | 0.50-0.95 ||| 0.62 | 95% | CI | 0.45-0.85 ||| 0.56 | 95% | CI | 0.40 | 1-2 ||| 0.66 | 95% | CI | 0.47-0.93 | 0.16 | 95% | CI | 0.04-0.65 ||| 0.11 | 95% | CI | 0.01 | DBP ||| 0.55 | 95% | CI | 0.35-0.86 ||| 0.33 | 95% | CI | 0.12-0.92 | COVID-19 ||| ||| COVID-19 [SUMMARY] |
|||||
Evaluation of sonic, ultrasonic, and laser irrigation activation systems to eliminate bacteria from the dentinal tubules of the root canal system. | 36417594 | Aiming to kill bacteria in dentin tubules of infected dental pulp cavities, we evaluated the effects of sodium hypochlorite (NaOCl) solution agitated by different irrigation protocols, i.e., conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), the EDDY tip, and the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser. The EDDY achieved good antibacterial effects as passive ultrasonic irrigation in the coronal and middle thirds. Nd:YAP laser irradiation and PUI were effective in the apical third of the root canal. | INTRODUCTION | Infected root canal models were established, and roots were randomly divided into six groups: negative control, positive control, CNI, PUI, sonic agitation with EDDY, and Nd:YAP laser groups. After irrigation, the teeth were split and stained using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacteria depth was evaluated by a confocal laser scanning microscopy and the temperature at the root surface was assessed using a thermal imaging camera during the irrigation process. | METHODOLOGY | In the coronal and middle thirds of the root canal, PUI and EDDY had stronger antibacterial effects than CNI (p<0.05); in the apical third, the antibacterial effects of PUI and Nd:YAP laser-activated irrigation were better than CNI (p<0.05). The maximum change in temperature was significantly greater during continuous Nd:YAP laser application compared with the other methods, but intermittent irrigation helped lessening this trend. | RESULTS | NaOCl agitated by EDDY tip and PUI exhibited a similar bacteria elimination effect in the coronal and middle root canal. Nd:YAP laser was effective in the apical third and intermittent irrigation reduced its thermal impact. | CONCLUSIONS | [
"Ultrasonics",
"Dental Pulp Cavity",
"Root Canal Therapy",
"Bacteria",
"Anti-Bacterial Agents"
] | 9724494 | Introduction | Chemo-mechanical cleaning and shaping of the root canal system to remove or reduce bacterial populations are essential for infection control, which is the main goal of root canal treatment.1 However, the complex anatomy of the root canal system limits the mechanical preparation, particularly in the apical third of the root.2 In infected root canals, bacteria can grow 400 μm or more into the dentinal tubules.3 Due to the strength and resistance of the remaining root canal wall, classic mechanical preparation generally only cuts the dentin at a depth of 150 μm, which is impossible to remove deep infection.4 Chemical irrigation is an effective supplemental method as it can reach more areas of the root canal surface.5 Techniques such as passive ultrasonic irrigation (PUI), sonic irrigation, and laser-activated irrigation enhance the disinfection effects of chemical irrigation and improve clinical outcomes.6
PUI activates irrigation via acoustic streaming and cavitation and its disinfecting effect is better than the conventional needle irrigation (CNI).7,8 The EDDY system (VDW, Munich, Germany) provides high-frequency sonic irrigation at 6000 Hz.9 Although previous studies have compared the antibacterial efficacies of EDDY, PUI, and CNI, the results have been inconsistent, presumably due to the different evaluation indicators employed.10–12 Most studies have quantified cultured residual bacteria, but some anaerobic bacteria are difficult to cultivate under normal conditions.13 Additionally, most studies12,14,15 used sterile paper points for sampling the root canals, however, this method only evaluates infection clearance for the overall root canal space, and not specifically for the dentinal tubules of the root canal wall.
Studies on the application of lasers to kill bacteria in root canals have been conducted since Fegan and Steiman16 (1995) evaluated the antibacterial effects of intracanal Nd:YAG laser irradiation, which may improve disinfection through a photothermal effect and by enhancing the chemical effects of irrigants.17
Commonly used lasers for root canal disinfection include the erbium-doped chromium-yttrium-scandium-gallium garnet (Er,Cr:YSGG) laser, the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser, and the photon-induced photoacoustic streaming laser.18 Wang, et al.17 (2018) compared the antibacterial effects of different lasers on deep regions of dentinal tubules in a prepared dentin block infected by Enterococcus faecalis, resulting in Nd:YAP laser irradiation without NaOCl having the weakest effect among all. This finding probably relates to differences in the working tip construction. The end of the Nd:YAP laser fiber is flat, thus the light can only propagate in a straight line, which may result in bacterial survival. The Er:YAG and Er,Cr:YSGG lasers have conically shaped, radial firing tips that irradiate the root canals in three dimensions, leading to more widespread bacterial death.19 The Nd:YAP laser is a near-infrared laser with a wavelength of 1340 nm and working fiber diameter of 200–320 μm.20 Liu, et al.21 (2019) reported that Nd:YAP laser agitating NaOCl at 280 and 360 mJ achieved effective antibacterial effects. However, the study evaluated only the proportion of dead bacteria in the entire root canal wall. Najah, Sid, Ghodbane22 (2016) found that the antibacterial effects of 2.5% NaOCl agitated by Nd:YAP laser and passive ultrasonic irrigation were similar, in which both irrigation protocols showed better effects than CNI with 2.5% NaOCl. However, the bacterial counting method they used to compare the numbers of residual microorganisms failed to assess the dead bacteria deep in the dentinal tubules. We currently lack research evaluating the dead bacteria depth in the dentinal tubules of the root canal wall with Nd:YAP lasers, especially regarding the apical region. The main limitation of the laser method is its thermal impact during application, which may damage the periodontal ligament. Namour, et al.20 (2016) found that given proper working parameters, the temperature increase caused by the Nd:YAP laser during continuously endodontic irrigation with 2.25% NaOCl would not damage periodontal tissue. Moreover, Zhang and Wang23 (2021) reported that when using the Nd:YAP laser ablating separated files in root canal, the increase in temperature at the root surface was <10°C. But Rochd, Calas, Roques24 (1998) found that the temperature of the outer surface of the tooth root could increase by approximately 25°C after continuous laser operation in bacterial suspension for 28 s, which may cause thermal damage. Although clinicians usually use this laser intermittently for root canal disinfection, we lack research concerning the temperature increase.
Overall, few studies have focused on the effects of NaOCl agitated by the EDDY tip and Nd:YAP laser on eliminating bacteria in dentinal tubules of infected root canal walls, especially in the apical region. Most of the research concerning the thermal impact of Nd:YAP laser compares it to other lasers (or its outcomes using different parameters), and comparisons with PUI and sonic irrigation are lacking. Therefore, the aim of this study was to compare the antimicrobial efficiency of NaOCl agitated by the EDDY, PUI, and Nd:YAP laser with CNI. We hypothesized that neither EDDY nor Nd:YAP laser-activated irrigation would exhibit better antibacterial effects than PUI. This study also compared the temperature increase on the root outer surface caused by the Nd:YAP laser with ultrasonic, sonic, and syringe irrigation devices in vitro, thus providing experimental evidence for the selection of safe and effective final irrigation protocols in root canal treatment. | Methodology | Tooth selection and preparation A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.
A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.
Establishment of E. faecalis infection To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.
The infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.
To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.
The infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.
Root canal irrigation protocols After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.
Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.
No root canal irrigation was performed in this group after bacterial incubation.
Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.
Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.
No root canal irrigation was performed in this group after bacterial incubation.
Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
CLSM evaluation After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).
After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).
Heat production evaluation Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.
Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.
Statistical analysis The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).
The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA). | Results | Establishment of root canal models infected with E. faecalis Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.
Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.
Dead bacteria depths of different irrigation protocols Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.
The red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).
In the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.
Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.
The red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).
In the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.
Evaluation of heat production All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.
SD, standard deviation.
The same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.
The mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.
Figure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.
All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.
SD, standard deviation.
The same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.
The mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.
Figure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups. | Conclusions | The NaOCl solution agitated by the EDDY system showed a potent bacterial killing effect in the dentinal tubules of coronal and middle thirds of root canal wall, but this effect was limited in the apical third. The NaOCl solution agitated by the Nd:YAP laser showed no advantage in terms of killing bacteria in dentinal tubules of coronal and middle thirds of the root canal compared to the PUI or EDDY but achieved similar antibacterial effect to the PUI in the apical third. Under the conditions used in this study, the increase in temperature at the root surface caused by the intermittent irrigation protocol is safe for clinical application. | [
"Tooth selection and preparation",
"Establishment of E. faecalis infection",
"Root canal irrigation protocols",
"Group 1: Negative control (n=2)",
"Group 2: Positive control (n=2)",
"Group 3: CNI (n=5)",
"Group 4: PUI (n=5)",
"Group 5: High-frequency sonic irrigation (n = 5)",
"Group 6: Nd:YAP laser (n=5)",
"CLSM evaluation",
"Heat production evaluation",
"Statistical analysis",
"Establishment of root canal models infected with E. faecalis",
"Dead bacteria depths of different irrigation protocols",
"Evaluation of heat production"
] | [
"A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.",
"To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.\nThe infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.",
"After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.\n Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.\nNo root canal irrigation was performed in this group after bacterial incubation.\n Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\nTeeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\n Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\nCNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\n Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\nThe canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\n Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\nThe activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\n Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.\nA 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.",
"No root canal irrigation was performed in this group after bacterial incubation.",
"Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.",
"CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.",
"The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.",
"The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.",
"A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.",
"After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).",
"Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.",
"The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).",
"Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.",
"Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.\nThe red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).\nIn the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.",
"All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.\nSD, standard deviation.\nThe same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.\nThe mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.\nFigure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups."
] | [
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] | [
"Introduction",
"Methodology",
"Tooth selection and preparation",
"Establishment of E. faecalis infection",
"Root canal irrigation protocols",
"Group 1: Negative control (n=2)",
"Group 2: Positive control (n=2)",
"Group 3: CNI (n=5)",
"Group 4: PUI (n=5)",
"Group 5: High-frequency sonic irrigation (n = 5)",
"Group 6: Nd:YAP laser (n=5)",
"CLSM evaluation",
"Heat production evaluation",
"Statistical analysis",
"Results",
"Establishment of root canal models infected with E. faecalis",
"Dead bacteria depths of different irrigation protocols",
"Evaluation of heat production",
"Discussion",
"Conclusions"
] | [
"Chemo-mechanical cleaning and shaping of the root canal system to remove or reduce bacterial populations are essential for infection control, which is the main goal of root canal treatment.1 However, the complex anatomy of the root canal system limits the mechanical preparation, particularly in the apical third of the root.2 In infected root canals, bacteria can grow 400 μm or more into the dentinal tubules.3 Due to the strength and resistance of the remaining root canal wall, classic mechanical preparation generally only cuts the dentin at a depth of 150 μm, which is impossible to remove deep infection.4 Chemical irrigation is an effective supplemental method as it can reach more areas of the root canal surface.5 Techniques such as passive ultrasonic irrigation (PUI), sonic irrigation, and laser-activated irrigation enhance the disinfection effects of chemical irrigation and improve clinical outcomes.6\nPUI activates irrigation via acoustic streaming and cavitation and its disinfecting effect is better than the conventional needle irrigation (CNI).7,8 The EDDY system (VDW, Munich, Germany) provides high-frequency sonic irrigation at 6000 Hz.9 Although previous studies have compared the antibacterial efficacies of EDDY, PUI, and CNI, the results have been inconsistent, presumably due to the different evaluation indicators employed.10–12 Most studies have quantified cultured residual bacteria, but some anaerobic bacteria are difficult to cultivate under normal conditions.13 Additionally, most studies12,14,15 used sterile paper points for sampling the root canals, however, this method only evaluates infection clearance for the overall root canal space, and not specifically for the dentinal tubules of the root canal wall.\nStudies on the application of lasers to kill bacteria in root canals have been conducted since Fegan and Steiman16 (1995) evaluated the antibacterial effects of intracanal Nd:YAG laser irradiation, which may improve disinfection through a photothermal effect and by enhancing the chemical effects of irrigants.17\nCommonly used lasers for root canal disinfection include the erbium-doped chromium-yttrium-scandium-gallium garnet (Er,Cr:YSGG) laser, the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser, and the photon-induced photoacoustic streaming laser.18 Wang, et al.17 (2018) compared the antibacterial effects of different lasers on deep regions of dentinal tubules in a prepared dentin block infected by Enterococcus faecalis, resulting in Nd:YAP laser irradiation without NaOCl having the weakest effect among all. This finding probably relates to differences in the working tip construction. The end of the Nd:YAP laser fiber is flat, thus the light can only propagate in a straight line, which may result in bacterial survival. The Er:YAG and Er,Cr:YSGG lasers have conically shaped, radial firing tips that irradiate the root canals in three dimensions, leading to more widespread bacterial death.19 The Nd:YAP laser is a near-infrared laser with a wavelength of 1340 nm and working fiber diameter of 200–320 μm.20 Liu, et al.21 (2019) reported that Nd:YAP laser agitating NaOCl at 280 and 360 mJ achieved effective antibacterial effects. However, the study evaluated only the proportion of dead bacteria in the entire root canal wall. Najah, Sid, Ghodbane22 (2016) found that the antibacterial effects of 2.5% NaOCl agitated by Nd:YAP laser and passive ultrasonic irrigation were similar, in which both irrigation protocols showed better effects than CNI with 2.5% NaOCl. However, the bacterial counting method they used to compare the numbers of residual microorganisms failed to assess the dead bacteria deep in the dentinal tubules. We currently lack research evaluating the dead bacteria depth in the dentinal tubules of the root canal wall with Nd:YAP lasers, especially regarding the apical region. The main limitation of the laser method is its thermal impact during application, which may damage the periodontal ligament. Namour, et al.20 (2016) found that given proper working parameters, the temperature increase caused by the Nd:YAP laser during continuously endodontic irrigation with 2.25% NaOCl would not damage periodontal tissue. Moreover, Zhang and Wang23 (2021) reported that when using the Nd:YAP laser ablating separated files in root canal, the increase in temperature at the root surface was <10°C. But Rochd, Calas, Roques24 (1998) found that the temperature of the outer surface of the tooth root could increase by approximately 25°C after continuous laser operation in bacterial suspension for 28 s, which may cause thermal damage. Although clinicians usually use this laser intermittently for root canal disinfection, we lack research concerning the temperature increase.\nOverall, few studies have focused on the effects of NaOCl agitated by the EDDY tip and Nd:YAP laser on eliminating bacteria in dentinal tubules of infected root canal walls, especially in the apical region. Most of the research concerning the thermal impact of Nd:YAP laser compares it to other lasers (or its outcomes using different parameters), and comparisons with PUI and sonic irrigation are lacking. Therefore, the aim of this study was to compare the antimicrobial efficiency of NaOCl agitated by the EDDY, PUI, and Nd:YAP laser with CNI. We hypothesized that neither EDDY nor Nd:YAP laser-activated irrigation would exhibit better antibacterial effects than PUI. This study also compared the temperature increase on the root outer surface caused by the Nd:YAP laser with ultrasonic, sonic, and syringe irrigation devices in vitro, thus providing experimental evidence for the selection of safe and effective final irrigation protocols in root canal treatment.",
" Tooth selection and preparation A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.\nA total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.\n Establishment of E. faecalis infection To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.\nThe infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.\nTo confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.\nThe infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.\n Root canal irrigation protocols After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.\n Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.\nNo root canal irrigation was performed in this group after bacterial incubation.\n Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\nTeeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\n Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\nCNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\n Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\nThe canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\n Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\nThe activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\n Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.\nA 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.\nAfter incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.\n Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.\nNo root canal irrigation was performed in this group after bacterial incubation.\n Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\nTeeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\n Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\nCNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\n Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\nThe canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\n Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\nThe activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\n Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.\nA 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.\n CLSM evaluation After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).\nAfter the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).\n Heat production evaluation Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.\nTen additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.\n Statistical analysis The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).\nThe normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).",
"A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.",
"To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.\nThe infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.",
"After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.\n Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.\nNo root canal irrigation was performed in this group after bacterial incubation.\n Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\nTeeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.\n Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\nCNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.\n Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\nThe canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.\n Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\nThe activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.\n Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.\nA 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.",
"No root canal irrigation was performed in this group after bacterial incubation.",
"Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.",
"CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.",
"The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.",
"The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.",
"A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.",
"After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).",
"Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.",
"The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).",
" Establishment of root canal models infected with E. faecalis Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.\nFigure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.\n Dead bacteria depths of different irrigation protocols Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.\nThe red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).\nIn the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.\nFigure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.\nThe red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).\nIn the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.\n Evaluation of heat production All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.\nSD, standard deviation.\nThe same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.\nThe mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.\nFigure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.\nAll samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.\nSD, standard deviation.\nThe same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.\nThe mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.\nFigure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.",
"Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.",
"Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.\nThe red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).\nIn the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.",
"All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.\nSD, standard deviation.\nThe same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.\nThe mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.\nFigure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.",
"This study compared the antibacterial effects of NaOCl agitated by four irrigation protocols on infected root canal walls. We calculated the depth of dead bacteria in the dentinal tubules of coronal, middle, and apical segments of the root via CLSM images analysis. The data shows the efficacies of different irrigation protocols for removing infections in the dentinal tubules.\nSeveral studies showed that the cleaning effects of dynamic irrigation are limited in the apical region.12,27,28 In our study, we found that the effects of NaOCl agitated by the PUI and EDDY showed a similar trend in the apical third. This finding probably relates to the small diameter of the root canal in the apical area and the short distance between the working tip and the root canal wall, which limits the effects of acoustic flow.6 However, we failed to find significant differences in the effects of the Nd:YAP laser in the coronal, middle, or apical thirds. The laser irradiation mainly inhibits bacterial growth via photothermal action. Its fiber head heat output can directly destroy the cell wall and the irrigants can absorb it, transferring heat into the dentinal tubules to kill bacteria.18 In this study, we moved the Nd:YAP laser fiber up and down 2 mm short of the WL, so that the irrigants in the root canal space could evenly absorb the heat. The uniform distribution of heat probably led to the relatively even depth of red fluorescence observed throughout the root canal.\nIn coronal and middle thirds, the antibacterial effect of the EDDY was equivalent to PUI. This finding indicates that the ultrasonic oscillation and sonic working tips could potentially achieve greater acoustic flow due to the unlimited space in middle and coronal parts of root canal.29 Although the EDDY frequency is lower than PUI, its vibration amplitude is larger. The irrigants velocity positively relates to the working tips amplitude30 Salas, et al.31 (2021) found that the penetration depth of 2% chlorhexidine irrigated by EDDY or PUI was similar in the cervical and middle region of the root canal. Meanwhile, the laser-activated irrigation was not significantly weaker than PUI or EDDY, nor significantly better than CNI. With CNI irrigation, irrigants have sufficient backflow space and the procedure is less affected by factors such as needle entry depth.32 These results suggest that, in the middle and coronal regions of the root canal, dynamic irrigation can achieve good results, whereas Nd:YAP lacks obvious advantages. However, because we removed more resistance in coronal third than what is commonly performed in clinical practice, coronal resistance may slightly differ between this study and an actual clinical situation, which may allow PUI and EDDY vibration to occur under less constrained conditions for potentially better effects.\nMeanwhile, in apical third, the Nd:YAP laser showed an effective antibacterial effect like PUI and both groups showed significantly better antibacterial effects compared to CNI. The flexible and small in diameter fiber of the Nd:YAP laser can enter the prepared apical root canal smoothly and kill bacteria through its photothermal effects.13 The EDDY was neither significantly weaker than PUI nor significantly better than CNI. Ahmad, et al.33 (1988) found that lateral displacement of the tip of an ultrasonic file can reach approximately 40 μm, less than the oscillation amplitude of sonic working tips. Hence, tips used in the EDDY are more likely to contact the root canal wall than those of PUI. Walmsley, Lumley, Laird34 (1989) found that when the movement of the sonic file is constrained, its sideway oscillation disappears and its movement pattern can be converted into pure longitudinal vibration; thus reducing the irrigants penetration depth and clearing infections inside the dentinal tubules.\nIn this study, we choose a single-species biofilm model E. faecalis to establish an infection model. This model has been used in many studies35,36 and is considered effective for evaluating root canal disinfection. However, multi-species bacterial biofilm has been used in recent studies of infected root canals. Hoedke, et al.37 (2018) inoculated the root canal with E. faecalis, Streptococcus oralis (S. oralis), and Prevotella intermedia (P. intermedia) to analyze the antibacterial effect of photodynamic therapy. Swimberghe, et al.38 (2021) found that, when grown in a multispecies biofilm, E. faecalis showed significantly less susceptibility to NaOCl than a monospecies biofilm. However, the cultivation conditions for the multi-species bacterial biofilm may be stricter. For further and future research, our goal is to establish a multi-species bacterial biofilm infection model.\nNajah, Sid and Ghodbane22 (2016) used the bacterial count method to compare the reduction of bacterial load in root canals after PUI and Nd:YAP laser-activated irrigation. The results failed to show significant differences between the groups. Calculating the reduction in bacterial load is a better solution because it represents a quantitative measure. However, this method mainly detects bacteria suspended in the root canal space and is not used to detect bacterial biofilm on the root canal wall or in the dentinal tubules. It also requires bacterial sampling, inoculation, and incubation increasing the chances of introducing microorganisms.39 In our study, we evaluated the depth of dead bacteria via CLSM. This step can be performed directly after staining the samples, which may reduce the potential of bacterial contamination.40 We focused on killing the bacteria in dentinal tubules, as we think that the depth of dead bacteria better reflects the antibacterial effect.\nThis study also showed that intermittent laser irradiation significantly reduced its heat-generation effect. The main limitation of laser is its thermal impact during application, which may damage the periodontal ligament41 and may cause postoperative pain.42 In this study, the maximum increases in temperature in the sonic irrigation, PUI, and intermittent Nd:YAP groups were all <10°C during the 30 s irrigation interval, which agrees with previous results.20 However, the increase in temperature in the Nd:YAP continuous irrigation group exceeded 10°C within 6–8 s; and its highest temperature during irrigation reached approximately 55°C. These results indicate that the heat generated by the laser is absorbed by water molecules in the dentin and then transmitted to the root outer surface. Therefore, to avoid damage to periodontal tissue when irrigating root canals, the Nd:YAP laser should be limited to intermittent use with short pulses.\nThis study had some limitations. First, the sample size was relatively small; and to avoid anatomical differences between groups and validate the results more strongly, further research with larger samples is needed. Second, the roots of teeth were exposed directly to air, whereas in another study the teeth were in 37°C water bath condition to simulate human body temperature.20 Under water bath conditions, the temperature at the roots outer surface could only be measured at specific points using a thermocouple, thus it may not represent its highest temperature reached.",
"The NaOCl solution agitated by the EDDY system showed a potent bacterial killing effect in the dentinal tubules of coronal and middle thirds of root canal wall, but this effect was limited in the apical third. The NaOCl solution agitated by the Nd:YAP laser showed no advantage in terms of killing bacteria in dentinal tubules of coronal and middle thirds of the root canal compared to the PUI or EDDY but achieved similar antibacterial effect to the PUI in the apical third. Under the conditions used in this study, the increase in temperature at the root surface caused by the intermittent irrigation protocol is safe for clinical application."
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"Dentinal tubules",
"EDDY",
"Nd:YAP laser",
"Root canal disinfection"
] | Introduction:
Chemo-mechanical cleaning and shaping of the root canal system to remove or reduce bacterial populations are essential for infection control, which is the main goal of root canal treatment.1 However, the complex anatomy of the root canal system limits the mechanical preparation, particularly in the apical third of the root.2 In infected root canals, bacteria can grow 400 μm or more into the dentinal tubules.3 Due to the strength and resistance of the remaining root canal wall, classic mechanical preparation generally only cuts the dentin at a depth of 150 μm, which is impossible to remove deep infection.4 Chemical irrigation is an effective supplemental method as it can reach more areas of the root canal surface.5 Techniques such as passive ultrasonic irrigation (PUI), sonic irrigation, and laser-activated irrigation enhance the disinfection effects of chemical irrigation and improve clinical outcomes.6
PUI activates irrigation via acoustic streaming and cavitation and its disinfecting effect is better than the conventional needle irrigation (CNI).7,8 The EDDY system (VDW, Munich, Germany) provides high-frequency sonic irrigation at 6000 Hz.9 Although previous studies have compared the antibacterial efficacies of EDDY, PUI, and CNI, the results have been inconsistent, presumably due to the different evaluation indicators employed.10–12 Most studies have quantified cultured residual bacteria, but some anaerobic bacteria are difficult to cultivate under normal conditions.13 Additionally, most studies12,14,15 used sterile paper points for sampling the root canals, however, this method only evaluates infection clearance for the overall root canal space, and not specifically for the dentinal tubules of the root canal wall.
Studies on the application of lasers to kill bacteria in root canals have been conducted since Fegan and Steiman16 (1995) evaluated the antibacterial effects of intracanal Nd:YAG laser irradiation, which may improve disinfection through a photothermal effect and by enhancing the chemical effects of irrigants.17
Commonly used lasers for root canal disinfection include the erbium-doped chromium-yttrium-scandium-gallium garnet (Er,Cr:YSGG) laser, the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser, and the photon-induced photoacoustic streaming laser.18 Wang, et al.17 (2018) compared the antibacterial effects of different lasers on deep regions of dentinal tubules in a prepared dentin block infected by Enterococcus faecalis, resulting in Nd:YAP laser irradiation without NaOCl having the weakest effect among all. This finding probably relates to differences in the working tip construction. The end of the Nd:YAP laser fiber is flat, thus the light can only propagate in a straight line, which may result in bacterial survival. The Er:YAG and Er,Cr:YSGG lasers have conically shaped, radial firing tips that irradiate the root canals in three dimensions, leading to more widespread bacterial death.19 The Nd:YAP laser is a near-infrared laser with a wavelength of 1340 nm and working fiber diameter of 200–320 μm.20 Liu, et al.21 (2019) reported that Nd:YAP laser agitating NaOCl at 280 and 360 mJ achieved effective antibacterial effects. However, the study evaluated only the proportion of dead bacteria in the entire root canal wall. Najah, Sid, Ghodbane22 (2016) found that the antibacterial effects of 2.5% NaOCl agitated by Nd:YAP laser and passive ultrasonic irrigation were similar, in which both irrigation protocols showed better effects than CNI with 2.5% NaOCl. However, the bacterial counting method they used to compare the numbers of residual microorganisms failed to assess the dead bacteria deep in the dentinal tubules. We currently lack research evaluating the dead bacteria depth in the dentinal tubules of the root canal wall with Nd:YAP lasers, especially regarding the apical region. The main limitation of the laser method is its thermal impact during application, which may damage the periodontal ligament. Namour, et al.20 (2016) found that given proper working parameters, the temperature increase caused by the Nd:YAP laser during continuously endodontic irrigation with 2.25% NaOCl would not damage periodontal tissue. Moreover, Zhang and Wang23 (2021) reported that when using the Nd:YAP laser ablating separated files in root canal, the increase in temperature at the root surface was <10°C. But Rochd, Calas, Roques24 (1998) found that the temperature of the outer surface of the tooth root could increase by approximately 25°C after continuous laser operation in bacterial suspension for 28 s, which may cause thermal damage. Although clinicians usually use this laser intermittently for root canal disinfection, we lack research concerning the temperature increase.
Overall, few studies have focused on the effects of NaOCl agitated by the EDDY tip and Nd:YAP laser on eliminating bacteria in dentinal tubules of infected root canal walls, especially in the apical region. Most of the research concerning the thermal impact of Nd:YAP laser compares it to other lasers (or its outcomes using different parameters), and comparisons with PUI and sonic irrigation are lacking. Therefore, the aim of this study was to compare the antimicrobial efficiency of NaOCl agitated by the EDDY, PUI, and Nd:YAP laser with CNI. We hypothesized that neither EDDY nor Nd:YAP laser-activated irrigation would exhibit better antibacterial effects than PUI. This study also compared the temperature increase on the root outer surface caused by the Nd:YAP laser with ultrasonic, sonic, and syringe irrigation devices in vitro, thus providing experimental evidence for the selection of safe and effective final irrigation protocols in root canal treatment.
Methodology:
Tooth selection and preparation A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.
A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.
Establishment of E. faecalis infection To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.
The infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.
To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.
The infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.
Root canal irrigation protocols After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.
Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.
No root canal irrigation was performed in this group after bacterial incubation.
Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.
Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.
No root canal irrigation was performed in this group after bacterial incubation.
Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
CLSM evaluation After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).
After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).
Heat production evaluation Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.
Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.
Statistical analysis The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).
The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).
Tooth selection and preparation:
A total of thirty-eight mature single-root-canal premolars were collected from the clinic of the Department of Oral and Maxillofacial Surgery at Peking University School and Hospital of Stomatology. The study was approved by the institutional review board (approval No. PKUSSIRB-202058173). The sample size was determined using the program PASS for Microsoft Windows (ver. 15.0; NCSS Inc, Kaysville., UT, USA) and a randomized design.25 With a confidence coefficient of 0.95 (α=0.05) and a power of 0.9 (β=0.1), the minimum sample size for confocal laser scanning microscopy (CLSM) analysis was n=5 per group. Another ten teeth were used for the temperature increase experiment. To avoid any influence of root anatomy on temperature measurement, these teeth were reused. Only intact premolars with straight root canals were considered. The exclusion criteria were teeth with caries, periodontal defects, calcifications, apical resorption, or more than one canal, and root curvature >15°. The collected teeth were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use. Root canal preparation was conducted before bacterial infection according to a previously described protocol.26 Teeth were decoronated using a water-cooled high-speed bur (MANI, Tochigi, Japan) and observed under a dental microscope to confirm whether only one canal existed in each root. A #10 K-file (MANI) was inserted into the canal until the file tip was visualized at the apical foramen. Then, the roots were shortened to 12 mm via a K-file stopper. The working length (WL) of the root canal was set as 11mm, 1 mm shorter than the apical foramen. After removing the pulp tissue using a barbed broach (MANI), the root canals were prepared using ProTaper Universal instruments (Dentsply Maillefer, Baillagues, Switzerland), beginning with Sx and progressing to S1, S2, F1, F2, and F3. During root canal preparation, the canals were irrigated with 2 mL 5.25% NaOCl solution (Peking University School and Hospital of Stomatology, Beijing, China) for 1 min using a 27-gauge side-vented needle (Dentsply Tulsa Dental, Tulsa, OK, USA) within 2 mm from the WL after each instrument change. After the last instrument change, the canals were irrigated with 2 mL 5.25% NaOCl solution, 2 mL 17% ethylenediaminetetraacetic acid (EDTA; Peking University School and Hospital of Stomatology, Beijing, China) solution, and 2 mL sterilized water. The irrigation time for each solution was 1 min (1 mL/30 s). After preparation, the roots were autoclaved at 121°C and 15 MPa for 20 min and stored in sterile water at 4°C for later use.
Establishment of E. faecalis infection:
To confirm the cleanliness of the surface and exposure of dentinal tubules (which would allow bacterial incubation), two roots that had been subjected to root canal preparation were observed by scanning electron microscopy before the infection. The specimens were split in half using a chisel along the long axis and then fixed in 2.5% glutaraldehyde solution for 1 week. Thereafter, they were dehydrated in a graded series of ethanol solutions to a critical dried point, coated with gold, and examined via scanning electron microscopy (S2500; Hitachi, Tokyo, Japan). Each root canal was divided into apical, middle, and coronal thirds. A randomly selected location in the apical third was photographed at ×1,000 magnification, 5.0 kV. Then, two additional images were captured at 1 mm proximal and 1 mm distal to this site. Using the same protocol, three images of both the middle and coronal thirds were captured.
The infection protocol was like a published method.27 Briefly, a standard suspension (1×108 cells/mL) of E. faecalis (ATCC29212; American Type Culture Collection, Manassas, VA, USA) was prepared in brain heart infusion medium (BHI) (Oxoid, Basingstoke, UK) at 37°C for 24 h. The E. faecalis suspension was filled to the root orifice level using a 27-gauge side-vented needle (Dentsply Tulsa Dental), showing a growth plateau after 12 h. The E. faecalis were cultured in 10 mL BHI broth at 37°C for 3 weeks and BHI medium was replaced every 48h to allow bacteria to grow into dentinal tubules. After incubation, the apical foramens were sealed with flowable composite (Ivoclar Vivadent, Schaan, Liechtenstein). Two additional roots were selected and observed via scanning electron microscopy to confirm that the infection deeply penetrated the dentinal tubules.
Root canal irrigation protocols:
After incubation, all roots were randomly divided into a negative control group (n=2), a positive control group (n=2), and four experimental groups (n=5 per group). In each experimental group, irrigation was performed using a distinct irrigation protocol. All canals were irrigated with 3 mL 5.25% NaOCl according to the following cycle: 30 s of 1 mL 5.25% NaOCl (1 mL/30 s), followed by 30 s of no irrigation. Then, 2 mL 17% EDTA (1 mL/30 s) and 2 mL sterilized water (1 mL/30 s) were delivered into the root canal and activated to remove residual irrigants.
Group 1: Negative control (n=2) No root canal irrigation was performed in this group after bacterial incubation.
No root canal irrigation was performed in this group after bacterial incubation.
Group 2: Positive control (n=2) Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Group 3: CNI (n=5) CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
Group 4: PUI (n=5) The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
Group 5: High-frequency sonic irrigation (n = 5) The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
Group 6: Nd:YAP laser (n=5) A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
Group 1: Negative control (n=2):
No root canal irrigation was performed in this group after bacterial incubation.
Group 2: Positive control (n=2):
Teeth were autoclaved at 121°C and 15 MPa for 20 min after bacterial incubation.
Group 3: CNI (n=5):
CNI was performed with a 27-gauge side-vented needle (Dentsply Tulsa Dental). Each canal was flushed with a continuous flow of 1 mL NaOCl for 30 s (1 mL/30 s) within 2 mm from the WL using a vertical motion and a 30-s soaking interval. This irrigating-soaking cycle was repeated twice (totaling three cycles). A total of 3 mL 5.25% NaOCl was used during this procedure. Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL with no soaking interval. Finally, 2 mL sterilized water was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm from the WL, with no soaking interval. A rubber stopper was used to control the WL.
Group 4: PUI (n=5):
The canal was passively filled with 5.25% NaOCl. To irrigate, a 27-gauge side-vented irrigation needle was placed at the orifice level. The irrigant in the canal was activated using a PUI device (Satelec Acteon Group, Merignac, France) at the power setting of 7. A #25 ultrasonic file (Satelec Acteon Group) was placed 2 mm from the WL. Each canal was irrigated for 30 s with 1 mL 5.25% NaOCl (1 mL/30 s) using an ultrasonic device, followed by a 30-s soaking interval. This irrigating-soaking cycle was also repeated twice (total of three cycles). Then, 2 mL 17% EDTA was continuously flushed into the canal for 1 min (1 mL/30 s) within 2 mm of the WL under ultrasonic activation. Finally, 2 mL sterilized water.
Group 5: High-frequency sonic irrigation (n = 5):
The activation procedure in the high-frequency sonic irrigation group was like the PUI group. A #20 EDDY tip (VDW) was placed in the canal at 2 mm from the WL and operated in vertical motion.
Group 6: Nd:YAP laser (n=5):
A 200-μm Lokki Dt2 (Lobel Medical, Les Roches-de-Condrieu, France) tip was placed 2 mm from the WL after the canal had been filled with irrigant. The laser tip was operated at 280 mJ, 5 Hz, and 1.4 W with the intermittent irrigation protocol. First, the canal was passively filled with 5.25% NaOCl, as described above. The laser fiber was pulled up and down from the position 2 mm of the WL to the orifice level, and activated at 0–4 s, 13–17 s, and 26–30 s. No upward/downward movement or laser activation was performed, but only irrigation with 5.25% NaOCl solution was applied at 4–13 s and 17–26 s. A 27-gauge side-vented irrigation needle was placed at the orifice level to provide 1 mL 5.25% NaOCl during the 30-s (1 mL/30 s) intermittent irrigation. A 30-s soaking interval was also needed and this irrigating-soaking cycle was repeated twice (totaling three cycles). Then, 2 mL 17% EDTA was delivered continuously into the root canal for 1 min. Laser activation was performed twice according to the above mentioned 30 s intermittent irrigation protocol. Thereafter, 2 mL sterile water was irrigated according to the process.
CLSM evaluation:
After the irrigation, the roots were split in half longitudinally and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, USA) for 15 min, according to the manufacturer's protocol. Then, the samples were observed via a CLSM device (LSM 710; Carl Zeiss, Oberkochen, Germany). For localization, each sample was observed at low magnification (×2). The apical, middle, and coronal thirds of the canal were defined 0–4, 4–8, and 8–11 mm, respectively, from the apical foramen, as the WL was 11 mm. The field of view was located near the longitudinal midpoint of each third, and three images of each sample were photographed at ×20 magnification. To observe live and dead bacteria, the wavelength was set at 480/500 nm for SYTO 9 (green fluorescent nucleic acid stain) and at 490/635 nm for propidium iodide (red fluorescent nucleic acid stain), respectively. Bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. The width of red fluorescence at 300 μm was measured using ImageJ program (National Institutes of Health, Bethesda, MD, USA) and then used to calculate the depth of dead bacteria in each third of the canal. Each CLSM image was divided into 10 equal parts and the depth of red fluorescence was measured at the midpoint of each part. Data are expressed as mean ± standard deviation (μm).
Heat production evaluation:
Ten additional teeth were obtained and prepared as described above. The roots were irrigated using CNI, PUI, and EDDY with 1 mL 5.25% NaOCl for 30 s (1 mL/30 s), using the device parameters described above. The Nd:YAP laser was operated at 280 mJ, 5 Hz and 1.4 W with a 200-μm tip. Like the procedure described earlier, the canal was first passively filled with 5.25% NaOCl with constant irrigation. For the intermittent group, the laser fiber was pulled up and down from the apical third to the coronal third and activated at 0–4, 13–17, and 26–30 s. No upward/downward movement or laser activation was performed, but constant irrigation was applied with 5.25% NaOCl solution at 4–13 and 17–26 s. For the continuous irrigation group, the laser parameters were the same as for the intermittent group, though the activation procedure was applied continuously for 30 s, consuming 1 mL 5.25% NaOCl with no interruption. The roots were exposed to room temperature air and its outer surface temperature was measured over the entire 30-s period via a FORTRIC230 thermal imaging camera located 10 cm away from the roots. Via AnalyzIR program, the initial temperature of roots external surface and its highest temperature during the irrigation process were recorded. Then, the change in temperature was calculated. The same ten roots were used in each group to avoid errors caused by anatomical differences. For at least 30 min before reuse, roots were left to cool and maintain their initial temperatures.
Statistical analysis:
The normality of the data was assessed using a P-P plot. If the data had a normal distribution, one-way analysis of variance was used for the analysis, with the Games–Howell test applied for pairwise comparisons. For data not showing a normal distribution, the Kruskal–Wallis nonparametric test was applied. The significance level for all statistical analyses was set at α=0.05. Statistical analyses were performed via SPSS version 20.0 for Microsoft Windows (SPSS Inc, Chicago, IL, USA).
Results:
Establishment of root canal models infected with E. faecalis Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.
Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.
Dead bacteria depths of different irrigation protocols Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.
The red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).
In the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.
Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.
The red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).
In the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.
Evaluation of heat production All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.
SD, standard deviation.
The same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.
The mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.
Figure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.
All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.
SD, standard deviation.
The same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.
The mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.
Figure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.
Establishment of root canal models infected with E. faecalis:
Figure 1 shows representative images from the coronal, middle, and apical regions of each group. Before infection, the dentinal tubules of the canals were all open. After the 21-day infection process, the tubules were covered by bacterial colonies, confirming that the models had been successfully established.
Dead bacteria depths of different irrigation protocols:
Figure 2 shows representative images of live and dead bacteria distributions. The negative control group showed only green fluorescence (live bacteria) in all three fields of view with no red fluorescence (dead bacteria) in the dentinal tubules, indicating that nearly all bacteria remained alive. On the other hand, the positive control group showed only red fluorescence in all three fields of view, indicating that nearly all bacteria were dead. We observed several depths of red fluorescence for the test groups. Figure 3 shows the results of our quantitative analysis of dead bacteria.
The red fluorescence depths of the CNI, PUI, and EDDY groups showed a decreasing trend from the coronal to the apical third and we observed significant differences between the coronal and apical third in all three groups (p<0.05). The Nd:YAP laser group showed similar red fluorescence depths in the coronal, middle, and apical thirds (p>0.05).
In the coronal third, the red fluorescence depth in the PUI group (147.14±42.89 μm) preceded the high-frequency sonic irrigation group (145.38±39.96 μm), with no significant difference observed between groups (p>0.05). Both depths significantly differed from the CNI group depth (78.51±13.12 μm, p<0.05). The red fluorescence depth in Nd:YAP laser group was 94.80±13.28 μm, which was not significantly different compared to the PUI, EDDY, and CNI groups (p>0.05). We observed the same trends in the middle third. However, in apical third, the laser and PUI groups had good dead bacteria depth (74.93±8.08 μm vs. 86.12±33.18 μm, p>0.05). These were significantly better than the CNI group depth (27.34±9.73 μm, p<0.05). The bacterial killing effects of high-frequency sonic irrigation group in apical third was similar (49.94±20.72 μm, p>0.05) to the Nd:YAP laser, PUI, and CNI groups.
Evaluation of heat production:
All samples began at the same temperature (Table 1). Except for the CNI group, the temperature increased during the procedure in all of them.
SD, standard deviation.
The same superscript letter in a particular column indicates differences that are not statistically significant (p>0.05) according to one-way analysis of variance.
The mean increase in temperature failed to significantly differ between the EDDY (3.78±2.83°C) and PUI groups (6.53±3.59°C). Between the two laser-assisted irrigation groups, the intermittent irrigation group (8.28±3.45 °C) showed significantly smaller mean increase than continuous irrigation group (29.06±4.45°C). The intermittent group showed a significantly greater mean increase than the EDDY group. Overall, the continuous group showed a significantly greater mean increase than the other three groups. Thus, the laser groups were more likely to cause an increase in temperature than ultrasonic and sonic irrigation. However, intermittent irrigation may help to reduce thermal damage caused by the Nd:YAP laser.
Figure 4 shows the changes in temperature of each group recorded with 1-s intervals during the 30 s irrigation procedure. In the EDDY, PUI, and continuous laser groups, the temperature increased gradually over time. In the intermittent laser group, it increased at 0–4 s, 13–17 s, and 26–30 s, and decreased at 4–13 s and 17–26 s. The increase in temperature in the continuous irrigation group exceeded 10°C after 7 s and remained below 10°C in all other groups.
Discussion:
This study compared the antibacterial effects of NaOCl agitated by four irrigation protocols on infected root canal walls. We calculated the depth of dead bacteria in the dentinal tubules of coronal, middle, and apical segments of the root via CLSM images analysis. The data shows the efficacies of different irrigation protocols for removing infections in the dentinal tubules.
Several studies showed that the cleaning effects of dynamic irrigation are limited in the apical region.12,27,28 In our study, we found that the effects of NaOCl agitated by the PUI and EDDY showed a similar trend in the apical third. This finding probably relates to the small diameter of the root canal in the apical area and the short distance between the working tip and the root canal wall, which limits the effects of acoustic flow.6 However, we failed to find significant differences in the effects of the Nd:YAP laser in the coronal, middle, or apical thirds. The laser irradiation mainly inhibits bacterial growth via photothermal action. Its fiber head heat output can directly destroy the cell wall and the irrigants can absorb it, transferring heat into the dentinal tubules to kill bacteria.18 In this study, we moved the Nd:YAP laser fiber up and down 2 mm short of the WL, so that the irrigants in the root canal space could evenly absorb the heat. The uniform distribution of heat probably led to the relatively even depth of red fluorescence observed throughout the root canal.
In coronal and middle thirds, the antibacterial effect of the EDDY was equivalent to PUI. This finding indicates that the ultrasonic oscillation and sonic working tips could potentially achieve greater acoustic flow due to the unlimited space in middle and coronal parts of root canal.29 Although the EDDY frequency is lower than PUI, its vibration amplitude is larger. The irrigants velocity positively relates to the working tips amplitude30 Salas, et al.31 (2021) found that the penetration depth of 2% chlorhexidine irrigated by EDDY or PUI was similar in the cervical and middle region of the root canal. Meanwhile, the laser-activated irrigation was not significantly weaker than PUI or EDDY, nor significantly better than CNI. With CNI irrigation, irrigants have sufficient backflow space and the procedure is less affected by factors such as needle entry depth.32 These results suggest that, in the middle and coronal regions of the root canal, dynamic irrigation can achieve good results, whereas Nd:YAP lacks obvious advantages. However, because we removed more resistance in coronal third than what is commonly performed in clinical practice, coronal resistance may slightly differ between this study and an actual clinical situation, which may allow PUI and EDDY vibration to occur under less constrained conditions for potentially better effects.
Meanwhile, in apical third, the Nd:YAP laser showed an effective antibacterial effect like PUI and both groups showed significantly better antibacterial effects compared to CNI. The flexible and small in diameter fiber of the Nd:YAP laser can enter the prepared apical root canal smoothly and kill bacteria through its photothermal effects.13 The EDDY was neither significantly weaker than PUI nor significantly better than CNI. Ahmad, et al.33 (1988) found that lateral displacement of the tip of an ultrasonic file can reach approximately 40 μm, less than the oscillation amplitude of sonic working tips. Hence, tips used in the EDDY are more likely to contact the root canal wall than those of PUI. Walmsley, Lumley, Laird34 (1989) found that when the movement of the sonic file is constrained, its sideway oscillation disappears and its movement pattern can be converted into pure longitudinal vibration; thus reducing the irrigants penetration depth and clearing infections inside the dentinal tubules.
In this study, we choose a single-species biofilm model E. faecalis to establish an infection model. This model has been used in many studies35,36 and is considered effective for evaluating root canal disinfection. However, multi-species bacterial biofilm has been used in recent studies of infected root canals. Hoedke, et al.37 (2018) inoculated the root canal with E. faecalis, Streptococcus oralis (S. oralis), and Prevotella intermedia (P. intermedia) to analyze the antibacterial effect of photodynamic therapy. Swimberghe, et al.38 (2021) found that, when grown in a multispecies biofilm, E. faecalis showed significantly less susceptibility to NaOCl than a monospecies biofilm. However, the cultivation conditions for the multi-species bacterial biofilm may be stricter. For further and future research, our goal is to establish a multi-species bacterial biofilm infection model.
Najah, Sid and Ghodbane22 (2016) used the bacterial count method to compare the reduction of bacterial load in root canals after PUI and Nd:YAP laser-activated irrigation. The results failed to show significant differences between the groups. Calculating the reduction in bacterial load is a better solution because it represents a quantitative measure. However, this method mainly detects bacteria suspended in the root canal space and is not used to detect bacterial biofilm on the root canal wall or in the dentinal tubules. It also requires bacterial sampling, inoculation, and incubation increasing the chances of introducing microorganisms.39 In our study, we evaluated the depth of dead bacteria via CLSM. This step can be performed directly after staining the samples, which may reduce the potential of bacterial contamination.40 We focused on killing the bacteria in dentinal tubules, as we think that the depth of dead bacteria better reflects the antibacterial effect.
This study also showed that intermittent laser irradiation significantly reduced its heat-generation effect. The main limitation of laser is its thermal impact during application, which may damage the periodontal ligament41 and may cause postoperative pain.42 In this study, the maximum increases in temperature in the sonic irrigation, PUI, and intermittent Nd:YAP groups were all <10°C during the 30 s irrigation interval, which agrees with previous results.20 However, the increase in temperature in the Nd:YAP continuous irrigation group exceeded 10°C within 6–8 s; and its highest temperature during irrigation reached approximately 55°C. These results indicate that the heat generated by the laser is absorbed by water molecules in the dentin and then transmitted to the root outer surface. Therefore, to avoid damage to periodontal tissue when irrigating root canals, the Nd:YAP laser should be limited to intermittent use with short pulses.
This study had some limitations. First, the sample size was relatively small; and to avoid anatomical differences between groups and validate the results more strongly, further research with larger samples is needed. Second, the roots of teeth were exposed directly to air, whereas in another study the teeth were in 37°C water bath condition to simulate human body temperature.20 Under water bath conditions, the temperature at the roots outer surface could only be measured at specific points using a thermocouple, thus it may not represent its highest temperature reached.
Conclusions:
The NaOCl solution agitated by the EDDY system showed a potent bacterial killing effect in the dentinal tubules of coronal and middle thirds of root canal wall, but this effect was limited in the apical third. The NaOCl solution agitated by the Nd:YAP laser showed no advantage in terms of killing bacteria in dentinal tubules of coronal and middle thirds of the root canal compared to the PUI or EDDY but achieved similar antibacterial effect to the PUI in the apical third. Under the conditions used in this study, the increase in temperature at the root surface caused by the intermittent irrigation protocol is safe for clinical application.
| Background:
Aiming to kill bacteria in dentin tubules of infected dental pulp cavities, we evaluated the effects of sodium hypochlorite (NaOCl) solution agitated by different irrigation protocols, i.e., conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), the EDDY tip, and the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser. The EDDY achieved good antibacterial effects as passive ultrasonic irrigation in the coronal and middle thirds. Nd:YAP laser irradiation and PUI were effective in the apical third of the root canal.
Methods:
Infected root canal models were established, and roots were randomly divided into six groups: negative control, positive control, CNI, PUI, sonic agitation with EDDY, and Nd:YAP laser groups. After irrigation, the teeth were split and stained using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacteria depth was evaluated by a confocal laser scanning microscopy and the temperature at the root surface was assessed using a thermal imaging camera during the irrigation process.
Results:
In the coronal and middle thirds of the root canal, PUI and EDDY had stronger antibacterial effects than CNI (p<0.05); in the apical third, the antibacterial effects of PUI and Nd:YAP laser-activated irrigation were better than CNI (p<0.05). The maximum change in temperature was significantly greater during continuous Nd:YAP laser application compared with the other methods, but intermittent irrigation helped lessening this trend.
Conclusions:
NaOCl agitated by EDDY tip and PUI exhibited a similar bacteria elimination effect in the coronal and middle root canal. Nd:YAP laser was effective in the apical third and intermittent irrigation reduced its thermal impact. | Introduction:
Chemo-mechanical cleaning and shaping of the root canal system to remove or reduce bacterial populations are essential for infection control, which is the main goal of root canal treatment.1 However, the complex anatomy of the root canal system limits the mechanical preparation, particularly in the apical third of the root.2 In infected root canals, bacteria can grow 400 μm or more into the dentinal tubules.3 Due to the strength and resistance of the remaining root canal wall, classic mechanical preparation generally only cuts the dentin at a depth of 150 μm, which is impossible to remove deep infection.4 Chemical irrigation is an effective supplemental method as it can reach more areas of the root canal surface.5 Techniques such as passive ultrasonic irrigation (PUI), sonic irrigation, and laser-activated irrigation enhance the disinfection effects of chemical irrigation and improve clinical outcomes.6
PUI activates irrigation via acoustic streaming and cavitation and its disinfecting effect is better than the conventional needle irrigation (CNI).7,8 The EDDY system (VDW, Munich, Germany) provides high-frequency sonic irrigation at 6000 Hz.9 Although previous studies have compared the antibacterial efficacies of EDDY, PUI, and CNI, the results have been inconsistent, presumably due to the different evaluation indicators employed.10–12 Most studies have quantified cultured residual bacteria, but some anaerobic bacteria are difficult to cultivate under normal conditions.13 Additionally, most studies12,14,15 used sterile paper points for sampling the root canals, however, this method only evaluates infection clearance for the overall root canal space, and not specifically for the dentinal tubules of the root canal wall.
Studies on the application of lasers to kill bacteria in root canals have been conducted since Fegan and Steiman16 (1995) evaluated the antibacterial effects of intracanal Nd:YAG laser irradiation, which may improve disinfection through a photothermal effect and by enhancing the chemical effects of irrigants.17
Commonly used lasers for root canal disinfection include the erbium-doped chromium-yttrium-scandium-gallium garnet (Er,Cr:YSGG) laser, the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser, and the photon-induced photoacoustic streaming laser.18 Wang, et al.17 (2018) compared the antibacterial effects of different lasers on deep regions of dentinal tubules in a prepared dentin block infected by Enterococcus faecalis, resulting in Nd:YAP laser irradiation without NaOCl having the weakest effect among all. This finding probably relates to differences in the working tip construction. The end of the Nd:YAP laser fiber is flat, thus the light can only propagate in a straight line, which may result in bacterial survival. The Er:YAG and Er,Cr:YSGG lasers have conically shaped, radial firing tips that irradiate the root canals in three dimensions, leading to more widespread bacterial death.19 The Nd:YAP laser is a near-infrared laser with a wavelength of 1340 nm and working fiber diameter of 200–320 μm.20 Liu, et al.21 (2019) reported that Nd:YAP laser agitating NaOCl at 280 and 360 mJ achieved effective antibacterial effects. However, the study evaluated only the proportion of dead bacteria in the entire root canal wall. Najah, Sid, Ghodbane22 (2016) found that the antibacterial effects of 2.5% NaOCl agitated by Nd:YAP laser and passive ultrasonic irrigation were similar, in which both irrigation protocols showed better effects than CNI with 2.5% NaOCl. However, the bacterial counting method they used to compare the numbers of residual microorganisms failed to assess the dead bacteria deep in the dentinal tubules. We currently lack research evaluating the dead bacteria depth in the dentinal tubules of the root canal wall with Nd:YAP lasers, especially regarding the apical region. The main limitation of the laser method is its thermal impact during application, which may damage the periodontal ligament. Namour, et al.20 (2016) found that given proper working parameters, the temperature increase caused by the Nd:YAP laser during continuously endodontic irrigation with 2.25% NaOCl would not damage periodontal tissue. Moreover, Zhang and Wang23 (2021) reported that when using the Nd:YAP laser ablating separated files in root canal, the increase in temperature at the root surface was <10°C. But Rochd, Calas, Roques24 (1998) found that the temperature of the outer surface of the tooth root could increase by approximately 25°C after continuous laser operation in bacterial suspension for 28 s, which may cause thermal damage. Although clinicians usually use this laser intermittently for root canal disinfection, we lack research concerning the temperature increase.
Overall, few studies have focused on the effects of NaOCl agitated by the EDDY tip and Nd:YAP laser on eliminating bacteria in dentinal tubules of infected root canal walls, especially in the apical region. Most of the research concerning the thermal impact of Nd:YAP laser compares it to other lasers (or its outcomes using different parameters), and comparisons with PUI and sonic irrigation are lacking. Therefore, the aim of this study was to compare the antimicrobial efficiency of NaOCl agitated by the EDDY, PUI, and Nd:YAP laser with CNI. We hypothesized that neither EDDY nor Nd:YAP laser-activated irrigation would exhibit better antibacterial effects than PUI. This study also compared the temperature increase on the root outer surface caused by the Nd:YAP laser with ultrasonic, sonic, and syringe irrigation devices in vitro, thus providing experimental evidence for the selection of safe and effective final irrigation protocols in root canal treatment.
Conclusions:
The NaOCl solution agitated by the EDDY system showed a potent bacterial killing effect in the dentinal tubules of coronal and middle thirds of root canal wall, but this effect was limited in the apical third. The NaOCl solution agitated by the Nd:YAP laser showed no advantage in terms of killing bacteria in dentinal tubules of coronal and middle thirds of the root canal compared to the PUI or EDDY but achieved similar antibacterial effect to the PUI in the apical third. Under the conditions used in this study, the increase in temperature at the root surface caused by the intermittent irrigation protocol is safe for clinical application. | Background:
Aiming to kill bacteria in dentin tubules of infected dental pulp cavities, we evaluated the effects of sodium hypochlorite (NaOCl) solution agitated by different irrigation protocols, i.e., conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), the EDDY tip, and the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser. The EDDY achieved good antibacterial effects as passive ultrasonic irrigation in the coronal and middle thirds. Nd:YAP laser irradiation and PUI were effective in the apical third of the root canal.
Methods:
Infected root canal models were established, and roots were randomly divided into six groups: negative control, positive control, CNI, PUI, sonic agitation with EDDY, and Nd:YAP laser groups. After irrigation, the teeth were split and stained using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacteria depth was evaluated by a confocal laser scanning microscopy and the temperature at the root surface was assessed using a thermal imaging camera during the irrigation process.
Results:
In the coronal and middle thirds of the root canal, PUI and EDDY had stronger antibacterial effects than CNI (p<0.05); in the apical third, the antibacterial effects of PUI and Nd:YAP laser-activated irrigation were better than CNI (p<0.05). The maximum change in temperature was significantly greater during continuous Nd:YAP laser application compared with the other methods, but intermittent irrigation helped lessening this trend.
Conclusions:
NaOCl agitated by EDDY tip and PUI exhibited a similar bacteria elimination effect in the coronal and middle root canal. Nd:YAP laser was effective in the apical third and intermittent irrigation reduced its thermal impact. | 14,299 | 320 | [
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] | [CONTENT] Dentinal tubules | EDDY | Nd:YAP laser | Root canal disinfection [SUMMARY] | [CONTENT] Dentinal tubules | EDDY | Nd:YAP laser | Root canal disinfection [SUMMARY] | [CONTENT] Dentinal tubules | EDDY | Nd:YAP laser | Root canal disinfection [SUMMARY] | [CONTENT] Dentinal tubules | EDDY | Nd:YAP laser | Root canal disinfection [SUMMARY] | [CONTENT] Dentinal tubules | EDDY | Nd:YAP laser | Root canal disinfection [SUMMARY] | [CONTENT] Dentinal tubules | EDDY | Nd:YAP laser | Root canal disinfection [SUMMARY] | [CONTENT] Ultrasonics | Dental Pulp Cavity | Root Canal Therapy | Bacteria | Anti-Bacterial Agents [SUMMARY] | [CONTENT] Ultrasonics | Dental Pulp Cavity | Root Canal Therapy | Bacteria | Anti-Bacterial Agents [SUMMARY] | [CONTENT] Ultrasonics | Dental Pulp Cavity | Root Canal Therapy | Bacteria | Anti-Bacterial Agents [SUMMARY] | [CONTENT] Ultrasonics | Dental Pulp Cavity | Root Canal Therapy | Bacteria | Anti-Bacterial Agents [SUMMARY] | [CONTENT] Ultrasonics | Dental Pulp Cavity | Root Canal Therapy | Bacteria | Anti-Bacterial Agents [SUMMARY] | [CONTENT] Ultrasonics | Dental Pulp Cavity | Root Canal Therapy | Bacteria | Anti-Bacterial Agents [SUMMARY] | [CONTENT] root canal compared | control root canal | bacteria root canals | sonic syringe irrigation | root canal disinfection [SUMMARY] | [CONTENT] root canal compared | control root canal | bacteria root canals | sonic syringe irrigation | root canal disinfection [SUMMARY] | [CONTENT] root canal compared | control root canal | bacteria root canals | sonic syringe irrigation | root canal disinfection [SUMMARY] | [CONTENT] root canal compared | control root canal | bacteria root canals | sonic syringe irrigation | root canal disinfection [SUMMARY] | [CONTENT] root canal compared | control root canal | bacteria root canals | sonic syringe irrigation | root canal disinfection [SUMMARY] | [CONTENT] root canal compared | control root canal | bacteria root canals | sonic syringe irrigation | root canal disinfection [SUMMARY] | [CONTENT] ml | canal | irrigation | group | 30 | laser | root | naocl | mm | 25 [SUMMARY] | [CONTENT] ml | canal | irrigation | group | 30 | laser | root | naocl | mm | 25 [SUMMARY] | [CONTENT] ml | canal | irrigation | group | 30 | laser | root | naocl | mm | 25 [SUMMARY] | [CONTENT] ml | canal | irrigation | group | 30 | laser | root | naocl | mm | 25 [SUMMARY] | [CONTENT] ml | canal | irrigation | group | 30 | laser | root | naocl | mm | 25 [SUMMARY] | [CONTENT] ml | canal | irrigation | group | 30 | laser | root | naocl | mm | 25 [SUMMARY] | [CONTENT] root | laser | nd | nd yap | yap | root canal | nd yap laser | yap laser | effects | lasers [SUMMARY] | [CONTENT] ml | 30 | canal | 25 | mm | group | 25 naocl | wl | naocl | soaking [SUMMARY] | [CONTENT] group | groups | 05 | fluorescence | significantly | red fluorescence | red | showed | laser | μm [SUMMARY] | [CONTENT] effect | solution agitated | middle thirds root canal | tubules coronal middle thirds | thirds root canal | thirds root | coronal middle thirds root | naocl solution agitated | middle thirds root | tubules coronal [SUMMARY] | [CONTENT] ml | group | canal | 30 | irrigation | root | laser | naocl | root canal | 25 [SUMMARY] | [CONTENT] ml | group | canal | 30 | irrigation | root | laser | naocl | root canal | 25 [SUMMARY] | [CONTENT] CNI | PUI | EDDY | YAP ||| EDDY ||| YAP | PUI | third [SUMMARY] | [CONTENT] six | CNI | PUI | EDDY | YAP ||| ||| [SUMMARY] | [CONTENT] PUI | EDDY | CNI | p<0.05 | third | PUI | YAP | CNI | p<0.05 ||| YAP [SUMMARY] | [CONTENT] EDDY | PUI ||| YAP | third [SUMMARY] | [CONTENT] CNI | PUI | EDDY | YAP ||| EDDY ||| YAP | PUI | third ||| six | CNI | PUI | EDDY | YAP ||| ||| ||| ||| PUI | EDDY | CNI | p<0.05 | third | PUI | YAP | CNI | p<0.05 ||| YAP ||| EDDY | PUI ||| YAP | third [SUMMARY] | [CONTENT] CNI | PUI | EDDY | YAP ||| EDDY ||| YAP | PUI | third ||| six | CNI | PUI | EDDY | YAP ||| ||| ||| ||| PUI | EDDY | CNI | p<0.05 | third | PUI | YAP | CNI | p<0.05 ||| YAP ||| EDDY | PUI ||| YAP | third [SUMMARY] |
||||||
Enhanced antibacterial activity of tilmicosin against | 34931502 | The poor bioadhesion capacity of tilmicosin resulting in treatment failure for Staphylococcus aureus small colony variants (SASCVs) mastitis. | BACKGROUND | Tilmicosin-loaded chitosan oligosaccharide (COS)-sodium carboxymethyl cellulose (CMC) composite nanogels were formulated by an electrostatic interaction between COS (positive charge) and CMC (negative charge) using sodium tripolyphosphate (TPP) (ionic crosslinkers). The formation mechanism, structural characteristics, bioadhesion, and antibacterial activity of tilmicosin composite nanogels were studied systematically. | METHODS | The optimized formulation was comprised of 50 mg/mL (COS), 32 mg/mL (CMC), and 0.25 mg/mL (TPP). The size, encapsulation efficiency, loading capacity, polydispersity index, and zeta potential of the optimized tilmicosin composite nanogels were 357.4 ± 2.6 nm, 65.4 ± 0.4%, 21.9 ± 0.4%, 0.11 ± 0.01, and -37.1 ± 0.4 mV, respectively; the sedimentation rate was one. Scanning electron microscopy showed that tilmicosin might be incorporated in nano-sized crosslinked polymeric networks. Moreover, adhesive studies suggested that tilmicosin composite nanogels could enhance the bioadhesion capacity of tilmicosin for the SASCVs strain. The inhibition zone of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2.13 ± 0.07, 3.35 ± 0.11, and 1.46 ± 0.04 cm, respectively. The minimum inhibitory concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2, 1, and 1 µg/mL, respectively. The in vitro time-killing curves showed that the tilmicosin composite nanogels increased the antibacterial activity against the SASCVs strain. | RESULTS | This study provides a potential strategy for developing tilmicosin composite nanogels to treat cow mastitis caused by the SASCVs strain. | CONCLUSIONS | [
"Animals",
"Anti-Bacterial Agents",
"Carboxymethylcellulose Sodium",
"Cattle",
"Chitosan",
"Female",
"Mastitis, Bovine",
"Nanogels",
"Oligosaccharides",
"Staphylococcal Infections",
"Staphylococcus aureus",
"Tylosin"
] | 8799941 | INTRODUCTION | Staphylococcus aureus seriously threatens human health and results in substantial economic losses for livestock worldwide and has aroused considerable concern [1]. In particular, the presence of Staphylococcus aureus small colony variants (SASCVs) in the host cells could establish reservoirs from which reinfection can occur and result in long-term and repeated infection [2]. Because tilmicosin concentrates in the udder efficiently, it has been suggested to treat cow mastitis. The minimum inhibitory concentration (MIC) of tilmicosin against S. aureus isolated from cow mastitis was reported to be 2.2 µg/mL [3]. Tilmicosin was suggested to have strong antibacterial activity against SASCVs because of the strong antibacterial activity of tilmicosin against S. aureus. Thus, tilmicosin is a candidate drug for treating cow mastitis caused by SASCVs owing to its excellent pharmacodynamics (PD) and pharmacokinetics (PK). On the other hand, tilmicosin might be ineffective for SASCVs mastitis because of its inadequate therapeutic drug concentrations and insufficient residence time of the antibiotic in the mammary gland [4]. The therapeutic drug concentrations and residence time in the mammary gland could be increased by modifying the bioadhesive ability of tilmicosin to SASCVs [5]. Therefore, there is an urgent need to develop novel smart delivery systems to modify the bioadhesive ability of tilmicosin to SASCVs, increase the therapeutic drug concentrations and residence time in the mammary gland, and enhance the efficacy of tilmicosin against cow mastitis caused by SASCVs.
Nanogels (crosslinked polymeric networks) have the advantages of both nanoparticles and hydrogel [6]. They are used to incorporate more than one drug, have excellent structural stability and bioadhesion performance for bacteria [7]. Thus, nanogels might be effective delivery systems to improve the bioadhesion capacity of antibacterial agents and enhance their treatment against SASCVs. The bioadhesion capacity is mainly up to the mucoadhesive materials [8]. Some research has shown that gel materials have excellent bioadhesion capacity. For example, sodium carboxymethyl cellulose (CMC), as a polysaccharide carrier material, is commonly used to deliver drugs, proteins, and genes because of its non-toxicity, excellent biocompatibility, and biodegradable [910]. In addition, chitosan with a low molecular weight, called chitosan oligosaccharide (COS), which has a positive charge, better water-solubility, and antibacterial function, has been used in the nanogel drug delivery systems [11].
In the present study, novel smart delivery composite nanosystems were designed. The designed composite nanogels might improve the bioadhesion capacity of tilmicosin to SASCVs and enhance the antibacterial ability of antibacterial agents by COS-CMC composite nanogels (Fig. 1). The formation mechanism, structural characteristics, bioadhesion, and antibacterial activity of the tilmicosin composite nanogels were studied systematically. | null | null | RESULTS | Optimization of tilmicosin-loaded COS-CMC composite nanogels Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).
COS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.
Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).
COS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.
Properties of the tilmicosin composite nanogels The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.
PDI, polydispersity index.
The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.
PDI, polydispersity index.
In vitro release study Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.
Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.
Adhesive studies The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.
The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.
Antibacterial activity test in vitro
Fig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.
SASCVs = Staphylococcus aureus small colony variants.
Fig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.
SASCVs = Staphylococcus aureus small colony variants. | null | null | [
"Materials",
"Quantitative measurement",
"Formulation of tilmicosin loaded COS-CMC composite nanogels",
"Characterization of the optimal tilmicosin loaded COS-CMC composite nanogels",
"Surface morphology determination",
"Detection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI)",
"Determination of sedimentation rate (F)",
"\nIn vitro release",
"Mucoadhesive studies",
"Antibacterial activity studies",
"The disk diffusion test",
"Broth macrodilution method",
"Time-killing curves",
"Statistical analysis",
"Optimization of tilmicosin-loaded COS-CMC composite nanogels",
"Properties of the tilmicosin composite nanogels",
"\nIn vitro release study",
"Adhesive studies",
"Antibacterial activity test in vitro\n"
] | [
"Tilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.",
"The tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.",
"The tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.",
"Surface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nThe appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nDetection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nThe particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nDetermination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\nThe original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\n\nIn vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.\nThe optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.",
"The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.",
"The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.",
"The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.",
"The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.",
"The tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.",
"The disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nThe agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nBroth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nThe antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nTime-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].\nThe in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].",
"The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.",
"The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.",
"The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].",
"The experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.",
"Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).\nCOS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.",
"The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.\nPDI, polydispersity index.",
"Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.",
"The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.",
"\nFig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.\nSASCVs = Staphylococcus aureus small colony variants."
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] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Materials",
"Quantitative measurement",
"Formulation of tilmicosin loaded COS-CMC composite nanogels",
"Characterization of the optimal tilmicosin loaded COS-CMC composite nanogels",
"Surface morphology determination",
"Detection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI)",
"Determination of sedimentation rate (F)",
"\nIn vitro release",
"Mucoadhesive studies",
"Antibacterial activity studies",
"The disk diffusion test",
"Broth macrodilution method",
"Time-killing curves",
"Statistical analysis",
"RESULTS",
"Optimization of tilmicosin-loaded COS-CMC composite nanogels",
"Properties of the tilmicosin composite nanogels",
"\nIn vitro release study",
"Adhesive studies",
"Antibacterial activity test in vitro\n",
"DISCUSSION"
] | [
"Staphylococcus aureus seriously threatens human health and results in substantial economic losses for livestock worldwide and has aroused considerable concern [1]. In particular, the presence of Staphylococcus aureus small colony variants (SASCVs) in the host cells could establish reservoirs from which reinfection can occur and result in long-term and repeated infection [2]. Because tilmicosin concentrates in the udder efficiently, it has been suggested to treat cow mastitis. The minimum inhibitory concentration (MIC) of tilmicosin against S. aureus isolated from cow mastitis was reported to be 2.2 µg/mL [3]. Tilmicosin was suggested to have strong antibacterial activity against SASCVs because of the strong antibacterial activity of tilmicosin against S. aureus. Thus, tilmicosin is a candidate drug for treating cow mastitis caused by SASCVs owing to its excellent pharmacodynamics (PD) and pharmacokinetics (PK). On the other hand, tilmicosin might be ineffective for SASCVs mastitis because of its inadequate therapeutic drug concentrations and insufficient residence time of the antibiotic in the mammary gland [4]. The therapeutic drug concentrations and residence time in the mammary gland could be increased by modifying the bioadhesive ability of tilmicosin to SASCVs [5]. Therefore, there is an urgent need to develop novel smart delivery systems to modify the bioadhesive ability of tilmicosin to SASCVs, increase the therapeutic drug concentrations and residence time in the mammary gland, and enhance the efficacy of tilmicosin against cow mastitis caused by SASCVs.\nNanogels (crosslinked polymeric networks) have the advantages of both nanoparticles and hydrogel [6]. They are used to incorporate more than one drug, have excellent structural stability and bioadhesion performance for bacteria [7]. Thus, nanogels might be effective delivery systems to improve the bioadhesion capacity of antibacterial agents and enhance their treatment against SASCVs. The bioadhesion capacity is mainly up to the mucoadhesive materials [8]. Some research has shown that gel materials have excellent bioadhesion capacity. For example, sodium carboxymethyl cellulose (CMC), as a polysaccharide carrier material, is commonly used to deliver drugs, proteins, and genes because of its non-toxicity, excellent biocompatibility, and biodegradable [910]. In addition, chitosan with a low molecular weight, called chitosan oligosaccharide (COS), which has a positive charge, better water-solubility, and antibacterial function, has been used in the nanogel drug delivery systems [11].\nIn the present study, novel smart delivery composite nanosystems were designed. The designed composite nanogels might improve the bioadhesion capacity of tilmicosin to SASCVs and enhance the antibacterial ability of antibacterial agents by COS-CMC composite nanogels (Fig. 1). The formation mechanism, structural characteristics, bioadhesion, and antibacterial activity of the tilmicosin composite nanogels were studied systematically.",
"Materials Tilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.\nTilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.\nQuantitative measurement The tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.\nThe tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.\nFormulation of tilmicosin loaded COS-CMC composite nanogels The tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.\nThe tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.\nCharacterization of the optimal tilmicosin loaded COS-CMC composite nanogels Surface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nThe appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nDetection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nThe particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nDetermination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\nThe original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\n\nIn vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.\nThe optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.\nSurface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nThe appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nDetection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nThe particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nDetermination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\nThe original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\n\nIn vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.\nThe optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.\nMucoadhesive studies The tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.\nThe tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.\nAntibacterial activity studies The disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nThe agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nBroth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nThe antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nTime-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].\nThe in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].\nThe disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nThe agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nBroth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nThe antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nTime-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].\nThe in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].\nStatistical analysis The experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.\nThe experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.",
"Tilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.",
"The tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.",
"The tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.",
"Surface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nThe appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.\nDetection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nThe particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.\nDetermination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\nThe original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.\n\nIn vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.\nThe optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.",
"The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.",
"The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.",
"The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.",
"The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.",
"The tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.",
"The disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nThe agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.\nBroth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nThe antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.\nTime-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].\nThe in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].",
"The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.",
"The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.",
"The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].",
"The experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.",
"Optimization of tilmicosin-loaded COS-CMC composite nanogels Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).\nCOS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.\nTilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).\nCOS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.\nProperties of the tilmicosin composite nanogels The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.\nPDI, polydispersity index.\nThe tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.\nPDI, polydispersity index.\n\nIn vitro release study Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.\nConsidering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.\nAdhesive studies The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.\nThe co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.\nAntibacterial activity test in vitro\n \nFig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.\nSASCVs = Staphylococcus aureus small colony variants.\n\nFig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.\nSASCVs = Staphylococcus aureus small colony variants.",
"Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).\nCOS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.",
"The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.\nPDI, polydispersity index.",
"Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.",
"The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.",
"\nFig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.\nSASCVs = Staphylococcus aureus small colony variants.",
"Combining the merits of nanoparticles and hydrogels to prepare composite nanogels to enhance the bioadhesion capacity of antibacterial drugs to bacteria and increase the therapeutic drug concentration and efficacy of antibacterial drugs against bacteria has attracted considerable interest [1314]. In this study, tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (ionic crosslinkers). The COS and CMC concentrations were selected as variables to optimize the tilmicosin-loaded loaded COS-CMC composite nanogels by a single factor test using EE and LC as assessment indices. The formation of tilmicosin composite nanogels was more successful when the EE and LC of tilmicosin-loaded COS-CMC composite nanogels were greater. Therefore, the final optimal formula was comprised of 50 mg/mL (COS), 32 mg/mL (CMC), and 0.25 mg/mL (TPP). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape, which agrees with previous studies [2]. The appearance of lyophilized nanogels showed a uniform crosslinked polymeric network. Moreover, SEM showed that tilmicosin might be incorporated in nano-sized crosslinked polymeric networks, suggesting that tilmicosin-loaded COS-CMC composite nanogels had been formulated successfully by electrostatic interactions between COS and CMC using TPP. This result is consistent with the schematic diagram of tilmicosin-loaded COS-CMC composite nanogels. On the other hand, the particle size of the tilmicosin-loaded loaded COS-CMC composite nanogels detected by SEM was smaller than that determined using the Zeta sizer ZX3600. The difference might be because the particle size was tested using a Zeta sizer ZX3600 in the aqueous state. Free water and some hydrated water were evaporated, so the size measured by the Zeta sizer was usually larger than the real size. On the other hand, the particle sizes measured by SEM were smaller than the genuine diameters. This result is consistent with previous studies [5].\nFor nanogels, the drug is dispersed/dissolved uniformly in the matrix. Hence, release occurs mainly by a combination of drug diffusion, polymer swelling, surface, and bulk erosion of the matrix. In the in vitro release study, 44.5% ± 1.1% and 36.8% ± 2.1% tilmicosin were released from the tilmicosin-loaded COS-CMC composite nanogels in the pH 6.5 and 7.4 PBS at 48 h, respectively. Thus, the in vitro release study indicated that the tilmicosin-loaded COS-CMC composite nanogels at both pH (6.5 and 7.4) had prominent sustained-release performance and might be delivered effectively to the mammary gland in the intact state. The co-culture of SASCVs strain and tilmicosin-loaded COS-CMC composite nanogels and tilmicosin solution for 30 min was observed by SEM to determine if the bioadhesion capacity of tilmicosin to bacteria was enhanced. A large number of tilmicosin-loaded COS-CMC composite nanogels were contacted with or absorbed on the surface of the SASCVs strains, but a tilmicosin solution was not or rarely contacted with or absorbed on the surface of SASCVs strains. These results suggest that the tilmicosin composite nanogels might enhance the bioadhesion capacity of tilmicosin to the SASCVs strain because the cationic nature of COS interacts with the negative charge of SASCVs strain and the excellent adhesion of CMC. This might influence the integrity of bacterial cell membranes or increase the drugs accessing bacteria, hence, increase the antibacterial activity of tilmicosin.\n\nIn vitro antibacterial tests were performed to determine if the tilmicosin composite nanogels could improve the in vitro antibacterial activity of tilmicosin, compare the antibacterial activity of the nanogel with the commercial preparation, and determine if the preparation methods affect the antibacterial activity of tilmicosin. The disk diffusion test showed that the tilmicosin composite nanogels did not improve the antibacterial activity of tilmicosin in vitro, and the antibacterial activity was slightly weak compared to commercial preparations. On the other hand, the MIC of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain showed that the tilmicosin composite nanogels improve the antibacterial activity of tilmicosin in vitro compared to commercial preparations. This might be because the diffusion of the tilmicosin composite nanogels in agar was more difficult than the tilmicosin solution, but the diffusion of the tilmicosin composite nanogels in the Mueller-Hinton broth was easier. Although the in vitro antibacterial activity of tilmicosin was not improved, the MIC of the tilmicosin standard and tilmicosin composite nanogels was similar. This suggests that the structure of tilmicosin was not changed in the process of preparing the tilmicosin composite nanogels. For the time-killing curves, tilmicosin composite nanogels displayed more effective bactericidal activity against the SASCVs strain than the tilmicosin standard, particularly native tilmicosin. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels exhibited bactericidal activity against the SASCVs strain, demonstrating that tilmicosin is concentration-dependent. Thus, the area under concentration-time curve/minimum inhibitory concentration (AUC/MIC) might be the most appropriate parameter to formulate the dosage regimen of the three different preparations of tilmicosin against cow mastitis caused by the SASCVs strain because of their concentration-dependent bactericidal effect. The tilmicosin composite nanogels could fully inhibit the SASCVs strain. This might be because the designed composite nanogels could be more effective in modifying the bioadhesive ability of tilmicosin to the SASCVs strain, thus displaying a stronger curative effect. These results suggest that tilmicosin composite nanogels might be a potential strategy to solve the therapy difficulty of cow mastitis caused by SASCVs strains.\nTilmicosin-loaded COS-CMC composite nanogels were successfully formulated by an electrostatic interaction between COS (positive charge) and CMC (negative charge) using TPP (ionic crosslinkers) to enhance the efficacy of tilmicosin against cow mastitis caused by the SASCVs strain. The tilmicosin-loaded COS-CMC composite nanogels were a homogenous canary yellow uniform suspension with a sedimentation rate of one. Tilmicosin was incorporated in nano-sized crosslinked polymeric networks, and particle size distributions of 357.4±2.6 nm, PDI of 0.11 ± 0.01, ZP of -37.1 ± 0.4 mV, EE of 65.4% ± 0.4%, LC of 21.9% ± 0.4%, and a sedimentation rate of one. More importantly, the tilmicosin-loaded COS-CMC composite nanogels could enhance the bioadhesion capacity of tilmicosin to the SASCVs strain. Furthermore, the tilmicosin composite nanogels displayed more effective bactericidal activity against SASCVs strain than the tilmicosin standard, especially native tilmicosin. Therefore, the prepared tilmicosin composite nanogels might help solve the therapeutic difficulty of cow mastitis caused by SASCVs strains."
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] | [
"Tilmicosin",
"\nStaphylococcus aureus\n",
"nanogels",
"adhesives",
"mastitis"
] | INTRODUCTION:
Staphylococcus aureus seriously threatens human health and results in substantial economic losses for livestock worldwide and has aroused considerable concern [1]. In particular, the presence of Staphylococcus aureus small colony variants (SASCVs) in the host cells could establish reservoirs from which reinfection can occur and result in long-term and repeated infection [2]. Because tilmicosin concentrates in the udder efficiently, it has been suggested to treat cow mastitis. The minimum inhibitory concentration (MIC) of tilmicosin against S. aureus isolated from cow mastitis was reported to be 2.2 µg/mL [3]. Tilmicosin was suggested to have strong antibacterial activity against SASCVs because of the strong antibacterial activity of tilmicosin against S. aureus. Thus, tilmicosin is a candidate drug for treating cow mastitis caused by SASCVs owing to its excellent pharmacodynamics (PD) and pharmacokinetics (PK). On the other hand, tilmicosin might be ineffective for SASCVs mastitis because of its inadequate therapeutic drug concentrations and insufficient residence time of the antibiotic in the mammary gland [4]. The therapeutic drug concentrations and residence time in the mammary gland could be increased by modifying the bioadhesive ability of tilmicosin to SASCVs [5]. Therefore, there is an urgent need to develop novel smart delivery systems to modify the bioadhesive ability of tilmicosin to SASCVs, increase the therapeutic drug concentrations and residence time in the mammary gland, and enhance the efficacy of tilmicosin against cow mastitis caused by SASCVs.
Nanogels (crosslinked polymeric networks) have the advantages of both nanoparticles and hydrogel [6]. They are used to incorporate more than one drug, have excellent structural stability and bioadhesion performance for bacteria [7]. Thus, nanogels might be effective delivery systems to improve the bioadhesion capacity of antibacterial agents and enhance their treatment against SASCVs. The bioadhesion capacity is mainly up to the mucoadhesive materials [8]. Some research has shown that gel materials have excellent bioadhesion capacity. For example, sodium carboxymethyl cellulose (CMC), as a polysaccharide carrier material, is commonly used to deliver drugs, proteins, and genes because of its non-toxicity, excellent biocompatibility, and biodegradable [910]. In addition, chitosan with a low molecular weight, called chitosan oligosaccharide (COS), which has a positive charge, better water-solubility, and antibacterial function, has been used in the nanogel drug delivery systems [11].
In the present study, novel smart delivery composite nanosystems were designed. The designed composite nanogels might improve the bioadhesion capacity of tilmicosin to SASCVs and enhance the antibacterial ability of antibacterial agents by COS-CMC composite nanogels (Fig. 1). The formation mechanism, structural characteristics, bioadhesion, and antibacterial activity of the tilmicosin composite nanogels were studied systematically.
MATERIALS AND METHODS:
Materials Tilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.
Tilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.
Quantitative measurement The tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.
The tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.
Formulation of tilmicosin loaded COS-CMC composite nanogels The tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.
The tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.
Characterization of the optimal tilmicosin loaded COS-CMC composite nanogels Surface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
Detection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
Determination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
In vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
Surface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
Detection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
Determination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
In vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
Mucoadhesive studies The tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.
The tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.
Antibacterial activity studies The disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
Broth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
Time-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
The disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
Broth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
Time-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
Statistical analysis The experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.
The experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.
Materials:
Tilmicosin solution and tilmicosin were obtained from ChuangXin Pharmaceutical Co., Ltd. (China) and Jinan Xinbao Star Animal Pharmaceutical Co., Ltd. (China), respectively. Tilmicosin standard was purchased from the China Institute of Veterinary Drugs Control (China). COS, CMC, and TPP were obtained from Tada Gene Co. Ltd (China). The SASCVs strain was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China). The water was prepared using a Milli-Q system (Millipore, USA). The other reagents in the text were of analytical grade or equivalent.
Quantitative measurement:
The tilmicosin concentration was detected using a Waters 2695 series reverse-phase high-performance liquid chromatography (HPLC). A Symmetry® C18 column (250 mm × 4.6 mm i.d., 5 µm) was used for separation. The mobile phase was 0.1 mol/L ammonium formate, acetonitrile, and methyl alcohol at 61: 29: 10 proportions. The standard curves of tilmicosin ranged from 0.05 to 100 µg/mL, R2 = 0.9988 with a recovery rate > 87% and relative standard deviations below 7.5% for the intra-day and inter-day variation. The specificity of the method was good for the target substances without endogenous interference on the chromatograms.
Formulation of tilmicosin loaded COS-CMC composite nanogels:
The tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions using an ionic crosslinker. Briefly, CMC (600, 800, or 1000 mg) were added to 15 mL of ultrapure water with magnetic stirring for complete dissolution. Subsequently, 0.4 mL of TPP (0.25 mg/mL) was added dropwise to the CMC solution with magnetic stirring at 1200 RPM. Concurrently, COS (300, 450, 600, 750, or 900 mg) was dissolved in 25 mL of ultrapure water. Ten milliliters of absolute alcohol containing 600 mg tilmicosin was added to the 25 mL COS solution at 0.5 mL/min under magnetic stirring. Finally, the COS solution mixture was added dropwise to the CMC mixture solution under magnetic stirring at 1,200 RPM to form the tilmicosin-loaded COS-CMC composite nanogels. The optimal amounts of CMC and CSO were evaluated by the loading capacity (LC) and encapsulation efficiency (EE). Briefly, different tilmicosin-loaded COS-CMC composite nanogels were centrifuged at 14,000 RPM for 60 min. The tilmicosin in the supernatant was then measured by HPLC to calculate the EE. The precipitate was lyophilized into a powder after re-suspending to calculate the LC. Each sample was formulated three times. The data are expressed as the mean ± SD.
Characterization of the optimal tilmicosin loaded COS-CMC composite nanogels:
Surface morphology determination The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
Detection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI) The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
Determination of sedimentation rate (F) The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
In vitro release The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
Surface morphology determination:
The appearance and optical microscopy images of the optimal tilmicosin-loaded COS-CMC composite nanogels were observed. The optimal tilmicosin composite nanogels were freeze-dried using a lyophilizer and characterized by optical microscopy. The optimal tilmicosin composite nanogels were then evaluated by scanning electron microscopy (SEM). Briefly, 1 mg tilmicosin composite nanogels was suspended in 1 mL of distilled water, and 2 μL of the suspension was placed on a coverslip. The samples were coated with gold by ion sputtering and examined at an accelerating voltage of 20 kV after oven-drying.
Detection of the particle diameter, zeta potential (ZP), and polydispersity index (PDI):
The particle diameter, ZP, and PDI of the optimal tilmicosin-loaded COS-CMC composite nanogels were measured using Zeta sizer ZX3600. Before the test, the tilmicosin composite nanogels were appropriately diluted to obtain the optimal kilo counts per second.
Determination of sedimentation rate (F):
The original height (H0) of the 30 mL tilmicosin nanogels sample in a 100 mL graduated cylinder after shaking was measured. The height (H) of the sediment in the cylinder after standing for 3 h was recorded. F was calculated using the equation, F= H/H0.
In vitro release:
The optimal tilmicosin-loaded COS-CMC composite nanogels were added to a dialysis bag that was then placed into 500 mL phosphate-buffered saline (PBS, pH 6.5 and 7.4) at 37 ± 0.5°C after sealing. At different time points (0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h), 1 mL of the dialysate was removed, and HPLC measured the drug concentration. Subsequently, the cumulative release percentage was calculated, and the cumulative release curve was plotted.
Mucoadhesive studies:
The tilmicosin composite nanogels were placed into a liquid medium containing the SASCVs strain of the logarithmic growth phase of 106 CFU/mL at the final tilmicosin concentrations of 10 μg/mL. The liquid culture was drawn on the copper mesh after incubation for 30 min. SEM was performed to determine if the tilmicosin composite nanogels were adsorbed on the bacteria surface.
Antibacterial activity studies:
The disk diffusion test The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
Broth macrodilution method The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
Time-killing curves The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
The disk diffusion test:
The agar diffusion method was used to estimate the in vitro antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels. Briefly, 15 mL of agar medium was first poured into an aseptic plate. Once the agar had solidified, another 5 mL agar medium containing 0.1 mL of bacterial fluid (contained 105–106 CFU SASCVs strain) was poured over the 15 mL agar medium. When the agar was solid, four holes in the solid agar were made using a straw. Subsequently, 50 µL physiological saline, tilmicosin standard, native tilmicosin, and composite nanogels (tilmicosin content: 10 µg/mL) were added. Physiological saline was used as the control. The plates incubating the SASCVs strain were placed in an incubator (5% CO2, 37°C) for 24 h, and the size of the inhibition zones was measured and recorded.
Broth macrodilution method:
The antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels was studied using the broth macrodilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, serial dilutions of the three different tilmicosin formulations in Mueller-Hinton broth were prepared. Subsequently, the SASCVs strain was added to each tube to achieve a final inoculum of 1 × 105 CFU/mL. The minimum inhibitory concentration of tilmicosin was defined as the lowest concentration inhibiting the visible growth of bacteria after 24 h incubation of the cultures at 37°C.
Time-killing curves:
The in vitro killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were obtained by plotting time as a function of log10 CFU/mL. The SASCVs strain was added to 2 ml of MH broth, giving a starting inoculum of 106 CFU/mL. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were added to obtain a serial concentration corresponding to 1/2×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC, and 16×MIC. The tubes were placed at 37°C, and the bacterial count (CFU/mL) was determined using the agar dilution method for each tube after incubating for 1, 2, 4, 8, 12, 24, 48, and 72 h. Briefly, 10-fold serial dilutions from each culture sample were performed, and 100 μL of each dilution was then spread over the agar plates and incubated at 37°C. The viable colonies were then counted after 24 h. Each concentration was performed in triplicate. The limit of detection was 10 CFU/mL [5].
Statistical analysis:
The experimental data are expressed as the mean ± SD and analyzed by one-way analysis of variance using SPSS software; p values < 0.05 were considered significant.
RESULTS:
Optimization of tilmicosin-loaded COS-CMC composite nanogels Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).
COS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.
Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).
COS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.
Properties of the tilmicosin composite nanogels The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.
PDI, polydispersity index.
The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.
PDI, polydispersity index.
In vitro release study Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.
Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.
Adhesive studies The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.
The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.
Antibacterial activity test in vitro
Fig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.
SASCVs = Staphylococcus aureus small colony variants.
Fig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.
SASCVs = Staphylococcus aureus small colony variants.
Optimization of tilmicosin-loaded COS-CMC composite nanogels:
Tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (crosslinkers). The amount of COS and CMC were chosen as variables. LC and EE are the crucial parameters as far as the formulation of drugs are significant. Thus, the tilmicosin composite nanogels were optimized by a single factor test using EE and LC as the assessment indices. The amount of COS and CMC affected the mean LC and EE of tilmicosin composite nanogels considerably (Table 1). The optimal tilmicosin-loaded COS-CMC composite nanogels were obtained when the EE and LC of the composite nanogels were the largest. The mean LC and EE of the optimal tilmicosin-loaded COS-CMC composite nanogels was 21.9% ± 0.4% and 65.4% ± 0.4%. Hence, the final optimal formula was comprised of 50 mg/mL (COS) and 32 mg/mL (CMC).
COS, chitosan oligosaccharide; CMC, sodium carboxymethyl cellulose; TPP, sodium tripolyphosphate; EE, loading capacity; LC, encapsulation efficiency.
Properties of the tilmicosin composite nanogels:
The tilmicosin-loaded COS-CMC composite nanogels had the appearance of a homogenous canary yellow uniform suspension with a sedimentation rate of 1 (Fig. 2A). After the tilmicosin-loaded COS-CMC composite nanogels were freeze-dried using a lyophilizer, the lyophilized nanogels exhibited a uniform across-linked polymeric network (Fig. 2B). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape (Fig. 2C). The mean sizes, PDI, and ZP of the optimal tilmicosin composite were 357.4 ± 2.6 nm (Fig. 2D), 0.11 ± 0.01, and -37.1 ± 0.4 mV (Fig. 2E), respectively. SEM revealed a nano-sized crosslinked polymeric network (Fig. 2F). This may be due to the electrostatic interactions between COS and CMC with the help of TPP. More importantly, tilmicosin might be incorporated in the nano-sized crosslinked polymeric networks. After the tilmicosin-loaded COS-CMC composite nanogels were lyophilized, the image also revealed crosslinked polymeric networks. Thus, the tilmicosin-loaded COS-CMC composite nanogel was prepared successfully by electrostatic interactions using an ionic crosslinker.
PDI, polydispersity index.
In vitro release study:
Considering that the pH of S. aureus infection sites (mammary gland) is usually 6.5 and the pH of mammary gland cells is 7.4 [12], the release of the tilmicosin-loaded COS-CMC composite nanogels in pH 6.5 and 7.4 were measured to determine the environmental pH-responsive release. At pH 6.5, 44.5% ± 1.1% was released from the tilmicosin-loaded COS-CMC composite nanogels at 48 h, while 36.8% ± 2.1% was released in the pH 7.4 PBS at 48 h (Fig. 3). These results suggest that the designed composite nanogels at both pHs exhibited prominent sustained-release performance.
Adhesive studies:
The co-culture of SASCVs and tilmicosin-loaded COS-CMC composite nanogels for 30 min were observed by SEM. SEM showed that the tilmicosin composite nanogels were in contact with or absorbed on the SASCVs strains (Fig. 4). A large number of tilmicosin-loaded COS-CMC composite nanogels were in contact with or absorbed on the surface of SASCVs strains (Fig. 4A). On the other hand, the tilmicosin solution was not or rarely in contact with or absorbed on the surface of the SASCVs strains (Fig. 4B). Thus, the designed composite nanogels might improve the bioadhesion capacity of tilmicosin to the SASCVs and enhance the treatment effects of tilmicosin against SASCVs infections.
Antibacterial activity test in vitro
:
Fig.5 shows the antibacterial activity of native tilmicosin, tilmicosin standard, and tilmicosin-loaded COS-CMC composite nanogels. From the size of the inhibition zone, the tilmicosin composite nanogels exhibited excellent antibacterial activity against the SASCVs strain. The inhibition zone of the control, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 0.0 cm, 2.13 ± 0.07 cm, 3.35 ± 0.11 cm, and 1.46 ± 0.04 cm, respectively (Fig. 5A). The MICs of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2 µg/mL, 1 µg/mL, and 1 µg/mL, respectively. In vitro time-killing curves of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain were illustrated in Fig. 5B-D. According to these profiles, native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels showed a concentration-dependent bactericidal effect as increasing the drug concentrations induced swifter and more radical killing effects. The bactericidal effect of tilmicosin was observed when the concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2×MIC (4 µg/mL, 2 µg/mL, and 2 µg/mL). With increasing tilmicosin concentration, obvious inhibition of bacterial growth was observed in a very short period. Hence, the bactericidal activity increased with increasing drug concentration.
SASCVs = Staphylococcus aureus small colony variants.
DISCUSSION:
Combining the merits of nanoparticles and hydrogels to prepare composite nanogels to enhance the bioadhesion capacity of antibacterial drugs to bacteria and increase the therapeutic drug concentration and efficacy of antibacterial drugs against bacteria has attracted considerable interest [1314]. In this study, tilmicosin-loaded COS-CMC composite nanogels were formulated by electrostatic interactions between COS (positive charge) and CMC (negative charge) using TPP (ionic crosslinkers). The COS and CMC concentrations were selected as variables to optimize the tilmicosin-loaded loaded COS-CMC composite nanogels by a single factor test using EE and LC as assessment indices. The formation of tilmicosin composite nanogels was more successful when the EE and LC of tilmicosin-loaded COS-CMC composite nanogels were greater. Therefore, the final optimal formula was comprised of 50 mg/mL (COS), 32 mg/mL (CMC), and 0.25 mg/mL (TPP). Optical microscopy showed that the tilmicosin composite nanogels were well dispersed with good particle size distributions and a spherical shape, which agrees with previous studies [2]. The appearance of lyophilized nanogels showed a uniform crosslinked polymeric network. Moreover, SEM showed that tilmicosin might be incorporated in nano-sized crosslinked polymeric networks, suggesting that tilmicosin-loaded COS-CMC composite nanogels had been formulated successfully by electrostatic interactions between COS and CMC using TPP. This result is consistent with the schematic diagram of tilmicosin-loaded COS-CMC composite nanogels. On the other hand, the particle size of the tilmicosin-loaded loaded COS-CMC composite nanogels detected by SEM was smaller than that determined using the Zeta sizer ZX3600. The difference might be because the particle size was tested using a Zeta sizer ZX3600 in the aqueous state. Free water and some hydrated water were evaporated, so the size measured by the Zeta sizer was usually larger than the real size. On the other hand, the particle sizes measured by SEM were smaller than the genuine diameters. This result is consistent with previous studies [5].
For nanogels, the drug is dispersed/dissolved uniformly in the matrix. Hence, release occurs mainly by a combination of drug diffusion, polymer swelling, surface, and bulk erosion of the matrix. In the in vitro release study, 44.5% ± 1.1% and 36.8% ± 2.1% tilmicosin were released from the tilmicosin-loaded COS-CMC composite nanogels in the pH 6.5 and 7.4 PBS at 48 h, respectively. Thus, the in vitro release study indicated that the tilmicosin-loaded COS-CMC composite nanogels at both pH (6.5 and 7.4) had prominent sustained-release performance and might be delivered effectively to the mammary gland in the intact state. The co-culture of SASCVs strain and tilmicosin-loaded COS-CMC composite nanogels and tilmicosin solution for 30 min was observed by SEM to determine if the bioadhesion capacity of tilmicosin to bacteria was enhanced. A large number of tilmicosin-loaded COS-CMC composite nanogels were contacted with or absorbed on the surface of the SASCVs strains, but a tilmicosin solution was not or rarely contacted with or absorbed on the surface of SASCVs strains. These results suggest that the tilmicosin composite nanogels might enhance the bioadhesion capacity of tilmicosin to the SASCVs strain because the cationic nature of COS interacts with the negative charge of SASCVs strain and the excellent adhesion of CMC. This might influence the integrity of bacterial cell membranes or increase the drugs accessing bacteria, hence, increase the antibacterial activity of tilmicosin.
In vitro antibacterial tests were performed to determine if the tilmicosin composite nanogels could improve the in vitro antibacterial activity of tilmicosin, compare the antibacterial activity of the nanogel with the commercial preparation, and determine if the preparation methods affect the antibacterial activity of tilmicosin. The disk diffusion test showed that the tilmicosin composite nanogels did not improve the antibacterial activity of tilmicosin in vitro, and the antibacterial activity was slightly weak compared to commercial preparations. On the other hand, the MIC of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against SASCVs strain showed that the tilmicosin composite nanogels improve the antibacterial activity of tilmicosin in vitro compared to commercial preparations. This might be because the diffusion of the tilmicosin composite nanogels in agar was more difficult than the tilmicosin solution, but the diffusion of the tilmicosin composite nanogels in the Mueller-Hinton broth was easier. Although the in vitro antibacterial activity of tilmicosin was not improved, the MIC of the tilmicosin standard and tilmicosin composite nanogels was similar. This suggests that the structure of tilmicosin was not changed in the process of preparing the tilmicosin composite nanogels. For the time-killing curves, tilmicosin composite nanogels displayed more effective bactericidal activity against the SASCVs strain than the tilmicosin standard, particularly native tilmicosin. The native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels exhibited bactericidal activity against the SASCVs strain, demonstrating that tilmicosin is concentration-dependent. Thus, the area under concentration-time curve/minimum inhibitory concentration (AUC/MIC) might be the most appropriate parameter to formulate the dosage regimen of the three different preparations of tilmicosin against cow mastitis caused by the SASCVs strain because of their concentration-dependent bactericidal effect. The tilmicosin composite nanogels could fully inhibit the SASCVs strain. This might be because the designed composite nanogels could be more effective in modifying the bioadhesive ability of tilmicosin to the SASCVs strain, thus displaying a stronger curative effect. These results suggest that tilmicosin composite nanogels might be a potential strategy to solve the therapy difficulty of cow mastitis caused by SASCVs strains.
Tilmicosin-loaded COS-CMC composite nanogels were successfully formulated by an electrostatic interaction between COS (positive charge) and CMC (negative charge) using TPP (ionic crosslinkers) to enhance the efficacy of tilmicosin against cow mastitis caused by the SASCVs strain. The tilmicosin-loaded COS-CMC composite nanogels were a homogenous canary yellow uniform suspension with a sedimentation rate of one. Tilmicosin was incorporated in nano-sized crosslinked polymeric networks, and particle size distributions of 357.4±2.6 nm, PDI of 0.11 ± 0.01, ZP of -37.1 ± 0.4 mV, EE of 65.4% ± 0.4%, LC of 21.9% ± 0.4%, and a sedimentation rate of one. More importantly, the tilmicosin-loaded COS-CMC composite nanogels could enhance the bioadhesion capacity of tilmicosin to the SASCVs strain. Furthermore, the tilmicosin composite nanogels displayed more effective bactericidal activity against SASCVs strain than the tilmicosin standard, especially native tilmicosin. Therefore, the prepared tilmicosin composite nanogels might help solve the therapeutic difficulty of cow mastitis caused by SASCVs strains.
| Background:
The poor bioadhesion capacity of tilmicosin resulting in treatment failure for Staphylococcus aureus small colony variants (SASCVs) mastitis.
Methods:
Tilmicosin-loaded chitosan oligosaccharide (COS)-sodium carboxymethyl cellulose (CMC) composite nanogels were formulated by an electrostatic interaction between COS (positive charge) and CMC (negative charge) using sodium tripolyphosphate (TPP) (ionic crosslinkers). The formation mechanism, structural characteristics, bioadhesion, and antibacterial activity of tilmicosin composite nanogels were studied systematically.
Results:
The optimized formulation was comprised of 50 mg/mL (COS), 32 mg/mL (CMC), and 0.25 mg/mL (TPP). The size, encapsulation efficiency, loading capacity, polydispersity index, and zeta potential of the optimized tilmicosin composite nanogels were 357.4 ± 2.6 nm, 65.4 ± 0.4%, 21.9 ± 0.4%, 0.11 ± 0.01, and -37.1 ± 0.4 mV, respectively; the sedimentation rate was one. Scanning electron microscopy showed that tilmicosin might be incorporated in nano-sized crosslinked polymeric networks. Moreover, adhesive studies suggested that tilmicosin composite nanogels could enhance the bioadhesion capacity of tilmicosin for the SASCVs strain. The inhibition zone of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2.13 ± 0.07, 3.35 ± 0.11, and 1.46 ± 0.04 cm, respectively. The minimum inhibitory concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2, 1, and 1 µg/mL, respectively. The in vitro time-killing curves showed that the tilmicosin composite nanogels increased the antibacterial activity against the SASCVs strain.
Conclusions:
This study provides a potential strategy for developing tilmicosin composite nanogels to treat cow mastitis caused by the SASCVs strain. | null | null | 12,239 | 329 | [
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| nanogels | adhesives | mastitis [SUMMARY] | null | [CONTENT] Tilmicosin |
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| nanogels | adhesives | mastitis [SUMMARY] | null | [CONTENT] Animals | Anti-Bacterial Agents | Carboxymethylcellulose Sodium | Cattle | Chitosan | Female | Mastitis, Bovine | Nanogels | Oligosaccharides | Staphylococcal Infections | Staphylococcus aureus | Tylosin [SUMMARY] | null | [CONTENT] Animals | Anti-Bacterial Agents | Carboxymethylcellulose Sodium | Cattle | Chitosan | Female | Mastitis, Bovine | Nanogels | Oligosaccharides | Staphylococcal Infections | Staphylococcus aureus | Tylosin [SUMMARY] | null | [CONTENT] Animals | Anti-Bacterial Agents | Carboxymethylcellulose Sodium | Cattle | Chitosan | Female | Mastitis, Bovine | Nanogels | Oligosaccharides | Staphylococcal Infections | Staphylococcus aureus | Tylosin [SUMMARY] | null | [CONTENT] tilmicosin compare antibacterial | aureus tilmicosin candidate | activity tilmicosin aureus | efficacy tilmicosin cow | tilmicosin cow mastitis [SUMMARY] | null | [CONTENT] tilmicosin compare antibacterial | aureus tilmicosin candidate | activity tilmicosin aureus | efficacy tilmicosin cow | tilmicosin cow mastitis [SUMMARY] | null | [CONTENT] tilmicosin compare antibacterial | aureus tilmicosin candidate | activity tilmicosin aureus | efficacy tilmicosin cow | tilmicosin cow mastitis [SUMMARY] | null | [CONTENT] tilmicosin | nanogels | composite | composite nanogels | ml | cmc | cos | tilmicosin composite | tilmicosin composite nanogels | cos cmc [SUMMARY] | null | [CONTENT] tilmicosin | nanogels | composite | composite nanogels | ml | cmc | cos | tilmicosin composite | tilmicosin composite nanogels | cos cmc [SUMMARY] | null | [CONTENT] tilmicosin | nanogels | composite | composite nanogels | ml | cmc | cos | tilmicosin composite | tilmicosin composite nanogels | cos cmc [SUMMARY] | null | [CONTENT] bioadhesion | mastitis | sascvs | delivery | antibacterial | drug | cow mastitis | cow | tilmicosin | residence time [SUMMARY] | null | [CONTENT] tilmicosin | composite | composite nanogels | nanogels | fig | cos | cmc | cos cmc | loaded cos cmc composite | tilmicosin loaded cos cmc [SUMMARY] | null | [CONTENT] tilmicosin | composite | nanogels | composite nanogels | ml | cmc | cos | tilmicosin composite | tilmicosin composite nanogels | sascvs [SUMMARY] | null | [CONTENT] Staphylococcus [SUMMARY] | null | [CONTENT] 50 mg/mL (COS | 32 | 0.25 | TPP ||| 357.4 | 2.6 | 65.4 | 0.4% | 21.9 | 0.4% | 0.11 | 0.01 | 0.4 ||| ||| tilmicosin ||| tilmicosin | tilmicosin | 2.13 | 0.07 | 3.35 | 0.11 | 1.46 ± | 0.04 cm ||| tilmicosin | 2 | 1 | 1 ||| [SUMMARY] | null | [CONTENT] Staphylococcus ||| Tilmicosin | CMC | COS | CMC | TPP ||| tilmicosin ||| ||| 50 mg/mL (COS | 32 | 0.25 | TPP ||| 357.4 | 2.6 | 65.4 | 0.4% | 21.9 | 0.4% | 0.11 | 0.01 | 0.4 ||| ||| tilmicosin ||| tilmicosin | tilmicosin | 2.13 | 0.07 | 3.35 | 0.11 | 1.46 ± | 0.04 cm ||| tilmicosin | 2 | 1 | 1 ||| ||| [SUMMARY] | null |
|||
Assessment of comprehensive HIV/AIDS knowledge level among in-school adolescents in eastern Ethiopia. | 23517715 | In Ethiopia, more adolescents are in school today than ever before; however, there are no studies that have assessed their comprehensive knowledge of HIV/AIDS. Thus, this study tried to assess the level of this knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia. | INTRODUCTION | A cross-sectional school-based study was conducted using a facilitator-guided self-administered questionnaire. The respondents were students attending regular school in 14 high schools located in 14 different districts in eastern Ethiopia. The proportion of in-school adolescents with comprehensive HIV/AIDS knowledge was computed and compared by sex. The factors that were associated with the comprehensive HIV/AIDS knowledge were assessed using bivariate and multivariable logistic regression. | METHODS | Only about one in four, 677 (24.5%), in-school adolescents have comprehensive HIV/AIDS knowledge. The knowledge was better among in-school adolescents from families with a relatively middle or high wealth index (adjusted OR [95% CI]=1.39 [1.03-1.87] and 1.75 [1.24-2.48], respectively), who got HIV/AIDS information mainly from friends or mass media (adjusted OR [95% CI]=1.63 [1.17-2.27] and 1.55 [1.14-2.11], respectively) and who received education on HIV/AIDS and sexual matters at school (adjusted OR [95% CI]=1.59 [1.22-2.08]). The females were less likely to have comprehensive HIV/AIDS knowledge compared to males (adjusted OR and [95% CI]=0.60 [0.49-0.75]). | RESULTS | In general, only about a quarter of in-school adolescents had comprehensive HIV/AIDS knowledge. Although the female adolescents are highly vulnerable to HIV infection and its effects, they were by far less likely to have comprehensive HIV/AIDS knowledge. HIV/AIDS information, education and communication activities need to be intensified in high schools. | CONCLUSIONS | [
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] | 3605405 | Introduction | In Ethiopia, a large number of adolescents are enrolled in high schools, and a significant proportion of rural students attend high school away from their home village. The level of comprehensive HIV/AIDS knowledge and access to HIV/AIDS information and services have been matters of great concern [1]. In Ethiopia, an awareness of HIV/AIDS among adult population has been found to be 97.6% for men and 96.2% for women, while the knowledge of preventive strategies is estimated to be 62.0% for men and 48.7% for women. The levels of overall (57%) and comprehensive (18.5%) knowledge of HIV/AIDS among different population groups including adolescents were lower [2,3]. Similarly, the comprehensive knowledge of modes of HIV transmission of in-school adolescents was lower than that of the general awareness or the separate modes of transmission [4,5]. Studies from other African countries and eastern India also revealed that comprehensive knowledge of HIV/AIDS ranged from 9% to 42% [6–8]; however, studies from Brazil and Europe showed a higher (more than 90%) degree of HIV/AIDS and related issues awareness [9,10].
Previous studies conducted in Ethiopia revealed that residing in urban areas, higher educational attainment and male gender are positively associated with increased awareness of HIV prevention methods [2]. Studies from other countries have also found out that comprehensive HIV/AIDS knowledge is associated with communication with guardians or parents and peers about sexual topics, while living in poor households and disadvantaged neighbourhoods is associated with inaccurate knowledge of the transmission and prevention methods of HIV [6,8].
In Ethiopia, there are only a few studies that have assessed the level of the comprehensive HIV/AIDS knowledge of in-school adolescents. The available studies revealed that sexual debut during adolescence is associated with the risk of being HIV positive at later ages and that secondary school adolescents have the highest HIV positive proportion among the youth age groups in Ethiopia [5,11]. Furthermore, after three decades of AIDS pandemic, it is believed that measuring knowledge of HIV/AIDS by a single awareness question (asking a question such as “Have you ever heard of HIV/AIDS?”) is simply misleading and inappropriate. This study tried to assess the level of the comprehensive HIV/AIDS knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia. | Methods | The study design was a cross-sectional school-based survey with internal comparison. The study was conducted in eastern Ethiopia, and it involved 14 randomly selected high schools found in 14 different districts. The sample size (N=2860) was determined by OpenEpi web-based epidemiological calculator based on the assumptions of 95% significance level; 25% males and 19% females had the outcome [3], considering 3:1 male–female proportion. All the students who were attending regular classes in the selected high schools were eligible for the study, and the respondents were randomly selected by the 3:1 male–female proportion (72% male and 28% female) based on the enrolment data for that academic year. Data were collected by a facilitator-guided self-administered structured questionnaire adapted from WHO sexual and reproductive health questionnaires [12]. In each school, students who were selected for the study were summoned by gender to designated classrooms, and data were collected simultaneously to overcome information contamination. The data collection process was facilitated by gender-matched facilitators. Facilitators were university lecturers who received training on the study procedures and spoke the local language fluently. Two facilitators per classroom were assigned to facilitate the data collection process. To check its consistency, the questionnaire was prepared in English and translated into Afan Oromo and Amharic and then back to English by independent bilingual language experts.
The dependent variable was comprehensive HIV/AIDS knowledge measured by correct answers to HIV/AIDS diagnosis and treatment, HIV transmission modes and HIV prevention methods; comprehensive knowledge was then redefined by the ability to identify correctly at least two major ways of preventing sexual transmission of HIV, to reject at least two most common local misconceptions about HIV transmission and by the correct knowledge of HIV diagnosis method. The independent variables were sex, age, area of residence, wealth index, parents’ vital status, father's educational status, mother's educational status, major source of HIV/AIDS information, discussion on sexual topics with parents or other family members and ever having been taught HIV/AIDS and related issues at school.
Data were double-entered onto the EPI-data Version 3.1 software by defining legal values for each variable and setting skip patterns. The double-entered data were verified and the cleaned version was exported to Stata/SE 11.0 software for analysis. The level of knowledge was computed and compared for males and females. The factors associated with comprehensive HIV/AIDS knowledge at bivariate were identified, and the variables with P value of 0.25 and less were taken to multivariable analysis. The model was built with backward elimination.
The study was conducted after obtaining approval from the Institutional Review Committee of Haramaya University and the necessary permission from other concerned educational authorities. The confidentiality of the information was maintained by excluding personal identifiers, and data were collected after getting informed consent and/or assent from the teacher–parent joint committee and/or every respondent. | Results | Of the 2860 students invited to fill out the facilitator-guided self-administered questionnaire, 2766 students responded adequately, making the response rate 96.7%. The majority, 1985 (71.8%), of the respondents were male. The majority of in-school students, 1901 (68.7%), had families in rural areas (Table 1). The age of the respondents ranged from 14 to 19 years, and the mean was 17.1. Only about one in four, 677 (24.5%), in-school adolescents had comprehensive HIV/AIDS knowledge, while the males had more comprehensive HIV/AIDS knowledge (27.3%) compared to the females (17.3%) (P<0.001). The combined comprehensive HIV/AIDS and pregnancy knowledge was very low, 139 (5%). However, the males were more likely to have the combined comprehensive knowledge (5.7%) compared to the females (3.2%) (P=0.006) (Table 2).
Background characteristics of in-school adolescents and their families, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia, 2011
Comprehensive HIV/AIDS and pregnancy knowledge by gender among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia, 2011
Predictors of comprehensive HIV/AIDS knowledge The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).
Logistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011
Bold values are to indicate the corresponding P-value<0.05.
The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).
Logistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011
Bold values are to indicate the corresponding P-value<0.05. | null | null | [
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] | [
"In Ethiopia, a large number of adolescents are enrolled in high schools, and a significant proportion of rural students attend high school away from their home village. The level of comprehensive HIV/AIDS knowledge and access to HIV/AIDS information and services have been matters of great concern [1]. In Ethiopia, an awareness of HIV/AIDS among adult population has been found to be 97.6% for men and 96.2% for women, while the knowledge of preventive strategies is estimated to be 62.0% for men and 48.7% for women. The levels of overall (57%) and comprehensive (18.5%) knowledge of HIV/AIDS among different population groups including adolescents were lower [2,3]. Similarly, the comprehensive knowledge of modes of HIV transmission of in-school adolescents was lower than that of the general awareness or the separate modes of transmission [4,5]. Studies from other African countries and eastern India also revealed that comprehensive knowledge of HIV/AIDS ranged from 9% to 42% [6–8]; however, studies from Brazil and Europe showed a higher (more than 90%) degree of HIV/AIDS and related issues awareness [9,10].\nPrevious studies conducted in Ethiopia revealed that residing in urban areas, higher educational attainment and male gender are positively associated with increased awareness of HIV prevention methods [2]. Studies from other countries have also found out that comprehensive HIV/AIDS knowledge is associated with communication with guardians or parents and peers about sexual topics, while living in poor households and disadvantaged neighbourhoods is associated with inaccurate knowledge of the transmission and prevention methods of HIV [6,8].\nIn Ethiopia, there are only a few studies that have assessed the level of the comprehensive HIV/AIDS knowledge of in-school adolescents. The available studies revealed that sexual debut during adolescence is associated with the risk of being HIV positive at later ages and that secondary school adolescents have the highest HIV positive proportion among the youth age groups in Ethiopia [5,11]. Furthermore, after three decades of AIDS pandemic, it is believed that measuring knowledge of HIV/AIDS by a single awareness question (asking a question such as “Have you ever heard of HIV/AIDS?”) is simply misleading and inappropriate. This study tried to assess the level of the comprehensive HIV/AIDS knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia.",
"The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).\nLogistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011\nBold values are to indicate the corresponding P-value<0.05."
] | [
null,
null
] | [
"Introduction",
"Methods",
"Results",
"Predictors of comprehensive HIV/AIDS knowledge",
"Discussion"
] | [
"In Ethiopia, a large number of adolescents are enrolled in high schools, and a significant proportion of rural students attend high school away from their home village. The level of comprehensive HIV/AIDS knowledge and access to HIV/AIDS information and services have been matters of great concern [1]. In Ethiopia, an awareness of HIV/AIDS among adult population has been found to be 97.6% for men and 96.2% for women, while the knowledge of preventive strategies is estimated to be 62.0% for men and 48.7% for women. The levels of overall (57%) and comprehensive (18.5%) knowledge of HIV/AIDS among different population groups including adolescents were lower [2,3]. Similarly, the comprehensive knowledge of modes of HIV transmission of in-school adolescents was lower than that of the general awareness or the separate modes of transmission [4,5]. Studies from other African countries and eastern India also revealed that comprehensive knowledge of HIV/AIDS ranged from 9% to 42% [6–8]; however, studies from Brazil and Europe showed a higher (more than 90%) degree of HIV/AIDS and related issues awareness [9,10].\nPrevious studies conducted in Ethiopia revealed that residing in urban areas, higher educational attainment and male gender are positively associated with increased awareness of HIV prevention methods [2]. Studies from other countries have also found out that comprehensive HIV/AIDS knowledge is associated with communication with guardians or parents and peers about sexual topics, while living in poor households and disadvantaged neighbourhoods is associated with inaccurate knowledge of the transmission and prevention methods of HIV [6,8].\nIn Ethiopia, there are only a few studies that have assessed the level of the comprehensive HIV/AIDS knowledge of in-school adolescents. The available studies revealed that sexual debut during adolescence is associated with the risk of being HIV positive at later ages and that secondary school adolescents have the highest HIV positive proportion among the youth age groups in Ethiopia [5,11]. Furthermore, after three decades of AIDS pandemic, it is believed that measuring knowledge of HIV/AIDS by a single awareness question (asking a question such as “Have you ever heard of HIV/AIDS?”) is simply misleading and inappropriate. This study tried to assess the level of the comprehensive HIV/AIDS knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia.",
"The study design was a cross-sectional school-based survey with internal comparison. The study was conducted in eastern Ethiopia, and it involved 14 randomly selected high schools found in 14 different districts. The sample size (N=2860) was determined by OpenEpi web-based epidemiological calculator based on the assumptions of 95% significance level; 25% males and 19% females had the outcome [3], considering 3:1 male–female proportion. All the students who were attending regular classes in the selected high schools were eligible for the study, and the respondents were randomly selected by the 3:1 male–female proportion (72% male and 28% female) based on the enrolment data for that academic year. Data were collected by a facilitator-guided self-administered structured questionnaire adapted from WHO sexual and reproductive health questionnaires [12]. In each school, students who were selected for the study were summoned by gender to designated classrooms, and data were collected simultaneously to overcome information contamination. The data collection process was facilitated by gender-matched facilitators. Facilitators were university lecturers who received training on the study procedures and spoke the local language fluently. Two facilitators per classroom were assigned to facilitate the data collection process. To check its consistency, the questionnaire was prepared in English and translated into Afan Oromo and Amharic and then back to English by independent bilingual language experts.\nThe dependent variable was comprehensive HIV/AIDS knowledge measured by correct answers to HIV/AIDS diagnosis and treatment, HIV transmission modes and HIV prevention methods; comprehensive knowledge was then redefined by the ability to identify correctly at least two major ways of preventing sexual transmission of HIV, to reject at least two most common local misconceptions about HIV transmission and by the correct knowledge of HIV diagnosis method. The independent variables were sex, age, area of residence, wealth index, parents’ vital status, father's educational status, mother's educational status, major source of HIV/AIDS information, discussion on sexual topics with parents or other family members and ever having been taught HIV/AIDS and related issues at school.\nData were double-entered onto the EPI-data Version 3.1 software by defining legal values for each variable and setting skip patterns. The double-entered data were verified and the cleaned version was exported to Stata/SE 11.0 software for analysis. The level of knowledge was computed and compared for males and females. The factors associated with comprehensive HIV/AIDS knowledge at bivariate were identified, and the variables with P value of 0.25 and less were taken to multivariable analysis. The model was built with backward elimination.\nThe study was conducted after obtaining approval from the Institutional Review Committee of Haramaya University and the necessary permission from other concerned educational authorities. The confidentiality of the information was maintained by excluding personal identifiers, and data were collected after getting informed consent and/or assent from the teacher–parent joint committee and/or every respondent.",
"Of the 2860 students invited to fill out the facilitator-guided self-administered questionnaire, 2766 students responded adequately, making the response rate 96.7%. The majority, 1985 (71.8%), of the respondents were male. The majority of in-school students, 1901 (68.7%), had families in rural areas (Table 1). The age of the respondents ranged from 14 to 19 years, and the mean was 17.1. Only about one in four, 677 (24.5%), in-school adolescents had comprehensive HIV/AIDS knowledge, while the males had more comprehensive HIV/AIDS knowledge (27.3%) compared to the females (17.3%) (P<0.001). The combined comprehensive HIV/AIDS and pregnancy knowledge was very low, 139 (5%). However, the males were more likely to have the combined comprehensive knowledge (5.7%) compared to the females (3.2%) (P=0.006) (Table 2).\nBackground characteristics of in-school adolescents and their families, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia, 2011\nComprehensive HIV/AIDS and pregnancy knowledge by gender among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia, 2011\n Predictors of comprehensive HIV/AIDS knowledge The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).\nLogistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011\nBold values are to indicate the corresponding P-value<0.05.\nThe logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).\nLogistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011\nBold values are to indicate the corresponding P-value<0.05.",
"The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).\nLogistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011\nBold values are to indicate the corresponding P-value<0.05.",
"Only about a quarter of the in-school adolescents had comprehensive HIV/AIDS knowledge. The knowledge was more common among in-school adolescents from families with a middle or higher wealth index, who got HIV/AIDS information mainly from friends or mass media and who received HIV/AIDS and sexual matters education at school. Although the females are highly vulnerable to HIV infection and its effects, they were less likely to have comprehensive HIV/AIDS knowledge compared to males. They were also less likely to have comprehensive pregnancy knowledge, even though they had more knowledge on pregnancy occurrence dates related to the menstrual cycle.\nThe major source of bias in this study might emerge from the self-administered data collection technique in which respondents might have failed to understand the questions correctly. To overcome this bias, data were collected by a facilitator-guided self-administered method (one facilitator read the questions while respondents worked on their questionnaire and other facilitators monitored whether all the students were progressing at equal pace with the facilitator). The respondents were also provided with questionnaires prepared in all possible languages respondents might understand well. Even though it may be difficult to totally overcome the bias which arises from such methods of data collection, its effect on the findings of this study is negligible.\nThe level of comprehensive HIV/AIDS knowledge in this study was lower than the previous AIDS awareness and prevention strategy knowledge estimates [2]. The reason might be that, as it has been more than 30 years since the first discovery of AIDS, the awareness should have been evidently high. The comprehensive HIV/AIDS knowledge in this study is slightly lower than the previous prevention strategy knowledge and slightly higher than previous comprehensive knowledge of HIV/AIDS reported by another study [3]. This could be due to the difference in the study populations, as this study was conducted on in-school students while the previous study covered wide population groups.\nComprehensive HIV/AIDS knowledge was associated with the sex of the respondents. The females were less likely to have comprehensive HIV/AIDS knowledge compared to the males. This finding is consistent with the Ethiopian DHS report on HIV prevention strategy knowledge and previous in-school adolescents study which reported low HIV transmission modes knowledge among females [2,4,10]. This may be due to the cultural double standards placed on males and females, which encourage males to discuss HIV/AIDS and related sexual matters issues more openly and discourage or even restrict females from discussing sexual related issues. Similarly, as some cultures in Ethiopia encourage or tolerate male adolescents’ pre-marital sexual intercourse but expect females to remain virgins until marriage, female adolescents will often shy away from discussing sexual issues or refrain from asking questions related to it.\nFamily wealth index was associated with comprehensive HIV/AIDS knowledge. The adolescents from middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index. This is consistent with a finding from another study which reported an increase in mean-knowledge score by increasing socio-economic class [13]. This may be because wealthier families can afford mass media items like televisions, radios, etc. giving their adolescent children access to different HIV/AIDS information sources, particularly as the positive effect was stronger and significant in this study for in-school adolescents whose families reside in rural areas. Furthermore, adolescents from urban families might have different sources of information other than the family-based resources.\nThose who cited friends and mass media as their major sources of HIV/AIDS information were more likely to have comprehensive HIV/AIDS knowledge compared to those who reported their parents or other family members as their major sources. This was not consistent with other study findings [6]. This is probably because adolescents may openly discuss more with their friends about sexual matters than with their parents or other family members. This is confirmed by a previous study in Ethiopia [14]. Similarly, mass media may also address such topics more openly in a matter that attracts adolescents’ attention.\nAttending classes on HIV/AIDS and sexual matters at school was significantly associated with comprehensive HIV/AIDS knowledge, in that the respondents who reported that they had attended such classes were more likely to have comprehensive HIV/AIDS knowledge compared to those who did not attend such classes. This may be because, even though such topics were integrated into some subjects in schools, some schools and/or teachers may not teach these topics as they might feel they are not well trained on the topic, while some other schools and teachers may teach such topics by making extra effort themselves or by inviting other relevant professionals.\nDiscussing sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge. This finding is not consistent with the report of another study [6]. This could be due to the limited knowledge of parents or other family members on HIV/AIDS. Furthermore, what the study participants reported as discussion might not be the open bi-lateral discussion; it might simply be the restrictive order of traditional parents or other family members to make their adolescents stay away from peers and not to listen to sexual related discussion, further limiting their access to other information sources [15].\nIn conclusion, only about one in four of the in-school adolescents had comprehensive HIV/AIDS knowledge. The factors associated with comprehensive HIV/AIDS knowledge of in-school adolescents were both individual factors (sex) and contextual factors (family wealth index, major source of HIV/AIDS information and ever been taught HIV/AIDS and sexual matters at school). Although the female adolescents are highly vulnerable to HIV infection and its effects, they were by far the less likely to have comprehensive HIV/AIDS knowledge. Thus, HIV/AIDS information, education and communication activities need to be intensified in high schools, including further attention being put on gender, the family wealth disparity, the positive influences of peers, mass media and teaching methods of HIV/AIDS and related issues at schools."
] | [
null,
"methods",
"results",
null,
"discussion"
] | [
"in-school",
"adolescents",
"HIV/AIDS",
"comprehensive knowledge"
] | Introduction:
In Ethiopia, a large number of adolescents are enrolled in high schools, and a significant proportion of rural students attend high school away from their home village. The level of comprehensive HIV/AIDS knowledge and access to HIV/AIDS information and services have been matters of great concern [1]. In Ethiopia, an awareness of HIV/AIDS among adult population has been found to be 97.6% for men and 96.2% for women, while the knowledge of preventive strategies is estimated to be 62.0% for men and 48.7% for women. The levels of overall (57%) and comprehensive (18.5%) knowledge of HIV/AIDS among different population groups including adolescents were lower [2,3]. Similarly, the comprehensive knowledge of modes of HIV transmission of in-school adolescents was lower than that of the general awareness or the separate modes of transmission [4,5]. Studies from other African countries and eastern India also revealed that comprehensive knowledge of HIV/AIDS ranged from 9% to 42% [6–8]; however, studies from Brazil and Europe showed a higher (more than 90%) degree of HIV/AIDS and related issues awareness [9,10].
Previous studies conducted in Ethiopia revealed that residing in urban areas, higher educational attainment and male gender are positively associated with increased awareness of HIV prevention methods [2]. Studies from other countries have also found out that comprehensive HIV/AIDS knowledge is associated with communication with guardians or parents and peers about sexual topics, while living in poor households and disadvantaged neighbourhoods is associated with inaccurate knowledge of the transmission and prevention methods of HIV [6,8].
In Ethiopia, there are only a few studies that have assessed the level of the comprehensive HIV/AIDS knowledge of in-school adolescents. The available studies revealed that sexual debut during adolescence is associated with the risk of being HIV positive at later ages and that secondary school adolescents have the highest HIV positive proportion among the youth age groups in Ethiopia [5,11]. Furthermore, after three decades of AIDS pandemic, it is believed that measuring knowledge of HIV/AIDS by a single awareness question (asking a question such as “Have you ever heard of HIV/AIDS?”) is simply misleading and inappropriate. This study tried to assess the level of the comprehensive HIV/AIDS knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia.
Methods:
The study design was a cross-sectional school-based survey with internal comparison. The study was conducted in eastern Ethiopia, and it involved 14 randomly selected high schools found in 14 different districts. The sample size (N=2860) was determined by OpenEpi web-based epidemiological calculator based on the assumptions of 95% significance level; 25% males and 19% females had the outcome [3], considering 3:1 male–female proportion. All the students who were attending regular classes in the selected high schools were eligible for the study, and the respondents were randomly selected by the 3:1 male–female proportion (72% male and 28% female) based on the enrolment data for that academic year. Data were collected by a facilitator-guided self-administered structured questionnaire adapted from WHO sexual and reproductive health questionnaires [12]. In each school, students who were selected for the study were summoned by gender to designated classrooms, and data were collected simultaneously to overcome information contamination. The data collection process was facilitated by gender-matched facilitators. Facilitators were university lecturers who received training on the study procedures and spoke the local language fluently. Two facilitators per classroom were assigned to facilitate the data collection process. To check its consistency, the questionnaire was prepared in English and translated into Afan Oromo and Amharic and then back to English by independent bilingual language experts.
The dependent variable was comprehensive HIV/AIDS knowledge measured by correct answers to HIV/AIDS diagnosis and treatment, HIV transmission modes and HIV prevention methods; comprehensive knowledge was then redefined by the ability to identify correctly at least two major ways of preventing sexual transmission of HIV, to reject at least two most common local misconceptions about HIV transmission and by the correct knowledge of HIV diagnosis method. The independent variables were sex, age, area of residence, wealth index, parents’ vital status, father's educational status, mother's educational status, major source of HIV/AIDS information, discussion on sexual topics with parents or other family members and ever having been taught HIV/AIDS and related issues at school.
Data were double-entered onto the EPI-data Version 3.1 software by defining legal values for each variable and setting skip patterns. The double-entered data were verified and the cleaned version was exported to Stata/SE 11.0 software for analysis. The level of knowledge was computed and compared for males and females. The factors associated with comprehensive HIV/AIDS knowledge at bivariate were identified, and the variables with P value of 0.25 and less were taken to multivariable analysis. The model was built with backward elimination.
The study was conducted after obtaining approval from the Institutional Review Committee of Haramaya University and the necessary permission from other concerned educational authorities. The confidentiality of the information was maintained by excluding personal identifiers, and data were collected after getting informed consent and/or assent from the teacher–parent joint committee and/or every respondent.
Results:
Of the 2860 students invited to fill out the facilitator-guided self-administered questionnaire, 2766 students responded adequately, making the response rate 96.7%. The majority, 1985 (71.8%), of the respondents were male. The majority of in-school students, 1901 (68.7%), had families in rural areas (Table 1). The age of the respondents ranged from 14 to 19 years, and the mean was 17.1. Only about one in four, 677 (24.5%), in-school adolescents had comprehensive HIV/AIDS knowledge, while the males had more comprehensive HIV/AIDS knowledge (27.3%) compared to the females (17.3%) (P<0.001). The combined comprehensive HIV/AIDS and pregnancy knowledge was very low, 139 (5%). However, the males were more likely to have the combined comprehensive knowledge (5.7%) compared to the females (3.2%) (P=0.006) (Table 2).
Background characteristics of in-school adolescents and their families, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia, 2011
Comprehensive HIV/AIDS and pregnancy knowledge by gender among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia, 2011
Predictors of comprehensive HIV/AIDS knowledge The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).
Logistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011
Bold values are to indicate the corresponding P-value<0.05.
The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).
Logistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011
Bold values are to indicate the corresponding P-value<0.05.
Predictors of comprehensive HIV/AIDS knowledge:
The logistic regression showed that the females were 40% less likely to have comprehensive HIV/AIDS knowledge compared to the males (adjusted OR [95% CI]=0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index (adjusted OR [95% CI]=1.39 [1.03–1.87] and 1.75 [1.24–2.47], respectively). The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas (Crude OR and [95% CI]=2.00 [1.24–3.20]; and 1.38 [0.77–2.45], respectively). The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source (adjusted OR [95% CI] =1.63 [1.17–2.27] and 1.55 [1.14–2.11], respectively). Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) (Table 3).
Logistic regression indicating factors associated with comprehensive HIV/AIDS knowledge among in-school adolescents, Eastern Hararge Zone, Oromia Regional State, Eastern Ethiopia 2011
Bold values are to indicate the corresponding P-value<0.05.
Discussion:
Only about a quarter of the in-school adolescents had comprehensive HIV/AIDS knowledge. The knowledge was more common among in-school adolescents from families with a middle or higher wealth index, who got HIV/AIDS information mainly from friends or mass media and who received HIV/AIDS and sexual matters education at school. Although the females are highly vulnerable to HIV infection and its effects, they were less likely to have comprehensive HIV/AIDS knowledge compared to males. They were also less likely to have comprehensive pregnancy knowledge, even though they had more knowledge on pregnancy occurrence dates related to the menstrual cycle.
The major source of bias in this study might emerge from the self-administered data collection technique in which respondents might have failed to understand the questions correctly. To overcome this bias, data were collected by a facilitator-guided self-administered method (one facilitator read the questions while respondents worked on their questionnaire and other facilitators monitored whether all the students were progressing at equal pace with the facilitator). The respondents were also provided with questionnaires prepared in all possible languages respondents might understand well. Even though it may be difficult to totally overcome the bias which arises from such methods of data collection, its effect on the findings of this study is negligible.
The level of comprehensive HIV/AIDS knowledge in this study was lower than the previous AIDS awareness and prevention strategy knowledge estimates [2]. The reason might be that, as it has been more than 30 years since the first discovery of AIDS, the awareness should have been evidently high. The comprehensive HIV/AIDS knowledge in this study is slightly lower than the previous prevention strategy knowledge and slightly higher than previous comprehensive knowledge of HIV/AIDS reported by another study [3]. This could be due to the difference in the study populations, as this study was conducted on in-school students while the previous study covered wide population groups.
Comprehensive HIV/AIDS knowledge was associated with the sex of the respondents. The females were less likely to have comprehensive HIV/AIDS knowledge compared to the males. This finding is consistent with the Ethiopian DHS report on HIV prevention strategy knowledge and previous in-school adolescents study which reported low HIV transmission modes knowledge among females [2,4,10]. This may be due to the cultural double standards placed on males and females, which encourage males to discuss HIV/AIDS and related sexual matters issues more openly and discourage or even restrict females from discussing sexual related issues. Similarly, as some cultures in Ethiopia encourage or tolerate male adolescents’ pre-marital sexual intercourse but expect females to remain virgins until marriage, female adolescents will often shy away from discussing sexual issues or refrain from asking questions related to it.
Family wealth index was associated with comprehensive HIV/AIDS knowledge. The adolescents from middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index. This is consistent with a finding from another study which reported an increase in mean-knowledge score by increasing socio-economic class [13]. This may be because wealthier families can afford mass media items like televisions, radios, etc. giving their adolescent children access to different HIV/AIDS information sources, particularly as the positive effect was stronger and significant in this study for in-school adolescents whose families reside in rural areas. Furthermore, adolescents from urban families might have different sources of information other than the family-based resources.
Those who cited friends and mass media as their major sources of HIV/AIDS information were more likely to have comprehensive HIV/AIDS knowledge compared to those who reported their parents or other family members as their major sources. This was not consistent with other study findings [6]. This is probably because adolescents may openly discuss more with their friends about sexual matters than with their parents or other family members. This is confirmed by a previous study in Ethiopia [14]. Similarly, mass media may also address such topics more openly in a matter that attracts adolescents’ attention.
Attending classes on HIV/AIDS and sexual matters at school was significantly associated with comprehensive HIV/AIDS knowledge, in that the respondents who reported that they had attended such classes were more likely to have comprehensive HIV/AIDS knowledge compared to those who did not attend such classes. This may be because, even though such topics were integrated into some subjects in schools, some schools and/or teachers may not teach these topics as they might feel they are not well trained on the topic, while some other schools and teachers may teach such topics by making extra effort themselves or by inviting other relevant professionals.
Discussing sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge. This finding is not consistent with the report of another study [6]. This could be due to the limited knowledge of parents or other family members on HIV/AIDS. Furthermore, what the study participants reported as discussion might not be the open bi-lateral discussion; it might simply be the restrictive order of traditional parents or other family members to make their adolescents stay away from peers and not to listen to sexual related discussion, further limiting their access to other information sources [15].
In conclusion, only about one in four of the in-school adolescents had comprehensive HIV/AIDS knowledge. The factors associated with comprehensive HIV/AIDS knowledge of in-school adolescents were both individual factors (sex) and contextual factors (family wealth index, major source of HIV/AIDS information and ever been taught HIV/AIDS and sexual matters at school). Although the female adolescents are highly vulnerable to HIV infection and its effects, they were by far the less likely to have comprehensive HIV/AIDS knowledge. Thus, HIV/AIDS information, education and communication activities need to be intensified in high schools, including further attention being put on gender, the family wealth disparity, the positive influences of peers, mass media and teaching methods of HIV/AIDS and related issues at schools.
| Background:
In Ethiopia, more adolescents are in school today than ever before; however, there are no studies that have assessed their comprehensive knowledge of HIV/AIDS. Thus, this study tried to assess the level of this knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia.
Methods:
A cross-sectional school-based study was conducted using a facilitator-guided self-administered questionnaire. The respondents were students attending regular school in 14 high schools located in 14 different districts in eastern Ethiopia. The proportion of in-school adolescents with comprehensive HIV/AIDS knowledge was computed and compared by sex. The factors that were associated with the comprehensive HIV/AIDS knowledge were assessed using bivariate and multivariable logistic regression.
Results:
Only about one in four, 677 (24.5%), in-school adolescents have comprehensive HIV/AIDS knowledge. The knowledge was better among in-school adolescents from families with a relatively middle or high wealth index (adjusted OR [95% CI]=1.39 [1.03-1.87] and 1.75 [1.24-2.48], respectively), who got HIV/AIDS information mainly from friends or mass media (adjusted OR [95% CI]=1.63 [1.17-2.27] and 1.55 [1.14-2.11], respectively) and who received education on HIV/AIDS and sexual matters at school (adjusted OR [95% CI]=1.59 [1.22-2.08]). The females were less likely to have comprehensive HIV/AIDS knowledge compared to males (adjusted OR and [95% CI]=0.60 [0.49-0.75]).
Conclusions:
In general, only about a quarter of in-school adolescents had comprehensive HIV/AIDS knowledge. Although the female adolescents are highly vulnerable to HIV infection and its effects, they were by far less likely to have comprehensive HIV/AIDS knowledge. HIV/AIDS information, education and communication activities need to be intensified in high schools. | null | null | 3,483 | 373 | [
459,
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] | null | null | [CONTENT] in-school | adolescents | HIV/AIDS | comprehensive knowledge [SUMMARY] | [CONTENT] in-school | adolescents | HIV/AIDS | comprehensive knowledge [SUMMARY] | [CONTENT] in-school | adolescents | HIV/AIDS | comprehensive knowledge [SUMMARY] | null | [CONTENT] in-school | adolescents | HIV/AIDS | comprehensive knowledge [SUMMARY] | null | [CONTENT] Acquired Immunodeficiency Syndrome | Adolescent | Cross-Sectional Studies | Ethiopia | Female | Health Knowledge, Attitudes, Practice | Humans | Male | Risk Assessment | Schools | Sex Factors | Students | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Acquired Immunodeficiency Syndrome | Adolescent | Cross-Sectional Studies | Ethiopia | Female | Health Knowledge, Attitudes, Practice | Humans | Male | Risk Assessment | Schools | Sex Factors | Students | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Acquired Immunodeficiency Syndrome | Adolescent | Cross-Sectional Studies | Ethiopia | Female | Health Knowledge, Attitudes, Practice | Humans | Male | Risk Assessment | Schools | Sex Factors | Students | Surveys and Questionnaires | Young Adult [SUMMARY] | null | [CONTENT] Acquired Immunodeficiency Syndrome | Adolescent | Cross-Sectional Studies | Ethiopia | Female | Health Knowledge, Attitudes, Practice | Humans | Male | Risk Assessment | Schools | Sex Factors | Students | Surveys and Questionnaires | Young Adult [SUMMARY] | null | [CONTENT] hiv ethiopia | aids knowledge study | increased awareness hiv | hiv ethiopia studies | aids knowledge adolescents [SUMMARY] | [CONTENT] hiv ethiopia | aids knowledge study | increased awareness hiv | hiv ethiopia studies | aids knowledge adolescents [SUMMARY] | [CONTENT] hiv ethiopia | aids knowledge study | increased awareness hiv | hiv ethiopia studies | aids knowledge adolescents [SUMMARY] | null | [CONTENT] hiv ethiopia | aids knowledge study | increased awareness hiv | hiv ethiopia studies | aids knowledge adolescents [SUMMARY] | null | [CONTENT] hiv | aids | hiv aids | knowledge | comprehensive | comprehensive hiv aids | comprehensive hiv | hiv aids knowledge | aids knowledge | adolescents [SUMMARY] | [CONTENT] hiv | aids | hiv aids | knowledge | comprehensive | comprehensive hiv aids | comprehensive hiv | hiv aids knowledge | aids knowledge | adolescents [SUMMARY] | [CONTENT] hiv | aids | hiv aids | knowledge | comprehensive | comprehensive hiv aids | comprehensive hiv | hiv aids knowledge | aids knowledge | adolescents [SUMMARY] | null | [CONTENT] hiv | aids | hiv aids | knowledge | comprehensive | comprehensive hiv aids | comprehensive hiv | hiv aids knowledge | aids knowledge | adolescents [SUMMARY] | null | [CONTENT] hiv | studies | aids | hiv aids | knowledge | awareness | adolescents | comprehensive | revealed | ethiopia [SUMMARY] | [CONTENT] data | hiv | study | selected | based | status | facilitators | female | educational | data collected [SUMMARY] | [CONTENT] hiv aids | hiv | aids | knowledge | comprehensive | comprehensive hiv | comprehensive hiv aids | 95 ci | ci | hiv aids knowledge [SUMMARY] | null | [CONTENT] hiv | aids | hiv aids | knowledge | comprehensive | comprehensive hiv aids | comprehensive hiv | aids knowledge | hiv aids knowledge | comprehensive hiv aids knowledge [SUMMARY] | null | [CONTENT] Ethiopia | today ||| Ethiopia [SUMMARY] | [CONTENT] ||| 14 | 14 | Ethiopia ||| ||| [SUMMARY] | [CONTENT] Only about one | four | 677 | 24.5% ||| ||| 95% | 1.03-1.87 ||| 1.75 | 1.24 ||| ||| 95% | 1.17 | 1.55 | 1.14 ||| 95% ||| 1.22-2.08 ||| 95% ||| CI]=0.60 | 0.49-0.75 [SUMMARY] | null | [CONTENT] Ethiopia | today ||| Ethiopia ||| ||| 14 | 14 | Ethiopia ||| ||| ||| Only about one | four | 677 | 24.5% ||| ||| 95% | 1.03-1.87 ||| 1.75 | 1.24 ||| ||| 95% | 1.17 | 1.55 | 1.14 ||| 95% ||| 1.22-2.08 ||| 95% ||| CI]=0.60 | 0.49-0.75 ||| only about a quarter ||| ||| [SUMMARY] | null |
||||
An evaluation of children with Kawasaki disease in Istanbul: a retrospective follow-up study. | 21340213 | Kawasaki disease (KD) is an acute, self-limiting vasculitis of unknown etiology. The incidence of KD is increasing world wide. However, the epidemiological data for KD in Turkey has not been well described. | BACKGROUND | Patients with KD were retrospectively identified from the hospital discharge records between 2002 and 2010. Atypical cases of KD were excluded. A standardized form was used to collect demographic data, clinical information, echocardiography and laboratory results. | METHOD | Thirty-five patients with KD, with a mean age of 2.5 + 1.9 years, were identified. Eighty-five point seven per cent of patients were under 5 years of age. A seasonal pattern favouring the winter months was noticed. In addition to fever and bilateral conjunctival injection, changes in the oral cavity and lips were the most commonly detected clinical signs in our cases. Coronary artery abnormalities were detected in nine patients. The majority of our patients had started treatment with intravenous immunoglobulin in the first 10 days of the onset of fever, and only one patient required systemic steroids for intravenous immunoglobulin-resistant KD. The coronary artery abnormalities resolved in all nine patients within 8 months. | RESULTS | This study is the most comprehensive series of children from Turkey with KD included in Medline. As adult-onset ischemic heart disease may be due to KD in childhood, further prospective clinical investigations are needed to understand the epidemiology, management and long-term follow-up of the disease. | CONCLUSION | [
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"Female",
"Follow-Up Studies",
"Humans",
"Immunoglobulins, Intravenous",
"Immunologic Factors",
"Infant",
"Male",
"Mucocutaneous Lymph Node Syndrome",
"Reference Values",
"Retrospective Studies",
"Turkey"
] | 3020335 | INTRODUCTION | Kawasaki disease (KD) is an acute, self‐limiting systemic vasculitis of unknown etiology, which mainly affects children aged <5 years. It was first described by Tomisaku Kawasaki in 1967 in Japan.1 KD has now replaced rheumatic fever as the leading cause of acquired heart disease in childhood in the developed world, and is the second most common childhood vasculitis.2,3 Although the incidence of KD varies among countries, it is much higher in children from Asian countries.4,5 The clinical signs of KD are similar to those of many other childhood illnesses. The disease is often complicated by coronary artery abnormalities (CAA), including dilatation and/or aneurysms, and thus is a leading cause of acquired heart disease in children.6,7 Some clinical features other than the classic diagnostic criteria are intense irritability, cough, diarrhea, sterile pyuria, arthritis, arthralgia, redness and induration at the site of a Bacille–Calmette–Guerin (BCG) scar. Patients with prolonged fever and fewer than four of the other principal criteria are diagnosed as atypical or incomplete KD if CAA are present.8
The incentive for this research came from the remarkable lack of knowledge about the epidemiology and features of KD in Turkey. In this article we present the demographic, clinical and laboratory features of children with KD, who were diagnosed and managed in our hospital. | null | null | RESULTS | Thirty‐five patients with KD were treated in the Istanbul American Hospital during the 8‐year period. The mean age of the patients was 2.5±1.9 years (range 2 months to 7 years). Eighty per cent of the cases were diagnosed within the first 10 days, with the longest time to diagnosis being 15 days. The demographic and clinical characteristics of patients are summarized in Table 2. Eighty six per cent (30 cases) of patients were aged <5 years and 14% (five cases) of patients were aged >5 years at the time of diagnosis. Fever was the main clinical sign in all patients and mean body temperature was 39.4±0.9°C at diagnosis. Although intense irritability is not part of the classic diagnostic criteria, it was present in all patients. In one patient there was redness and induration at the site of the BCG scar. Three patients had arthritis and one patient had abdominal pain and diarrhea. Laboratory findings showed that anemia (Hb <11 g/dl) was present in 14 (40%) patients who were all a year old. Elevated serum transaminases were detected in 10 (28.6%) patients. Laboratory values of the patients at diagnosis are shown in Table 3. No positive culture (blood, urine and throat) was found in our patients. Sterile pyuria was present in two patients.
Patients were receiving antibiotic therapy at diagnosis, with the exception of two patients. During the 8‐year period, the monthly number of cases was lowest between July and September and peaked in the winter months (Fig 1). The highest incidence was seen in February, with seven patients.
CAA were detected in nine (25.7%) cases (six males, three female). One patient had ectasia in the right coronary artery and two patients in the left coronary artery. Six children had coronary artery aneurysms (one in both right and left coronary arteries with pericardial effusion, three had left coronary artery aneurysms and two had right coronary artery aneurysms with mitral valve regurgitation). All children were treated with intravenous immunoglobulin (IVIg) at a dose of 2 g/kg for a period of 10–12 h in addition to high‐dose aspirin (100 mg/kg) during the febrile period, according to current consensus guidelines.10 In one patient, after a second infusion of IVIg, high‐dose (30 mg/kg) methylprednisolone pulse therapy was carried out for a period of 2 h. Symptoms resolved in this child after two intravenous doses of steroids. There was no recurrence in any patient. | CONCLUSIONS | None. | [
"CONCLUSIONS"
] | [
"In summary, it is our belief that KD is not a rare disease in Turkey. This study is the most comprehensive series of children from Turkey with KD included in Medline. As adult‐onset ischemic heart disease may be due to KD in childhood,9,18,28 further prospective clinical investigations are needed to understand the epidemiology, management and long‐term follow‐up the disease in Turkey."
] | [
null
] | [
"INTRODUCTION",
"MATERIAL AND METHODS",
"RESULTS",
"DISCUSSION",
"CONCLUSIONS"
] | [
"Kawasaki disease (KD) is an acute, self‐limiting systemic vasculitis of unknown etiology, which mainly affects children aged <5 years. It was first described by Tomisaku Kawasaki in 1967 in Japan.1 KD has now replaced rheumatic fever as the leading cause of acquired heart disease in childhood in the developed world, and is the second most common childhood vasculitis.2,3 Although the incidence of KD varies among countries, it is much higher in children from Asian countries.4,5 The clinical signs of KD are similar to those of many other childhood illnesses. The disease is often complicated by coronary artery abnormalities (CAA), including dilatation and/or aneurysms, and thus is a leading cause of acquired heart disease in children.6,7 Some clinical features other than the classic diagnostic criteria are intense irritability, cough, diarrhea, sterile pyuria, arthritis, arthralgia, redness and induration at the site of a Bacille–Calmette–Guerin (BCG) scar. Patients with prolonged fever and fewer than four of the other principal criteria are diagnosed as atypical or incomplete KD if CAA are present.8 \nThe incentive for this research came from the remarkable lack of knowledge about the epidemiology and features of KD in Turkey. In this article we present the demographic, clinical and laboratory features of children with KD, who were diagnosed and managed in our hospital.",
"Patients with KD were identified from hospital discharge records between 2002 and 2010. All the children were being followed up routinely at an outpatient clinic of the American Hospital, Istanbul, Turkey—a private hospital, generally relying on a high socioeconomic population from Istanbul. Diagnosis of KD was made according to American Heart Association guidelines.9 Table 1 shows the standard diagnostic criteria for KD. Medical charts of patients with KD were reviewed using a standardized form to collect demographic data, clinical information, and laboratory test results, retrospectively. All children diagnosed with atypical KD were excluded. Echocardiography was performed during hospitalization and follow‐up in all patients.\nDefinitions of CAA were based on the following criteria: for children aged <5 years, an internal lumen diameter (ILD) ≤3 mm was considered normal and for children aged ≥5 years, an ILD ≤4 mm was considered normal. An ILD of a coronary artery segment enlarged to <1.5 times the normal upper limit was defined as a dilatation, and an ILD enlarged to ≥1.5 times the normal upper limit was defined as an aneurysm. When a coronary artery was larger than normal (dilated) and without a segmental aneurysm, the vessel was considered ectasic.9 Echocardiography was usually repeated within 2 weeks of the onset of illness, during the fourth week, and thereafter depending on the initial findings. All patients underwent laboratory investigations for platelets, leukocyte (white blood cell) count, hemoglobin (Hb), C‐reactive protein (CRP), erythrocyte sedimentation rate (ESR), aspartate aminotransferase, alanine aminotransferase and underwent urine analysis.\nQualitative data are presented as frequencies with percentages and quantitative data as means with standard deviations (SD).",
"Thirty‐five patients with KD were treated in the Istanbul American Hospital during the 8‐year period. The mean age of the patients was 2.5±1.9 years (range 2 months to 7 years). Eighty per cent of the cases were diagnosed within the first 10 days, with the longest time to diagnosis being 15 days. The demographic and clinical characteristics of patients are summarized in Table 2. Eighty six per cent (30 cases) of patients were aged <5 years and 14% (five cases) of patients were aged >5 years at the time of diagnosis. Fever was the main clinical sign in all patients and mean body temperature was 39.4±0.9°C at diagnosis. Although intense irritability is not part of the classic diagnostic criteria, it was present in all patients. In one patient there was redness and induration at the site of the BCG scar. Three patients had arthritis and one patient had abdominal pain and diarrhea. Laboratory findings showed that anemia (Hb <11 g/dl) was present in 14 (40%) patients who were all a year old. Elevated serum transaminases were detected in 10 (28.6%) patients. Laboratory values of the patients at diagnosis are shown in Table 3. No positive culture (blood, urine and throat) was found in our patients. Sterile pyuria was present in two patients.\nPatients were receiving antibiotic therapy at diagnosis, with the exception of two patients. During the 8‐year period, the monthly number of cases was lowest between July and September and peaked in the winter months (Fig 1). The highest incidence was seen in February, with seven patients.\nCAA were detected in nine (25.7%) cases (six males, three female). One patient had ectasia in the right coronary artery and two patients in the left coronary artery. Six children had coronary artery aneurysms (one in both right and left coronary arteries with pericardial effusion, three had left coronary artery aneurysms and two had right coronary artery aneurysms with mitral valve regurgitation). All children were treated with intravenous immunoglobulin (IVIg) at a dose of 2 g/kg for a period of 10–12 h in addition to high‐dose aspirin (100 mg/kg) during the febrile period, according to current consensus guidelines.10 In one patient, after a second infusion of IVIg, high‐dose (30 mg/kg) methylprednisolone pulse therapy was carried out for a period of 2 h. Symptoms resolved in this child after two intravenous doses of steroids. There was no recurrence in any patient.",
"KD is an acute febrile vasculitis that has been reported with increasing incidence among all racial ethnic groups. While Japan has the highest and increasing annual incidence in the world (184.6 per 100,000 children aged <5 years between 2005 and 2006), the epidemiology of KD in Europe has not been well described.11,12 Likewise, the epidemiology of KD in Turkey is also not well known. A nationwide survey found that KD had an incidence of 9% of childhood vasculitides.13 \nThere are many unconfirmed hypotheses about the cause of KD. Although clinical, laboratory and epidemiologic features strongly suggest an infectious cause, the etiology of KD still remains unknown. One possibility is that a viral repiratory infection (represented by a seasonal pattern), juxtaposed with a subsequent secondary bacterial colonization, precipitates a cascade of events that result in an exaggerated immunologic response.14,15 Our study confirms the winter peak of KD (Figure 1), but no clear relationship with respiratory pathogens was shown as virologic and immunologic data were lacking. KD is more common in boys than girls and 85% of cases occur in children aged <5 years.11 In our study, we detected a male predominance and age distribution resembling that of previous studies.16 Studies from the United States also show that KD is more common during the winter and that 76% of children are <5 years old.17 \nIn KD, the fever is typically high‐spiking and remittent, with peak temperatures of >39°C and, in many cases, >40°C.18 Our patients' fever pattern was consistent with this classic finding, with a mean body temperature of 39.4±0.9°C at diagnosis. In addition to fever, bilateral conjunctival injection and changes in the oral cavity and lips were the most commonly detected clinical signs in our cases, as found in other studies from Turkey and other parts of the world.19-22 The occurrence of cervical lymphadenopathy in KD is variable. It is seen in only 50–75% of patients, whereas most of the other features are seen in approximately 90%.23 It is usually unilateral and confined to the anterior cervical triangle, and its classic criteria include ≥1 lymph node that is >1.5 cm in diameter.18 In our study, cervical lymphadenopathy was the least common of the principal clinical features (48.6%). The lymphadenopathies in our patients were typically classic, except for one patient who presented with massive cervical node enlargement.\nMultiple clinical findings may be observed in patients with KD. Arthritis or arthralgia can occur in the first week of the illness and tends to affect multiple joints, including the small interphalangeal joints and large weight‐bearing joints.18 In our series, two patients had arthritis in their knees and one patient had arthritis in his ankle.\nChildren with KD are often more irritable than the children with other febrile ilnesses,18 and all the patients in this study were irritable. Gastrointestinal complaints, including diarrhea, vomiting and abdominal pain, occur in about one‐third of patients.24 Rarely, KD can present as an acute surgical abdomen.24 Contrary to previous studies, only one of our 35 patients had abdominal pain and diarrhea. Erythema and induration at the site of BCG vaccination is common in Japan, where BCG is used widely.25 As in Japan, BCG is routinely performed in Turkey, but only one patient had redness and induration at the site of the BCG scar.\nKD is mainly a clinical diagnosis and there are no pathognomonic laboratory tests or findings. However, leukocytosis is typical during the acute stage of KD. Approximately 50% of patients had white blood cell counts >15,000/mm3.18 Similar to other reports,8,10,22 the mean leukocyte count of our study group was 15,896±6,383/mm3. Normocytic anemia may develop, particularly with more prolonged duration of active inflammation.8,18 Marked anemia (Hb <11 g/dl) on admission was noted in 14 (40%) patients who were all over 1 year old and who had prolonged fever for more than 7 days. Thrombocytosis is a characteristic feature of the later phases of the illness. It is rarely present in the first week of the illness, usually appearing in the second week, and peaking in the third week with a gradual return to normal by 4–8 weeks after onset in uncomplicated cases. Platelet counts are usually >450,000/mm3 in patients evaluated after day 7 of illness.18 In our series most of the patients were diagnosed in the second week and the mean platelet count was 496,889±208,503/mm3, which is consistent with other studies.18 Elevations of CRP and ESR are almost universal in KD, usually returning to normal values by 6–10 weeks after the onset of illness.18 Mean values of CRP and ESR were high in our patients. Burns et al.26 reported mild to moderate elevations in serum transaminases in ≤40% of patients. Although the mean values of transaminases were not high in our study group, the ratio of patients with high levels (28.6%) was consistent with this multicenter study.\nUrine analysis showed intermittent mild to moderate sterile pyuria in approximately 33% of patients, suggesting urethritis.18 Urine cultures of two patients with sterile pyuria were normal.\nAs the principal clinical findings that fulfill the diagnostic criteria are not specific for KD, patients with other diseases (Table 1) with similar clinical features were excluded from our study.\nThe major sequelae of KD are related to the cardiovascular, and more specifically, the coronary arterial system. Therefore cardiac imaging by echocardiography is a critical part of the evaluation of all patients with suspected KD. For uncomplicated cases, echocardiography is recommended at the time of diagnosis, at 2 weeks, and at 6–8 weeks after the onset of disease.27 We followed the above recommendations and also repeated the echocardiography beyond 8 weeks for all patients with previously normal findings, as other abnormalities in the coronary artery vasculature (aneurysms) and aortic root (dilatation with or without regurgitation) may develop, even among patients with normal baseline echocardiograms.27,28 Additionally, echocardiography was performed with higher frequency for patients with pericardial effusion, mitral valve regurgitation and IVIg‐resistant KD, for closer evaluation and follow‐up. None of our patients with normal baseline echocardiography was shown to develop CAA on follow‐up echocardiograms beyond the 8 weeks. CAA develop in approximately 15–25% of untreated children with the disease and may lead to ischemic heart disease, myocardial infarction or sudden death.18 In our patients, CAA were detected in nine (25.7%) cases. None of them led to myocardial infarction or ischemic heart disease. A recent study from Turkey reported the CAA rate as 33.3% in 24 children with KD.22 Burns et al. described the emergence of the peak mortality as 15–45 days after the onset of fever; and during this time, well‐established coronary vasculitis occurs concomitantly with a marked elevation of the platelet count and a hypercoagulable state.28 The mean duration of fever in our cases was 7.8±2.8 days (range 4–15). Our patients had fewer adverse sequelae, as all were treated before the peak mortality days, thus suggesting the importance of early diagnosis and treatment. The opportunity for early management occurred as the patients were being routinely followed up in our outpatient clinic.\nThe current medical management of KD is IVIg and high‐dose aspirin. IVIg is very effective when given in the first ten days of illness. It reduces the risk of CAA from 20–25% to 1–2%.6,9 A subgroup of patients with KD will be resistant to IVIg therapy; these patients are at greatest risk of developing coronary artery aneurysms and long‐term sequelae of the disease.29 Data have demonstrated the usefulness and safety of systemic steroids in patients resistant to IVIg.30 In our series, the majority of our patients were given IVIg before the tenth day and only one patient was IVIg resistant, who was successfully treated with pulse methylprednisolone. Newburger et al. reported that 50–67% of aneurysms resolve within 1–2 years.31 Echocardiographic evaluation of the nine children with CAA in our study was normal within 8 months (three within 6 months, four within 7 months, and two within 8 months). All patients are alive and receiving annual echocardiographic follow‐up.",
"In summary, it is our belief that KD is not a rare disease in Turkey. This study is the most comprehensive series of children from Turkey with KD included in Medline. As adult‐onset ischemic heart disease may be due to KD in childhood,9,18,28 further prospective clinical investigations are needed to understand the epidemiology, management and long‐term follow‐up the disease in Turkey."
] | [
"intro",
"materials|methods",
"results",
"discussion",
null
] | [
"Kawasaki disease",
"Coronary artery disease",
"Echocardiography",
"Steroids"
] | INTRODUCTION:
Kawasaki disease (KD) is an acute, self‐limiting systemic vasculitis of unknown etiology, which mainly affects children aged <5 years. It was first described by Tomisaku Kawasaki in 1967 in Japan.1 KD has now replaced rheumatic fever as the leading cause of acquired heart disease in childhood in the developed world, and is the second most common childhood vasculitis.2,3 Although the incidence of KD varies among countries, it is much higher in children from Asian countries.4,5 The clinical signs of KD are similar to those of many other childhood illnesses. The disease is often complicated by coronary artery abnormalities (CAA), including dilatation and/or aneurysms, and thus is a leading cause of acquired heart disease in children.6,7 Some clinical features other than the classic diagnostic criteria are intense irritability, cough, diarrhea, sterile pyuria, arthritis, arthralgia, redness and induration at the site of a Bacille–Calmette–Guerin (BCG) scar. Patients with prolonged fever and fewer than four of the other principal criteria are diagnosed as atypical or incomplete KD if CAA are present.8
The incentive for this research came from the remarkable lack of knowledge about the epidemiology and features of KD in Turkey. In this article we present the demographic, clinical and laboratory features of children with KD, who were diagnosed and managed in our hospital.
MATERIAL AND METHODS:
Patients with KD were identified from hospital discharge records between 2002 and 2010. All the children were being followed up routinely at an outpatient clinic of the American Hospital, Istanbul, Turkey—a private hospital, generally relying on a high socioeconomic population from Istanbul. Diagnosis of KD was made according to American Heart Association guidelines.9 Table 1 shows the standard diagnostic criteria for KD. Medical charts of patients with KD were reviewed using a standardized form to collect demographic data, clinical information, and laboratory test results, retrospectively. All children diagnosed with atypical KD were excluded. Echocardiography was performed during hospitalization and follow‐up in all patients.
Definitions of CAA were based on the following criteria: for children aged <5 years, an internal lumen diameter (ILD) ≤3 mm was considered normal and for children aged ≥5 years, an ILD ≤4 mm was considered normal. An ILD of a coronary artery segment enlarged to <1.5 times the normal upper limit was defined as a dilatation, and an ILD enlarged to ≥1.5 times the normal upper limit was defined as an aneurysm. When a coronary artery was larger than normal (dilated) and without a segmental aneurysm, the vessel was considered ectasic.9 Echocardiography was usually repeated within 2 weeks of the onset of illness, during the fourth week, and thereafter depending on the initial findings. All patients underwent laboratory investigations for platelets, leukocyte (white blood cell) count, hemoglobin (Hb), C‐reactive protein (CRP), erythrocyte sedimentation rate (ESR), aspartate aminotransferase, alanine aminotransferase and underwent urine analysis.
Qualitative data are presented as frequencies with percentages and quantitative data as means with standard deviations (SD).
RESULTS:
Thirty‐five patients with KD were treated in the Istanbul American Hospital during the 8‐year period. The mean age of the patients was 2.5±1.9 years (range 2 months to 7 years). Eighty per cent of the cases were diagnosed within the first 10 days, with the longest time to diagnosis being 15 days. The demographic and clinical characteristics of patients are summarized in Table 2. Eighty six per cent (30 cases) of patients were aged <5 years and 14% (five cases) of patients were aged >5 years at the time of diagnosis. Fever was the main clinical sign in all patients and mean body temperature was 39.4±0.9°C at diagnosis. Although intense irritability is not part of the classic diagnostic criteria, it was present in all patients. In one patient there was redness and induration at the site of the BCG scar. Three patients had arthritis and one patient had abdominal pain and diarrhea. Laboratory findings showed that anemia (Hb <11 g/dl) was present in 14 (40%) patients who were all a year old. Elevated serum transaminases were detected in 10 (28.6%) patients. Laboratory values of the patients at diagnosis are shown in Table 3. No positive culture (blood, urine and throat) was found in our patients. Sterile pyuria was present in two patients.
Patients were receiving antibiotic therapy at diagnosis, with the exception of two patients. During the 8‐year period, the monthly number of cases was lowest between July and September and peaked in the winter months (Fig 1). The highest incidence was seen in February, with seven patients.
CAA were detected in nine (25.7%) cases (six males, three female). One patient had ectasia in the right coronary artery and two patients in the left coronary artery. Six children had coronary artery aneurysms (one in both right and left coronary arteries with pericardial effusion, three had left coronary artery aneurysms and two had right coronary artery aneurysms with mitral valve regurgitation). All children were treated with intravenous immunoglobulin (IVIg) at a dose of 2 g/kg for a period of 10–12 h in addition to high‐dose aspirin (100 mg/kg) during the febrile period, according to current consensus guidelines.10 In one patient, after a second infusion of IVIg, high‐dose (30 mg/kg) methylprednisolone pulse therapy was carried out for a period of 2 h. Symptoms resolved in this child after two intravenous doses of steroids. There was no recurrence in any patient.
DISCUSSION:
KD is an acute febrile vasculitis that has been reported with increasing incidence among all racial ethnic groups. While Japan has the highest and increasing annual incidence in the world (184.6 per 100,000 children aged <5 years between 2005 and 2006), the epidemiology of KD in Europe has not been well described.11,12 Likewise, the epidemiology of KD in Turkey is also not well known. A nationwide survey found that KD had an incidence of 9% of childhood vasculitides.13
There are many unconfirmed hypotheses about the cause of KD. Although clinical, laboratory and epidemiologic features strongly suggest an infectious cause, the etiology of KD still remains unknown. One possibility is that a viral repiratory infection (represented by a seasonal pattern), juxtaposed with a subsequent secondary bacterial colonization, precipitates a cascade of events that result in an exaggerated immunologic response.14,15 Our study confirms the winter peak of KD (Figure 1), but no clear relationship with respiratory pathogens was shown as virologic and immunologic data were lacking. KD is more common in boys than girls and 85% of cases occur in children aged <5 years.11 In our study, we detected a male predominance and age distribution resembling that of previous studies.16 Studies from the United States also show that KD is more common during the winter and that 76% of children are <5 years old.17
In KD, the fever is typically high‐spiking and remittent, with peak temperatures of >39°C and, in many cases, >40°C.18 Our patients' fever pattern was consistent with this classic finding, with a mean body temperature of 39.4±0.9°C at diagnosis. In addition to fever, bilateral conjunctival injection and changes in the oral cavity and lips were the most commonly detected clinical signs in our cases, as found in other studies from Turkey and other parts of the world.19-22 The occurrence of cervical lymphadenopathy in KD is variable. It is seen in only 50–75% of patients, whereas most of the other features are seen in approximately 90%.23 It is usually unilateral and confined to the anterior cervical triangle, and its classic criteria include ≥1 lymph node that is >1.5 cm in diameter.18 In our study, cervical lymphadenopathy was the least common of the principal clinical features (48.6%). The lymphadenopathies in our patients were typically classic, except for one patient who presented with massive cervical node enlargement.
Multiple clinical findings may be observed in patients with KD. Arthritis or arthralgia can occur in the first week of the illness and tends to affect multiple joints, including the small interphalangeal joints and large weight‐bearing joints.18 In our series, two patients had arthritis in their knees and one patient had arthritis in his ankle.
Children with KD are often more irritable than the children with other febrile ilnesses,18 and all the patients in this study were irritable. Gastrointestinal complaints, including diarrhea, vomiting and abdominal pain, occur in about one‐third of patients.24 Rarely, KD can present as an acute surgical abdomen.24 Contrary to previous studies, only one of our 35 patients had abdominal pain and diarrhea. Erythema and induration at the site of BCG vaccination is common in Japan, where BCG is used widely.25 As in Japan, BCG is routinely performed in Turkey, but only one patient had redness and induration at the site of the BCG scar.
KD is mainly a clinical diagnosis and there are no pathognomonic laboratory tests or findings. However, leukocytosis is typical during the acute stage of KD. Approximately 50% of patients had white blood cell counts >15,000/mm3.18 Similar to other reports,8,10,22 the mean leukocyte count of our study group was 15,896±6,383/mm3. Normocytic anemia may develop, particularly with more prolonged duration of active inflammation.8,18 Marked anemia (Hb <11 g/dl) on admission was noted in 14 (40%) patients who were all over 1 year old and who had prolonged fever for more than 7 days. Thrombocytosis is a characteristic feature of the later phases of the illness. It is rarely present in the first week of the illness, usually appearing in the second week, and peaking in the third week with a gradual return to normal by 4–8 weeks after onset in uncomplicated cases. Platelet counts are usually >450,000/mm3 in patients evaluated after day 7 of illness.18 In our series most of the patients were diagnosed in the second week and the mean platelet count was 496,889±208,503/mm3, which is consistent with other studies.18 Elevations of CRP and ESR are almost universal in KD, usually returning to normal values by 6–10 weeks after the onset of illness.18 Mean values of CRP and ESR were high in our patients. Burns et al.26 reported mild to moderate elevations in serum transaminases in ≤40% of patients. Although the mean values of transaminases were not high in our study group, the ratio of patients with high levels (28.6%) was consistent with this multicenter study.
Urine analysis showed intermittent mild to moderate sterile pyuria in approximately 33% of patients, suggesting urethritis.18 Urine cultures of two patients with sterile pyuria were normal.
As the principal clinical findings that fulfill the diagnostic criteria are not specific for KD, patients with other diseases (Table 1) with similar clinical features were excluded from our study.
The major sequelae of KD are related to the cardiovascular, and more specifically, the coronary arterial system. Therefore cardiac imaging by echocardiography is a critical part of the evaluation of all patients with suspected KD. For uncomplicated cases, echocardiography is recommended at the time of diagnosis, at 2 weeks, and at 6–8 weeks after the onset of disease.27 We followed the above recommendations and also repeated the echocardiography beyond 8 weeks for all patients with previously normal findings, as other abnormalities in the coronary artery vasculature (aneurysms) and aortic root (dilatation with or without regurgitation) may develop, even among patients with normal baseline echocardiograms.27,28 Additionally, echocardiography was performed with higher frequency for patients with pericardial effusion, mitral valve regurgitation and IVIg‐resistant KD, for closer evaluation and follow‐up. None of our patients with normal baseline echocardiography was shown to develop CAA on follow‐up echocardiograms beyond the 8 weeks. CAA develop in approximately 15–25% of untreated children with the disease and may lead to ischemic heart disease, myocardial infarction or sudden death.18 In our patients, CAA were detected in nine (25.7%) cases. None of them led to myocardial infarction or ischemic heart disease. A recent study from Turkey reported the CAA rate as 33.3% in 24 children with KD.22 Burns et al. described the emergence of the peak mortality as 15–45 days after the onset of fever; and during this time, well‐established coronary vasculitis occurs concomitantly with a marked elevation of the platelet count and a hypercoagulable state.28 The mean duration of fever in our cases was 7.8±2.8 days (range 4–15). Our patients had fewer adverse sequelae, as all were treated before the peak mortality days, thus suggesting the importance of early diagnosis and treatment. The opportunity for early management occurred as the patients were being routinely followed up in our outpatient clinic.
The current medical management of KD is IVIg and high‐dose aspirin. IVIg is very effective when given in the first ten days of illness. It reduces the risk of CAA from 20–25% to 1–2%.6,9 A subgroup of patients with KD will be resistant to IVIg therapy; these patients are at greatest risk of developing coronary artery aneurysms and long‐term sequelae of the disease.29 Data have demonstrated the usefulness and safety of systemic steroids in patients resistant to IVIg.30 In our series, the majority of our patients were given IVIg before the tenth day and only one patient was IVIg resistant, who was successfully treated with pulse methylprednisolone. Newburger et al. reported that 50–67% of aneurysms resolve within 1–2 years.31 Echocardiographic evaluation of the nine children with CAA in our study was normal within 8 months (three within 6 months, four within 7 months, and two within 8 months). All patients are alive and receiving annual echocardiographic follow‐up.
CONCLUSIONS:
In summary, it is our belief that KD is not a rare disease in Turkey. This study is the most comprehensive series of children from Turkey with KD included in Medline. As adult‐onset ischemic heart disease may be due to KD in childhood,9,18,28 further prospective clinical investigations are needed to understand the epidemiology, management and long‐term follow‐up the disease in Turkey.
| Background:
Kawasaki disease (KD) is an acute, self-limiting vasculitis of unknown etiology. The incidence of KD is increasing world wide. However, the epidemiological data for KD in Turkey has not been well described.
Methods:
Patients with KD were retrospectively identified from the hospital discharge records between 2002 and 2010. Atypical cases of KD were excluded. A standardized form was used to collect demographic data, clinical information, echocardiography and laboratory results.
Results:
Thirty-five patients with KD, with a mean age of 2.5 + 1.9 years, were identified. Eighty-five point seven per cent of patients were under 5 years of age. A seasonal pattern favouring the winter months was noticed. In addition to fever and bilateral conjunctival injection, changes in the oral cavity and lips were the most commonly detected clinical signs in our cases. Coronary artery abnormalities were detected in nine patients. The majority of our patients had started treatment with intravenous immunoglobulin in the first 10 days of the onset of fever, and only one patient required systemic steroids for intravenous immunoglobulin-resistant KD. The coronary artery abnormalities resolved in all nine patients within 8 months.
Conclusions:
This study is the most comprehensive series of children from Turkey with KD included in Medline. As adult-onset ischemic heart disease may be due to KD in childhood, further prospective clinical investigations are needed to understand the epidemiology, management and long-term follow-up of the disease. | INTRODUCTION:
Kawasaki disease (KD) is an acute, self‐limiting systemic vasculitis of unknown etiology, which mainly affects children aged <5 years. It was first described by Tomisaku Kawasaki in 1967 in Japan.1 KD has now replaced rheumatic fever as the leading cause of acquired heart disease in childhood in the developed world, and is the second most common childhood vasculitis.2,3 Although the incidence of KD varies among countries, it is much higher in children from Asian countries.4,5 The clinical signs of KD are similar to those of many other childhood illnesses. The disease is often complicated by coronary artery abnormalities (CAA), including dilatation and/or aneurysms, and thus is a leading cause of acquired heart disease in children.6,7 Some clinical features other than the classic diagnostic criteria are intense irritability, cough, diarrhea, sterile pyuria, arthritis, arthralgia, redness and induration at the site of a Bacille–Calmette–Guerin (BCG) scar. Patients with prolonged fever and fewer than four of the other principal criteria are diagnosed as atypical or incomplete KD if CAA are present.8
The incentive for this research came from the remarkable lack of knowledge about the epidemiology and features of KD in Turkey. In this article we present the demographic, clinical and laboratory features of children with KD, who were diagnosed and managed in our hospital.
CONCLUSIONS:
None. | Background:
Kawasaki disease (KD) is an acute, self-limiting vasculitis of unknown etiology. The incidence of KD is increasing world wide. However, the epidemiological data for KD in Turkey has not been well described.
Methods:
Patients with KD were retrospectively identified from the hospital discharge records between 2002 and 2010. Atypical cases of KD were excluded. A standardized form was used to collect demographic data, clinical information, echocardiography and laboratory results.
Results:
Thirty-five patients with KD, with a mean age of 2.5 + 1.9 years, were identified. Eighty-five point seven per cent of patients were under 5 years of age. A seasonal pattern favouring the winter months was noticed. In addition to fever and bilateral conjunctival injection, changes in the oral cavity and lips were the most commonly detected clinical signs in our cases. Coronary artery abnormalities were detected in nine patients. The majority of our patients had started treatment with intravenous immunoglobulin in the first 10 days of the onset of fever, and only one patient required systemic steroids for intravenous immunoglobulin-resistant KD. The coronary artery abnormalities resolved in all nine patients within 8 months.
Conclusions:
This study is the most comprehensive series of children from Turkey with KD included in Medline. As adult-onset ischemic heart disease may be due to KD in childhood, further prospective clinical investigations are needed to understand the epidemiology, management and long-term follow-up of the disease. | 2,652 | 283 | [
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] | null | [CONTENT] Kawasaki disease | Coronary artery disease | Echocardiography | Steroids [SUMMARY] | null | [CONTENT] Kawasaki disease | Coronary artery disease | Echocardiography | Steroids [SUMMARY] | [CONTENT] Kawasaki disease | Coronary artery disease | Echocardiography | Steroids [SUMMARY] | [CONTENT] Kawasaki disease | Coronary artery disease | Echocardiography | Steroids [SUMMARY] | [CONTENT] Kawasaki disease | Coronary artery disease | Echocardiography | Steroids [SUMMARY] | [CONTENT] Child | Child, Preschool | Female | Follow-Up Studies | Humans | Immunoglobulins, Intravenous | Immunologic Factors | Infant | Male | Mucocutaneous Lymph Node Syndrome | Reference Values | Retrospective Studies | Turkey [SUMMARY] | null | [CONTENT] Child | Child, Preschool | Female | Follow-Up Studies | Humans | Immunoglobulins, Intravenous | Immunologic Factors | Infant | Male | Mucocutaneous Lymph Node Syndrome | Reference Values | Retrospective Studies | Turkey [SUMMARY] | [CONTENT] Child | Child, Preschool | Female | Follow-Up Studies | Humans | Immunoglobulins, Intravenous | Immunologic Factors | Infant | Male | Mucocutaneous Lymph Node Syndrome | Reference Values | Retrospective Studies | Turkey [SUMMARY] | [CONTENT] Child | Child, Preschool | Female | Follow-Up Studies | Humans | Immunoglobulins, Intravenous | Immunologic Factors | Infant | Male | Mucocutaneous Lymph Node Syndrome | Reference Values | Retrospective Studies | Turkey [SUMMARY] | [CONTENT] Child | Child, Preschool | Female | Follow-Up Studies | Humans | Immunoglobulins, Intravenous | Immunologic Factors | Infant | Male | Mucocutaneous Lymph Node Syndrome | Reference Values | Retrospective Studies | Turkey [SUMMARY] | [CONTENT] coronary vasculitis | kd related cardiovascular | disease kd childhood | introduction kawasaki disease | kawasaki disease [SUMMARY] | null | [CONTENT] coronary vasculitis | kd related cardiovascular | disease kd childhood | introduction kawasaki disease | kawasaki disease [SUMMARY] | [CONTENT] coronary vasculitis | kd related cardiovascular | disease kd childhood | introduction kawasaki disease | kawasaki disease [SUMMARY] | [CONTENT] coronary vasculitis | kd related cardiovascular | disease kd childhood | introduction kawasaki disease | kawasaki disease [SUMMARY] | [CONTENT] coronary vasculitis | kd related cardiovascular | disease kd childhood | introduction kawasaki disease | kawasaki disease [SUMMARY] | [CONTENT] patients | kd | children | clinical | coronary | normal | 18 | disease | cases | years [SUMMARY] | null | [CONTENT] patients | kd | children | clinical | coronary | normal | 18 | disease | cases | years [SUMMARY] | [CONTENT] patients | kd | children | clinical | coronary | normal | 18 | disease | cases | years [SUMMARY] | [CONTENT] patients | kd | children | clinical | coronary | normal | 18 | disease | cases | years [SUMMARY] | [CONTENT] patients | kd | children | clinical | coronary | normal | 18 | disease | cases | years [SUMMARY] | [CONTENT] kd | disease | features | childhood | acquired heart | leading cause | leading | cause acquired heart disease | cause acquired heart | cause acquired [SUMMARY] | null | [CONTENT] patients | period | cases | patient | coronary | diagnosis | 10 | left coronary | left | kg [SUMMARY] | [CONTENT] disease | disease turkey | turkey | kd | understand epidemiology management long | clinical investigations | term follow disease | term follow | understand | understand epidemiology [SUMMARY] | [CONTENT] patients | kd | disease | children | normal | turkey | cases | 18 | coronary | clinical [SUMMARY] | [CONTENT] patients | kd | disease | children | normal | turkey | cases | 18 | coronary | clinical [SUMMARY] | [CONTENT] Kawasaki | KD ||| KD ||| KD | Turkey [SUMMARY] | null | [CONTENT] Thirty-five | KD | 2.5 + 1.9 years ||| Eighty-five | under 5 years of age ||| the winter months ||| ||| nine ||| the first 10 days | only one | KD ||| nine | 8 months [SUMMARY] | [CONTENT] Turkey | KD | Medline ||| KD [SUMMARY] | [CONTENT] Kawasaki | KD ||| KD ||| KD | Turkey ||| KD | between 2002 and 2010 ||| KD ||| ||| Thirty-five | KD | 2.5 + 1.9 years ||| Eighty-five | under 5 years of age ||| the winter months ||| ||| nine ||| the first 10 days | only one | KD ||| nine | 8 months ||| Turkey | KD | Medline ||| KD [SUMMARY] | [CONTENT] Kawasaki | KD ||| KD ||| KD | Turkey ||| KD | between 2002 and 2010 ||| KD ||| ||| Thirty-five | KD | 2.5 + 1.9 years ||| Eighty-five | under 5 years of age ||| the winter months ||| ||| nine ||| the first 10 days | only one | KD ||| nine | 8 months ||| Turkey | KD | Medline ||| KD [SUMMARY] |
|||||
Cross-sectional analysis of health-related quality of life and elements of yoga practice. | 28143469 | Mind-body practices such as yoga have been studied for their generally positive effects on health-related quality of life (HRQOL). The association between how a person practices yoga and the person's HRQOL is not known. | BACKGROUND | Yoga practitioners were sent invitations to participate in an online survey via email. Yoga characteristics, HRQOL, and other sociodemographics were collected. Analyses of data from 309 consenting responders evaluated associations between yoga practice characteristics (use of yoga tools, length of practice, location, method, etc.) and the 10-item PROMIS Global Health scale for both physical and mental health components. | MATERIALS AND METHODS | Multivariable regression models demonstrated higher mental health scores were associated with regular meditation practice, higher income, and the method of practicing in a community group class (versus one-on-one). Higher physical health scores were associated with length of lifetime practice, teacher status, Krishnamacharya yoga style, and practicing in a yoga school/studio (versus at home). | RESULTS | Meditation practice in yoga is positively associated with mental health. Length of lifetime yoga practice was significantly associated with better physical health, suggesting yoga has a potential cumulative benefit over time. Different locations and methods of practice may be associated with varying effects on health outcomes. Comparative cross-sectional and longitudinal studies on the variations in yoga practice are needed to further characterize health benefits of yoga. | CONCLUSIONS | [
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] | 5282804 | Background | Mind-body practices are physical and mental exercises that are often used for health purposes [1]. Yoga, a mind-body practice originally derived from India, is one of the world’s most popular practices. In the United States, one out five adults practice yoga for health [1]. Individuals often report practicing yoga for general well-being [2]. Yoga uses three main tools for practice: movement, breathing, and meditation [3]. Practitioners use these tools to varying degrees. Many studies have shown improvements in health-related quality of life (HRQOL) from yoga practice including gains in both physical and mental health [4]. What is not clear is if characteristics of yoga practice, such as techniques used, yoga style, practice frequency, or level of experience, are associated with HRQOL. In the context of validating a new instrument to assess yoga self-efficacy, we collected additional data regarding how yoga was practiced by the participants, along with a measure of HRQOL [2]. The purpose of the present study was to perform a secondary analysis of that dataset to examine the associations between characteristics of yoga practice and HRQOL. We hypothesized that higher frequency of weekly yoga practice, length of practice, older age, and being a yoga teacher would be associated with higher physical and mental quality of life measures. Lastly, we expected no association between yoga style, location of practice, or method of practice with HRQOL. | null | null | null | null | Conclusion | The practice of yoga for recreation and wellness is very popular in the United States. Understanding the effects of specific features of yoga practice on physical and mental health quality of life may enable teachers to ask more pertinent questions in order to better understand its observed health outcomes. Similarly, teachers may offer counsel on mind-body therapies or tailor yoga practices based on the specific needs of the student. If more data regarding the differential effects of yoga was known, yoga teachers may be able to provide more direction on frequency, intensity, and type, similar to the guidance clinicians provide with other types of exercise. For example, regular use of meditation in yoga practice may have a positive effect on mental HRQOL, whereas practicing at a yoga school/studio may have a positive effect on physical HRQOL. More research is required to better understand the relationship between techniques, length of practice, and setting of yoga practice and the potential differential effects on quality of life in regards to physical and mental health. | [
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"The study sample’s characteristics have been previously published [5] and are summarized in Table 1. We selected the following measures for secondary analysis",
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"Data Source: We analyzed data collected during the development of the Yoga Self-Efficacy Scale which has been described in detail previously [5]. Yoga is represented by many different styles and traditions. Although our new measure was originally designed based on the competencies of a yoga practitioner according to the Krishnamacharya tradition (or Viniyoga), our sample included both practitioners of Viniyoga yoga and other yoga styles. The other types of yoga that were queried in the previous study include: Iyengar, Ashtanga, Bikram, Power, Kundalini, Sivananda, Kripalu, Anusara, and Hatha, and other. We dichotomized yoga styles into two groups: Viniyoga or other yoga styles. Yoga practitioners were recruited using national yoga association networks and the principal investigator’s personal contacts by email. Those who consented to participate were sent surveys through Research Electronic Data Capture (REDCap), a web application created at Vanderbilt for building and managing online surveys and databases for its use in research studies.\n The study sample’s characteristics have been previously published [5] and are summarized in Table 1. We selected the following measures for secondary analysis \nTable 1Sample characteristics (n = 309)\n*\nCharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)\n\nSample characteristics (n = 309)\n*\n\n Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\nThe 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\n\nTable 1Sample characteristics (n = 309)\n*\nCharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)\n\nSample characteristics (n = 309)\n*\n\n Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\nThe 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\n Yoga practice characteristics and sociodemographics A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.\nA series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.\n Analyses Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.\nAnalyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.",
"\nTable 1Sample characteristics (n = 309)\n*\nCharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)\n\nSample characteristics (n = 309)\n*\n\n Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\nThe 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.",
"The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.",
"A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.",
"Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.",
" Sample characteristics by global PROMIS subscale scores In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa\n\nN = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n\nGlobal PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics\n\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\nIn Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa\n\nN = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n\nGlobal PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics\n\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n Correlations of yoga tools and global PROMIS subscale scores Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**\n.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**\n.14*\n*p < 0.05 **p <0.01\n\nSpearman correlations of yoga tools with global PROMIS HRQOL subscale scores\n*p < 0.05 **p <0.01\nTable 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**\n.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**\n.14*\n*p < 0.05 **p <0.01\n\nSpearman correlations of yoga tools with global PROMIS HRQOL subscale scores\n*p < 0.05 **p <0.01\n Regression models predicting global PROMIS subscale scores Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R\n2 \n= 0.15, R\n2\nadjusted \n= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*\nR\n0.386\nR\n2\n0.149\nR\n2\nadjusted\n0.125ANOVA F(7,248)\n6.211**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal mental HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nThe parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R\n2 \n= 0.14, R\n2\nadjusted \n= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**\nR\n0.372\nR\n2\n0.138\nR\n2\nadjusted\n0.107ANOVA F(9,246)\n4.394**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal physical HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nTable 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R\n2 \n= 0.15, R\n2\nadjusted \n= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*\nR\n0.386\nR\n2\n0.149\nR\n2\nadjusted\n0.125ANOVA F(7,248)\n6.211**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal mental HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nThe parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R\n2 \n= 0.14, R\n2\nadjusted \n= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**\nR\n0.372\nR\n2\n0.138\nR\n2\nadjusted\n0.107ANOVA F(9,246)\n4.394**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal physical HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01",
"In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa\n\nN = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n\nGlobal PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics\n\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship",
"Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**\n.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**\n.14*\n*p < 0.05 **p <0.01\n\nSpearman correlations of yoga tools with global PROMIS HRQOL subscale scores\n*p < 0.05 **p <0.01",
"Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R\n2 \n= 0.15, R\n2\nadjusted \n= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*\nR\n0.386\nR\n2\n0.149\nR\n2\nadjusted\n0.125ANOVA F(7,248)\n6.211**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal mental HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nThe parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R\n2 \n= 0.14, R\n2\nadjusted \n= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**\nR\n0.372\nR\n2\n0.138\nR\n2\nadjusted\n0.107ANOVA F(9,246)\n4.394**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal physical HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01",
"Our analysis demonstrated that specific characteristics of yoga practice are associated with different components of HRQOL. Global mental health scores were associated with regular practice of meditation and community group instruction (versus one-on-one instruction), while global physical health scores were associated with longer length of yoga practice, Viniyoga style, being a certified yoga teacher, and practicing in a yoga studio/school. Of the three yoga tools (meditation, movement, and breathing), GMH had a positive correlation with breathing and meditation, whereas GPH had a positive correlation with breathing only. These results suggest that variations in yoga practice are associated with different physical and mental health statuses.\nThe specific effect of yoga on mental health has been a popular topic of research, with previous studies showing a positive relationship between the two [4, 7]. Yoga interventions have been shown to reduce stress, and yoga practitioners tend to demonstrate a higher level of mindfulness than non-practitioners [8–10]. Complimentary to these findings, our analyses demonstrated that participants who reported regular use of meditation or breathing in their yoga practice tended to have higher mental health scores. In the regression model predicting mental HRQOL, regular use of meditation was the only yoga tool to have a significant unique relationship with mental health status, albeit a small one. Because meditation has been demonstrated to improve mental health and is a common component of yoga practice, further research is needed to identify if meditation is the major causal mechanism for improving mental health status among yoga practitioners. The model predicting physical HRQOL scores was found to be positively associated with the length of yoga practice. We defined length of yoga practice as the total time a practitioner has been practicing yoga, whereas frequency of practice was defined as how often he/she practices in an average week. Previous research shows conflicting results. One study demonstrated that frequency of weekly yoga practice was better at defining health outcomes than length of yoga practice, whereas another study determined that frequency of practice (one versus two practice days) had little difference in health outcomes [11, 12]. In our analyses, length of practice had the highest correlation with physical HRQOL status among the variables tested. One possible hypothesis for this observation may be that many of our participants were yoga teachers who had been practicing for many years benefiting from a cumulative effect on overall physical HRQOL over time.\nOther findings to note include the significance of the location of practice. Practicing in a yoga school/class setting was positively associated with physical HRQOL. There may be multiple reasons for this association. Individuals who practice at a yoga school may benefit from group effects where they are socially motivated to participate, resulting in higher adherence and intensity. Secondly, attending class at a school may provide access to other physical exercise programs offered. Lastly, yoga classes in schools may emphasize physical fitness more than home or other places of practice. Further research is necessary to examine if classes in yoga schools are optimal for improving physical well-being. Method of practice was found to be important for mental HRQOL. Specifically, those who reported practicing yoga in community group classes had higher GMH scores. This requires further examination, as those individuals who seek community group instruction may especially benefit from group settings. Both of these findings suggest that location and method of practice may influence the HRQOL status of an individual. To our knowledge this has not been previously reported in the literature.\n Limitations Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.\nOur study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.",
"Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.",
"The practice of yoga for recreation and wellness is very popular in the United States. Understanding the effects of specific features of yoga practice on physical and mental health quality of life may enable teachers to ask more pertinent questions in order to better understand its observed health outcomes. Similarly, teachers may offer counsel on mind-body therapies or tailor yoga practices based on the specific needs of the student. If more data regarding the differential effects of yoga was known, yoga teachers may be able to provide more direction on frequency, intensity, and type, similar to the guidance clinicians provide with other types of exercise. For example, regular use of meditation in yoga practice may have a positive effect on mental HRQOL, whereas practicing at a yoga school/studio may have a positive effect on physical HRQOL. More research is required to better understand the relationship between techniques, length of practice, and setting of yoga practice and the potential differential effects on quality of life in regards to physical and mental health."
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null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"The study sample’s characteristics have been previously published [5] and are summarized in Table 1. We selected the following measures for secondary analysis",
"Health-related quality of life",
"Yoga practice characteristics and sociodemographics",
"Analyses",
"Results",
"Sample characteristics by global PROMIS subscale scores",
"Correlations of yoga tools and global PROMIS subscale scores",
"Regression models predicting global PROMIS subscale scores",
"Discussion",
"Limitations",
"Conclusion"
] | [
"Mind-body practices are physical and mental exercises that are often used for health purposes [1]. Yoga, a mind-body practice originally derived from India, is one of the world’s most popular practices. In the United States, one out five adults practice yoga for health [1]. Individuals often report practicing yoga for general well-being [2]. Yoga uses three main tools for practice: movement, breathing, and meditation [3]. Practitioners use these tools to varying degrees. Many studies have shown improvements in health-related quality of life (HRQOL) from yoga practice including gains in both physical and mental health [4]. What is not clear is if characteristics of yoga practice, such as techniques used, yoga style, practice frequency, or level of experience, are associated with HRQOL. In the context of validating a new instrument to assess yoga self-efficacy, we collected additional data regarding how yoga was practiced by the participants, along with a measure of HRQOL [2]. The purpose of the present study was to perform a secondary analysis of that dataset to examine the associations between characteristics of yoga practice and HRQOL. We hypothesized that higher frequency of weekly yoga practice, length of practice, older age, and being a yoga teacher would be associated with higher physical and mental quality of life measures. Lastly, we expected no association between yoga style, location of practice, or method of practice with HRQOL.",
"Data Source: We analyzed data collected during the development of the Yoga Self-Efficacy Scale which has been described in detail previously [5]. Yoga is represented by many different styles and traditions. Although our new measure was originally designed based on the competencies of a yoga practitioner according to the Krishnamacharya tradition (or Viniyoga), our sample included both practitioners of Viniyoga yoga and other yoga styles. The other types of yoga that were queried in the previous study include: Iyengar, Ashtanga, Bikram, Power, Kundalini, Sivananda, Kripalu, Anusara, and Hatha, and other. We dichotomized yoga styles into two groups: Viniyoga or other yoga styles. Yoga practitioners were recruited using national yoga association networks and the principal investigator’s personal contacts by email. Those who consented to participate were sent surveys through Research Electronic Data Capture (REDCap), a web application created at Vanderbilt for building and managing online surveys and databases for its use in research studies.\n The study sample’s characteristics have been previously published [5] and are summarized in Table 1. We selected the following measures for secondary analysis \nTable 1Sample characteristics (n = 309)\n*\nCharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)\n\nSample characteristics (n = 309)\n*\n\n Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\nThe 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\n\nTable 1Sample characteristics (n = 309)\n*\nCharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)\n\nSample characteristics (n = 309)\n*\n\n Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\nThe 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\n Yoga practice characteristics and sociodemographics A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.\nA series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.\n Analyses Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.\nAnalyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.",
"\nTable 1Sample characteristics (n = 309)\n*\nCharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)\n\nSample characteristics (n = 309)\n*\n\n Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.\nThe 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.",
"The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.",
"A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.",
"Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.",
" Sample characteristics by global PROMIS subscale scores In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa\n\nN = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n\nGlobal PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics\n\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\nIn Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa\n\nN = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n\nGlobal PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics\n\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n Correlations of yoga tools and global PROMIS subscale scores Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**\n.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**\n.14*\n*p < 0.05 **p <0.01\n\nSpearman correlations of yoga tools with global PROMIS HRQOL subscale scores\n*p < 0.05 **p <0.01\nTable 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**\n.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**\n.14*\n*p < 0.05 **p <0.01\n\nSpearman correlations of yoga tools with global PROMIS HRQOL subscale scores\n*p < 0.05 **p <0.01\n Regression models predicting global PROMIS subscale scores Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R\n2 \n= 0.15, R\n2\nadjusted \n= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*\nR\n0.386\nR\n2\n0.149\nR\n2\nadjusted\n0.125ANOVA F(7,248)\n6.211**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal mental HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nThe parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R\n2 \n= 0.14, R\n2\nadjusted \n= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**\nR\n0.372\nR\n2\n0.138\nR\n2\nadjusted\n0.107ANOVA F(9,246)\n4.394**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal physical HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nTable 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R\n2 \n= 0.15, R\n2\nadjusted \n= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*\nR\n0.386\nR\n2\n0.149\nR\n2\nadjusted\n0.125ANOVA F(7,248)\n6.211**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal mental HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nThe parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R\n2 \n= 0.14, R\n2\nadjusted \n= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**\nR\n0.372\nR\n2\n0.138\nR\n2\nadjusted\n0.107ANOVA F(9,246)\n4.394**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal physical HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01",
"In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa\n\nN = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship\n\nGlobal PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics\n\naMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship",
"Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**\n.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**\n.14*\n*p < 0.05 **p <0.01\n\nSpearman correlations of yoga tools with global PROMIS HRQOL subscale scores\n*p < 0.05 **p <0.01",
"Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R\n2 \n= 0.15, R\n2\nadjusted \n= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*\nR\n0.386\nR\n2\n0.149\nR\n2\nadjusted\n0.125ANOVA F(7,248)\n6.211**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal mental HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01\nThe parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R\n2 \n= 0.14, R\n2\nadjusted \n= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable\nr\na\nBSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**\nR\n0.372\nR\n2\n0.138\nR\n2\nadjusted\n0.107ANOVA F(9,246)\n4.394**\naCorrelation coefficient*p <0.05 **p < 0.01\n\nGlobal physical HRQOL subscale score correlations and regression model\n\naCorrelation coefficient\n*p <0.05 **p < 0.01",
"Our analysis demonstrated that specific characteristics of yoga practice are associated with different components of HRQOL. Global mental health scores were associated with regular practice of meditation and community group instruction (versus one-on-one instruction), while global physical health scores were associated with longer length of yoga practice, Viniyoga style, being a certified yoga teacher, and practicing in a yoga studio/school. Of the three yoga tools (meditation, movement, and breathing), GMH had a positive correlation with breathing and meditation, whereas GPH had a positive correlation with breathing only. These results suggest that variations in yoga practice are associated with different physical and mental health statuses.\nThe specific effect of yoga on mental health has been a popular topic of research, with previous studies showing a positive relationship between the two [4, 7]. Yoga interventions have been shown to reduce stress, and yoga practitioners tend to demonstrate a higher level of mindfulness than non-practitioners [8–10]. Complimentary to these findings, our analyses demonstrated that participants who reported regular use of meditation or breathing in their yoga practice tended to have higher mental health scores. In the regression model predicting mental HRQOL, regular use of meditation was the only yoga tool to have a significant unique relationship with mental health status, albeit a small one. Because meditation has been demonstrated to improve mental health and is a common component of yoga practice, further research is needed to identify if meditation is the major causal mechanism for improving mental health status among yoga practitioners. The model predicting physical HRQOL scores was found to be positively associated with the length of yoga practice. We defined length of yoga practice as the total time a practitioner has been practicing yoga, whereas frequency of practice was defined as how often he/she practices in an average week. Previous research shows conflicting results. One study demonstrated that frequency of weekly yoga practice was better at defining health outcomes than length of yoga practice, whereas another study determined that frequency of practice (one versus two practice days) had little difference in health outcomes [11, 12]. In our analyses, length of practice had the highest correlation with physical HRQOL status among the variables tested. One possible hypothesis for this observation may be that many of our participants were yoga teachers who had been practicing for many years benefiting from a cumulative effect on overall physical HRQOL over time.\nOther findings to note include the significance of the location of practice. Practicing in a yoga school/class setting was positively associated with physical HRQOL. There may be multiple reasons for this association. Individuals who practice at a yoga school may benefit from group effects where they are socially motivated to participate, resulting in higher adherence and intensity. Secondly, attending class at a school may provide access to other physical exercise programs offered. Lastly, yoga classes in schools may emphasize physical fitness more than home or other places of practice. Further research is necessary to examine if classes in yoga schools are optimal for improving physical well-being. Method of practice was found to be important for mental HRQOL. Specifically, those who reported practicing yoga in community group classes had higher GMH scores. This requires further examination, as those individuals who seek community group instruction may especially benefit from group settings. Both of these findings suggest that location and method of practice may influence the HRQOL status of an individual. To our knowledge this has not been previously reported in the literature.\n Limitations Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.\nOur study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.",
"Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.",
"The practice of yoga for recreation and wellness is very popular in the United States. Understanding the effects of specific features of yoga practice on physical and mental health quality of life may enable teachers to ask more pertinent questions in order to better understand its observed health outcomes. Similarly, teachers may offer counsel on mind-body therapies or tailor yoga practices based on the specific needs of the student. If more data regarding the differential effects of yoga was known, yoga teachers may be able to provide more direction on frequency, intensity, and type, similar to the guidance clinicians provide with other types of exercise. For example, regular use of meditation in yoga practice may have a positive effect on mental HRQOL, whereas practicing at a yoga school/studio may have a positive effect on physical HRQOL. More research is required to better understand the relationship between techniques, length of practice, and setting of yoga practice and the potential differential effects on quality of life in regards to physical and mental health."
] | [
"introduction",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Yoga",
"Health-related quality of life",
"Mind-body practices"
] | Background:
Mind-body practices are physical and mental exercises that are often used for health purposes [1]. Yoga, a mind-body practice originally derived from India, is one of the world’s most popular practices. In the United States, one out five adults practice yoga for health [1]. Individuals often report practicing yoga for general well-being [2]. Yoga uses three main tools for practice: movement, breathing, and meditation [3]. Practitioners use these tools to varying degrees. Many studies have shown improvements in health-related quality of life (HRQOL) from yoga practice including gains in both physical and mental health [4]. What is not clear is if characteristics of yoga practice, such as techniques used, yoga style, practice frequency, or level of experience, are associated with HRQOL. In the context of validating a new instrument to assess yoga self-efficacy, we collected additional data regarding how yoga was practiced by the participants, along with a measure of HRQOL [2]. The purpose of the present study was to perform a secondary analysis of that dataset to examine the associations between characteristics of yoga practice and HRQOL. We hypothesized that higher frequency of weekly yoga practice, length of practice, older age, and being a yoga teacher would be associated with higher physical and mental quality of life measures. Lastly, we expected no association between yoga style, location of practice, or method of practice with HRQOL.
Methods:
Data Source: We analyzed data collected during the development of the Yoga Self-Efficacy Scale which has been described in detail previously [5]. Yoga is represented by many different styles and traditions. Although our new measure was originally designed based on the competencies of a yoga practitioner according to the Krishnamacharya tradition (or Viniyoga), our sample included both practitioners of Viniyoga yoga and other yoga styles. The other types of yoga that were queried in the previous study include: Iyengar, Ashtanga, Bikram, Power, Kundalini, Sivananda, Kripalu, Anusara, and Hatha, and other. We dichotomized yoga styles into two groups: Viniyoga or other yoga styles. Yoga practitioners were recruited using national yoga association networks and the principal investigator’s personal contacts by email. Those who consented to participate were sent surveys through Research Electronic Data Capture (REDCap), a web application created at Vanderbilt for building and managing online surveys and databases for its use in research studies.
The study sample’s characteristics have been previously published [5] and are summarized in Table 1. We selected the following measures for secondary analysis
Table 1Sample characteristics (n = 309)
*
CharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)
Sample characteristics (n = 309)
*
Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
Table 1Sample characteristics (n = 309)
*
CharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)
Sample characteristics (n = 309)
*
Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
Yoga practice characteristics and sociodemographics A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.
A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.
Analyses Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.
Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.
The study sample’s characteristics have been previously published [5] and are summarized in Table 1. We selected the following measures for secondary analysis:
Table 1Sample characteristics (n = 309)
*
CharacteristicAge (mean, S.D.)51 (13)Female Sex (n, %)253 (82)Race (n, %) White262 (85) Black7 (2) Asian15 (5) Some other race or missing25 (8)Hispanic, Latino or Spanish in origin (n, %)15 (5)Annual income (U.S. dollars) (n, %) Less than $24,99934 (12) $25,000-$44,99950 (16) $50,000-$74,99948 (16) $75,000-$99,99946 (15) $100,000 or more78 (25) Decline to answer35 (11)Education (n, %) High school graduate/GED/ or less6 (2) Vocational/technical school8 (3) Associate degree/some college31 (10) Bachelor’s degree88 (29) Advanced degree158 (51)Most frequent yoga style practiced (n, %) Krishnamacharya or Viniyoga159 (52) Hatha53 (17) Anusara8 (3) Ashtanga9 (3) Kripalu4 (1) Bikram1 (<1) Kundalini1 (<1) Other31 (10)Certified yoga teacher (n, %)174 (56)On average, how many times a week do you practice yoga? (mean, S.D.)5 (2)When you practice yoga, on average how many minutes? (mean, S.D.)54 (20)How long have you been practicing yoga? (n, %) More than 3 years246 (80) 3 years or less53 (17)Health Related Quality of Life Sub-scores (mean, SD) Global Mental Health15.01 (2.75) Global Physical Health16.51 (2.23)
Sample characteristics (n = 309)
*
Health-related quality of life The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
Health-related quality of life:
The 10-item PROMIS Global Health scale was administered to assess the physical and mental components of health related quality of life [6]. This scale can be scored into Global Mental Health (GMH) and Global Physical Health (GPH) subscales. A sample question from the GMH subscale is, “In general, how would you rate your mental health, including your mood and your ability to think?”[Likert scale: 5 = excellent to 1 = poor]. In the previous study, Cronbach’s alpha was 0.831 for GMH scores. An example from the GPH subscale is, “In general, please rate how well you carry out your everyday physical activities such as walking, climbing stairs, carrying groceries, or moving a chair [Likert scale: 5 = completely to 1 = not at all]. In the previous study, Cronbach’s alpha was 0.729 for GPH scores.
Yoga practice characteristics and sociodemographics:
A series of questions about characteristics of yoga practice were asked in a series of questions, including information about adherence, length of practice, and the perceived importance and practice of the three tools of yoga (breathing, movement, and meditation). Items concerning the three tools of yoga were ranked on a 9-point Likert scale ranging from 1 =”strongly disagree” to 9 = “strongly agree”. Perceived importance of each tool was assessed by asking if the tool was an important part of the respondent’s regular yoga practice (e.g., “Meditation is an important part of my regular yoga practice”), while practice of each tool was asked in the following fashion, for example, “My regular yoga practice includes meditation”. Participants were also queried on the duration of their yoga practice [less than 1 month, 1–3 months, 4–11 months, 1–3 years, more than 3 years]; and how many days a week they practiced [1–7 days]. Participants also self-reported their age, sex, yoga style, and if they were a certified yoga teacher.
Analyses:
Analyses were conducted using IBM SPSS Statistics Version 23. Descriptive statistics were used to assess perceived importance and practice of the three yoga tools (movement, breathing, and meditation), practice frequency, and length of practice. Pearson Chi Square analyses were conducted with sociodemographic variables and quality of life measures (GPH and GMH). Correlations between the practice of the three yoga tools and quality of life measures were calculated using Spearman’s correlation. We constructed multivariable linear regression models with the practice of the three yoga tools, certified yoga teacher status, length of practice, practice frequency, location of practice method of practice, method of practice, and yoga style as the independent variables affecting the physical and mental components of HRQOL. Age, gender, education level, income, and race were included as co-variates in all models.
Results:
Sample characteristics by global PROMIS subscale scores In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa
N = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08
aMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship
Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics
aMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship
In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa
N = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08
aMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship
Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics
aMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship
Correlations of yoga tools and global PROMIS subscale scores Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**
.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**
.14*
*p < 0.05 **p <0.01
Spearman correlations of yoga tools with global PROMIS HRQOL subscale scores
*p < 0.05 **p <0.01
Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**
.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**
.14*
*p < 0.05 **p <0.01
Spearman correlations of yoga tools with global PROMIS HRQOL subscale scores
*p < 0.05 **p <0.01
Regression models predicting global PROMIS subscale scores Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R
2
= 0.15, R
2
adjusted
= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable
r
a
BSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*
R
0.386
R
2
0.149
R
2
adjusted
0.125ANOVA F(7,248)
6.211**
aCorrelation coefficient*p <0.05 **p < 0.01
Global mental HRQOL subscale score correlations and regression model
aCorrelation coefficient
*p <0.05 **p < 0.01
The parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R
2
= 0.14, R
2
adjusted
= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable
r
a
BSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**
R
0.372
R
2
0.138
R
2
adjusted
0.107ANOVA F(9,246)
4.394**
aCorrelation coefficient*p <0.05 **p < 0.01
Global physical HRQOL subscale score correlations and regression model
aCorrelation coefficient
*p <0.05 **p < 0.01
Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R
2
= 0.15, R
2
adjusted
= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable
r
a
BSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*
R
0.386
R
2
0.149
R
2
adjusted
0.125ANOVA F(7,248)
6.211**
aCorrelation coefficient*p <0.05 **p < 0.01
Global mental HRQOL subscale score correlations and regression model
aCorrelation coefficient
*p <0.05 **p < 0.01
The parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R
2
= 0.14, R
2
adjusted
= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable
r
a
BSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**
R
0.372
R
2
0.138
R
2
adjusted
0.107ANOVA F(9,246)
4.394**
aCorrelation coefficient*p <0.05 **p < 0.01
Global physical HRQOL subscale score correlations and regression model
aCorrelation coefficient
*p <0.05 **p < 0.01
Sample characteristics by global PROMIS subscale scores:
In Table 2, we report average GPH and GMH scores by the sociodemographic variables listed. GPH scores were significantly different by teacher status (certified teacher vs. not certified), with certified teachers scoring higher than non-certified practitioners. There was no significant difference between the two groups on GMH scores.Table 2Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristicsPROMIS Scoresa
N = 291Global PhysicalGlobal MentalAge 25-35 (n = 49)16.34 SD = 2.3514.12 SD = 3.30 36-45 (n = 57)16.21 SD = 2.2214.21 SD = 2.34 46-55 (n = 61)16.52 SD = 2.6314.97 SD = 2.74 56-65 (n = 84)16.93 SD = 1.9715.56 SD = 2.51 66-75 (n = 36)16.19 SD = 2.4116.17 SD = 2.42 > 75 (n = 4)16.63 SD = 3.9015.75 SD = 3.40Gender Male (n = 11)16.74 SD = 2.1614.74 SD = 3.16 Female (n = 253)16.48 SD = 2.2415.05 SD = 2.69Race White (n = 258)16.54 SD = 2.2615.04 SD = 2.74 Other (n = 33)16.30 SD = 2.0514.70 SD = 2.89Yoga Style Viniyoga Yoga (n = 158)16.80 SD = 2.0315.27 SD = 2.55 Other Yoga (n = 133)16.17 SD = 2.4114.69 SD = 2.95Teacher Status Certified Teacher (n = 170)16.89 SD = 2.0815.05 SD = 2.50 Not Certified Teacher (n = 121)15.98 SD = 2.3314.95 D = 3.08
aMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship
Global PROMIS HRQOL subscale scores by selective demographic and yoga practice characteristics
aMann-Whitney U tests were calculated for all sociodemographics, Teacher status vs. GPH (U = 7516.5, p < 0.01) was the only significant relationship
Correlations of yoga tools and global PROMIS subscale scores:
Table 3 shows the correlations between the degree of practice of the three yoga tools (meditation, movement, and breathing) and GMH and GPH scores). GMH had significant correlations with both breathing and meditation whereas GPH only had a significant correlation with breathing.Table 3Spearman correlations of yoga tools with global PROMIS HRQOL subscale scoresHRQOL Subscale scores N=291ToolsGlobal MentalGlobal PhysicalRegular Yoga Practice Includes Meditation.16**
.09Regular Yoga Practice Includes Movement.09.02Regular Yoga Practice Includes Breathing Technique.20**
.14*
*p < 0.05 **p <0.01
Spearman correlations of yoga tools with global PROMIS HRQOL subscale scores
*p < 0.05 **p <0.01
Regression models predicting global PROMIS subscale scores:
Table 4 reports the regression model we built to understand the relationship between GMH and selected independent variables. The regression model considers characteristics of yoga practice and sociodemographic factors as independent variables with GMH serving as the dependent variable. Significant predictors were: age, regular practice of the meditation, and method of practicing in a community group class (versus one-on-one). The model was found to explain a significant amount of variance in GMH scores (R
2
= 0.15, R
2
adjusted
= 0.13; F (7,248) = 6.21, p <0.05).Table 4Global mental HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable
r
a
BSE BβAge0.29**0.0560.0130.265**Race0.040.2010.5410.022Gender0.040.2930.5100.034Income0.21**0.1250.0980.083Education0.090.6180.4720.080Regular practice includes meditation (yoga tool)0.18**0.2310.0870.159**Method of Practice- community group class0.12*0.7970.3760.128*
R
0.386
R
2
0.149
R
2
adjusted
0.125ANOVA F(7,248)
6.211**
aCorrelation coefficient*p <0.05 **p < 0.01
Global mental HRQOL subscale score correlations and regression model
aCorrelation coefficient
*p <0.05 **p < 0.01
The parallel regression model for GPH as the dependent variable is shown in Table 5. Significant independent variables included length of practice, teacher status, yoga style, and practicing in a yoga school/studio (versus at home) were significantly associated with GPH. This model was also found to explain a significant amount of variance in GPH scores (R
2
= 0.14, R
2
adjusted
= 0.11; F (9, 246) = 4.39, p <0.05).Table 5Global physical HRQOL subscale score correlations and regression modelCorrelationsRegression modelVariable
r
a
BSE BβAge0.070.0020.0110.012Race0.040.1710.4420.023Gender0.03-0.5180.420-0.074Income0.070.0810.0800.066Education0.110.4800.3850.077Length of Yoga Practice0.22**0.5760.2140.183*Teacher Status0.20**0.8320.3340.180*Viniyoga0.14**0.6940.2830.153*Location of Practice- yoga studio/school0.070.9100.3310.178**
R
0.372
R
2
0.138
R
2
adjusted
0.107ANOVA F(9,246)
4.394**
aCorrelation coefficient*p <0.05 **p < 0.01
Global physical HRQOL subscale score correlations and regression model
aCorrelation coefficient
*p <0.05 **p < 0.01
Discussion:
Our analysis demonstrated that specific characteristics of yoga practice are associated with different components of HRQOL. Global mental health scores were associated with regular practice of meditation and community group instruction (versus one-on-one instruction), while global physical health scores were associated with longer length of yoga practice, Viniyoga style, being a certified yoga teacher, and practicing in a yoga studio/school. Of the three yoga tools (meditation, movement, and breathing), GMH had a positive correlation with breathing and meditation, whereas GPH had a positive correlation with breathing only. These results suggest that variations in yoga practice are associated with different physical and mental health statuses.
The specific effect of yoga on mental health has been a popular topic of research, with previous studies showing a positive relationship between the two [4, 7]. Yoga interventions have been shown to reduce stress, and yoga practitioners tend to demonstrate a higher level of mindfulness than non-practitioners [8–10]. Complimentary to these findings, our analyses demonstrated that participants who reported regular use of meditation or breathing in their yoga practice tended to have higher mental health scores. In the regression model predicting mental HRQOL, regular use of meditation was the only yoga tool to have a significant unique relationship with mental health status, albeit a small one. Because meditation has been demonstrated to improve mental health and is a common component of yoga practice, further research is needed to identify if meditation is the major causal mechanism for improving mental health status among yoga practitioners. The model predicting physical HRQOL scores was found to be positively associated with the length of yoga practice. We defined length of yoga practice as the total time a practitioner has been practicing yoga, whereas frequency of practice was defined as how often he/she practices in an average week. Previous research shows conflicting results. One study demonstrated that frequency of weekly yoga practice was better at defining health outcomes than length of yoga practice, whereas another study determined that frequency of practice (one versus two practice days) had little difference in health outcomes [11, 12]. In our analyses, length of practice had the highest correlation with physical HRQOL status among the variables tested. One possible hypothesis for this observation may be that many of our participants were yoga teachers who had been practicing for many years benefiting from a cumulative effect on overall physical HRQOL over time.
Other findings to note include the significance of the location of practice. Practicing in a yoga school/class setting was positively associated with physical HRQOL. There may be multiple reasons for this association. Individuals who practice at a yoga school may benefit from group effects where they are socially motivated to participate, resulting in higher adherence and intensity. Secondly, attending class at a school may provide access to other physical exercise programs offered. Lastly, yoga classes in schools may emphasize physical fitness more than home or other places of practice. Further research is necessary to examine if classes in yoga schools are optimal for improving physical well-being. Method of practice was found to be important for mental HRQOL. Specifically, those who reported practicing yoga in community group classes had higher GMH scores. This requires further examination, as those individuals who seek community group instruction may especially benefit from group settings. Both of these findings suggest that location and method of practice may influence the HRQOL status of an individual. To our knowledge this has not been previously reported in the literature.
Limitations Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.
Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.
Limitations:
Our study has several limitations. The survey responders consisted of a fairly homogenous group (predominantly white, female, and educated) [8]. Although these demographics are consistent with the general yoga practicing population in the United States, we have not assessed the association of these characteristics (white, female, and educated) with increased regular practice of yoga, which may factor into the higher GPH/GMH scores demonstrated in the study [13]. In addition, the method employed for recruiting survey responders may have led to selection bias as it was focused on a specific style of yoga (Krishnamacharya or Viniyoga), and most responders were experienced yoga teachers. Many of the relationships we found, although statistically significant, may not be considered as strong, thus limiting our interpretations of our results. Again, this may be attributed to the homogeneity of our study sample and warrants further investigation of these relationships. Thus, these findings may not generalize to different styles of yoga or to less experienced practitioners. Lastly, these data are cross-sectional, thus we cannot reliably infer any causal relationships from our secondary analyses.
Conclusion:
The practice of yoga for recreation and wellness is very popular in the United States. Understanding the effects of specific features of yoga practice on physical and mental health quality of life may enable teachers to ask more pertinent questions in order to better understand its observed health outcomes. Similarly, teachers may offer counsel on mind-body therapies or tailor yoga practices based on the specific needs of the student. If more data regarding the differential effects of yoga was known, yoga teachers may be able to provide more direction on frequency, intensity, and type, similar to the guidance clinicians provide with other types of exercise. For example, regular use of meditation in yoga practice may have a positive effect on mental HRQOL, whereas practicing at a yoga school/studio may have a positive effect on physical HRQOL. More research is required to better understand the relationship between techniques, length of practice, and setting of yoga practice and the potential differential effects on quality of life in regards to physical and mental health.
| Background:
Mind-body practices such as yoga have been studied for their generally positive effects on health-related quality of life (HRQOL). The association between how a person practices yoga and the person's HRQOL is not known.
Methods:
Yoga practitioners were sent invitations to participate in an online survey via email. Yoga characteristics, HRQOL, and other sociodemographics were collected. Analyses of data from 309 consenting responders evaluated associations between yoga practice characteristics (use of yoga tools, length of practice, location, method, etc.) and the 10-item PROMIS Global Health scale for both physical and mental health components.
Results:
Multivariable regression models demonstrated higher mental health scores were associated with regular meditation practice, higher income, and the method of practicing in a community group class (versus one-on-one). Higher physical health scores were associated with length of lifetime practice, teacher status, Krishnamacharya yoga style, and practicing in a yoga school/studio (versus at home).
Conclusions:
Meditation practice in yoga is positively associated with mental health. Length of lifetime yoga practice was significantly associated with better physical health, suggesting yoga has a potential cumulative benefit over time. Different locations and methods of practice may be associated with varying effects on health outcomes. Comparative cross-sectional and longitudinal studies on the variations in yoga practice are needed to further characterize health benefits of yoga. | Background:
Mind-body practices are physical and mental exercises that are often used for health purposes [1]. Yoga, a mind-body practice originally derived from India, is one of the world’s most popular practices. In the United States, one out five adults practice yoga for health [1]. Individuals often report practicing yoga for general well-being [2]. Yoga uses three main tools for practice: movement, breathing, and meditation [3]. Practitioners use these tools to varying degrees. Many studies have shown improvements in health-related quality of life (HRQOL) from yoga practice including gains in both physical and mental health [4]. What is not clear is if characteristics of yoga practice, such as techniques used, yoga style, practice frequency, or level of experience, are associated with HRQOL. In the context of validating a new instrument to assess yoga self-efficacy, we collected additional data regarding how yoga was practiced by the participants, along with a measure of HRQOL [2]. The purpose of the present study was to perform a secondary analysis of that dataset to examine the associations between characteristics of yoga practice and HRQOL. We hypothesized that higher frequency of weekly yoga practice, length of practice, older age, and being a yoga teacher would be associated with higher physical and mental quality of life measures. Lastly, we expected no association between yoga style, location of practice, or method of practice with HRQOL.
Conclusion:
The practice of yoga for recreation and wellness is very popular in the United States. Understanding the effects of specific features of yoga practice on physical and mental health quality of life may enable teachers to ask more pertinent questions in order to better understand its observed health outcomes. Similarly, teachers may offer counsel on mind-body therapies or tailor yoga practices based on the specific needs of the student. If more data regarding the differential effects of yoga was known, yoga teachers may be able to provide more direction on frequency, intensity, and type, similar to the guidance clinicians provide with other types of exercise. For example, regular use of meditation in yoga practice may have a positive effect on mental HRQOL, whereas practicing at a yoga school/studio may have a positive effect on physical HRQOL. More research is required to better understand the relationship between techniques, length of practice, and setting of yoga practice and the potential differential effects on quality of life in regards to physical and mental health. | Background:
Mind-body practices such as yoga have been studied for their generally positive effects on health-related quality of life (HRQOL). The association between how a person practices yoga and the person's HRQOL is not known.
Methods:
Yoga practitioners were sent invitations to participate in an online survey via email. Yoga characteristics, HRQOL, and other sociodemographics were collected. Analyses of data from 309 consenting responders evaluated associations between yoga practice characteristics (use of yoga tools, length of practice, location, method, etc.) and the 10-item PROMIS Global Health scale for both physical and mental health components.
Results:
Multivariable regression models demonstrated higher mental health scores were associated with regular meditation practice, higher income, and the method of practicing in a community group class (versus one-on-one). Higher physical health scores were associated with length of lifetime practice, teacher status, Krishnamacharya yoga style, and practicing in a yoga school/studio (versus at home).
Conclusions:
Meditation practice in yoga is positively associated with mental health. Length of lifetime yoga practice was significantly associated with better physical health, suggesting yoga has a potential cumulative benefit over time. Different locations and methods of practice may be associated with varying effects on health outcomes. Comparative cross-sectional and longitudinal studies on the variations in yoga practice are needed to further characterize health benefits of yoga. | 8,365 | 273 | [
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] | null | null | [CONTENT] Yoga | Health-related quality of life | Mind-body practices [SUMMARY] | null | null | [CONTENT] Yoga | Health-related quality of life | Mind-body practices [SUMMARY] | [CONTENT] Yoga | Health-related quality of life | Mind-body practices [SUMMARY] | [CONTENT] Yoga | Health-related quality of life | Mind-body practices [SUMMARY] | [CONTENT] Adult | Aged | Female | Humans | Male | Meditation | Mental Health | Middle Aged | Quality of Life | Regression Analysis | Social Class | Surveys and Questionnaires | Yoga [SUMMARY] | null | null | [CONTENT] Adult | Aged | Female | Humans | Male | Meditation | Mental Health | Middle Aged | Quality of Life | Regression Analysis | Social Class | Surveys and Questionnaires | Yoga [SUMMARY] | [CONTENT] Adult | Aged | Female | Humans | Male | Meditation | Mental Health | Middle Aged | Quality of Life | Regression Analysis | Social Class | Surveys and Questionnaires | Yoga [SUMMARY] | [CONTENT] Adult | Aged | Female | Humans | Male | Meditation | Mental Health | Middle Aged | Quality of Life | Regression Analysis | Social Class | Surveys and Questionnaires | Yoga [SUMMARY] | [CONTENT] life hrqol yoga | yoga practice hrqol | yoga mental health | yoga practice research | yoga practice characteristicspromis [SUMMARY] | null | null | [CONTENT] life hrqol yoga | yoga practice hrqol | yoga mental health | yoga practice research | yoga practice characteristicspromis [SUMMARY] | [CONTENT] life hrqol yoga | yoga practice hrqol | yoga mental health | yoga practice research | yoga practice characteristicspromis [SUMMARY] | [CONTENT] life hrqol yoga | yoga practice hrqol | yoga mental health | yoga practice research | yoga practice characteristicspromis [SUMMARY] | [CONTENT] yoga | practice | sd | scores | health | gph | yoga practice | 16 | global | gmh [SUMMARY] | null | null | [CONTENT] yoga | practice | sd | scores | health | gph | yoga practice | 16 | global | gmh [SUMMARY] | [CONTENT] yoga | practice | sd | scores | health | gph | yoga practice | 16 | global | gmh [SUMMARY] | [CONTENT] yoga | practice | sd | scores | health | gph | yoga practice | 16 | global | gmh [SUMMARY] | [CONTENT] yoga | practice | health | hrqol | practice hrqol | yoga practice | body | mind | mind body | physical mental [SUMMARY] | null | null | [CONTENT] yoga | effects | better understand | differential | differential effects | positive effect | teachers | practice | better | provide [SUMMARY] | [CONTENT] yoga | practice | sd | health | yoga practice | scores | physical | mental | 16 | scale [SUMMARY] | [CONTENT] yoga | practice | sd | health | yoga practice | scores | physical | mental | 16 | scale [SUMMARY] | [CONTENT] ||| HRQOL [SUMMARY] | null | null | [CONTENT] ||| ||| ||| [SUMMARY] | [CONTENT] ||| HRQOL ||| ||| HRQOL ||| 309 ||| 10 | Global Health ||| ||| one ||| Krishnamacharya ||| ||| ||| ||| [SUMMARY] | [CONTENT] ||| HRQOL ||| ||| HRQOL ||| 309 ||| 10 | Global Health ||| ||| one ||| Krishnamacharya ||| ||| ||| ||| [SUMMARY] |
||||
Molecular aspects of Chikungunya virus infections in cancer patients. | 35475905 | Chikungunya virus (CHIKV) is an arbovirus that can cause chronic and debilitating manifestations. The first autochthonous case in Rio de Janeiro state was diagnosed in 2015, and an outbreak was declared in 2016. | BACKGROUND | Paired serum, plasma and urine collected from 31 cancer patients were tested by real-time quantitative polymerase chain reaction (qPCR) and a segment of the CHIKV E1 gene was sequenced. | METHODS | We detected 11 CHIKV+ oncological patients. Paired samples analyses of nine patients showed a different pattern of detection. Also, a higher viral load in plasma (6.84 log10) and serum (6.07 log10) vs urine (3.76 log10) was found. Phylogenetic analysis and molecular characterisation revealed East/Central/Southern Africa (ECSA) genotype circulation and three amino acids substitutions (E1-K211T, E1-M269V, E1-T288I) in positive patients. | FINDINGS | The results indicate the bioequivalence of serum and plasma for CHIKV diagnosis, with urine being an important complement. ECSA genotype was circulating among patients in the period of the 2016 outbreak with K211T, M269V and T288I substitution. | MAIN CONCLUSION | [
"Brazil",
"Chikungunya Fever",
"Chikungunya virus",
"Humans",
"Neoplasms",
"Phylogeny"
] | 9037814 | null | null | null | null | RESULTS |
Laboratorial diagnosis of arbovirus infection and clinical characteristics of CHIKV+ oncological patients - A total of thirty-one participants were recruited at INCA when clinical staff had a suspicion of arbovirus infection. All participants presented all selection criteria and were tested for ZIKV, CHIKV and all DENV’s serotype using qPCR reactions. We found a 35.48% positivity (11/31) for CHIKV in oncological patients and no co-infections were observed.
From the 11 CHIKV positive oncological patients, six were adults (> 18 years old) and five were under 18 years old, with a median age of 34 years (1-69 years), being 54.54% male and 45.45% female. Others clinicals features of CHIKV positive individuals are described in Table I.
TABLE IClinical features of positive Chikungunya virus (CHIKV) individuals (n = 11)
Individuals CHIKV+
Oncological patients (> 18 years)n = 6
Oncological patients (< 18 years)n = 5
Age (years)
Median58.5014.00Mean ± SD54.6 ± 13.1711.6 ± 6.26Gender, n(%)
Female4 (66.6)1 (20)Male2 (33.3)4 (80)Oncological disease, n(%)
Chronic myeloid leukemia (CML)3 (50)0 (0)Acute myeloid leukemia (AML)1 (16.6)0 (0)Hodgkin’s lymphoma 2 (33.3)0 (0)Glioma0 (0)1 (20)Neuroblastoma III0 (0)1 (20)Sarcoma
*
0 (0)2 (40)PNET0 (0)1 (20)Symptoms, n(%)
Fever6 (100)5 (100)Rash6 (100)5 (100)Myalgia1 (16.6)0 (0)Arthralgia1 (16.6)2 (40)
* malignant neoplasm of long bones of the lower limbs or osteosarcoma; PNET: primitive neuroectodermal tumor; SD: standard deviation.
* malignant neoplasm of long bones of the lower limbs or osteosarcoma; PNET: primitive neuroectodermal tumor; SD: standard deviation.
Viral load quantification in blood and urine specimens - After CHIKV infection confirmation by qPCR, we decided to investigate possible differences in viral load in different samples obtained (serum, plasma and urine) in order to evaluate the best sample for CHIKV diagnosis in cancer patients.
From the 11 CHIKV positive cancer patients, the urine collection was impaired in two patients under 18 years old. So, considering nine patients with paired sample collection (serum, plasma and urine), 22.2% (2/9) had a positive result on the three specimens, 88.8% (8/9) had a positive result in serum, and 77.7% (7/9) cases had a positive result in plasma. Interestingly, just one patient had CHIKV detection only in urine (11.1%).
CHIKV viraemia analyses in cancer patients showed that viral loads were significantly higher in plasma (mean 6.84 log10, range 5.82 log10 to 7.91 log10) and serum (mean 6.07 log10, range 3.14 log10 to 7.95 log10,), than in urine (mean 3.76 log10, range 2.84 log10 to 5.53 log10) (Fig. 1).
Fig. 1:Chikungunya virus (CHIKV) viral load comparison between blood (serum and plasma) and urine specimens in oncological patients (n = 9). Data are expressed as averages ± standard deviations (scatter plot with bar). Each dot (●) represents one positive patient. Statistical analyses were performed using One-way ANOVA with multiple comparisons using Tukey test, p values < 0.05 were considered significant. Asterisks indicate significant differences (p = 0.0121).
Phylogenetic analysis of CHIKV positive samples from oncological patients in INCA-RJ - The phylogenetic analysis was done with representative strains (n = 11) of CHIKV detected in oncological patients from INCA (Table II).
Genotyping and phylogenetic reconstructions were carried out from a 474 bp fragment, revealing a similarity of 98-99% of the Rio de Janeiro circulating variants with the East/Centre/South Africa (ECSA) genotype (Fig. 2).
The analysis of E1 gene sequence revealed the existence of 12 synonymous mutations and three non-synonymous mutations at amino-acid level, namely K211T, M269V and T288I, when compared to HM045811 from Tanzania (1953) (Fig. 3). We also included in our analysis one isolate from India (2006) EU372006.1. We did not observe the A226V shift described for Indian isolates. All sequenced cases presented the same molecular pattern.
TABLE IIThe Chikungunya virus (CHIKV) strains from Rio de Janeiro from oncological patients (n = 11) for partial E1 gene sequencing, in 2016, BrazilID samplesYearCountry, city/stateOrigin of strainGenBank accession numberReferenceINCA1.RJ.0104162016Brazil, Rio de Janeiro/RJSerumMG262507This studyINCA2.RJ.2504162016Brazil, Rio de Janeiro/RJSerumMG262508This studyINCA4.RJ.0605162016Brazil, Rio de Janeiro/RJSerumMG262510This studyINCA5.RJ.1104162016Brazil, Rio de Janeiro/RJSerumMG262511This studyINCA6.RJ.1404162016Brazil, Rio de Janeiro/RJUrineMG262512This studyINCA7.RJ.1804162016Brazil, Rio de Janeiro/RJPlasmaMG262513This studyINCA8.RJ.2204162016Brazil, Rio de Janeiro/RJPlasmaMG262514This studyINCA9.RJ.2204162016Brazil, Rio de Janeiro/RJPlasmaMG262515This studyINCA10.RJ.2604162016Brazil, Rio de Janeiro/RJUrineMG262516This studyINCA12.RJ.2604162016Brazil, Rio de Janeiro/RJSerumMG262518This studyINCA13.RJ.0405162016Brazil, Rio de Janeiro/RJUrineMG262519This study
Fig. 2:phylogenetic analysis based on 474 nucleotides recovered from the E1 gene of Chikungunya virus (CHIKV) strains identified in Rio de Janeiro (n = 11) during 2016. The analysed CHIKV sequences in this study are represented by a pink circle (●) in oncological patients (n = 11). The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model, bootstrap of 1000 repetitions. The rate variation model allowed for some sites to be evolutionarily invariable (+I). The CHIKV strains were designated as follows: GenBank accession number/name or country/year). ECSA: East-Central-South African genotype. Evolutionary analyses were conducted in MEGA X (10.2.3 version).
Fig. 3:multiple amino acid alignment of the partial nucleotide sequences from Chikungunya virus (CHIKV ) E1 protein coding region showing non-synonym mutations. Amino acids positions numbered with respect to the strain Ross low-psg (GenBank Accession No. HM045811), strain DRDE-07 from India (GenBank Accession No. EU372006.1) and strain BHI3745/H804709 from Bahia state (Brazil, 2014) (GenBank Accession No. KP164570.1).
| null | null | [] | [] | [] | [
"SUBJECTS AND METHODS",
"RESULTS",
"DISCUSSION"
] | [
"\nStudy design, volunteers, and samples - This study included thirty-one oncological patients (adults and children) in cancer treatment and regular follow-up at Instituto Nacional do Câncer José Alencar Gomes da Silva (INCA), Rio de Janeiro, Brazil. Their recruitment was performed by INCA’s Hospital infection control committees, based on clinical signs and symptoms of arboviral infection, described as: exanthema and fever, with/without pruritus, non-purulent conjunctivitis, adenomegaly, headache, neurological symptoms, myalgia, arthralgia, blood dyscrasias, limb edema, and absence of flu suggestive respiratory manifestations. Recruitment was performed within three-seven days of symptoms onset, between February and July 2016. Serum, plasma and urine samples were collected, and all cases were screened for dengue virus (DENV) serotype 1 to 4, Zika virus (ZIKV), and CHIKV by molecular diagnosis at the Oncovirology Laboratory, Bone Marrow Transplantation Centre (CEMO).\n\nRNA extraction, molecular diagnosis, and viral load quantification - Viral RNA extraction was performed with the QIAamp VIRAL RNA kit (Qiagen) followed by with cDNA synthesis using 20 μL of RNA sample, with 300 ng/μL of Random hexameric primers, 40 U/μL of RNAseOUT and 200 U/μL of Superscript II Reverse Transcriptase (all from Invitrogen, Thermo Fisher Scientific). The reaction was incubated at 65ºC for 5 min and then at 25ºC for 10 min, followed by 42ºC for 1 h and 70ºC for 15 min, in a Veriti Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific).\nFor the differential diagnosis of arboviruses infections (CHIKV, ZIKV or DENV) a real-time quantitative polymerase chain reaction (qPCR) was performed, using a TaqMan-based system (Applied Biosystems) [Supplementary data (Table I)] specific for each virus.\n13\n\n,\n\n16\n\n,\n\n17\n\n,\n\n18\n\n\nThe qPCR reactions were performed in duplicate using PrimeTime Std qPCR Assay (IDT) and TaqMan Universal PCR Master mix (Applied Biosystems). As well, it was included primers and probes that amplify the HPRT1 human gene (assay ID Hs02800695_m1, Life Technologies). The results were contrasted with calibration curves performed by 6 log serial dilutions of oligo DNA template (IDT) in an enriched yeast-tRNA (Ambion) Tris-EDTA buffer. The thermocycling conditions consisted of 1 cycle of 95ºC, 10 min and 50 cycles of 95ºC, 15 s and 60ºC, 1 min.\n\nSequencing and phylogenetic analysis\n- CHIKV positive samples (serum, plasma or urine with Cycle threshold ≤ 37) by the qPCR method were selected for the study of viral variants by DNA sequencing. The amplification of the segment of interest of the gene encoding the viral envelope E1 glycoprotein was performed by nested-PCR using primers that amplified 1013 bp fragments in the first reaction and 555 bp in the second reaction [Supplementary data (Table II)].\n7\n\n,\n\n19\n\n\nThe fragments generated were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and PureLink PCR Purification Kit (Invitrogen) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems). The thermocycling conditions consisted of 40 cycles of denaturation (94ºC/10 s), annealing (50ºC/5 s) and extension (60ºC/4 min).\nThe sequences analysis was performed using BioEdit Sequence Alignment Editor (7.0.5.3 version) (http://www.mbio.ncsu.edu/bioedit/bioedit.htmL), sequences’ identity was performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and phylogenetic analysis was made using ClustalW multiple alignment with an external group (O’Nyong-nyong virus - M20303.1). See Supplementary data (Table III) for strains used. The phylogenetic tree was generated with the aid of the MEGA X Software (10.2.3 version) (http://www.megasoftware.net/) using Maximum Likelihood method and Kimura 2-parameter model, with a bootstrap of 1000 repetitions and a rate variation model allowed for some sites to be evolutionarily invariable ([+I]). Partial CHIKV genome sequences were deposited in GenBank and accession numbers were as follows: MG262507, MG262508, MG262510, MG262511, MG262512, MG262513, MG262514, MG262515, MG262516, MG262518, MG262519.\n\nStatistics analyses\n- The statistical analyses were performed using One-Way ANOVA with multiple comparison using Tukey test and Kruskal-Wallis test, p values < 0.05 were considered significant. For analyses, GraphPad PRISM 6 (version 6.1) (GraphPad Software) was used.\n\nStudy approval - This study was approved by the INCA’s Ethics Committee (CAAE 53571116.4.0000.5274).",
"\nLaboratorial diagnosis of arbovirus infection and clinical characteristics of CHIKV+ oncological patients - A total of thirty-one participants were recruited at INCA when clinical staff had a suspicion of arbovirus infection. All participants presented all selection criteria and were tested for ZIKV, CHIKV and all DENV’s serotype using qPCR reactions. We found a 35.48% positivity (11/31) for CHIKV in oncological patients and no co-infections were observed.\nFrom the 11 CHIKV positive oncological patients, six were adults (> 18 years old) and five were under 18 years old, with a median age of 34 years (1-69 years), being 54.54% male and 45.45% female. Others clinicals features of CHIKV positive individuals are described in Table I.\n\nTABLE IClinical features of positive Chikungunya virus (CHIKV) individuals (n = 11)\nIndividuals CHIKV+\nOncological patients (> 18 years)n = 6\nOncological patients (< 18 years)n = 5\nAge (years)\n\nMedian58.5014.00Mean ± SD54.6 ± 13.1711.6 ± 6.26Gender, n(%)\n\nFemale4 (66.6)1 (20)Male2 (33.3)4 (80)Oncological disease, n(%)\n\nChronic myeloid leukemia (CML)3 (50)0 (0)Acute myeloid leukemia (AML)1 (16.6)0 (0)Hodgkin’s lymphoma 2 (33.3)0 (0)Glioma0 (0)1 (20)Neuroblastoma III0 (0)1 (20)Sarcoma\n*\n\n0 (0)2 (40)PNET0 (0)1 (20)Symptoms, n(%)\n\nFever6 (100)5 (100)Rash6 (100)5 (100)Myalgia1 (16.6)0 (0)Arthralgia1 (16.6)2 (40)\n* malignant neoplasm of long bones of the lower limbs or osteosarcoma; PNET: primitive neuroectodermal tumor; SD: standard deviation.\n\n\n* malignant neoplasm of long bones of the lower limbs or osteosarcoma; PNET: primitive neuroectodermal tumor; SD: standard deviation.\n\nViral load quantification in blood and urine specimens - After CHIKV infection confirmation by qPCR, we decided to investigate possible differences in viral load in different samples obtained (serum, plasma and urine) in order to evaluate the best sample for CHIKV diagnosis in cancer patients.\nFrom the 11 CHIKV positive cancer patients, the urine collection was impaired in two patients under 18 years old. So, considering nine patients with paired sample collection (serum, plasma and urine), 22.2% (2/9) had a positive result on the three specimens, 88.8% (8/9) had a positive result in serum, and 77.7% (7/9) cases had a positive result in plasma. Interestingly, just one patient had CHIKV detection only in urine (11.1%).\nCHIKV viraemia analyses in cancer patients showed that viral loads were significantly higher in plasma (mean 6.84 log10, range 5.82 log10 to 7.91 log10) and serum (mean 6.07 log10, range 3.14 log10 to 7.95 log10,), than in urine (mean 3.76 log10, range 2.84 log10 to 5.53 log10) (Fig. 1).\n\nFig. 1:Chikungunya virus (CHIKV) viral load comparison between blood (serum and plasma) and urine specimens in oncological patients (n = 9). Data are expressed as averages ± standard deviations (scatter plot with bar). Each dot (●) represents one positive patient. Statistical analyses were performed using One-way ANOVA with multiple comparisons using Tukey test, p values < 0.05 were considered significant. Asterisks indicate significant differences (p = 0.0121).\n\n\nPhylogenetic analysis of CHIKV positive samples from oncological patients in INCA-RJ - The phylogenetic analysis was done with representative strains (n = 11) of CHIKV detected in oncological patients from INCA (Table II).\nGenotyping and phylogenetic reconstructions were carried out from a 474 bp fragment, revealing a similarity of 98-99% of the Rio de Janeiro circulating variants with the East/Centre/South Africa (ECSA) genotype (Fig. 2).\nThe analysis of E1 gene sequence revealed the existence of 12 synonymous mutations and three non-synonymous mutations at amino-acid level, namely K211T, M269V and T288I, when compared to HM045811 from Tanzania (1953) (Fig. 3). We also included in our analysis one isolate from India (2006) EU372006.1. We did not observe the A226V shift described for Indian isolates. All sequenced cases presented the same molecular pattern.\n\nTABLE IIThe Chikungunya virus (CHIKV) strains from Rio de Janeiro from oncological patients (n = 11) for partial E1 gene sequencing, in 2016, BrazilID samplesYearCountry, city/stateOrigin of strainGenBank accession numberReferenceINCA1.RJ.0104162016Brazil, Rio de Janeiro/RJSerumMG262507This studyINCA2.RJ.2504162016Brazil, Rio de Janeiro/RJSerumMG262508This studyINCA4.RJ.0605162016Brazil, Rio de Janeiro/RJSerumMG262510This studyINCA5.RJ.1104162016Brazil, Rio de Janeiro/RJSerumMG262511This studyINCA6.RJ.1404162016Brazil, Rio de Janeiro/RJUrineMG262512This studyINCA7.RJ.1804162016Brazil, Rio de Janeiro/RJPlasmaMG262513This studyINCA8.RJ.2204162016Brazil, Rio de Janeiro/RJPlasmaMG262514This studyINCA9.RJ.2204162016Brazil, Rio de Janeiro/RJPlasmaMG262515This studyINCA10.RJ.2604162016Brazil, Rio de Janeiro/RJUrineMG262516This studyINCA12.RJ.2604162016Brazil, Rio de Janeiro/RJSerumMG262518This studyINCA13.RJ.0405162016Brazil, Rio de Janeiro/RJUrineMG262519This study\n\n\nFig. 2:phylogenetic analysis based on 474 nucleotides recovered from the E1 gene of Chikungunya virus (CHIKV) strains identified in Rio de Janeiro (n = 11) during 2016. The analysed CHIKV sequences in this study are represented by a pink circle (●) in oncological patients (n = 11). The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model, bootstrap of 1000 repetitions. The rate variation model allowed for some sites to be evolutionarily invariable (+I). The CHIKV strains were designated as follows: GenBank accession number/name or country/year). ECSA: East-Central-South African genotype. Evolutionary analyses were conducted in MEGA X (10.2.3 version).\n\n\nFig. 3:multiple amino acid alignment of the partial nucleotide sequences from Chikungunya virus (CHIKV ) E1 protein coding region showing non-synonym mutations. Amino acids positions numbered with respect to the strain Ross low-psg (GenBank Accession No. HM045811), strain DRDE-07 from India (GenBank Accession No. EU372006.1) and strain BHI3745/H804709 from Bahia state (Brazil, 2014) (GenBank Accession No. KP164570.1).\n",
"Cancer is a situation in which patients suffer a systemic immunosuppression, that results from the particular characteristics of neoplastic diseases (i.e., some types of cancer are associated with a more severe state of immunodepression than others), the burden of disease on the body, and from the aggressiveness of current therapies like chemotherapy, radiotherapy, immunotherapy and in some cases, surgery.\n20\n As well it cannot be forgotten that the heterogeneity of patient populations (past infections, malnutrition, comorbidities, cancer status etc.) may impact the treatment and can increase the risk of bacterial, fungal and viral infection. Infection is a significant cause of morbidity and mortality in these patients.\n21\n\n\nCHIKV has a long history of emergence in urban transmission cycles from its enzootic and wild outbreaks in Africa, expanding to the Americas in 2013, across the Asian continent.\n22\n\n,\n\n23\n In Brazil, CHIKV had two introductions, one of the Asian genotype in Amapá state and the other of the ECSA genotype in Bahia state.\n2\n Driven by the presence of competent vectors (Aedes genus) and by an intense flow of people traveling, the number of cases in the whole country increased exponentially. Although the big burden of cases occurred in the 2016 outbreak, between 2017-2021 (until epidemiological week 29), a total of 551,393 cases were reported in Brazil and 134,209 cases in Rio de Janeiro state until today,\n2\n\n,\n\n24\n\n,\n\n25\n\n,\n\n26\n\n,\n\n27\n showing that CHIKV continues to cause large numbers of cases across the country.\nIn this work, we were able to diagnose and evaluate the viral load of CHIKV infection in cancer patients who were being followed up at INCA in Rio de Janeiro state. A total of 11/31 oncological patients were CHIKV positive by qPCR at least in one sample (plasma, serum or urine). Indeed, we believe that this number is underestimated, because samples collection was made at the time of CHIKV outbreak flourishing, and so a higher positive rate was expected. The goal of cancer treatment is based on selective killing of the cancer cells. Chemotherapy, for example, is the most common treatment used that is supposed to target just cancer cells, nonetheless, normal cells suffer damage too, and these damages lead to a range of manifestations and side-effects. Among them, headache, fatigue, weakness, hair loss, nausea, vomiting, diarrhea, abdominal cramps, fever, skin rash\n28\n can mask signs and symptoms of viral infections. Hence, when the patient is in the cycle chemotherapy and may have a viral infection, such as CHIKV infection, one could first suspect the treatment side-effects and afterwards the infections. Consequently, some patients may not have looked for medical care and viral diagnosis not made.\nSome viruses, like CHIKV, can cause high levels of viraemia in humans in acute phase.\n29\n\n,\n\n30\n In blood, CHIKV can reach up 108 or 109 genome copies/mL.\n31\n\n,\n\n32\n\n,\n\n33\n A case of series has analysed solid organ transplantation (SOT) recipients with CHIKV infection. Rosso et al. has observed that 80% of SOT patients presented high viral load (> 106 copies/mL) and none of them developed graft rejection, chronic inflammatory manifestations or fatal cases after CHIKV infection,\n34\n indicating that CHIKV infection may not have a negative impact on SOT patients. On other hand, another study suggested a negative impact on immune response in HIV-positive patients (elite controller) that live in a CHIKV endemic area in Martinique.\n35\n Therefore, more studies in immunosuppressed individuals are needed.\nIn our study, we proposed to quantify CHIKV viral load in cancer patients in blood and urine. Although the positive final dataset is small, we observed that our patients present a high viral load in plasma (mean 6.84 log10) and serum (mean 6.07 log10) similar to SOT patients described in a case of series by Rosso and collaborators.\n34\n Also, we were able to detect and quantify viral load in urine of 33.33% (3/9) patients (mean 3.76 log10) that was significantly lower than the viral load present in plasma. Of note, our detection rate in urine is much higher than that reported in French Polynesia outbreak.\n36\n\n\nStudies have been reporting the importance of using urine in arbovirus diagnosis.\n37\n Bandeira and collaborators in a case-report study detected CHIKV RNA in urine and semen for an extended period (30 days).\n38\n Moreover, another study has described that DENV RNA detection rate in urine, on acute phase, was 25% (2/8) on days 0 to 3 and 32% (7/22) on days 4 to 5. As the days went by, the detection rate increased until day 11 after the first symptom appearance and started to decline until the last day of detection (day 16). The authors suggested that urine can be an ally to molecular diagnosis when viremia disappears.\n39\n Herein, we did not evaluate the cancer patients in multiples collections and different days after the first CHIKV detection, so, we may speculate that the detection rate in urine could rise if we had multiple collections some days later.\nFurthermore, we found a similarity of 98-99% of the circulating variants in oncological patients from Rio de Janeiro state with ECSA. Other studies showed the circulation of the ECSA genotype in the state of Rio de Janeiro in 2016,\n40\n\n,\n\n41\n\n,\n\n42\n as the present study shows, but also the permanence of this strain until today.\n43\n\n\nIn addition, we made molecular characterisation of CHIKV strains. We found 12 synonym mutations and three non-synonym mutations (E1-K211T, E1-M269V and E1-T288I). Souza et al. showed two amino acids substitutions (E1-K211T and E1-V156A) which are exclusive to the CHIKV strains obtained also during the 2016 epidemic in Rio de Janeiro (Brazil).\n41\n More studies are needed to evaluate the consequences of these mutations on viral replication, in human immune response and in vector fitness (A. albopictus and A. aegypti). Knowing the vector competence of each species is important to understand the transmissibility potential of mosquitos, and therefore it is important to evaluate the potential amino acid substitution and its impact in virus replication to aid epidemiological studies and epidemics previsions.\n44\n\n\nWe conclude that CHIKV genome detection can be performed with equal efficiency for both serum and plasma. Viral loads found in the serum and plasma of oncological patients infected with CHIKV were greater than those found in the urine. Although we are not able to discard a late virus excretion, the detection of the single case with virus detection only in urine suggests that this compartment may be an important complement to diagnostics. Finally, phylogenetic analyses had shown that the circulating genotype among cancer patients in 2016 outbreak belonged to the ECSA genotype, carrying K211T, M269V and T288I substitutions in E1-CHIKV genome."
] | [
"materials|methods",
"results",
"discussion"
] | [
"CHIKV",
"oncological patients",
"viral load",
"ECSA"
] | SUBJECTS AND METHODS:
Study design, volunteers, and samples - This study included thirty-one oncological patients (adults and children) in cancer treatment and regular follow-up at Instituto Nacional do Câncer José Alencar Gomes da Silva (INCA), Rio de Janeiro, Brazil. Their recruitment was performed by INCA’s Hospital infection control committees, based on clinical signs and symptoms of arboviral infection, described as: exanthema and fever, with/without pruritus, non-purulent conjunctivitis, adenomegaly, headache, neurological symptoms, myalgia, arthralgia, blood dyscrasias, limb edema, and absence of flu suggestive respiratory manifestations. Recruitment was performed within three-seven days of symptoms onset, between February and July 2016. Serum, plasma and urine samples were collected, and all cases were screened for dengue virus (DENV) serotype 1 to 4, Zika virus (ZIKV), and CHIKV by molecular diagnosis at the Oncovirology Laboratory, Bone Marrow Transplantation Centre (CEMO).
RNA extraction, molecular diagnosis, and viral load quantification - Viral RNA extraction was performed with the QIAamp VIRAL RNA kit (Qiagen) followed by with cDNA synthesis using 20 μL of RNA sample, with 300 ng/μL of Random hexameric primers, 40 U/μL of RNAseOUT and 200 U/μL of Superscript II Reverse Transcriptase (all from Invitrogen, Thermo Fisher Scientific). The reaction was incubated at 65ºC for 5 min and then at 25ºC for 10 min, followed by 42ºC for 1 h and 70ºC for 15 min, in a Veriti Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific).
For the differential diagnosis of arboviruses infections (CHIKV, ZIKV or DENV) a real-time quantitative polymerase chain reaction (qPCR) was performed, using a TaqMan-based system (Applied Biosystems) [Supplementary data (Table I)] specific for each virus.
13
,
16
,
17
,
18
The qPCR reactions were performed in duplicate using PrimeTime Std qPCR Assay (IDT) and TaqMan Universal PCR Master mix (Applied Biosystems). As well, it was included primers and probes that amplify the HPRT1 human gene (assay ID Hs02800695_m1, Life Technologies). The results were contrasted with calibration curves performed by 6 log serial dilutions of oligo DNA template (IDT) in an enriched yeast-tRNA (Ambion) Tris-EDTA buffer. The thermocycling conditions consisted of 1 cycle of 95ºC, 10 min and 50 cycles of 95ºC, 15 s and 60ºC, 1 min.
Sequencing and phylogenetic analysis
- CHIKV positive samples (serum, plasma or urine with Cycle threshold ≤ 37) by the qPCR method were selected for the study of viral variants by DNA sequencing. The amplification of the segment of interest of the gene encoding the viral envelope E1 glycoprotein was performed by nested-PCR using primers that amplified 1013 bp fragments in the first reaction and 555 bp in the second reaction [Supplementary data (Table II)].
7
,
19
The fragments generated were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and PureLink PCR Purification Kit (Invitrogen) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems). The thermocycling conditions consisted of 40 cycles of denaturation (94ºC/10 s), annealing (50ºC/5 s) and extension (60ºC/4 min).
The sequences analysis was performed using BioEdit Sequence Alignment Editor (7.0.5.3 version) (http://www.mbio.ncsu.edu/bioedit/bioedit.htmL), sequences’ identity was performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and phylogenetic analysis was made using ClustalW multiple alignment with an external group (O’Nyong-nyong virus - M20303.1). See Supplementary data (Table III) for strains used. The phylogenetic tree was generated with the aid of the MEGA X Software (10.2.3 version) (http://www.megasoftware.net/) using Maximum Likelihood method and Kimura 2-parameter model, with a bootstrap of 1000 repetitions and a rate variation model allowed for some sites to be evolutionarily invariable ([+I]). Partial CHIKV genome sequences were deposited in GenBank and accession numbers were as follows: MG262507, MG262508, MG262510, MG262511, MG262512, MG262513, MG262514, MG262515, MG262516, MG262518, MG262519.
Statistics analyses
- The statistical analyses were performed using One-Way ANOVA with multiple comparison using Tukey test and Kruskal-Wallis test, p values < 0.05 were considered significant. For analyses, GraphPad PRISM 6 (version 6.1) (GraphPad Software) was used.
Study approval - This study was approved by the INCA’s Ethics Committee (CAAE 53571116.4.0000.5274).
RESULTS:
Laboratorial diagnosis of arbovirus infection and clinical characteristics of CHIKV+ oncological patients - A total of thirty-one participants were recruited at INCA when clinical staff had a suspicion of arbovirus infection. All participants presented all selection criteria and were tested for ZIKV, CHIKV and all DENV’s serotype using qPCR reactions. We found a 35.48% positivity (11/31) for CHIKV in oncological patients and no co-infections were observed.
From the 11 CHIKV positive oncological patients, six were adults (> 18 years old) and five were under 18 years old, with a median age of 34 years (1-69 years), being 54.54% male and 45.45% female. Others clinicals features of CHIKV positive individuals are described in Table I.
TABLE IClinical features of positive Chikungunya virus (CHIKV) individuals (n = 11)
Individuals CHIKV+
Oncological patients (> 18 years)n = 6
Oncological patients (< 18 years)n = 5
Age (years)
Median58.5014.00Mean ± SD54.6 ± 13.1711.6 ± 6.26Gender, n(%)
Female4 (66.6)1 (20)Male2 (33.3)4 (80)Oncological disease, n(%)
Chronic myeloid leukemia (CML)3 (50)0 (0)Acute myeloid leukemia (AML)1 (16.6)0 (0)Hodgkin’s lymphoma 2 (33.3)0 (0)Glioma0 (0)1 (20)Neuroblastoma III0 (0)1 (20)Sarcoma
*
0 (0)2 (40)PNET0 (0)1 (20)Symptoms, n(%)
Fever6 (100)5 (100)Rash6 (100)5 (100)Myalgia1 (16.6)0 (0)Arthralgia1 (16.6)2 (40)
* malignant neoplasm of long bones of the lower limbs or osteosarcoma; PNET: primitive neuroectodermal tumor; SD: standard deviation.
* malignant neoplasm of long bones of the lower limbs or osteosarcoma; PNET: primitive neuroectodermal tumor; SD: standard deviation.
Viral load quantification in blood and urine specimens - After CHIKV infection confirmation by qPCR, we decided to investigate possible differences in viral load in different samples obtained (serum, plasma and urine) in order to evaluate the best sample for CHIKV diagnosis in cancer patients.
From the 11 CHIKV positive cancer patients, the urine collection was impaired in two patients under 18 years old. So, considering nine patients with paired sample collection (serum, plasma and urine), 22.2% (2/9) had a positive result on the three specimens, 88.8% (8/9) had a positive result in serum, and 77.7% (7/9) cases had a positive result in plasma. Interestingly, just one patient had CHIKV detection only in urine (11.1%).
CHIKV viraemia analyses in cancer patients showed that viral loads were significantly higher in plasma (mean 6.84 log10, range 5.82 log10 to 7.91 log10) and serum (mean 6.07 log10, range 3.14 log10 to 7.95 log10,), than in urine (mean 3.76 log10, range 2.84 log10 to 5.53 log10) (Fig. 1).
Fig. 1:Chikungunya virus (CHIKV) viral load comparison between blood (serum and plasma) and urine specimens in oncological patients (n = 9). Data are expressed as averages ± standard deviations (scatter plot with bar). Each dot (●) represents one positive patient. Statistical analyses were performed using One-way ANOVA with multiple comparisons using Tukey test, p values < 0.05 were considered significant. Asterisks indicate significant differences (p = 0.0121).
Phylogenetic analysis of CHIKV positive samples from oncological patients in INCA-RJ - The phylogenetic analysis was done with representative strains (n = 11) of CHIKV detected in oncological patients from INCA (Table II).
Genotyping and phylogenetic reconstructions were carried out from a 474 bp fragment, revealing a similarity of 98-99% of the Rio de Janeiro circulating variants with the East/Centre/South Africa (ECSA) genotype (Fig. 2).
The analysis of E1 gene sequence revealed the existence of 12 synonymous mutations and three non-synonymous mutations at amino-acid level, namely K211T, M269V and T288I, when compared to HM045811 from Tanzania (1953) (Fig. 3). We also included in our analysis one isolate from India (2006) EU372006.1. We did not observe the A226V shift described for Indian isolates. All sequenced cases presented the same molecular pattern.
TABLE IIThe Chikungunya virus (CHIKV) strains from Rio de Janeiro from oncological patients (n = 11) for partial E1 gene sequencing, in 2016, BrazilID samplesYearCountry, city/stateOrigin of strainGenBank accession numberReferenceINCA1.RJ.0104162016Brazil, Rio de Janeiro/RJSerumMG262507This studyINCA2.RJ.2504162016Brazil, Rio de Janeiro/RJSerumMG262508This studyINCA4.RJ.0605162016Brazil, Rio de Janeiro/RJSerumMG262510This studyINCA5.RJ.1104162016Brazil, Rio de Janeiro/RJSerumMG262511This studyINCA6.RJ.1404162016Brazil, Rio de Janeiro/RJUrineMG262512This studyINCA7.RJ.1804162016Brazil, Rio de Janeiro/RJPlasmaMG262513This studyINCA8.RJ.2204162016Brazil, Rio de Janeiro/RJPlasmaMG262514This studyINCA9.RJ.2204162016Brazil, Rio de Janeiro/RJPlasmaMG262515This studyINCA10.RJ.2604162016Brazil, Rio de Janeiro/RJUrineMG262516This studyINCA12.RJ.2604162016Brazil, Rio de Janeiro/RJSerumMG262518This studyINCA13.RJ.0405162016Brazil, Rio de Janeiro/RJUrineMG262519This study
Fig. 2:phylogenetic analysis based on 474 nucleotides recovered from the E1 gene of Chikungunya virus (CHIKV) strains identified in Rio de Janeiro (n = 11) during 2016. The analysed CHIKV sequences in this study are represented by a pink circle (●) in oncological patients (n = 11). The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model, bootstrap of 1000 repetitions. The rate variation model allowed for some sites to be evolutionarily invariable (+I). The CHIKV strains were designated as follows: GenBank accession number/name or country/year). ECSA: East-Central-South African genotype. Evolutionary analyses were conducted in MEGA X (10.2.3 version).
Fig. 3:multiple amino acid alignment of the partial nucleotide sequences from Chikungunya virus (CHIKV ) E1 protein coding region showing non-synonym mutations. Amino acids positions numbered with respect to the strain Ross low-psg (GenBank Accession No. HM045811), strain DRDE-07 from India (GenBank Accession No. EU372006.1) and strain BHI3745/H804709 from Bahia state (Brazil, 2014) (GenBank Accession No. KP164570.1).
DISCUSSION:
Cancer is a situation in which patients suffer a systemic immunosuppression, that results from the particular characteristics of neoplastic diseases (i.e., some types of cancer are associated with a more severe state of immunodepression than others), the burden of disease on the body, and from the aggressiveness of current therapies like chemotherapy, radiotherapy, immunotherapy and in some cases, surgery.
20
As well it cannot be forgotten that the heterogeneity of patient populations (past infections, malnutrition, comorbidities, cancer status etc.) may impact the treatment and can increase the risk of bacterial, fungal and viral infection. Infection is a significant cause of morbidity and mortality in these patients.
21
CHIKV has a long history of emergence in urban transmission cycles from its enzootic and wild outbreaks in Africa, expanding to the Americas in 2013, across the Asian continent.
22
,
23
In Brazil, CHIKV had two introductions, one of the Asian genotype in Amapá state and the other of the ECSA genotype in Bahia state.
2
Driven by the presence of competent vectors (Aedes genus) and by an intense flow of people traveling, the number of cases in the whole country increased exponentially. Although the big burden of cases occurred in the 2016 outbreak, between 2017-2021 (until epidemiological week 29), a total of 551,393 cases were reported in Brazil and 134,209 cases in Rio de Janeiro state until today,
2
,
24
,
25
,
26
,
27
showing that CHIKV continues to cause large numbers of cases across the country.
In this work, we were able to diagnose and evaluate the viral load of CHIKV infection in cancer patients who were being followed up at INCA in Rio de Janeiro state. A total of 11/31 oncological patients were CHIKV positive by qPCR at least in one sample (plasma, serum or urine). Indeed, we believe that this number is underestimated, because samples collection was made at the time of CHIKV outbreak flourishing, and so a higher positive rate was expected. The goal of cancer treatment is based on selective killing of the cancer cells. Chemotherapy, for example, is the most common treatment used that is supposed to target just cancer cells, nonetheless, normal cells suffer damage too, and these damages lead to a range of manifestations and side-effects. Among them, headache, fatigue, weakness, hair loss, nausea, vomiting, diarrhea, abdominal cramps, fever, skin rash
28
can mask signs and symptoms of viral infections. Hence, when the patient is in the cycle chemotherapy and may have a viral infection, such as CHIKV infection, one could first suspect the treatment side-effects and afterwards the infections. Consequently, some patients may not have looked for medical care and viral diagnosis not made.
Some viruses, like CHIKV, can cause high levels of viraemia in humans in acute phase.
29
,
30
In blood, CHIKV can reach up 108 or 109 genome copies/mL.
31
,
32
,
33
A case of series has analysed solid organ transplantation (SOT) recipients with CHIKV infection. Rosso et al. has observed that 80% of SOT patients presented high viral load (> 106 copies/mL) and none of them developed graft rejection, chronic inflammatory manifestations or fatal cases after CHIKV infection,
34
indicating that CHIKV infection may not have a negative impact on SOT patients. On other hand, another study suggested a negative impact on immune response in HIV-positive patients (elite controller) that live in a CHIKV endemic area in Martinique.
35
Therefore, more studies in immunosuppressed individuals are needed.
In our study, we proposed to quantify CHIKV viral load in cancer patients in blood and urine. Although the positive final dataset is small, we observed that our patients present a high viral load in plasma (mean 6.84 log10) and serum (mean 6.07 log10) similar to SOT patients described in a case of series by Rosso and collaborators.
34
Also, we were able to detect and quantify viral load in urine of 33.33% (3/9) patients (mean 3.76 log10) that was significantly lower than the viral load present in plasma. Of note, our detection rate in urine is much higher than that reported in French Polynesia outbreak.
36
Studies have been reporting the importance of using urine in arbovirus diagnosis.
37
Bandeira and collaborators in a case-report study detected CHIKV RNA in urine and semen for an extended period (30 days).
38
Moreover, another study has described that DENV RNA detection rate in urine, on acute phase, was 25% (2/8) on days 0 to 3 and 32% (7/22) on days 4 to 5. As the days went by, the detection rate increased until day 11 after the first symptom appearance and started to decline until the last day of detection (day 16). The authors suggested that urine can be an ally to molecular diagnosis when viremia disappears.
39
Herein, we did not evaluate the cancer patients in multiples collections and different days after the first CHIKV detection, so, we may speculate that the detection rate in urine could rise if we had multiple collections some days later.
Furthermore, we found a similarity of 98-99% of the circulating variants in oncological patients from Rio de Janeiro state with ECSA. Other studies showed the circulation of the ECSA genotype in the state of Rio de Janeiro in 2016,
40
,
41
,
42
as the present study shows, but also the permanence of this strain until today.
43
In addition, we made molecular characterisation of CHIKV strains. We found 12 synonym mutations and three non-synonym mutations (E1-K211T, E1-M269V and E1-T288I). Souza et al. showed two amino acids substitutions (E1-K211T and E1-V156A) which are exclusive to the CHIKV strains obtained also during the 2016 epidemic in Rio de Janeiro (Brazil).
41
More studies are needed to evaluate the consequences of these mutations on viral replication, in human immune response and in vector fitness (A. albopictus and A. aegypti). Knowing the vector competence of each species is important to understand the transmissibility potential of mosquitos, and therefore it is important to evaluate the potential amino acid substitution and its impact in virus replication to aid epidemiological studies and epidemics previsions.
44
We conclude that CHIKV genome detection can be performed with equal efficiency for both serum and plasma. Viral loads found in the serum and plasma of oncological patients infected with CHIKV were greater than those found in the urine. Although we are not able to discard a late virus excretion, the detection of the single case with virus detection only in urine suggests that this compartment may be an important complement to diagnostics. Finally, phylogenetic analyses had shown that the circulating genotype among cancer patients in 2016 outbreak belonged to the ECSA genotype, carrying K211T, M269V and T288I substitutions in E1-CHIKV genome.
| Background:
Chikungunya virus (CHIKV) is an arbovirus that can cause chronic and debilitating manifestations. The first autochthonous case in Rio de Janeiro state was diagnosed in 2015, and an outbreak was declared in 2016.
Methods:
Paired serum, plasma and urine collected from 31 cancer patients were tested by real-time quantitative polymerase chain reaction (qPCR) and a segment of the CHIKV E1 gene was sequenced.
Results:
We detected 11 CHIKV+ oncological patients. Paired samples analyses of nine patients showed a different pattern of detection. Also, a higher viral load in plasma (6.84 log10) and serum (6.07 log10) vs urine (3.76 log10) was found. Phylogenetic analysis and molecular characterisation revealed East/Central/Southern Africa (ECSA) genotype circulation and three amino acids substitutions (E1-K211T, E1-M269V, E1-T288I) in positive patients.
Conclusions:
The results indicate the bioequivalence of serum and plasma for CHIKV diagnosis, with urine being an important complement. ECSA genotype was circulating among patients in the period of the 2016 outbreak with K211T, M269V and T288I substitution. | null | null | 3,387 | 217 | [] | 3 | [
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Delays in presentation of intussusception and development of gangrene in Zimbabwe. | 34548895 | prompt diagnosis and treatment are considered key to successful management of intussusception. We examined pre-treatment delay among intussusception cases in Zimbabwe and conducted an exploratory analysis of factors associated with intraoperative finding of gangrene. | INTRODUCTION | data were prospectively collected as part of the African Intussusception Network using a questionnaire administered on consecutive patients with intussusception managed at Harare Children´s Hospital. Delays were classified using the Three-Delays-Model: care-seeking delay (time from onset of symptoms to first presentation for health care), health-system delay (referral time from presentation to first facility to treatment facility) and treatment delay (time from presentation at treatment facility to treatment). | METHODS | ninety-two patients were enrolled from August 2014 to December 2016. The mean care-seeking interval was 1.9 days, the mean health-system interval was 1.5 days, and the mean treatment interval was 1.1 days. Mean total time from symptom onset to treatment was 4.4 days. Being transferred from another institution added 1.4 days to the patient journey. Gangrene was found in 2 (25%) of children who received treatment within 1 day, 13 (41%) of children who received treatment 2-3 days, and 26 (50%) of children who received treatment more than 3 days after symptom onset (p = 0.34). | RESULTS | significant care-seeking and health-system delays are encountered by intussusception patients in Zimbabwe. Our findings highlight the need to explore approaches to improve the early diagnosis of intussusception and prompt referral of patients for treatment. | CONCLUSION | [
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] | 8437425 | Introduction | Intussusception is an enteric invagination into an adjacent segment of bowel. Some intussusception cases have been associated with infection with various enteric viruses causing Peyer´s patch hypertrophy [1, 2]. This assertion was bolstered by some studies finding a seasonal pattern of intussusception cases [3, 4]. A slightly increased risk of intussusception of 1 to 6 excess cases per 100,000 vaccinated infants has been observed following rotavirus vaccination in clinical trials in high- and middle-income countries [5]; however, no association was found between rotavirus vaccine and intussusception in a multi-country analysis in sub-Saharan Africa [6]. Intussusception is the most common cause of childhood intestinal obstruction in Zimbabwe [3], and is also the most frequently encountered paediatric surgical emergency [3]. This is similar to the experience in other African countries [7]. It was found to be the most common cause of childhood intestinal obstruction in Nigeria and of acute mechanical obstruction in children in Niger [8, 9].
Intussusception is managed surgically, with manual reduction or resection, or nonoperatively by air, hydrostatic or contrast enema. In Africa rates of surgical intervention are higher than for non-operative reduction [6, 7, 10]. Ekenze et al. reported that in south eastern Nigeria surgical management was performed routinely in cases of intussusception [11]. In contrast, 81% of intussusception patients in a study in Europe had non-operative reduction [12].
Delays in presentation and treatment of serious surgical diseases, including intussusception, are common in low-resource countries due to limited access to care [7, 10, 13, 14]. In a study from Nigeria only 7.7% of patients presented within 24 hours of onset of intussusception symptoms [15]. Late presentation of intussusception cases is considered a risk factor for gangrene and death, increasing the need for surgery [16-18] and predicting the failure of non-operative reduction [16, 19-20]. It also increases the chances of sepsis, multiple organ dysfunction and death. [21, 22] In this analysis we describe the time intervals from onset of symptoms to definitive treatment of infants with intussusception in Zimbabwe. As an exploratory analysis, we considered the relationship between delayed presentation and gangrene. | Methods | Patient population All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.
study participants flow chart
All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.
study participants flow chart
Study setting The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.
The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.
Data collection Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).
Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).
Description of surgical procedure Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.
Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.
Definitions of time intervals The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.
The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.
Statistical analysis We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.
Ethical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.
Disclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.
We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.
Ethical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.
Disclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention. | Results | Demographics Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.
Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.
Geographic factors Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.
home addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown
Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.
home addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown
Time intervals in the patient journey eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.
time intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results
eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.
time intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results
Relationship with development of gangrene Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).
relationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene
1Chi-square statistic; 2Fisher´s exact test
Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).
relationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene
1Chi-square statistic; 2Fisher´s exact test
Complications Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.
Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction. | Conclusion | Time to hospital for treatment of intussusception in Zimbabwe is longer than commonly accepted benchmarks. Low sample size in this study may not have provided enough statistical power to show significant associations between gangrene and pre-hospital and pre-treatment duration although these may have existed. Advocacy and training among primary care providers to improve timeliness and accuracy of diagnosis and capacitating small peripheral health institutions as well as health education in parents to improve healthcare-seeking behaviour are potential targets for reducing delays in the pre-treatment interval. Future research should investigate mechanical factors and the morphology of the bowel in intussusception.
What is known about this topic
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
What this study adds
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive. | [
"Patient population",
"Study setting",
"Data collection",
"Description of surgical procedure",
"Definitions of time intervals",
"Statistical analysis",
"Demographics",
"Geographic factors",
"Time intervals in the patient journey",
"Relationship with development of gangrene",
"Complications",
"Limitations",
"What is known about this topic",
"What this study adds"
] | [
"All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.\nstudy participants flow chart",
"The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.",
"Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).",
"Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.",
"The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.",
"We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.\nEthical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.\nDisclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.",
"Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.",
"Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.\nhome addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown",
"eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.\ntime intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results",
"Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).\nrelationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene\n1Chi-square statistic; 2Fisher´s exact test",
"Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.",
"A major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.\nThe data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.",
"\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.\n\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;\nData on the epidemiology of intussusception in sub-Saharan African are sparse.",
"\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.\n\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;\nChildren with intussusception who die are more likely to present later for treatment than children who survive."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Patient population",
"Study setting",
"Data collection",
"Description of surgical procedure",
"Definitions of time intervals",
"Statistical analysis",
"Results",
"Demographics",
"Geographic factors",
"Time intervals in the patient journey",
"Relationship with development of gangrene",
"Complications",
"Discussion",
"Limitations",
"Conclusion",
"What is known about this topic",
"What this study adds"
] | [
"Intussusception is an enteric invagination into an adjacent segment of bowel. Some intussusception cases have been associated with infection with various enteric viruses causing Peyer´s patch hypertrophy [1, 2]. This assertion was bolstered by some studies finding a seasonal pattern of intussusception cases [3, 4]. A slightly increased risk of intussusception of 1 to 6 excess cases per 100,000 vaccinated infants has been observed following rotavirus vaccination in clinical trials in high- and middle-income countries [5]; however, no association was found between rotavirus vaccine and intussusception in a multi-country analysis in sub-Saharan Africa [6]. Intussusception is the most common cause of childhood intestinal obstruction in Zimbabwe [3], and is also the most frequently encountered paediatric surgical emergency [3]. This is similar to the experience in other African countries [7]. It was found to be the most common cause of childhood intestinal obstruction in Nigeria and of acute mechanical obstruction in children in Niger [8, 9].\nIntussusception is managed surgically, with manual reduction or resection, or nonoperatively by air, hydrostatic or contrast enema. In Africa rates of surgical intervention are higher than for non-operative reduction [6, 7, 10]. Ekenze et al. reported that in south eastern Nigeria surgical management was performed routinely in cases of intussusception [11]. In contrast, 81% of intussusception patients in a study in Europe had non-operative reduction [12].\nDelays in presentation and treatment of serious surgical diseases, including intussusception, are common in low-resource countries due to limited access to care [7, 10, 13, 14]. In a study from Nigeria only 7.7% of patients presented within 24 hours of onset of intussusception symptoms [15]. Late presentation of intussusception cases is considered a risk factor for gangrene and death, increasing the need for surgery [16-18] and predicting the failure of non-operative reduction [16, 19-20]. It also increases the chances of sepsis, multiple organ dysfunction and death. [21, 22] In this analysis we describe the time intervals from onset of symptoms to definitive treatment of infants with intussusception in Zimbabwe. As an exploratory analysis, we considered the relationship between delayed presentation and gangrene.",
"Patient population All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.\nstudy participants flow chart\nAll patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.\nstudy participants flow chart\nStudy setting The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.\nThe study was performed at Harare Children´s Hospital, a public, teaching referral hospital.\nData collection Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).\nData were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).\nDescription of surgical procedure Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.\nPatients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.\nDefinitions of time intervals The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.\nThe time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.\nStatistical analysis We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.\nEthical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.\nDisclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.\nWe used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.\nEthical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.\nDisclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.",
"All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.\nstudy participants flow chart",
"The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.",
"Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).",
"Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.",
"The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.",
"We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.\nEthical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.\nDisclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.",
"Demographics Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.\nNinety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.\nGeographic factors Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.\nhome addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown\nHome addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.\nhome addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown\nTime intervals in the patient journey eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.\ntime intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results\neighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.\ntime intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results\nRelationship with development of gangrene Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).\nrelationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene\n1Chi-square statistic; 2Fisher´s exact test\nOf the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).\nrelationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene\n1Chi-square statistic; 2Fisher´s exact test\nComplications Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.\nFive patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.",
"Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.",
"Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.\nhome addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown",
"eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.\ntime intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results",
"Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).\nrelationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene\n1Chi-square statistic; 2Fisher´s exact test",
"Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.",
"We found significant delays between the onset of intussusception symptoms and reduction among children < 12 months old in Zimbabwe. The mean care-seeking interval was slightly higher than the mean health-system interval but this difference was not statistically significant. Therefore, both intervals likely contributed equally to delays in reaching definitive treatment. The evidence to date would suggest that diagnostic delay plays a large part in late presentation rather than socioeconomic factors, which has been reported by other evaluations [27-29]. Barriers to timely care in paediatric surgery were explored by Pilkington et al and include transport and cost on the part of the patient as well as shortcomings in hospital infrastructure and resources [30]. The mean treatment interval was 1.1 days in our study and was comparable to guidelines for wait times in paediatric surgical patients formulated by the Canadian Paediatric Surgical Wait Times Taskforce [31]. It was also much shorter than average treatment interval in Uganda [30]. This is a surrogate quality measure and shows that, definitive management is instituted quickly once the decision has been made.\nSurgery was used to manage intussusception for 100% of this study population because of lack of facilities required for enema reduction during the study period. Additionally, when duration of symptoms is more than 24 hours, surgeons may be tempted to forgo non-operative reduction because of a presumed high rate of failure in these patients. The percentage of patients who received surgery is very high when compared to the much lower rates observed in Europe (19%) [12] and Vietnam (8%) [2]. The provision of facilities for non-operative reduction should be prioritised since a sizeable percentage of patients may be amenable to this method of treatment even when they present late.\nWhile we observed a trend toward increasing rates of gangrene with increasing intervals from intussusception onset to treatment, the results were not statistically significant likely because of our small sample size. Although some previous studies have found such a relationship [18-20], other studies have not found a relationship between duration of symptoms and success of non-operative reduction or need for surgery [17, 32-38]. Gangrene is the major reason for failure of nonoperative reduction and failure of reduction may be considered a proxy for gangrene. This suggests there may be additional factors that influence the development of gangrene. Mechanical factors have been suggested that influence the tension or pressure on mesenteric blood vessels including abnormalities of intestinal fixation [39-41]. The assertion by Brereton [42], Gil-Vargas [40] and others [43] that an excessively long, loose mesentery may be an etiological factor for intussusception is plausible. It may also protect the bowel from the development of gangrene. Furthermore, rectal protrusion of intussusception has been thought to represent an excessive delay in presentation [44, 45], but equally could reflect excessive laxity of the mesentery of normally fixed retroperitoneal structures [46]. One patient from Nigeria with rectal protrusion reported presented after 28 days and had no gangrene or perforation [47]. Similarly, in our study one patient received definitive treatment 33 days after onset of symptoms and had viable bowel requiring only manual reduction. Further research is needed in this area.\nLimitations A major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.\nThe data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.\nA major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.\nThe data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.",
"A major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.\nThe data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.",
"Time to hospital for treatment of intussusception in Zimbabwe is longer than commonly accepted benchmarks. Low sample size in this study may not have provided enough statistical power to show significant associations between gangrene and pre-hospital and pre-treatment duration although these may have existed. Advocacy and training among primary care providers to improve timeliness and accuracy of diagnosis and capacitating small peripheral health institutions as well as health education in parents to improve healthcare-seeking behaviour are potential targets for reducing delays in the pre-treatment interval. Future research should investigate mechanical factors and the morphology of the bowel in intussusception.\nWhat is known about this topic \nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.\n\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;\nData on the epidemiology of intussusception in sub-Saharan African are sparse.\n\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.\n\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;\nData on the epidemiology of intussusception in sub-Saharan African are sparse.\nWhat this study adds \nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.\n\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;\nChildren with intussusception who die are more likely to present later for treatment than children who survive.\n\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.\n\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;\nChildren with intussusception who die are more likely to present later for treatment than children who survive.",
"\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.\n\nRotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;\nData on the epidemiology of intussusception in sub-Saharan African are sparse.",
"\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.\n\nIntussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;\nChildren with intussusception who die are more likely to present later for treatment than children who survive."
] | [
"intro",
"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
null,
"conclusions",
null,
null
] | [
"Intussusception",
"gangrene",
"intestinal obstruction",
"delay",
"developing countries",
"global paediatric surgery"
] | Introduction:
Intussusception is an enteric invagination into an adjacent segment of bowel. Some intussusception cases have been associated with infection with various enteric viruses causing Peyer´s patch hypertrophy [1, 2]. This assertion was bolstered by some studies finding a seasonal pattern of intussusception cases [3, 4]. A slightly increased risk of intussusception of 1 to 6 excess cases per 100,000 vaccinated infants has been observed following rotavirus vaccination in clinical trials in high- and middle-income countries [5]; however, no association was found between rotavirus vaccine and intussusception in a multi-country analysis in sub-Saharan Africa [6]. Intussusception is the most common cause of childhood intestinal obstruction in Zimbabwe [3], and is also the most frequently encountered paediatric surgical emergency [3]. This is similar to the experience in other African countries [7]. It was found to be the most common cause of childhood intestinal obstruction in Nigeria and of acute mechanical obstruction in children in Niger [8, 9].
Intussusception is managed surgically, with manual reduction or resection, or nonoperatively by air, hydrostatic or contrast enema. In Africa rates of surgical intervention are higher than for non-operative reduction [6, 7, 10]. Ekenze et al. reported that in south eastern Nigeria surgical management was performed routinely in cases of intussusception [11]. In contrast, 81% of intussusception patients in a study in Europe had non-operative reduction [12].
Delays in presentation and treatment of serious surgical diseases, including intussusception, are common in low-resource countries due to limited access to care [7, 10, 13, 14]. In a study from Nigeria only 7.7% of patients presented within 24 hours of onset of intussusception symptoms [15]. Late presentation of intussusception cases is considered a risk factor for gangrene and death, increasing the need for surgery [16-18] and predicting the failure of non-operative reduction [16, 19-20]. It also increases the chances of sepsis, multiple organ dysfunction and death. [21, 22] In this analysis we describe the time intervals from onset of symptoms to definitive treatment of infants with intussusception in Zimbabwe. As an exploratory analysis, we considered the relationship between delayed presentation and gangrene.
Methods:
Patient population All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.
study participants flow chart
All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.
study participants flow chart
Study setting The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.
The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.
Data collection Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).
Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).
Description of surgical procedure Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.
Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.
Definitions of time intervals The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.
The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.
Statistical analysis We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.
Ethical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.
Disclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.
We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.
Ethical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.
Disclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.
Patient population:
All patients < 12 months old admitted and treated for intussusception at Harare Children´s Hospital from August 2014 to December 2016 and enrolled as part of the African Intussusception Surveillance Network were included in this analysis. Patients were included if they fulfilled level 1 of the Brighton Collaboration Intussusception Working Group criteria of diagnostic certainty [23]. For this analysis, patients were excluded if they did not have an ileocolic intussusception (Figure 1). Non-ileocolic intussusception is frequently caused by a distinct lead point [24, 25], which would confound the effect of embryological mechanical factors.
study participants flow chart
Study setting:
The study was performed at Harare Children´s Hospital, a public, teaching referral hospital.
Data collection:
Data were collected using a structured questionnaire on admission and during hospital stay. Information regarding age, sex, home address, pertinent dates in the referral journey, method of definitive treatment, intraoperative findings, and procedure performed was collected. Patient codes were used to anonymize the data. Patients with missing time interval and intraoperative data were excluded from statistical analysis (Figure 1).
Description of surgical procedure:
Patients were operated by the paediatric surgical team of 10 experienced surgeons and surgical trainees at Harare Children´s Hospital paediatric theatre. The surgical procedure was performed as per institutional standard and involved initial exploratory laparotomy with an attempt at reduction made if bowel was assessed to be viable. Bowel was considered to be viable if bowel had good colour, contractility and consistency as well as strong mesenteric pulsations. Bowel was resected with primary anastomosis if it was judged to be gangrenous, based on these four parameters. The viability of the unresected intestines was confirmed by post-operative follow-up. Gangrene of resected intestines was corroborated on histological examination of resected specimens which is performed routinely for all resections.
Definitions of time intervals:
The time from symptom onset to definitive management was split into three time intervals using a modification of Three Delays Model [26]. This includes: care-seeking interval, health-system interval and treatment interval. Composite intervals were added to this model as described below. The care-seeking interval was calculated as the time in days from the date of first symptoms to the date of first contact with the health system at a conventional medical institution. The health-system interval was calculated from the date of first contact with the health system until the date of admission to Harare Children´s Hospital. Treatment interval was calculated as the time in days from the date of admission at Harare Children´s Hospital to the date of definitive management. Total time to hospital (TTH) was calculated as the time in days from the date of symptom onset to the date of admission at Harare Children´s Hospital, in cases where the child was not transferred from another facility and the first contact with the healthcare system was Harare Children´s Hospital, the care-seeking interval and time to hospital were equal. Total time to treatment (TTT) was calculated from the date of symptom onset to date of definitive treatment.
Statistical analysis:
We used descriptive statistics to describe the demographic characteristics and the patient journey time intervals. Sample means, and standard deviations were calculated for each interval. A dependent t-test was used to determine whether the care-seeking interval and health-system intervals were significantly different from one another. We used chi-square or Fisher´s exact tests to investigate whether a relationship existed between time to hospital; time to treatment; referral status and the intraoperative finding of gangrene. P-values of < 0.05 were considered significant.
Ethical approval: ethical approval for this publication has been waived by the Medical Research Council of Zimbabwe.
Disclaimer: the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.
Results:
Demographics Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.
Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.
Geographic factors Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.
home addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown
Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.
home addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown
Time intervals in the patient journey eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.
time intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results
eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.
time intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results
Relationship with development of gangrene Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).
relationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene
1Chi-square statistic; 2Fisher´s exact test
Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).
relationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene
1Chi-square statistic; 2Fisher´s exact test
Complications Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.
Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.
Demographics:
Ninety two (92) patients with intussusception were included in this analysis. 59 (64%) were male with a male to female ratio of 1.8:1. The median age was 6 months and interquartile range was 5-9 months. All patients were treated with surgery and 41 (45%) developed gangrene.
Geographic factors:
Home addresses were used to determine where patients lived at the time of illness onset. Figure 2 shows the distribution of patients according to home address in Zimbabwe and the mean delay for each province. The prevalence ranged from 25.8 per 100,000 live births in Harare to 3.3 per 100,000 live births in Mashonaland Central. There were no cases admitted to Harare Children´s Hospital from the provinces of Matabeleland North, Matabeleland South, or Bulawayo for intussusception during the surveillance period. The shortest mean time to hospital was 2.5 days among children from Harare and Midlands provinces. The longest mean time to hospital was 14.3 days among children from Mashonaland Central.
home addresses of patients with intussusception: a map of Zimbabwe with level one administrative boundaries (provinces) showing the distribution of intussusception cases as cases per 100,000 live births; the location of Harare Children´s Hospital is shown; mean time to hospital (TTH) and time to treatment (TTT) in days for each province are also shown
Time intervals in the patient journey:
eighty two (82) patients (89%) were transferred to Harare Children´s Hospital from another health institution and 10 patients (11%) came directly from home. Of those who were transferred from another hospital, mean care-seeking interval, health-system interval and treatment interval were 1.9 days (SD: 3.6), 1.5 days (SD: 1.9) and 1.1 days (SD: 1.2) respectively. No significant difference was observed between the care-seeking interval and health-system interval (p = 0.501). For patients admitted from home, mean care-seeking interval was 2.0 days (SD 2.3) and treatment interval was 1.1 days (SD: 0.3) (Figure 3). For all patients, the mean treatment interval was 1.1 days (SD: 1.2). Mean time to hospital was 3.3 days (SD: 3.6) and mean time to treatment was 4.4 days (SD: 3.8). Children who were transferred from another facility to Harare Children´s Hospital had an average of 1.4 days longer time to hospital compared to children who were not transferred.
time intervals involved in the patient journey to treatment: table shows how time intervals were measured and the calculation of composite time intervals; mean time (in days) for each component of the patient journey as well as composite time intervals are shown from the results
Relationship with development of gangrene:
Of the patients that were transferred from another facility, 44% (n = 36) developed gangrene and 56% (n = 46) did not (p = 0.75) (Table 1). Gangrene was found intraoperatively in 38% (n = 9) of children who arrived to hospital within 1 day, 42% (n = 16) of those who arrived to hospital 2-3 days, and 53% of those who arrived at hospital more than 3 days of symptom onset (p = 0.47). Similarly, gangrene was found intraoperatively in 25% (n = 2) of children who received treatment within 1 day, 41% (n = 13) of children who received treatment 2-3 days, and 50% (n = 26) of children who received treatment more than 3 days after symptom onset (p = 0.34).
relationship between transfer status, time to hospital, time to treatment with intraoperative finding of gangrene
1Chi-square statistic; 2Fisher´s exact test
Complications:
Five patients died postoperatively due to multi-organ dysfunction. Three patients died after hospital discharge from unrelated causes. One patient required another laparotomy 1 month postoperatively for adhesive small bowel obstruction.
Discussion:
We found significant delays between the onset of intussusception symptoms and reduction among children < 12 months old in Zimbabwe. The mean care-seeking interval was slightly higher than the mean health-system interval but this difference was not statistically significant. Therefore, both intervals likely contributed equally to delays in reaching definitive treatment. The evidence to date would suggest that diagnostic delay plays a large part in late presentation rather than socioeconomic factors, which has been reported by other evaluations [27-29]. Barriers to timely care in paediatric surgery were explored by Pilkington et al and include transport and cost on the part of the patient as well as shortcomings in hospital infrastructure and resources [30]. The mean treatment interval was 1.1 days in our study and was comparable to guidelines for wait times in paediatric surgical patients formulated by the Canadian Paediatric Surgical Wait Times Taskforce [31]. It was also much shorter than average treatment interval in Uganda [30]. This is a surrogate quality measure and shows that, definitive management is instituted quickly once the decision has been made.
Surgery was used to manage intussusception for 100% of this study population because of lack of facilities required for enema reduction during the study period. Additionally, when duration of symptoms is more than 24 hours, surgeons may be tempted to forgo non-operative reduction because of a presumed high rate of failure in these patients. The percentage of patients who received surgery is very high when compared to the much lower rates observed in Europe (19%) [12] and Vietnam (8%) [2]. The provision of facilities for non-operative reduction should be prioritised since a sizeable percentage of patients may be amenable to this method of treatment even when they present late.
While we observed a trend toward increasing rates of gangrene with increasing intervals from intussusception onset to treatment, the results were not statistically significant likely because of our small sample size. Although some previous studies have found such a relationship [18-20], other studies have not found a relationship between duration of symptoms and success of non-operative reduction or need for surgery [17, 32-38]. Gangrene is the major reason for failure of nonoperative reduction and failure of reduction may be considered a proxy for gangrene. This suggests there may be additional factors that influence the development of gangrene. Mechanical factors have been suggested that influence the tension or pressure on mesenteric blood vessels including abnormalities of intestinal fixation [39-41]. The assertion by Brereton [42], Gil-Vargas [40] and others [43] that an excessively long, loose mesentery may be an etiological factor for intussusception is plausible. It may also protect the bowel from the development of gangrene. Furthermore, rectal protrusion of intussusception has been thought to represent an excessive delay in presentation [44, 45], but equally could reflect excessive laxity of the mesentery of normally fixed retroperitoneal structures [46]. One patient from Nigeria with rectal protrusion reported presented after 28 days and had no gangrene or perforation [47]. Similarly, in our study one patient received definitive treatment 33 days after onset of symptoms and had viable bowel requiring only manual reduction. Further research is needed in this area.
Limitations A major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.
The data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.
A major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.
The data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.
Limitations:
A major limitation of this study is that intraoperative clinical judgment was used in the determination of intestinal gangrene, which may have overestimated the presence of gangrene compared to other techniques such as fluorescence or laser Doppler ultrasound [48-50]. However, there was > 95% concordance between histological assessment and clinical judgment in this population suggesting that clinical judgment was an acceptable method for intraoperative gangrene assessment for this study. The dates of intussusception symptom onset were self-reported by each child´s caregiver and were not able to be verified. As a result, there may have been bias introduced into these findings.
The data shows a trend towards higher rates of gangrene when the pre-hospital and pre-treatment delay is longer. The inability to find a statistically significant relationship may have been related to inadequate power of the study to detect differences considering the low sample sizes in some cells. Future studies with larger sample sizes could help clarify this possibility. Because this was a single-centre study, it may not be generalizable to all of Zimbabwe. Harare Children´s Hospital is the only dedicated paediatric hospital in Zimbabwe, however a small number of patients from the south-west of the country are managed by general surgeons in the region.
Conclusion:
Time to hospital for treatment of intussusception in Zimbabwe is longer than commonly accepted benchmarks. Low sample size in this study may not have provided enough statistical power to show significant associations between gangrene and pre-hospital and pre-treatment duration although these may have existed. Advocacy and training among primary care providers to improve timeliness and accuracy of diagnosis and capacitating small peripheral health institutions as well as health education in parents to improve healthcare-seeking behaviour are potential targets for reducing delays in the pre-treatment interval. Future research should investigate mechanical factors and the morphology of the bowel in intussusception.
What is known about this topic
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
What this study adds
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive.
What is known about this topic:
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
What this study adds:
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive.
| Background:
prompt diagnosis and treatment are considered key to successful management of intussusception. We examined pre-treatment delay among intussusception cases in Zimbabwe and conducted an exploratory analysis of factors associated with intraoperative finding of gangrene.
Methods:
data were prospectively collected as part of the African Intussusception Network using a questionnaire administered on consecutive patients with intussusception managed at Harare Children´s Hospital. Delays were classified using the Three-Delays-Model: care-seeking delay (time from onset of symptoms to first presentation for health care), health-system delay (referral time from presentation to first facility to treatment facility) and treatment delay (time from presentation at treatment facility to treatment).
Results:
ninety-two patients were enrolled from August 2014 to December 2016. The mean care-seeking interval was 1.9 days, the mean health-system interval was 1.5 days, and the mean treatment interval was 1.1 days. Mean total time from symptom onset to treatment was 4.4 days. Being transferred from another institution added 1.4 days to the patient journey. Gangrene was found in 2 (25%) of children who received treatment within 1 day, 13 (41%) of children who received treatment 2-3 days, and 26 (50%) of children who received treatment more than 3 days after symptom onset (p = 0.34).
Conclusions:
significant care-seeking and health-system delays are encountered by intussusception patients in Zimbabwe. Our findings highlight the need to explore approaches to improve the early diagnosis of intussusception and prompt referral of patients for treatment. | Introduction:
Intussusception is an enteric invagination into an adjacent segment of bowel. Some intussusception cases have been associated with infection with various enteric viruses causing Peyer´s patch hypertrophy [1, 2]. This assertion was bolstered by some studies finding a seasonal pattern of intussusception cases [3, 4]. A slightly increased risk of intussusception of 1 to 6 excess cases per 100,000 vaccinated infants has been observed following rotavirus vaccination in clinical trials in high- and middle-income countries [5]; however, no association was found between rotavirus vaccine and intussusception in a multi-country analysis in sub-Saharan Africa [6]. Intussusception is the most common cause of childhood intestinal obstruction in Zimbabwe [3], and is also the most frequently encountered paediatric surgical emergency [3]. This is similar to the experience in other African countries [7]. It was found to be the most common cause of childhood intestinal obstruction in Nigeria and of acute mechanical obstruction in children in Niger [8, 9].
Intussusception is managed surgically, with manual reduction or resection, or nonoperatively by air, hydrostatic or contrast enema. In Africa rates of surgical intervention are higher than for non-operative reduction [6, 7, 10]. Ekenze et al. reported that in south eastern Nigeria surgical management was performed routinely in cases of intussusception [11]. In contrast, 81% of intussusception patients in a study in Europe had non-operative reduction [12].
Delays in presentation and treatment of serious surgical diseases, including intussusception, are common in low-resource countries due to limited access to care [7, 10, 13, 14]. In a study from Nigeria only 7.7% of patients presented within 24 hours of onset of intussusception symptoms [15]. Late presentation of intussusception cases is considered a risk factor for gangrene and death, increasing the need for surgery [16-18] and predicting the failure of non-operative reduction [16, 19-20]. It also increases the chances of sepsis, multiple organ dysfunction and death. [21, 22] In this analysis we describe the time intervals from onset of symptoms to definitive treatment of infants with intussusception in Zimbabwe. As an exploratory analysis, we considered the relationship between delayed presentation and gangrene.
Conclusion:
Time to hospital for treatment of intussusception in Zimbabwe is longer than commonly accepted benchmarks. Low sample size in this study may not have provided enough statistical power to show significant associations between gangrene and pre-hospital and pre-treatment duration although these may have existed. Advocacy and training among primary care providers to improve timeliness and accuracy of diagnosis and capacitating small peripheral health institutions as well as health education in parents to improve healthcare-seeking behaviour are potential targets for reducing delays in the pre-treatment interval. Future research should investigate mechanical factors and the morphology of the bowel in intussusception.
What is known about this topic
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;Data on the epidemiology of intussusception in sub-Saharan African are sparse.
Rotavirus vaccines have been associated with an increased risk of intussusception in some high and middle income countries but not in countries in sub-Saharan Africa;
Data on the epidemiology of intussusception in sub-Saharan African are sparse.
What this study adds
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;Children with intussusception who die are more likely to present later for treatment than children who survive.
Intussusception rarely occurs in the first three months of life in Ethiopia when rotavirus vaccine doses are given;
Children with intussusception who die are more likely to present later for treatment than children who survive. | Background:
prompt diagnosis and treatment are considered key to successful management of intussusception. We examined pre-treatment delay among intussusception cases in Zimbabwe and conducted an exploratory analysis of factors associated with intraoperative finding of gangrene.
Methods:
data were prospectively collected as part of the African Intussusception Network using a questionnaire administered on consecutive patients with intussusception managed at Harare Children´s Hospital. Delays were classified using the Three-Delays-Model: care-seeking delay (time from onset of symptoms to first presentation for health care), health-system delay (referral time from presentation to first facility to treatment facility) and treatment delay (time from presentation at treatment facility to treatment).
Results:
ninety-two patients were enrolled from August 2014 to December 2016. The mean care-seeking interval was 1.9 days, the mean health-system interval was 1.5 days, and the mean treatment interval was 1.1 days. Mean total time from symptom onset to treatment was 4.4 days. Being transferred from another institution added 1.4 days to the patient journey. Gangrene was found in 2 (25%) of children who received treatment within 1 day, 13 (41%) of children who received treatment 2-3 days, and 26 (50%) of children who received treatment more than 3 days after symptom onset (p = 0.34).
Conclusions:
significant care-seeking and health-system delays are encountered by intussusception patients in Zimbabwe. Our findings highlight the need to explore approaches to improve the early diagnosis of intussusception and prompt referral of patients for treatment. | 6,814 | 302 | [
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] | [CONTENT] Intussusception | gangrene | intestinal obstruction | delay | developing countries | global paediatric surgery [SUMMARY] | [CONTENT] Intussusception | gangrene | intestinal obstruction | delay | developing countries | global paediatric surgery [SUMMARY] | [CONTENT] Intussusception | gangrene | intestinal obstruction | delay | developing countries | global paediatric surgery [SUMMARY] | [CONTENT] Intussusception | gangrene | intestinal obstruction | delay | developing countries | global paediatric surgery [SUMMARY] | [CONTENT] Intussusception | gangrene | intestinal obstruction | delay | developing countries | global paediatric surgery [SUMMARY] | [CONTENT] Intussusception | gangrene | intestinal obstruction | delay | developing countries | global paediatric surgery [SUMMARY] | [CONTENT] Child | Female | Gangrene | Hospitals, Pediatric | Humans | Infant | Intussusception | Male | Patient Acceptance of Health Care | Prospective Studies | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Zimbabwe [SUMMARY] | [CONTENT] Child | Female | Gangrene | Hospitals, Pediatric | Humans | Infant | Intussusception | Male | Patient Acceptance of Health Care | Prospective Studies | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Zimbabwe [SUMMARY] | [CONTENT] Child | Female | Gangrene | Hospitals, Pediatric | Humans | Infant | Intussusception | Male | Patient Acceptance of Health Care | Prospective Studies | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Zimbabwe [SUMMARY] | [CONTENT] Child | Female | Gangrene | Hospitals, Pediatric | Humans | Infant | Intussusception | Male | Patient Acceptance of Health Care | Prospective Studies | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Zimbabwe [SUMMARY] | [CONTENT] Child | Female | Gangrene | Hospitals, Pediatric | Humans | Infant | Intussusception | Male | Patient Acceptance of Health Care | Prospective Studies | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Zimbabwe [SUMMARY] | [CONTENT] Child | Female | Gangrene | Hospitals, Pediatric | Humans | Infant | Intussusception | Male | Patient Acceptance of Health Care | Prospective Studies | Surveys and Questionnaires | Time Factors | Time-to-Treatment | Zimbabwe [SUMMARY] | [CONTENT] vaccine intussusception | infants intussusception zimbabwe | saharan africa intussusception | intestinal obstruction nigeria | rotavirus vaccine intussusception [SUMMARY] | [CONTENT] vaccine intussusception | infants intussusception zimbabwe | saharan africa intussusception | intestinal obstruction nigeria | rotavirus vaccine intussusception [SUMMARY] | [CONTENT] vaccine intussusception | infants intussusception zimbabwe | saharan africa intussusception | intestinal obstruction nigeria | rotavirus vaccine intussusception [SUMMARY] | [CONTENT] vaccine intussusception | infants intussusception zimbabwe | saharan africa intussusception | intestinal obstruction nigeria | rotavirus vaccine intussusception [SUMMARY] | [CONTENT] vaccine intussusception | infants intussusception zimbabwe | saharan africa intussusception | intestinal obstruction nigeria | rotavirus vaccine intussusception [SUMMARY] | [CONTENT] vaccine intussusception | infants intussusception zimbabwe | saharan africa intussusception | intestinal obstruction nigeria | rotavirus vaccine intussusception [SUMMARY] | [CONTENT] hospital | time | intussusception | children | treatment | days | patients | interval | gangrene | harare [SUMMARY] | [CONTENT] hospital | time | intussusception | children | treatment | days | patients | interval | gangrene | harare [SUMMARY] | [CONTENT] hospital | time | intussusception | children | treatment | days | patients | interval | gangrene | harare [SUMMARY] | [CONTENT] hospital | time | intussusception | children | treatment | days | patients | interval | gangrene | harare [SUMMARY] | [CONTENT] hospital | time | intussusception | children | treatment | days | patients | interval | gangrene | harare [SUMMARY] | [CONTENT] hospital | time | intussusception | children | treatment | days | patients | interval | gangrene | harare [SUMMARY] | [CONTENT] intussusception | cases | common | reduction | surgical | non operative reduction | nigeria | non operative | operative reduction | presentation [SUMMARY] | [CONTENT] date | time | interval | calculated | hospital | system | harare children | harare children hospital | harare | children hospital [SUMMARY] | [CONTENT] days | time | mean | sd | days sd | hospital | patients | mean time | children | interval [SUMMARY] | [CONTENT] intussusception | countries | saharan | sub | sub saharan | rotavirus | children intussusception die | die likely present later | rarely | vaccines [SUMMARY] | [CONTENT] intussusception | hospital | time | days | children | patients | interval | treatment | gangrene | harare [SUMMARY] | [CONTENT] intussusception | hospital | time | days | children | patients | interval | treatment | gangrene | harare [SUMMARY] | [CONTENT] ||| Zimbabwe [SUMMARY] | [CONTENT] the African Intussusception Network | Harare Children´s Hospital ||| Three | first | first [SUMMARY] | [CONTENT] ninety-two | August 2014 to December 2016 ||| 1.9 days | 1.5 days | 1.1 days ||| 4.4 days ||| 1.4 days ||| Gangrene | 2 | 25% | 1 day | 13 | 41% | 2-3 days | 26 | 50% | more than 3 days | 0.34 [SUMMARY] | [CONTENT] Zimbabwe ||| [SUMMARY] | [CONTENT] ||| Zimbabwe ||| the African Intussusception Network | Harare Children´s Hospital ||| Three | first | first ||| ninety-two | August 2014 to December 2016 ||| 1.9 days | 1.5 days | 1.1 days ||| 4.4 days ||| 1.4 days ||| Gangrene | 2 | 25% | 1 day | 13 | 41% | 2-3 days | 26 | 50% | more than 3 days | 0.34 ||| Zimbabwe ||| [SUMMARY] | [CONTENT] ||| Zimbabwe ||| the African Intussusception Network | Harare Children´s Hospital ||| Three | first | first ||| ninety-two | August 2014 to December 2016 ||| 1.9 days | 1.5 days | 1.1 days ||| 4.4 days ||| 1.4 days ||| Gangrene | 2 | 25% | 1 day | 13 | 41% | 2-3 days | 26 | 50% | more than 3 days | 0.34 ||| Zimbabwe ||| [SUMMARY] |
||||||
Social Stigma and Depression among Asymptomatic COVID-19 Carriers in Shanghai, China: The Mediating Role of Entrapment and Decadence. | 36293585 | Since the advent of 2019 novel coronavirus (COVID-19), the coexistence between social stigma and depression symptoms (depression hereafter) in COVID-19 patients has been mentioned, but the mechanisms involved remains unclear. This study aimed to explore how the stigma affects depression during the mid-pandemic period. | INTRODUCTION | A cross-sectional survey using non-probability sampling was conducted among asymptomatic COVID-19 carriers in Shanghai, China (April 2022). An online questionnaire was used to obtain information on demographic characteristics and psychological traits. Logistic regression and path analysis were performed to analyze the depression risk factors and examine the mediation model, respectively. | METHODS | A total of 1283 participants (59.6% men) were involved in this study, in which 44.7% of carriers reported having depression. Univariate analyses found that education level (OR 0.575; 95% CI 0.448-0.737) and doses of vaccine (OR 1.693; 95% CI 1.042-2.750), were significantly associated with depression among asymptomatic carriers. The association between social stigma and depression was fully mediated by their feelings of entrapment and decadence (indirect effect = 0.204, p < 0.001; direct effect = -0.059, p = 0.058). The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). | RESULTS | Mental health issues resulting from the COVID-19 pandemic are increasingly apparent in China and require urgent attention and responses. These findings provide new perspectives for the early prevention of depression in asymptomatic carriers. | CONCLUSION | [
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"Social Stigma",
"Pandemics",
"COVID-19",
"Depression",
"Cross-Sectional Studies",
"China",
"Anxiety"
] | 9602397 | 1. Introduction | The pandemic of 2019 novel coronavirus (COVID-19) has become a major global public health challenge [1,2]. It is self-evident that it not only caused the burden of physical illness but has also triggered a wide range of declines in mental well-being [3,4]. A meta-analysis suggested that the global prevalence of major depressive disorder increased the most among a series of negative psychology issues with the emergence of COVID-19. The prevalence of depression increased by 27.6% (25.1% to 30.3%), with a loss of disability-adjusted life-years of 4.94 million (33.6 to 68.7) [5]. Global levels of depression in the general population, health care providers and patients during the COVID-19 pandemic went up to 24.0% (21.0–27.1%) [6]. A meta-analysis revealed that psychological problems in infected patients were prominent, with a prevalence of 45% depression [7].
Although risk factors for depression in vulnerable groups have been documented [8,9,10], current information regarding how the factors affect depression is lacking. Evidence suggested that COVID-19 survivors experienced higher stigma levels compared with healthy controls [11]. COVID-19-related stigma refers to a disagreeable or negative self-attitude that develops after infection or close contact with COVID-19 patients [12], including the negative deflection of social identity, with self-concept thus leading to a “spoiled identity” [12,13]. Additional evidence suggests a correlation between depression and social stigma. Liu et al. emphasized the importance of stigma in exacerbating the emotional impact of infected patients [9]. A prospective cohort study of COVID-19 survivors found that discrimination may partly account for the high incidence of depression after infection remission [14]. The identity-threat model of stigma raised by Brenda Major et al., which views stigma as a stressor, attempts to explain the effects of stigma on psychological well-being such as depression through coping strategies [15,16].
The economic consequences and psychosocial impacts of the COVID-19 pandemic are pervasive and profound [17,18,19]. Consequently, to avoid long-term mental health issues, there is an urgent need to clarify the mechanisms of the development of depression for COVID-19 patients [20,21]. The Social Rank Theory (SRT) proposes that depression evolves as a natural result of prolonged involuntary subordination [22]. Faced with an unfavorable situation (e.g., unachievable objectives, attempted change or challenge inhibited), individuals involuntarily yield adaptive responses through an automatic shutdown strategy [23]. If the situations persist and cannot be changed or escaped, the adaptive response will progressively change to maladjustment and eventually lead to depression [24,25]. Entrapment and decadence are precisely the key maladaptive defensive responses to this process. Entrapment is described as a common situation when there is a strong motivation to escape from an unsatisfying status, but that expectation cannot be met [22,26]. It could activate the Involuntary Defeat Strategy that, along with the feelings of decadence, forms a “depressogenic feedback loop” and ultimately contributes to the development of depression [27].
To our knowledge, little is known about the exact mechanisms on how stigma affects depression. Aiming to address this gap in understanding, we hypothesized and constructed a mediating model in which entrapment and decadence are mediators between social stigma and depression. The reasons are as follows: First, the identity-threat model of stigma posits that intrusive thinking and rumination about an event or issues occur when stigmatized individuals respond to stigma with involuntary engagement [15,28]. These two responses may have potential connection points with the assumed prerequisites of the SRT, that adverse situations are uncomfortable for individuals but cannot yet be accepted or escaped. That is, the identity-threat model of stigma implies that stigma affects entrapment and decadence. Second, entrapment and decadence are psychological signals prior to the onset of depression based on the SRT. Finally, a stigma-related stressor begins with external stimuli, while entrapment or decadence are inherently intrinsic psychological products, which satisfies the theoretical requirement of chronological order.
The primary objective of this study was to explore potential mediations between social stigma and depression via entrapment or decadence based on quantitative data from asymptomatic COVID-19 carriers in Shanghai, China. These findings may help us further understand the impact of the pandemic on COVID-19 patients’ mental health and inform subsequent intervention strategies and services for depression related to COVID-19. | 2. Methods | 2.1. Study Design and Participants This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.
Inclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).
This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.
Inclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).
2.2. Data Collection To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.
To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.
2.3. Measures 2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
2.4. Statistical Analysis The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.
The Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.
After adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.
To enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.
Descriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.
The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.
The Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.
After adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.
To enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.
Descriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant. | 3. Results | 3.1. Participants Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.
Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.
3.2. Sociodemographic Characteristics Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.
In the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.
Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.
In the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.
3.3. Covariate Selection and Correlation Analysis Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).
As shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.
Education level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.
Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).
As shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.
Education level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.
3.4. Hierarchical Regression Analysis Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).
Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).
3.5. Path Analysis Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).
As shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).
The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).
Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).
As shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).
The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).
3.6. Temporal Validation We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).
We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9). | 5. Conclusions | This is one of the few studies to provide insights into the relationship between social stigma and depression among asymptomatic COVID-19 carriers in the post-epidemic era. Asymptomatic carriers were more likely to exhibit depression in the mid-stage of the pandemic. The current data showed the linkage between social stigma and depression through the mediating effects of entrapment and decadence. The routine assessment of stigma and screening for entrapment and decadence should be incorporated into early intervention strategies for depression among carriers to alleviate the psychological burden of the outbreak on society. | [
"2.1. Study Design and Participants",
"2.2. Data Collection ",
"2.3. Measures",
"2.3.1. Background Characteristics ",
"2.3.2. Social Stigma",
"2.3.3. Entrapment and Decadence",
"2.3.4. Depression",
"2.4. Statistical Analysis",
"3.2. Sociodemographic Characteristics",
"3.3. Covariate Selection and Correlation Analysis",
"3.4. Hierarchical Regression Analysis",
"3.5. Path Analysis",
"3.6. Temporal Validation"
] | [
"This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.\nInclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).",
"To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.",
" 2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\nInformation on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\n 2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\nStigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\n 2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\nFeelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\n 2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].\nThe Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].",
"Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.",
"Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.",
"Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.",
"The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].",
"The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.\nThe Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.\nAfter adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.\nTo enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.\nDescriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.",
"Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.\nIn the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.",
"Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).\nAs shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.\nEducation level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.",
"Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).",
"Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).",
"We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9)."
] | [
"subjects",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"1. Introduction",
"2. Methods ",
"2.1. Study Design and Participants",
"2.2. Data Collection ",
"2.3. Measures",
"2.3.1. Background Characteristics ",
"2.3.2. Social Stigma",
"2.3.3. Entrapment and Decadence",
"2.3.4. Depression",
"2.4. Statistical Analysis",
"3. Results",
"3.1. Participants",
"3.2. Sociodemographic Characteristics",
"3.3. Covariate Selection and Correlation Analysis",
"3.4. Hierarchical Regression Analysis",
"3.5. Path Analysis",
"3.6. Temporal Validation",
"4. Discussion",
"5. Conclusions"
] | [
"The pandemic of 2019 novel coronavirus (COVID-19) has become a major global public health challenge [1,2]. It is self-evident that it not only caused the burden of physical illness but has also triggered a wide range of declines in mental well-being [3,4]. A meta-analysis suggested that the global prevalence of major depressive disorder increased the most among a series of negative psychology issues with the emergence of COVID-19. The prevalence of depression increased by 27.6% (25.1% to 30.3%), with a loss of disability-adjusted life-years of 4.94 million (33.6 to 68.7) [5]. Global levels of depression in the general population, health care providers and patients during the COVID-19 pandemic went up to 24.0% (21.0–27.1%) [6]. A meta-analysis revealed that psychological problems in infected patients were prominent, with a prevalence of 45% depression [7].\nAlthough risk factors for depression in vulnerable groups have been documented [8,9,10], current information regarding how the factors affect depression is lacking. Evidence suggested that COVID-19 survivors experienced higher stigma levels compared with healthy controls [11]. COVID-19-related stigma refers to a disagreeable or negative self-attitude that develops after infection or close contact with COVID-19 patients [12], including the negative deflection of social identity, with self-concept thus leading to a “spoiled identity” [12,13]. Additional evidence suggests a correlation between depression and social stigma. Liu et al. emphasized the importance of stigma in exacerbating the emotional impact of infected patients [9]. A prospective cohort study of COVID-19 survivors found that discrimination may partly account for the high incidence of depression after infection remission [14]. The identity-threat model of stigma raised by Brenda Major et al., which views stigma as a stressor, attempts to explain the effects of stigma on psychological well-being such as depression through coping strategies [15,16].\nThe economic consequences and psychosocial impacts of the COVID-19 pandemic are pervasive and profound [17,18,19]. Consequently, to avoid long-term mental health issues, there is an urgent need to clarify the mechanisms of the development of depression for COVID-19 patients [20,21]. The Social Rank Theory (SRT) proposes that depression evolves as a natural result of prolonged involuntary subordination [22]. Faced with an unfavorable situation (e.g., unachievable objectives, attempted change or challenge inhibited), individuals involuntarily yield adaptive responses through an automatic shutdown strategy [23]. If the situations persist and cannot be changed or escaped, the adaptive response will progressively change to maladjustment and eventually lead to depression [24,25]. Entrapment and decadence are precisely the key maladaptive defensive responses to this process. Entrapment is described as a common situation when there is a strong motivation to escape from an unsatisfying status, but that expectation cannot be met [22,26]. It could activate the Involuntary Defeat Strategy that, along with the feelings of decadence, forms a “depressogenic feedback loop” and ultimately contributes to the development of depression [27].\nTo our knowledge, little is known about the exact mechanisms on how stigma affects depression. Aiming to address this gap in understanding, we hypothesized and constructed a mediating model in which entrapment and decadence are mediators between social stigma and depression. The reasons are as follows: First, the identity-threat model of stigma posits that intrusive thinking and rumination about an event or issues occur when stigmatized individuals respond to stigma with involuntary engagement [15,28]. These two responses may have potential connection points with the assumed prerequisites of the SRT, that adverse situations are uncomfortable for individuals but cannot yet be accepted or escaped. That is, the identity-threat model of stigma implies that stigma affects entrapment and decadence. Second, entrapment and decadence are psychological signals prior to the onset of depression based on the SRT. Finally, a stigma-related stressor begins with external stimuli, while entrapment or decadence are inherently intrinsic psychological products, which satisfies the theoretical requirement of chronological order.\nThe primary objective of this study was to explore potential mediations between social stigma and depression via entrapment or decadence based on quantitative data from asymptomatic COVID-19 carriers in Shanghai, China. These findings may help us further understand the impact of the pandemic on COVID-19 patients’ mental health and inform subsequent intervention strategies and services for depression related to COVID-19.",
" 2.1. Study Design and Participants This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.\nInclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).\nThis is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.\nInclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).\n 2.2. Data Collection To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.\nTo comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.\n 2.3. Measures 2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\nInformation on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\n 2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\nStigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\n 2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\nFeelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\n 2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].\nThe Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].\n 2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\nInformation on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\n 2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\nStigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\n 2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\nFeelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\n 2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].\nThe Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].\n 2.4. Statistical Analysis The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.\nThe Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.\nAfter adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.\nTo enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.\nDescriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.\nThe normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.\nThe Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.\nAfter adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.\nTo enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.\nDescriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.",
"This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.\nInclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).",
"To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.",
" 2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\nInformation on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.\n 2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\nStigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.\n 2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\nFeelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.\n 2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].\nThe Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].",
"Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.",
"Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.",
"Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.\nFeelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.",
"The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].",
"The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.\nThe Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.\nAfter adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.\nTo enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.\nDescriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.",
" 3.1. Participants Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.\nOf 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.\n 3.2. Sociodemographic Characteristics Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.\nIn the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.\nTable 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.\nIn the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.\n 3.3. Covariate Selection and Correlation Analysis Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).\nAs shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.\nEducation level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.\nConsidering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).\nAs shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.\nEducation level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.\n 3.4. Hierarchical Regression Analysis Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).\nOur data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).\n 3.5. Path Analysis Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).\nAdjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).\n 3.6. Temporal Validation We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).\nWe additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).",
"Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.",
"Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.\nIn the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.",
"Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).\nAs shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.\nEducation level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.",
"Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).",
"Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).\nAs shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).\nThe mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).",
"We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).",
"We conducted a quantitative study to explore the potential mechanism of how stigma affects the likelihood of depression in asymptomatic carriers during the mid-stage of the COVID-19 outbreak in Shanghai. In the current study, depression was reported by 44.7% of participants. A high education level was a protective factor for depression. This study characterized the psychological status of COVID-19 asymptomatic carriers, underscoring the importance of psychiatric screening and early interventions in this subgroup. Moreover, we found a mediating role of entrapment and decadence on the relationship between social stigma and depression, which offers a new way to mitigate the high prevalence of depression among asymptomatic carriers in China.\nWe found that the prevalence of depression reported by asymptomatic carriers was comparable to the rate in COVID-19 patients in previous studies [7,43]. Since physicians prioritize the physical illnesses of hospital patients, it is not surprising that the mental health issues of this subpopulation did not receive attention. Asymptomatic carriers account for a large proportion of COVID-19 patients in China, which highlights the importance of raising awareness of psychiatric screening in infected patients. In addition, the final model showed that a higher education level was a protective factor for depression. Inconsistent findings were reported for the association between education level and psychological symptoms [7,9]. One explanation for our finding is that better-educated individuals tend to acquire the correct information and awareness about COVID-19, therefore being more likely to avoid some adverse psychological health outcomes.\nIn line with our hypothesis, entrapment and decadence mediate the relationship between stigma and depression in asymptomatic carriers during the epidemic. People who suffered from stigma were more likely to feel trapped and decadent, which, in turn, was associated with an increase depressive symptoms. Two conceptual frameworks described at the beginning of the article guided this mediation assumption complementarily. Moreover, we found that the indirect relationship between stigma and depression through entrapment was moderated by age group. This further supports the finding that stigma can affect depression via entrapment.\nMediation analysis is essentially a correlation analysis [44]. Based on accumulated theory and research, we hypothesized that entrapment mediates the relationship between stigma and depression. However, the existence of a correlation between stigma and depression may also be the result of reverse causality or the presence of confounding factors. We found a significant difference in the correlation coefficients between stigma and depression in the two age groups, which at least suggests that the correlation is not exactly the result of the two possible causes mentioned above (reverse causality and confounding factors) [44,45]. Otherwise, their correlation would not differ. Thus, the mediation effect exists.\nOur findings have three implications for the early detection of depression and effective mental health services for asymptomatic carriers. First and foremost, the timely assessment of stigmatizing stressors associated with COVID-19 must be carried out. Prior studies suggest that social stigma is not only a product of structural inequalities caused by bias and discrimination, but also the result of individual failures to cope adaptively with the stressors [15]. Ding K et al. also suggested that personal-level factors are more likely to be related to depression [46,47]. Therefore, the consideration of individuals’ stigmatizing stressors in early depression interventions is clearly warranted. This study focused on stigmatizing stressors related to job refusals and other overlooked situations. In future research, we believe there is a need to help infected individuals identify the well-defined type of stigma-related stress (e.g., being quarantined or treatment factors) they encounter to effectively help them cope with it successfully.\nSecond, response efforts to alleviate entrapment and decadence can be a crucial early intervention point for depression. Our main results shows that entrapment and decadence fully mediated the association between COVID-19-related stigma and depression. It appears that improving entrapment and decadence may effectively block the effects of stigma on depression. Specifically, an assessment of the psychological status of entrapment and decadence can be included in the early detection of depressive symptoms. Additionally, hospitals can implement remote mental health psychiatric counseling and screening programs through telemedicine or online mental health interventions [48,49]. Preventive strategies and early interventions are needed to adequately address early psychological abnormal states and avoid long-term mental health problems.\nThird, this research provides a basis for implementing individualized mental health intervention strategies. Our findings revealed that the indirect link between stigmatization and depression via entrapment is stronger in younger age groups, which implies we could give priority to younger infected individuals with respect to entrapment-reducing interventions. Meanwhile, we also need to find other influential factors of the mental state of entrapment and decadence for relatively high age groups in the future.\nThe current study involves several limitations. First, participants were recruited by non-probability sampling approaches, which may be subject to sampling bias. The non-response rate in this study was 18.4%, which may underestimate the prevalence of depression. Second, participants were recruited from one location, and we need to be cautious when applying the results to other areas. However, we did use a larger sample in our study, so the results may provide a worthy reference for the exploration of depressive mechanisms. Third, the cross-sectional study design limits our ability to establish causal mediation relationships. The mediating effect was developed and temporally validated based on the observational data. Future studies should trace changes in the psychological status of carriers at 376 later stages to further verify the causal mechanism. Fourth, other stigma-related stressors such as isolation factors or treatment factors were not studied. Future research could consider more stressors into account to understand the mechanism in depth.",
"This is one of the few studies to provide insights into the relationship between social stigma and depression among asymptomatic COVID-19 carriers in the post-epidemic era. Asymptomatic carriers were more likely to exhibit depression in the mid-stage of the pandemic. The current data showed the linkage between social stigma and depression through the mediating effects of entrapment and decadence. The routine assessment of stigma and screening for entrapment and decadence should be incorporated into early intervention strategies for depression among carriers to alleviate the psychological burden of the outbreak on society."
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"asymptomatic COVID-19 carriers",
"social stigma",
"depression",
"entrapment",
"decadence",
"mediating effect"
] | 1. Introduction:
The pandemic of 2019 novel coronavirus (COVID-19) has become a major global public health challenge [1,2]. It is self-evident that it not only caused the burden of physical illness but has also triggered a wide range of declines in mental well-being [3,4]. A meta-analysis suggested that the global prevalence of major depressive disorder increased the most among a series of negative psychology issues with the emergence of COVID-19. The prevalence of depression increased by 27.6% (25.1% to 30.3%), with a loss of disability-adjusted life-years of 4.94 million (33.6 to 68.7) [5]. Global levels of depression in the general population, health care providers and patients during the COVID-19 pandemic went up to 24.0% (21.0–27.1%) [6]. A meta-analysis revealed that psychological problems in infected patients were prominent, with a prevalence of 45% depression [7].
Although risk factors for depression in vulnerable groups have been documented [8,9,10], current information regarding how the factors affect depression is lacking. Evidence suggested that COVID-19 survivors experienced higher stigma levels compared with healthy controls [11]. COVID-19-related stigma refers to a disagreeable or negative self-attitude that develops after infection or close contact with COVID-19 patients [12], including the negative deflection of social identity, with self-concept thus leading to a “spoiled identity” [12,13]. Additional evidence suggests a correlation between depression and social stigma. Liu et al. emphasized the importance of stigma in exacerbating the emotional impact of infected patients [9]. A prospective cohort study of COVID-19 survivors found that discrimination may partly account for the high incidence of depression after infection remission [14]. The identity-threat model of stigma raised by Brenda Major et al., which views stigma as a stressor, attempts to explain the effects of stigma on psychological well-being such as depression through coping strategies [15,16].
The economic consequences and psychosocial impacts of the COVID-19 pandemic are pervasive and profound [17,18,19]. Consequently, to avoid long-term mental health issues, there is an urgent need to clarify the mechanisms of the development of depression for COVID-19 patients [20,21]. The Social Rank Theory (SRT) proposes that depression evolves as a natural result of prolonged involuntary subordination [22]. Faced with an unfavorable situation (e.g., unachievable objectives, attempted change or challenge inhibited), individuals involuntarily yield adaptive responses through an automatic shutdown strategy [23]. If the situations persist and cannot be changed or escaped, the adaptive response will progressively change to maladjustment and eventually lead to depression [24,25]. Entrapment and decadence are precisely the key maladaptive defensive responses to this process. Entrapment is described as a common situation when there is a strong motivation to escape from an unsatisfying status, but that expectation cannot be met [22,26]. It could activate the Involuntary Defeat Strategy that, along with the feelings of decadence, forms a “depressogenic feedback loop” and ultimately contributes to the development of depression [27].
To our knowledge, little is known about the exact mechanisms on how stigma affects depression. Aiming to address this gap in understanding, we hypothesized and constructed a mediating model in which entrapment and decadence are mediators between social stigma and depression. The reasons are as follows: First, the identity-threat model of stigma posits that intrusive thinking and rumination about an event or issues occur when stigmatized individuals respond to stigma with involuntary engagement [15,28]. These two responses may have potential connection points with the assumed prerequisites of the SRT, that adverse situations are uncomfortable for individuals but cannot yet be accepted or escaped. That is, the identity-threat model of stigma implies that stigma affects entrapment and decadence. Second, entrapment and decadence are psychological signals prior to the onset of depression based on the SRT. Finally, a stigma-related stressor begins with external stimuli, while entrapment or decadence are inherently intrinsic psychological products, which satisfies the theoretical requirement of chronological order.
The primary objective of this study was to explore potential mediations between social stigma and depression via entrapment or decadence based on quantitative data from asymptomatic COVID-19 carriers in Shanghai, China. These findings may help us further understand the impact of the pandemic on COVID-19 patients’ mental health and inform subsequent intervention strategies and services for depression related to COVID-19.
2. Methods :
2.1. Study Design and Participants This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.
Inclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).
This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.
Inclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).
2.2. Data Collection To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.
To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.
2.3. Measures 2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
2.4. Statistical Analysis The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.
The Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.
After adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.
To enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.
Descriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.
The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.
The Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.
After adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.
To enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.
Descriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.
2.1. Study Design and Participants:
This is a cross-sectional study of asymptomatic COVID-19 carriers admitted to the Ruijin Jiahe Fangcang shelter hospital (Shanghai, China) in April 2022. Non-probability sampling was used to recruit participants for this study. Health care providers were responsible for advocacy and inviting asymptomatic patients, aiming to cover over 80% of patients admitted. According to previous studies, the expected prevalence of depression among COVID-19 carriers was 45% [7]. We used α of 0.05 and a permissible error of 0.03. Considering a non-response rate of 10%, the required sample size was 1209.
Inclusion criteria included: (1) older than 18 years. (2) A diagnosis of asymptomatic COVID-19 infection, which was judged by the threshold Cycle (Ct) value obtained from the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. The test for SARS-CoV-2 RNA in the nasopharynx is considered positive at a CT value below 35. (3) Able to use WeChat to complete the questionnaire independently. (4) Capable of providing informed consent. The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (protocol code LL202070).
2.2. Data Collection :
To comply with social distancing guidelines, an electronic questionnaire was selected for assessment in this study. Health care providers asked participants to answer questions based on their feelings and thoughts in the recent period when the diagnosis of COVID-19 was confirmed and presented QR codes of the questionnaire to the participants. Self-designed questionnaires were administered to collect information about demographic characteristics and psychological traits (social stigma, entrapment, decadence and depression). The information was gathered via the online survey platform “Questionnaire Star”. All survey respondents provided their informed written consent before the survey.
2.3. Measures:
2.3.1. Background Characteristics Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
2.3.2. Social Stigma Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
2.3.3. Entrapment and Decadence Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
2.3.4. Depression The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
2.3.1. Background Characteristics :
Information on background characteristics was collected, including age, sex, education level, marriage status, length of time from diagnosis, and doses of COVID-19 vaccines.
2.3.2. Social Stigma:
Stigma from COVID-19 was measured via two subscales of the Social Impact Scale (SIS) [29], a generic stigma scale initially applied to patients with cancer or infectious diseases (e.g., human immunodeficiency virus, HIV) (Cronbach’s α = 0.85–0.90). Sixteen items assessed the social stigma related to COVID-19 at the mid-stage of the outbreak. (1) Social rejection measures stigma-related stressors, including traditional infectious disease’s role beliefs (perceiving others to treat them with less respect, no longer consider them competent or avoid them), and experiencing a major life event (being denied employment, education or being otherwise neglected) [16,29]. It gives the individual a sense of being discriminated against at work and in society [29]. (2) Social isolation signifies a sense of anomie in the traditional sociological sense, including feelings of loneliness, inequality with others and uselessness, accompanied by a devalued social identity [16,29]. Compared to HIV stigma, the stigmatization of COVID-19 has less of a moral link and perhaps less self-blame than HIV [30]. In addition, the trajectory of people diagnosed as positive needs to be disclosed in order to reduce public panic. We therefore believe that internalized shame from the SIS is not sufficiently consistent with the content of the social stigma of individuals at the early stage of diagnosis. Thus, only two subscales of the SIS were included. Sample items included “my employer/co-workers have discriminated against me because of my illness”, “some people act as though I am less competent than usual.” The options were rated from 1 (strongly disagree) to 4 (strongly agree). Higher total scores suggested higher levels of COVID-19-related stigma. A score lower than the 75th percentile—a score of 35—was defined as a low level. The Chinese version of the SIS has been found to have good psychological traits [31]. The SIS was applied to measure COVID-19-related stigma in Chinese COVID-19 survivors in 2020 [11]. The Cronbach’s α of the SIS in this study was 0.967, with factor loadings of 0.511–0.745. Psychometrics for the current study sample are enclosed in the Supplementary Materials.
2.3.3. Entrapment and Decadence:
Feelings of entrapment was assessed by the Chinese version of Entrapment Scale (ES) [32] (Cronbach’s α = 0.96). The ES is a 16-item self-report scale used to identify the subjective experiences of entrapment [22]., e.g., “I am in a situation I feel trapped in”. Options for each item range from 0 (not at all), 1 (light), 2 (medium), 3 (heavy), and 4 (serious) [33]. The total possible score can be between 0 and 64. A level above the 75th percentile was defined as a high level of sense of entrapment.
Feelings of decadence were quantified with the Chinese version of the subscale of Defeat Scale (DS) (Cronbach’s α = 0.93), which is designed to assess personal perceptions of failed struggles and loss of rank within the last week [22]., e.g., “I feel defeated by life”. The response options are 0 (never), 1 (seldom), 2 (sometimes), 3 (often), and 4 (always). The overall scores for this 13-item scale range from 0 to 52. A total score over the 75th percentile was defined as a high level of decadence. Both scales have demonstrated validity and reliability among Chinese populations [32,34]. In the present study, the internal consistency coefficients of ES and DS were 0.973 and 0.904, respectively, and the factor loadings of each item in confirmatory factor analysis were roughly above 0.7. Details are presented in the Supplementary Materials.
2.3.4. Depression:
The Zung Self-Rating Depression Scale (SDS), which consists of 20 self-report items, was used to assess participants’ depression based on their feelings [35]. The SDS scale includes physiological and psychological symptoms which have been identified in factor analysis studies of depression [35]. Ten items reflect the negative aspect., e.g., “I feel down-hearted and blue”. Ten items express positive aspects and are reverse scored., e.g., “morning is when I feel the best”. Each question has four response options, from 1 (none, or a little of the time) to 4 (most, or all of the time). The final raw score is the sum of the 20 items, ranging from 20 to 80. A higher score indicates a higher level of depressive severity. The total raw score ≥ 42 is taken as presenting with depression. Additionally, the cut-off value has also been adopted in other Chinese studies [36,37]. The Chinese version of the SDS has been shown to be a valid tool for screening depression (Cronbach’s α = 0.82) [36,38].
2.4. Statistical Analysis:
The normally distributed variable (age) and skewedly distributed variables (stigma, entrapment, decadence, and depression) were described as the mean with standard deviation (SD) and median (interquartile range (IQR)), respectively. Categorical variables (e.g., sex and education level) were expressed as counts and percentages. The univariate logistic regression model was used to identify the characteristics associated with depression.
The Change-in-Estimate procedure was used for confounder identification and selection [39]. All socio-demographic variables and the target variable (stigma) were included in a multivariate logistic regression to build the initial full model. Covariates were selected by backward elimination. In this procedure, the covariate for which removal caused a change of less than 10% in the OR of stigma was removed [39]. The correlation coefficients between variables in final model were based on Spearman correlation analysis. We performed three hierarchical regression analyses to initially test the mediating effect. In step 1, the association of significant sociodemographic characteristics to depression was tested. The stigma factor was added in the step 2. Entrapment; decadence; and entrapment and decadence were added separately in step 3 to test for changes in the effect of stigma on depression.
After adjusting for covariates, path analysis was conducted to examine the relationships among stigma, entrapment, decadence, and depression. Four psychosocial variables are expressed as quantitative data in the mediation model. Two product-of-coefficients strategies (bias-corrected and percentile methods) were performed to examine the mediating role of entrapment and decadence in the association between stigma and depression [40]. In this process, 5000 bootstrap samples were used. The 95% confidence intervals (CI) that do not contain zero indicate the significance of the indirect effects [40,41]. Finally, a bias-corrected bootstrapping procedure based on 5000 resamples was employed to examine the significance of the age-moderated mediation effect. Using the mean value as the cut-off point, age was divided into high and low groups in the moderation analysis. The high age group mentioned in this study is only relative to the low group.
To enhance the credibility of our analysis, a temporal validation was conducted [42]. The data were randomized to the training group and validation group in a 3:1 ratio. Then, the bootstrapping method was used for temporal validation.
Descriptive analyses, logistic regression, correlation analysis and hierarchical regression were conducted using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA). Path analysis was conducted using R software (V. 4.2.1; http://www.Rproject.org; URL (accessed on 1 June 2022)). p values < 0.05 were considered as statistically significant.
3. Results:
3.1. Participants Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.
Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.
3.2. Sociodemographic Characteristics Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.
In the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.
Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.
In the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.
3.3. Covariate Selection and Correlation Analysis Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).
As shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.
Education level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.
Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).
As shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.
Education level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.
3.4. Hierarchical Regression Analysis Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).
Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).
3.5. Path Analysis Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).
As shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).
The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).
Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).
As shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).
The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).
3.6. Temporal Validation We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).
We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).
3.1. Participants:
Of 1750 individuals referred to hospital, 1425 completed the questionnaire. The response rate was 81.4%. After excluding 142 participants who did not meet the inclusion criteria, a total of 1283 participants were included, as shown in Figure 1.
3.2. Sociodemographic Characteristics:
Table 1 shows the demographic and psychosocial characteristics of 1283 asymptomatic COVID-19 carriers during the COVID-19 outbreak in Shanghai. The mean age of participants was 39.6 ± 11.1 years with a range from 18 to 71, and 765 (59.6%) were men. The majority of participants were married (948 (73.9%)) and graduated from senior secondary or below (906 [70.6%]). More than eighty percent of participants (1097) were diagnosed as asymptomatic carriers for greater than or equal to 8 days, among which, 646 (50.4%) tested positive for SARS-CoV-2 RNA within two weeks. Regarding COVID-19 vaccination, a large number of participants received the vaccine, of which, 808 (63.0%) received three doses, 355 (27.7%) received two doses, and less than ten percent of participants (9.4%) did not receive the vaccine or only received a single dose.
In the univariate analysis (Table 1), education level (OR 0.575; 95% CI 0.448–0.737) and doses of vaccine (OR 1.693; 95% CI 1.042–2.750) were significantly associated with depression among asymptomatic COVID-19 carriers. Higher educational attainment was a protective factor, whereas three doses of vaccine was a risk factor for depression.
3.3. Covariate Selection and Correlation Analysis:
Considering the significant effect of education level and vaccination dose on depression in both univariate and multivariate logistic regressions, these two covariates were included in the subsequent analysis (Supplementary Materials Table S7). The Change-in-Estimate procedure showed that by sequentially removing the socio-demographic variables (the four remaining variables), none of the ORs of the stigma changed by more than 10%; thus, all met the criteria for exclusion (Supplementary Materials Table S8).
As shown in Table 2, the median perceived stigmatization score for all participants was 29.0 (IQR = 18.0), with 305 participants (23.8%) reaching a high level. Participants’ median entrapment and decadence scores were 1.0 (IQR = 10.0) and 2.0 (IQR = 12.0), among which, 318 (24.8%) and 304 (23.7%) reported high levels, respectively. With respect to depression, the median score of the SDS was 40.0 (IQR = 19.0), and nearly half of the participants (574 (44.7%)) reported having depression.
Education level was significantly associated with all other variables. Doses of vaccine was significantly associated with stigma. Stigma was positively correlated with depression, entrapment and decadence. Depression was positively correlated with entrapment and decadence, and entrapment was positively correlated with decadence.
3.4. Hierarchical Regression Analysis:
Our data met the assumption for mediation. Significant sociodemographic characteristics, including education level and doses of vaccine tested in step 1, explained 2.0% of the variance in depression. Stigma scores, added in step 2, had significant effects on depression (β = 0.148, t = 5.551, p < 0.001). However, the effect was no longer significant after the addition of entrapment and decadence in step 3 (p > 0.05) (Table 3).
3.5. Path Analysis:
Adjusted for covariates (i.e., education level, doses of vaccine), the results of the path analysis are detailed in Table 4. The indirect path from perceived stigma to depression through entrapment (p < 0.001) and decadence (p = 0.001) were significant, while the direct path between them was not significant (p = 0.058). In another word, entrapment and decadence completely mediated the relationship between stigma and depression (estimate = 0.204, Delta z = 10.912, p < 0.001).
As shown in Figure 2, stigma among COVID-19 carriers was positively associated with their feelings of entrapment (estimate = 0.507, p < 0.001), which in turn was positively associated with their risk of depression (estimate = 0.247, p < 0.001). Similarly, the social stigmatization of participants was significantly linked to the sense of decadence (estimate = 0.461, p < 0.001), which in turn was positively related to depressive symptoms (estimate = 0.171, p < 0.001). When comparing indirect effects in the mediator models of entrapment and decadence, the differences were not significant (p = 0.254) (Table 3).
The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008). For participants aged 18–39 years, the indirect effect of entrapment was significant (estimate = 0.182, Delta z = 4.962, p < 0.001). For subjects aged 40–71 years, the mediating role of entrapment was still positively correlated with stigma and depression, but much weaker (estimate = 0.066, Delta z = 2.754, p = 0.006). However, the moderating role of age group in the decadence path was not significant (estimate = −0.114, p = 0.762) (Table 3).
3.6. Temporal Validation:
We additionally considered the relationship between one variable and all other variables in the model in the training set; the path plot is shown in Supplementary Materials Figure S1. The results of the validation set showed that entrapment and decadence partially mediated the relationship between stigma and depression (Supplementary Materials Table S9).
4. Discussion:
We conducted a quantitative study to explore the potential mechanism of how stigma affects the likelihood of depression in asymptomatic carriers during the mid-stage of the COVID-19 outbreak in Shanghai. In the current study, depression was reported by 44.7% of participants. A high education level was a protective factor for depression. This study characterized the psychological status of COVID-19 asymptomatic carriers, underscoring the importance of psychiatric screening and early interventions in this subgroup. Moreover, we found a mediating role of entrapment and decadence on the relationship between social stigma and depression, which offers a new way to mitigate the high prevalence of depression among asymptomatic carriers in China.
We found that the prevalence of depression reported by asymptomatic carriers was comparable to the rate in COVID-19 patients in previous studies [7,43]. Since physicians prioritize the physical illnesses of hospital patients, it is not surprising that the mental health issues of this subpopulation did not receive attention. Asymptomatic carriers account for a large proportion of COVID-19 patients in China, which highlights the importance of raising awareness of psychiatric screening in infected patients. In addition, the final model showed that a higher education level was a protective factor for depression. Inconsistent findings were reported for the association between education level and psychological symptoms [7,9]. One explanation for our finding is that better-educated individuals tend to acquire the correct information and awareness about COVID-19, therefore being more likely to avoid some adverse psychological health outcomes.
In line with our hypothesis, entrapment and decadence mediate the relationship between stigma and depression in asymptomatic carriers during the epidemic. People who suffered from stigma were more likely to feel trapped and decadent, which, in turn, was associated with an increase depressive symptoms. Two conceptual frameworks described at the beginning of the article guided this mediation assumption complementarily. Moreover, we found that the indirect relationship between stigma and depression through entrapment was moderated by age group. This further supports the finding that stigma can affect depression via entrapment.
Mediation analysis is essentially a correlation analysis [44]. Based on accumulated theory and research, we hypothesized that entrapment mediates the relationship between stigma and depression. However, the existence of a correlation between stigma and depression may also be the result of reverse causality or the presence of confounding factors. We found a significant difference in the correlation coefficients between stigma and depression in the two age groups, which at least suggests that the correlation is not exactly the result of the two possible causes mentioned above (reverse causality and confounding factors) [44,45]. Otherwise, their correlation would not differ. Thus, the mediation effect exists.
Our findings have three implications for the early detection of depression and effective mental health services for asymptomatic carriers. First and foremost, the timely assessment of stigmatizing stressors associated with COVID-19 must be carried out. Prior studies suggest that social stigma is not only a product of structural inequalities caused by bias and discrimination, but also the result of individual failures to cope adaptively with the stressors [15]. Ding K et al. also suggested that personal-level factors are more likely to be related to depression [46,47]. Therefore, the consideration of individuals’ stigmatizing stressors in early depression interventions is clearly warranted. This study focused on stigmatizing stressors related to job refusals and other overlooked situations. In future research, we believe there is a need to help infected individuals identify the well-defined type of stigma-related stress (e.g., being quarantined or treatment factors) they encounter to effectively help them cope with it successfully.
Second, response efforts to alleviate entrapment and decadence can be a crucial early intervention point for depression. Our main results shows that entrapment and decadence fully mediated the association between COVID-19-related stigma and depression. It appears that improving entrapment and decadence may effectively block the effects of stigma on depression. Specifically, an assessment of the psychological status of entrapment and decadence can be included in the early detection of depressive symptoms. Additionally, hospitals can implement remote mental health psychiatric counseling and screening programs through telemedicine or online mental health interventions [48,49]. Preventive strategies and early interventions are needed to adequately address early psychological abnormal states and avoid long-term mental health problems.
Third, this research provides a basis for implementing individualized mental health intervention strategies. Our findings revealed that the indirect link between stigmatization and depression via entrapment is stronger in younger age groups, which implies we could give priority to younger infected individuals with respect to entrapment-reducing interventions. Meanwhile, we also need to find other influential factors of the mental state of entrapment and decadence for relatively high age groups in the future.
The current study involves several limitations. First, participants were recruited by non-probability sampling approaches, which may be subject to sampling bias. The non-response rate in this study was 18.4%, which may underestimate the prevalence of depression. Second, participants were recruited from one location, and we need to be cautious when applying the results to other areas. However, we did use a larger sample in our study, so the results may provide a worthy reference for the exploration of depressive mechanisms. Third, the cross-sectional study design limits our ability to establish causal mediation relationships. The mediating effect was developed and temporally validated based on the observational data. Future studies should trace changes in the psychological status of carriers at 376 later stages to further verify the causal mechanism. Fourth, other stigma-related stressors such as isolation factors or treatment factors were not studied. Future research could consider more stressors into account to understand the mechanism in depth.
5. Conclusions:
This is one of the few studies to provide insights into the relationship between social stigma and depression among asymptomatic COVID-19 carriers in the post-epidemic era. Asymptomatic carriers were more likely to exhibit depression in the mid-stage of the pandemic. The current data showed the linkage between social stigma and depression through the mediating effects of entrapment and decadence. The routine assessment of stigma and screening for entrapment and decadence should be incorporated into early intervention strategies for depression among carriers to alleviate the psychological burden of the outbreak on society.
| Background:
Since the advent of 2019 novel coronavirus (COVID-19), the coexistence between social stigma and depression symptoms (depression hereafter) in COVID-19 patients has been mentioned, but the mechanisms involved remains unclear. This study aimed to explore how the stigma affects depression during the mid-pandemic period.
Methods:
A cross-sectional survey using non-probability sampling was conducted among asymptomatic COVID-19 carriers in Shanghai, China (April 2022). An online questionnaire was used to obtain information on demographic characteristics and psychological traits. Logistic regression and path analysis were performed to analyze the depression risk factors and examine the mediation model, respectively.
Results:
A total of 1283 participants (59.6% men) were involved in this study, in which 44.7% of carriers reported having depression. Univariate analyses found that education level (OR 0.575; 95% CI 0.448-0.737) and doses of vaccine (OR 1.693; 95% CI 1.042-2.750), were significantly associated with depression among asymptomatic carriers. The association between social stigma and depression was fully mediated by their feelings of entrapment and decadence (indirect effect = 0.204, p < 0.001; direct effect = -0.059, p = 0.058). The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008).
Conclusions:
Mental health issues resulting from the COVID-19 pandemic are increasingly apparent in China and require urgent attention and responses. These findings provide new perspectives for the early prevention of depression in asymptomatic carriers. | 1. Introduction:
The pandemic of 2019 novel coronavirus (COVID-19) has become a major global public health challenge [1,2]. It is self-evident that it not only caused the burden of physical illness but has also triggered a wide range of declines in mental well-being [3,4]. A meta-analysis suggested that the global prevalence of major depressive disorder increased the most among a series of negative psychology issues with the emergence of COVID-19. The prevalence of depression increased by 27.6% (25.1% to 30.3%), with a loss of disability-adjusted life-years of 4.94 million (33.6 to 68.7) [5]. Global levels of depression in the general population, health care providers and patients during the COVID-19 pandemic went up to 24.0% (21.0–27.1%) [6]. A meta-analysis revealed that psychological problems in infected patients were prominent, with a prevalence of 45% depression [7].
Although risk factors for depression in vulnerable groups have been documented [8,9,10], current information regarding how the factors affect depression is lacking. Evidence suggested that COVID-19 survivors experienced higher stigma levels compared with healthy controls [11]. COVID-19-related stigma refers to a disagreeable or negative self-attitude that develops after infection or close contact with COVID-19 patients [12], including the negative deflection of social identity, with self-concept thus leading to a “spoiled identity” [12,13]. Additional evidence suggests a correlation between depression and social stigma. Liu et al. emphasized the importance of stigma in exacerbating the emotional impact of infected patients [9]. A prospective cohort study of COVID-19 survivors found that discrimination may partly account for the high incidence of depression after infection remission [14]. The identity-threat model of stigma raised by Brenda Major et al., which views stigma as a stressor, attempts to explain the effects of stigma on psychological well-being such as depression through coping strategies [15,16].
The economic consequences and psychosocial impacts of the COVID-19 pandemic are pervasive and profound [17,18,19]. Consequently, to avoid long-term mental health issues, there is an urgent need to clarify the mechanisms of the development of depression for COVID-19 patients [20,21]. The Social Rank Theory (SRT) proposes that depression evolves as a natural result of prolonged involuntary subordination [22]. Faced with an unfavorable situation (e.g., unachievable objectives, attempted change or challenge inhibited), individuals involuntarily yield adaptive responses through an automatic shutdown strategy [23]. If the situations persist and cannot be changed or escaped, the adaptive response will progressively change to maladjustment and eventually lead to depression [24,25]. Entrapment and decadence are precisely the key maladaptive defensive responses to this process. Entrapment is described as a common situation when there is a strong motivation to escape from an unsatisfying status, but that expectation cannot be met [22,26]. It could activate the Involuntary Defeat Strategy that, along with the feelings of decadence, forms a “depressogenic feedback loop” and ultimately contributes to the development of depression [27].
To our knowledge, little is known about the exact mechanisms on how stigma affects depression. Aiming to address this gap in understanding, we hypothesized and constructed a mediating model in which entrapment and decadence are mediators between social stigma and depression. The reasons are as follows: First, the identity-threat model of stigma posits that intrusive thinking and rumination about an event or issues occur when stigmatized individuals respond to stigma with involuntary engagement [15,28]. These two responses may have potential connection points with the assumed prerequisites of the SRT, that adverse situations are uncomfortable for individuals but cannot yet be accepted or escaped. That is, the identity-threat model of stigma implies that stigma affects entrapment and decadence. Second, entrapment and decadence are psychological signals prior to the onset of depression based on the SRT. Finally, a stigma-related stressor begins with external stimuli, while entrapment or decadence are inherently intrinsic psychological products, which satisfies the theoretical requirement of chronological order.
The primary objective of this study was to explore potential mediations between social stigma and depression via entrapment or decadence based on quantitative data from asymptomatic COVID-19 carriers in Shanghai, China. These findings may help us further understand the impact of the pandemic on COVID-19 patients’ mental health and inform subsequent intervention strategies and services for depression related to COVID-19.
5. Conclusions:
This is one of the few studies to provide insights into the relationship between social stigma and depression among asymptomatic COVID-19 carriers in the post-epidemic era. Asymptomatic carriers were more likely to exhibit depression in the mid-stage of the pandemic. The current data showed the linkage between social stigma and depression through the mediating effects of entrapment and decadence. The routine assessment of stigma and screening for entrapment and decadence should be incorporated into early intervention strategies for depression among carriers to alleviate the psychological burden of the outbreak on society. | Background:
Since the advent of 2019 novel coronavirus (COVID-19), the coexistence between social stigma and depression symptoms (depression hereafter) in COVID-19 patients has been mentioned, but the mechanisms involved remains unclear. This study aimed to explore how the stigma affects depression during the mid-pandemic period.
Methods:
A cross-sectional survey using non-probability sampling was conducted among asymptomatic COVID-19 carriers in Shanghai, China (April 2022). An online questionnaire was used to obtain information on demographic characteristics and psychological traits. Logistic regression and path analysis were performed to analyze the depression risk factors and examine the mediation model, respectively.
Results:
A total of 1283 participants (59.6% men) were involved in this study, in which 44.7% of carriers reported having depression. Univariate analyses found that education level (OR 0.575; 95% CI 0.448-0.737) and doses of vaccine (OR 1.693; 95% CI 1.042-2.750), were significantly associated with depression among asymptomatic carriers. The association between social stigma and depression was fully mediated by their feelings of entrapment and decadence (indirect effect = 0.204, p < 0.001; direct effect = -0.059, p = 0.058). The mediating role of entrapment between stigma and depression was moderated by age group (estimate = 0.116, p = 0.008).
Conclusions:
Mental health issues resulting from the COVID-19 pandemic are increasingly apparent in China and require urgent attention and responses. These findings provide new perspectives for the early prevention of depression in asymptomatic carriers. | 14,772 | 296 | [
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decadence | participants | entrapment decadence | social [SUMMARY] | [CONTENT] depression | stigma | entrapment | covid | covid 19 | 19 | decadence | participants | entrapment decadence | social [SUMMARY] | [CONTENT] 2019 | COVID-19 | COVID-19 ||| [SUMMARY] | [CONTENT] COVID-19 | Shanghai | China | April 2022 ||| ||| [SUMMARY] | [CONTENT] 1283 | 59.6% | 44.7% ||| 0.575 | 95% | CI | 0.448-0.737 | 1.693 | 95% | CI | 1.042 ||| 0.204, p < | 0.001 | 0.058 ||| 0.116 | 0.008 [SUMMARY] | [CONTENT] COVID-19 | China ||| [SUMMARY] | [CONTENT] 2019 | COVID-19 | COVID-19 ||| ||| COVID-19 | Shanghai | China | April 2022 ||| ||| ||| 1283 | 59.6% | 44.7% ||| 0.575 | 95% | CI | 0.448-0.737 | 1.693 | 95% | CI | 1.042 ||| 0.204, p < | 0.001 | 0.058 ||| 0.116 | 0.008 ||| COVID-19 | China ||| [SUMMARY] | [CONTENT] 2019 | COVID-19 | COVID-19 ||| ||| COVID-19 | Shanghai | China | April 2022 ||| ||| ||| 1283 | 59.6% | 44.7% ||| 0.575 | 95% | CI | 0.448-0.737 | 1.693 | 95% | CI | 1.042 ||| 0.204, p < | 0.001 | 0.058 ||| 0.116 | 0.008 ||| COVID-19 | China ||| [SUMMARY] |
||||||
Trends in hospital discharges, management and in-hospital mortality from acute myocardial infarction in Switzerland between 1998 and 2008. | 23530470 | Since the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap. | BACKGROUND | Swiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed. | METHODS | Overall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered. | RESULTS | In Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates. | CONCLUSION | [
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] | 3626665 | Background | Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.
Switzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug
therapies and revascularization interventions. Indeed, management of AMI, namely
regarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].
There are few data on the evolution of AMI management and outcome in Switzerland. A
study published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus
register (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in
invasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if
the findings from this study also apply to non participating centers, and whether
these trends are equally applied in all Swiss regions. Indeed, in Switzerland,
health policies rarely go down to clinical guidelines and, if they exist, they are
decided at the hospital or canton level, with little or no intervention from the
federal bodies. The purpose of this study was to assess the trends in AMI management
and outcome for the whole country and for the main administrative regions, using
data from the Swiss hospital discharge database for the period 1998–2008. | Methods | Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The
database was provided by the Swiss federal office of statistics
(http://www.bfs.admin.ch) and covers 99% of public and private
hospitals within Switzerland [11]. Data providing is compulsory and the information collected includes
gender, age, length of stay, discharge status (main and secondary diagnoses,
vital status) and procedures. Four types of stays could be obtained:
“passing through”, patients who were admitted from another hospital
and transferred to another hospital; “inbound”, patients who were
admitted from another hospital and managed on-site; “outbound”,
patients who were managed on-site and transferred to another hospital and
“in-house”, patients who were admitted and managed without being
transferred to another hospital.
Overall length of stay was indicated in days and length of stay in an intensive
care unit (ICU) in hours. When the length of stay in the ICU was zero, it was
considered as no stay in ICU. Vital status at discharge was indicated as dead or
alive. Overall and seven-day in-hospital all-cause mortalities were computed;
for seven-day mortality, patients who died after seven days were considered as
being alive.
Main and secondary diagnoses at discharge were coded using the International
Classification of Diseases, 10th revision (ICD10) of the World Health
Organization. Acute myocardial infarction was defined as ICD10 code I21X, where
X = any number. The ICD10 coding does not distinguish between STEMI
and non-STEMI. Procedures were coded using the International Classification of
Diseases, 9th Revision, Clinical Modification (ICD9-CM). The
following revascularization procedures (and corresponding IDC9-CM codes) were
assessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)
stent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting
(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and
thrombolysis (3602, 3604 and 991); and 379X for other procedures. All these
procedures were coded as binary (yes/no) variables.
Inclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the
main discharge diagnosis and 2) age ≥18 years. Exclusion criterion
was cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial
infarction (ICD10 code I22), was also excluded, as it was not possible to trace
back to the initial event (and corresponding management) due to the
anonymization of the data.
Population statistics by age were also obtained from the Swiss federal office of
statistics and used to calculate a discharge rate, expressed as the number of
discharges per 100,000 adult (≥18 years) inhabitants.
Data from the hospital discharge database for years 1998 to 2008 was used. The
database was provided by the Swiss federal office of statistics
(http://www.bfs.admin.ch) and covers 99% of public and private
hospitals within Switzerland [11]. Data providing is compulsory and the information collected includes
gender, age, length of stay, discharge status (main and secondary diagnoses,
vital status) and procedures. Four types of stays could be obtained:
“passing through”, patients who were admitted from another hospital
and transferred to another hospital; “inbound”, patients who were
admitted from another hospital and managed on-site; “outbound”,
patients who were managed on-site and transferred to another hospital and
“in-house”, patients who were admitted and managed without being
transferred to another hospital.
Overall length of stay was indicated in days and length of stay in an intensive
care unit (ICU) in hours. When the length of stay in the ICU was zero, it was
considered as no stay in ICU. Vital status at discharge was indicated as dead or
alive. Overall and seven-day in-hospital all-cause mortalities were computed;
for seven-day mortality, patients who died after seven days were considered as
being alive.
Main and secondary diagnoses at discharge were coded using the International
Classification of Diseases, 10th revision (ICD10) of the World Health
Organization. Acute myocardial infarction was defined as ICD10 code I21X, where
X = any number. The ICD10 coding does not distinguish between STEMI
and non-STEMI. Procedures were coded using the International Classification of
Diseases, 9th Revision, Clinical Modification (ICD9-CM). The
following revascularization procedures (and corresponding IDC9-CM codes) were
assessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)
stent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting
(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and
thrombolysis (3602, 3604 and 991); and 379X for other procedures. All these
procedures were coded as binary (yes/no) variables.
Inclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the
main discharge diagnosis and 2) age ≥18 years. Exclusion criterion
was cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial
infarction (ICD10 code I22), was also excluded, as it was not possible to trace
back to the initial event (and corresponding management) due to the
anonymization of the data.
Population statistics by age were also obtained from the Swiss federal office of
statistics and used to calculate a discharge rate, expressed as the number of
discharges per 100,000 adult (≥18 years) inhabitants.
Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,
Cary, NC, USA). Results were expressed as average ± standard deviation and
as number of subjects and (percentage). Two sets of analyses were performed: the
main analysis for trends in revascularization procedures included all hospital
discharges, a supplementary analysis was carried out to limit the impact of
transfers on significance levels and included only patients who were treated in
one hospital (in-hospital).
Trends in the annual use of revascularisation procedures were assessed by
Cochran-Armitage test for trend and further confirmed by multivariate logistic
regression adjusting for age, gender and type of transfer; when assessing trends
for Switzerland, a further adjustment was performed on region. Trends for
seven-day and overall mortality rates were assessed among inbound and in-house
patients only, as among “passing through” and outbound patients
mortality was zero. Multivariate models assessing trends for seven-day and
overall hospital mortality rates were adjusted for age, gender, ICU stay
(yes/no), hemodynamic assistance (yes/no) and revascularization procedures
(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends
for Switzerland, a further adjustment was performed on region. Results were
expressed as Odds ratio (OR) and (95% confidence interval). Statistical
significance was assessed for p < 0.05.
Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,
Cary, NC, USA). Results were expressed as average ± standard deviation and
as number of subjects and (percentage). Two sets of analyses were performed: the
main analysis for trends in revascularization procedures included all hospital
discharges, a supplementary analysis was carried out to limit the impact of
transfers on significance levels and included only patients who were treated in
one hospital (in-hospital).
Trends in the annual use of revascularisation procedures were assessed by
Cochran-Armitage test for trend and further confirmed by multivariate logistic
regression adjusting for age, gender and type of transfer; when assessing trends
for Switzerland, a further adjustment was performed on region. Trends for
seven-day and overall mortality rates were assessed among inbound and in-house
patients only, as among “passing through” and outbound patients
mortality was zero. Multivariate models assessing trends for seven-day and
overall hospital mortality rates were adjusted for age, gender, ICU stay
(yes/no), hemodynamic assistance (yes/no) and revascularization procedures
(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends
for Switzerland, a further adjustment was performed on region. Results were
expressed as Odds ratio (OR) and (95% confidence interval). Statistical
significance was assessed for p < 0.05. | Results | Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The
characteristics of the patients discharged according to year are summarized in
Table 1. The percentage of women tended to
decrease, while the mean age of patients at discharge increased by one year.
Discharge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to
221.3 in 2008 (Table 1).
Characteristics of patients discharged from hospital with a main
diagnosis of acute myocardial infarction, Switzerland, for period
1998–2008
Results are expressed as percentage or as
mean ± standard deviation. Trends assessed by
Cochran-Armitage test or linear regression: p < 0.001
for gender and age. § defined as the ratio between the number
of discharges and the adult (≥18 years) population,
expressed per 100,000 inhabitants.
The number of discharges from AMI rose considerably from 5530 in 1998 to 13,834
in 2008 (Figures 1, 2, 3 and 4). Most of this increase was
due to between-hospital transfers (inbound, outbound and passing through,
Figures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for
Switzerland). In 2008, over half of hospital discharges for AMI were due to
transfers in Mittelland (52%) and Ticino (68%).
Number of patients discharged with a main diagnosis of acute
myocardial infarction who were managed in a single hospital
(in-house), in Switzerland and by region, 1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were admitted from another hospital and managed
on-site (inbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were managed on-site and transferred to another
hospital (outbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial admitted
from another hospital and transferred to another hospital (passing
through), in Switzerland and by region, 1998–2008.
Overall, data from 102,729 hospital discharges from AMI were analyzed. The
characteristics of the patients discharged according to year are summarized in
Table 1. The percentage of women tended to
decrease, while the mean age of patients at discharge increased by one year.
Discharge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to
221.3 in 2008 (Table 1).
Characteristics of patients discharged from hospital with a main
diagnosis of acute myocardial infarction, Switzerland, for period
1998–2008
Results are expressed as percentage or as
mean ± standard deviation. Trends assessed by
Cochran-Armitage test or linear regression: p < 0.001
for gender and age. § defined as the ratio between the number
of discharges and the adult (≥18 years) population,
expressed per 100,000 inhabitants.
The number of discharges from AMI rose considerably from 5530 in 1998 to 13,834
in 2008 (Figures 1, 2, 3 and 4). Most of this increase was
due to between-hospital transfers (inbound, outbound and passing through,
Figures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for
Switzerland). In 2008, over half of hospital discharges for AMI were due to
transfers in Mittelland (52%) and Ticino (68%).
Number of patients discharged with a main diagnosis of acute
myocardial infarction who were managed in a single hospital
(in-house), in Switzerland and by region, 1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were admitted from another hospital and managed
on-site (inbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were managed on-site and transferred to another
hospital (outbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial admitted
from another hospital and transferred to another hospital (passing
through), in Switzerland and by region, 1998–2008.
Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit
decreased from 38.0% in 1998 to 36.2% in 2008 (p for
trend < 0.001, Figure 5). This trend
was further confirmed after multivariate adjustment for Switzerland. Differing
trends were found according to the region considered: increase in Leman, Eastern
and Central Switzerland; decrease in Northwest, Zurich and Ticino and no change
in Mittelland (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results (Additional file
1: Figure S1).
Trends in intensive care unit (ICU) utilization for acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of hospital
discharges with a diagnosis of acute myocardial infarction.
Multivariate analysis of the trends in revascularisation procedures
and in in-hospital mortality for patients discharged from hospital
with a main diagnosis of acute myocardial infarction, Switzerland
and Swiss regions, for period 1998–2008
Results are expressed as multivariate adjusted Odds ratio and (95%
confidence interval) for a one-year increase. ICU intensive
care unit; CABG coronary artery bypass graft; DNC
model did not converge. Statistical analysis by multivariate
logistic regression. For revascularization procedures, analysis was
conducted adjusting for age (continuous), gender and transfer type
(in-house, inbound, outbound and passing through) for each Swiss
region; for Switzerland, a further adjustment was performed on
region. For in-hospital mortality, analysis was conducted adjusting
for age (continuous), gender, ICU stay (yes/no), hemodynamic
assistance (yes/no) and revascularization procedures (yes/no code:
bare stent, drug eluting stent and CABG) for each Swiss region; for
Switzerland, a further adjustment was performed on region. §,
patients not transferred to another hospital (inboud or in-house);
§§, patients managed in a single hospital (in-house).
The percentage of hospitalizations with a stay in an Intensive Care Unit
decreased from 38.0% in 1998 to 36.2% in 2008 (p for
trend < 0.001, Figure 5). This trend
was further confirmed after multivariate adjustment for Switzerland. Differing
trends were found according to the region considered: increase in Leman, Eastern
and Central Switzerland; decrease in Northwest, Zurich and Ticino and no change
in Mittelland (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results (Additional file
1: Figure S1).
Trends in intensive care unit (ICU) utilization for acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of hospital
discharges with a diagnosis of acute myocardial infarction.
Multivariate analysis of the trends in revascularisation procedures
and in in-hospital mortality for patients discharged from hospital
with a main diagnosis of acute myocardial infarction, Switzerland
and Swiss regions, for period 1998–2008
Results are expressed as multivariate adjusted Odds ratio and (95%
confidence interval) for a one-year increase. ICU intensive
care unit; CABG coronary artery bypass graft; DNC
model did not converge. Statistical analysis by multivariate
logistic regression. For revascularization procedures, analysis was
conducted adjusting for age (continuous), gender and transfer type
(in-house, inbound, outbound and passing through) for each Swiss
region; for Switzerland, a further adjustment was performed on
region. For in-hospital mortality, analysis was conducted adjusting
for age (continuous), gender, ICU stay (yes/no), hemodynamic
assistance (yes/no) and revascularization procedures (yes/no code:
bare stent, drug eluting stent and CABG) for each Swiss region; for
Switzerland, a further adjustment was performed on region. §,
patients not transferred to another hospital (inboud or in-house);
§§, patients managed in a single hospital (in-house).
Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients
discharged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in
2008 (p for trend < 0.001, Figure 6).
This trend was further confirmed after multivariate adjustment for Switzerland
and all regions considered (Table 2).
Trends in the use of drug-eluting and non-drug-eluting stents for
acute myocardial infarction in Switzerland, overall and by region,
for the period 1998–2008. Results are expressed as
percentage of hospital discharges with a diagnosis of acute myocardial
infarction.
Bare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,
further decreasing to 16.6% in 2008 (p for trend < 0.001,
Figure 6). This trend was further confirmed after
multivariate adjustment for Switzerland and all Swiss regions considered
(Table 2).
DES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease
to 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment
for Switzerland and all regions concerned, although stronger for Central,
Ticino, Eastern and Zurich than for Leman, Mittelland or Northwest
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 2: Figure S2).
CABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to
3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment
for Switzerland and all Swiss regions, with the exception of Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 3: Figure S3).
Trends in the use of coronary artery bypass graft (CABG) for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008. Results are expressed as percentage of
hospital discharges with a diagnosis of acute myocardial infarction.
Circulatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and
decreased to 1.71% in 2008 (p for trend < 0.001). This trend was
further confirmed after multivariate adjustment for Switzerland and all Swiss
regions, with the exception of Ticino (Table 2).
Finally, no significant trends were found for thrombolysis, which increased from
0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for
trend = 0.09). This trend was further confirmed after multivariate
adjustment for Switzerland. Conversely, differing trends were found according to
the region considered: increase in Leman, Zurich and Eastern Switzerland,
decrease in Northwest and Ticino and no change in Mittelland and Central
Switzerland (Table 2).
Percutaneous revascularizations increased sevenfold, from 6.0% of all patients
discharged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in
2008 (p for trend < 0.001, Figure 6).
This trend was further confirmed after multivariate adjustment for Switzerland
and all regions considered (Table 2).
Trends in the use of drug-eluting and non-drug-eluting stents for
acute myocardial infarction in Switzerland, overall and by region,
for the period 1998–2008. Results are expressed as
percentage of hospital discharges with a diagnosis of acute myocardial
infarction.
Bare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,
further decreasing to 16.6% in 2008 (p for trend < 0.001,
Figure 6). This trend was further confirmed after
multivariate adjustment for Switzerland and all Swiss regions considered
(Table 2).
DES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease
to 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment
for Switzerland and all regions concerned, although stronger for Central,
Ticino, Eastern and Zurich than for Leman, Mittelland or Northwest
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 2: Figure S2).
CABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to
3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment
for Switzerland and all Swiss regions, with the exception of Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 3: Figure S3).
Trends in the use of coronary artery bypass graft (CABG) for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008. Results are expressed as percentage of
hospital discharges with a diagnosis of acute myocardial infarction.
Circulatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and
decreased to 1.71% in 2008 (p for trend < 0.001). This trend was
further confirmed after multivariate adjustment for Switzerland and all Swiss
regions, with the exception of Ticino (Table 2).
Finally, no significant trends were found for thrombolysis, which increased from
0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for
trend = 0.09). This trend was further confirmed after multivariate
adjustment for Switzerland. Conversely, differing trends were found according to
the region considered: increase in Leman, Zurich and Eastern Switzerland,
decrease in Northwest and Ticino and no change in Mittelland and Central
Switzerland (Table 2).
In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital
mortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for
trend < 0.01, Figure 8), while total
hospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for
trend < 0.01, Figure 9). After
multivariate adjustment on age, gender, ICU and revascularization procedures, no
change in seven-day in-hospital mortality was found for Switzerland and all
Swiss regions (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results, with the exception
of an increase in mortality in Mittelland and Central Switzerland
(Table 2 and Additional file 4: Figure S4).
Trends in seven-day in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
Trends in overall in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
For overall in-hospital death, no significant trend was found for Switzerland,
while differing trends were found for regions: decrease in Eastern; increase in
Mittelland and Central and no change in Leman, Northwest, Zurich and Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results, except that the decrease in
Eastern Switzerland was no longer statistically significant (Table 2).
Among patients not transferred to another hospital, seven-day in-hospital
mortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for
trend < 0.01, Figure 8), while total
hospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for
trend < 0.01, Figure 9). After
multivariate adjustment on age, gender, ICU and revascularization procedures, no
change in seven-day in-hospital mortality was found for Switzerland and all
Swiss regions (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results, with the exception
of an increase in mortality in Mittelland and Central Switzerland
(Table 2 and Additional file 4: Figure S4).
Trends in seven-day in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
Trends in overall in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
For overall in-hospital death, no significant trend was found for Switzerland,
while differing trends were found for regions: decrease in Eastern; increase in
Mittelland and Central and no change in Leman, Northwest, Zurich and Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results, except that the decrease in
Eastern Switzerland was no longer statistically significant (Table 2). | Conclusion | In Switzerland, a steep rise in hospital discharges and in revascularization
procedures for AMI occurred between 1998 and 2008. | [
"Background",
"Databases and available data",
"Statistical analysis",
"Characteristics of the patients",
"Intensive care unit",
"Revascularisation procedures",
"In-hospital mortality",
"Intensive care unit",
"Revascularisation procedures",
"In-hospital mortality",
"Study limitations",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008.",
"Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.",
"Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.",
"Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.",
"The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).",
"Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).",
"Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).",
"The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.",
"Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.",
"An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.",
"This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].",
"The authors declare that they have no competing interests.",
"CI made part of the statistical analyses and wrote most of the article; PMV collected\ndata, made part of the statistical analysis and wrote part of the article; FP\nrevised the article for important intellectual content. PMV had full access to the\ndata and is the guarantor of the study. All authors read and approved the final\nmanuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\n"
] | [
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] | [
"Background",
"Methods",
"Databases and available data",
"Statistical analysis",
"Results",
"Characteristics of the patients",
"Intensive care unit",
"Revascularisation procedures",
"In-hospital mortality",
"Discussion",
"Intensive care unit",
"Revascularisation procedures",
"In-hospital mortality",
"Study limitations",
"Conclusion",
"Competing interests",
"Authors’ contributions",
"Pre-publication history",
"Supplementary Material"
] | [
"Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.\nSwitzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug\ntherapies and revascularization interventions. Indeed, management of AMI, namely\nregarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].\nThere are few data on the evolution of AMI management and outcome in Switzerland. A\nstudy published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus\nregister (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in\ninvasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if\nthe findings from this study also apply to non participating centers, and whether\nthese trends are equally applied in all Swiss regions. Indeed, in Switzerland,\nhealth policies rarely go down to clinical guidelines and, if they exist, they are\ndecided at the hospital or canton level, with little or no intervention from the\nfederal bodies. The purpose of this study was to assess the trends in AMI management\nand outcome for the whole country and for the main administrative regions, using\ndata from the Swiss hospital discharge database for the period 1998–2008.",
" Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\nData from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.\n Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.\nStatistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.",
"Data from the hospital discharge database for years 1998 to 2008 was used. The\ndatabase was provided by the Swiss federal office of statistics\n(http://www.bfs.admin.ch) and covers 99% of public and private\nhospitals within Switzerland [11]. Data providing is compulsory and the information collected includes\ngender, age, length of stay, discharge status (main and secondary diagnoses,\nvital status) and procedures. Four types of stays could be obtained:\n“passing through”, patients who were admitted from another hospital\nand transferred to another hospital; “inbound”, patients who were\nadmitted from another hospital and managed on-site; “outbound”,\npatients who were managed on-site and transferred to another hospital and\n“in-house”, patients who were admitted and managed without being\ntransferred to another hospital.\nOverall length of stay was indicated in days and length of stay in an intensive\ncare unit (ICU) in hours. When the length of stay in the ICU was zero, it was\nconsidered as no stay in ICU. Vital status at discharge was indicated as dead or\nalive. Overall and seven-day in-hospital all-cause mortalities were computed;\nfor seven-day mortality, patients who died after seven days were considered as\nbeing alive.\nMain and secondary diagnoses at discharge were coded using the International\nClassification of Diseases, 10th revision (ICD10) of the World Health\nOrganization. Acute myocardial infarction was defined as ICD10 code I21X, where\nX = any number. The ICD10 coding does not distinguish between STEMI\nand non-STEMI. Procedures were coded using the International Classification of\nDiseases, 9th Revision, Clinical Modification (ICD9-CM). The\nfollowing revascularization procedures (and corresponding IDC9-CM codes) were\nassessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)\nstent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting\n(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and\nthrombolysis (3602, 3604 and 991); and 379X for other procedures. All these\nprocedures were coded as binary (yes/no) variables.\nInclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the\nmain discharge diagnosis and 2) age ≥18 years. Exclusion criterion\nwas cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial\ninfarction (ICD10 code I22), was also excluded, as it was not possible to trace\nback to the initial event (and corresponding management) due to the\nanonymization of the data.\nPopulation statistics by age were also obtained from the Swiss federal office of\nstatistics and used to calculate a discharge rate, expressed as the number of\ndischarges per 100,000 adult (≥18 years) inhabitants.",
"Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,\nCary, NC, USA). Results were expressed as average ± standard deviation and\nas number of subjects and (percentage). Two sets of analyses were performed: the\nmain analysis for trends in revascularization procedures included all hospital\ndischarges, a supplementary analysis was carried out to limit the impact of\ntransfers on significance levels and included only patients who were treated in\none hospital (in-hospital).\nTrends in the annual use of revascularisation procedures were assessed by\nCochran-Armitage test for trend and further confirmed by multivariate logistic\nregression adjusting for age, gender and type of transfer; when assessing trends\nfor Switzerland, a further adjustment was performed on region. Trends for\nseven-day and overall mortality rates were assessed among inbound and in-house\npatients only, as among “passing through” and outbound patients\nmortality was zero. Multivariate models assessing trends for seven-day and\noverall hospital mortality rates were adjusted for age, gender, ICU stay\n(yes/no), hemodynamic assistance (yes/no) and revascularization procedures\n(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends\nfor Switzerland, a further adjustment was performed on region. Results were\nexpressed as Odds ratio (OR) and (95% confidence interval). Statistical\nsignificance was assessed for p < 0.05.",
" Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\nOverall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.\n Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\nThe percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).\n Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\nPercutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).\n In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).\nAmong patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).",
"Overall, data from 102,729 hospital discharges from AMI were analyzed. The\ncharacteristics of the patients discharged according to year are summarized in\nTable 1. The percentage of women tended to\ndecrease, while the mean age of patients at discharge increased by one year.\nDischarge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to\n221.3 in 2008 (Table 1).\nCharacteristics of patients discharged from hospital with a main\ndiagnosis of acute myocardial infarction, Switzerland, for period\n1998–2008\nResults are expressed as percentage or as\nmean ± standard deviation. Trends assessed by\nCochran-Armitage test or linear regression: p < 0.001\nfor gender and age. § defined as the ratio between the number\nof discharges and the adult (≥18 years) population,\nexpressed per 100,000 inhabitants.\nThe number of discharges from AMI rose considerably from 5530 in 1998 to 13,834\nin 2008 (Figures 1, 2, 3 and 4). Most of this increase was\ndue to between-hospital transfers (inbound, outbound and passing through,\nFigures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for\nSwitzerland). In 2008, over half of hospital discharges for AMI were due to\ntransfers in Mittelland (52%) and Ticino (68%).\nNumber of patients discharged with a main diagnosis of acute\nmyocardial infarction who were managed in a single hospital\n(in-house), in Switzerland and by region, 1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were admitted from another hospital and managed\non-site (inbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial\ninfarction who were managed on-site and transferred to another\nhospital (outbound), in Switzerland and by region,\n1998–2008.\nNumber of patients with a main diagnosis of acute myocardial admitted\nfrom another hospital and transferred to another hospital (passing\nthrough), in Switzerland and by region, 1998–2008.",
"The percentage of hospitalizations with a stay in an Intensive Care Unit\ndecreased from 38.0% in 1998 to 36.2% in 2008 (p for\ntrend < 0.001, Figure 5). This trend\nwas further confirmed after multivariate adjustment for Switzerland. Differing\ntrends were found according to the region considered: increase in Leman, Eastern\nand Central Switzerland; decrease in Northwest, Zurich and Ticino and no change\nin Mittelland (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results (Additional file\n1: Figure S1).\nTrends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of hospital\ndischarges with a diagnosis of acute myocardial infarction.\nMultivariate analysis of the trends in revascularisation procedures\nand in in-hospital mortality for patients discharged from hospital\nwith a main diagnosis of acute myocardial infarction, Switzerland\nand Swiss regions, for period 1998–2008\nResults are expressed as multivariate adjusted Odds ratio and (95%\nconfidence interval) for a one-year increase. ICU intensive\ncare unit; CABG coronary artery bypass graft; DNC\nmodel did not converge. Statistical analysis by multivariate\nlogistic regression. For revascularization procedures, analysis was\nconducted adjusting for age (continuous), gender and transfer type\n(in-house, inbound, outbound and passing through) for each Swiss\nregion; for Switzerland, a further adjustment was performed on\nregion. For in-hospital mortality, analysis was conducted adjusting\nfor age (continuous), gender, ICU stay (yes/no), hemodynamic\nassistance (yes/no) and revascularization procedures (yes/no code:\nbare stent, drug eluting stent and CABG) for each Swiss region; for\nSwitzerland, a further adjustment was performed on region. §,\npatients not transferred to another hospital (inboud or in-house);\n§§, patients managed in a single hospital (in-house).",
"Percutaneous revascularizations increased sevenfold, from 6.0% of all patients\ndischarged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in\n2008 (p for trend < 0.001, Figure 6).\nThis trend was further confirmed after multivariate adjustment for Switzerland\nand all regions considered (Table 2).\nTrends in the use of drug-eluting and non-drug-eluting stents for\nacute myocardial infarction in Switzerland, overall and by region,\nfor the period 1998–2008. Results are expressed as\npercentage of hospital discharges with a diagnosis of acute myocardial\ninfarction.\nBare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,\nfurther decreasing to 16.6% in 2008 (p for trend < 0.001,\nFigure 6). This trend was further confirmed after\nmultivariate adjustment for Switzerland and all Swiss regions considered\n(Table 2).\nDES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease\nto 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all regions concerned, although stronger for Central,\nTicino, Eastern and Zurich than for Leman, Mittelland or Northwest\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 2: Figure S2).\nCABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to\n3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment\nfor Switzerland and all Swiss regions, with the exception of Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results (Additional file 3: Figure S3).\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008. Results are expressed as percentage of\nhospital discharges with a diagnosis of acute myocardial infarction.\nCirculatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and\ndecreased to 1.71% in 2008 (p for trend < 0.001). This trend was\nfurther confirmed after multivariate adjustment for Switzerland and all Swiss\nregions, with the exception of Ticino (Table 2).\nFinally, no significant trends were found for thrombolysis, which increased from\n0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for\ntrend = 0.09). This trend was further confirmed after multivariate\nadjustment for Switzerland. Conversely, differing trends were found according to\nthe region considered: increase in Leman, Zurich and Eastern Switzerland,\ndecrease in Northwest and Ticino and no change in Mittelland and Central\nSwitzerland (Table 2).",
"Among patients not transferred to another hospital, seven-day in-hospital\nmortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for\ntrend < 0.01, Figure 8), while total\nhospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for\ntrend < 0.01, Figure 9). After\nmultivariate adjustment on age, gender, ICU and revascularization procedures, no\nchange in seven-day in-hospital mortality was found for Switzerland and all\nSwiss regions (Table 2). Restricting the analysis to\npatients managed in a single hospital led to similar results, with the exception\nof an increase in mortality in Mittelland and Central Switzerland\n(Table 2 and Additional file 4: Figure S4).\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nTrends in overall in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008. Results are expressed as percentage of patients\ninbound or in-house discharged with a diagnosis of acute myocardial\ninfarction.\nFor overall in-hospital death, no significant trend was found for Switzerland,\nwhile differing trends were found for regions: decrease in Eastern; increase in\nMittelland and Central and no change in Leman, Northwest, Zurich and Ticino\n(Table 2). Restricting the analysis to patients\nmanaged in a single hospital led to similar results, except that the decrease in\nEastern Switzerland was no longer statistically significant (Table 2).",
"Since the end of the MONICA study [12,13], few studies have assessed the management and outcome of AMI in\nSwitzerland. Our results show an increase in most revascularization procedures,\nnamely PCI, while CABG tended to stabilize or even to decrease in the most recent\nyears. Moreover, these trends strongly differ according to region. Overall, the\nchanges in revascularization procedure in Switzerland follow the ones observed in\nother countries, [6,7,14,15]. These findings are also in agreement with a previous study conducted on\n19,500 patients of the AMIS Plus registry [8], although the increase in PCI procedures observed [16] was considerably stronger than in ours. This difference is most likely\ndue to the overrepresentation of PCI procedures among the AMIS Plus registry\nhospitals as the Registry includes only selected voluntary teams.\nBetween 1998 and 2008, the number of hospital discharges for AMI increased over\ntwo-fold in Switzerland. This finding is in contradiction with some previous studies [17,18], but it should be noted that these studies showed declining rates of AMI\nrather than number of discharges. Part of this increase might be due to population\naging [2], although the observed increase in population age (from 66.5 years\nin 1998 to 67.6 years in 2008) would not lead to a doubling of the number of\nhospitalizations for AMI. Further, all analyses were adjusted for age. Another\npossibility would be a change in disease coding, but again this is unlikely as\nhospital discharge data has been shown to be reliable regarding the diagnosis of AMI [19] and as this study focused on a specific code. It is also possible that\nmore people are hospitalized for a second or even a third AMI (partly related to the\ndecrease in case fatality rate of the first AMI, leading to an increased incidence\nof 2nd or 3rd AMI). Another possibility is the change in\ncriteria defining AMI [20], which has been shown to increase the incidence of AMI [21]. The most likely explanation for the considerable increase in the number\nof hospital discharges for AMI in Switzerland is the steep rise in hospital\ntransfers, which accounted for almost half of hospital discharges for AMI in 2008.\nThe increase in hospital transfers might be due to the limited number of hospitals\ncapable of performing revascularization procedures, prompting smaller hospitals to\ntransfer their patients as recommended by European guidelines [22,23]. Still, considerable differences in the percentages of hospital\ndischarges due to transfers were noted: in 2008 the values ranged from 40% in Leman\nto 68% in Ticino. These differences might be due to local policies. As AMI\nmanagement is expensive [24], the increase in hospital discharges for AMI would add a considerable\npressure in Swiss health expenditures, which already increased from 9.6% in 1995 to\n11.4% of the Swiss GNP in 2009 [25].\n Intensive care unit The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\nThe percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.\n Revascularisation procedures Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\nStent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.\n In-hospital mortality An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\nAn overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.\n Study limitations This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].\nThis study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].",
"The percentage of hospitalizations with ICU stay remained stable between 1998 and\n2008 in Switzerland. This stabilization might be the consequence of improved\nmanagement, requiring less ICU admissions before or after a revascularization\nintervention. Another likely explanation is the increasing number of transfers,\nthe patients being stabilized before being transferred to another hospital for\nrevascularization. Still, considerable differences in the proportion of\nhospitalizations with ICU and corresponding trends were found between regions,\nsuggesting that other factors such as the number of ICU beds available or local\noptions for AMI management might be at play. Unfortunately there is no available\ninformation regarding the number of ICU beds in each region, so the precise\nreasons for the regional differences in ICU admissions for AMI remain to be\nfurther assessed.",
"Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,\na finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several\nreports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss\nregions were more responsive than others. For instance, in the Leman area DES\nuse equaled but never exceeded bare stent use, while in South Switzerland\n(Ticino) DES represented nearly all stents implanted since 2005. The reasons for\nsuch regional differences are more likely due to local preferences or to local\nagreements regarding DES cost than to decisions based on scientific\nevidence.\nThe rate of CABG remained stable at 3% throughout the study period, a finding\nalso reported in other countries [3,6]. As for stents, very different trends according to the region were\nfound. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,\nstarting in 1999 to reach an average rate of 7%, well above the Swiss average.\nThis trend might partly be explained by the creation in 1999 of new medical\ncenters with the capacity to carry out these interventions. Conversely, in\nEastern and Central Switzerland, hardly any CABG was performed until 2007,\nlikely the result of the lack of required infrastructure. Overall, our results\nshow that CABG rates evolved differently between Swiss regions, the most likely\nexplanations being the existence of infrastructures and local preferences for\ncertain types of revascularization procedures.",
"An overall decrease in seven-day in-hospital mortality was found for Switzerland,\na finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,\nsuggesting that most of the decrease in mortality is due to revascularization\ninterventions and the improved quality of treatment according to the newest\nguidelines [9,10]. Restricting the analysis to patients who survived at least three\ndays led to similar findings, although some regions presented a slight increase\nin mortality (Additional file 5: Table S1). The rising\nprevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the\nmultivariate model as they are not reported systematically in the database.\nSimilarly, it was not possible to assess whether the patients presented with a\nSTEMI or a non-STEMI, and which drug treatments were provided during hospital\nstay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of\nrevascularization interventions performed among patients discharged with a main\ndiagnosis of AMI do not suffice to decrease in-hospital mortality in\nSwitzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors\nassociated with in-hospital mortality from AMI in order to optimize patient\nmanagement.",
"This study has some limitations. Medicines are not included in the hospital\ndischarge database, thus precluding the assessment of their trends. This absence\nmight also explain the very low rates for thrombolysis, which are likely due to\nthe absence of coding rather than a true absence of use. Furthermore, we lack\ndata to assess whether the changes in thrombolytic therapy are related to\nchanges in reporting levels or are actually real changes. The ICD10 coding does\nnot distinguish STEMI from NSTEMI; hence, it is possible that part of the\nincrease in discharges from AMI during the study period might be related to the\nchange in the definition of NSTEMI populations due to the generalization of the\nuse of troponin measurements. Indeed, introduction of troponin measurements led\nto the reclassification of many unstable angina patients into NSTEMI patients,\nwith a 33% increase of the population of NSTEMIs from before to after the\nintroduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis\nled to similar findings, with the exception that the increase in CABG was no\nlonger significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of\nbetween-hospital transfers and the anonymization of the data (which precluded\nthe follow-up of the patients) complicated the interpretation of the trends.\nStill, restricting the analysis to patients fully managed in a single hospital\nled to similar results. It was not possible to assess “true” seven-\nand 30-day mortality rates as no follow-up data was available for the patients\ndischarged. The AMIS registry, which collects vital status data for AMI patients\nhospitalized in the participating institutions, will be able to provide such\ninformation [35]. Finally, there is no published information on the validity of\nhospital discharge data in Switzerland regarding the diagnosis of AMI. Still,\nusing the results from an ongoing study (CoLaus), of 159 hospitalizations for\nAMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,\na value in agreement with the literature [36,37].",
"In Switzerland, a steep rise in hospital discharges and in revascularization\nprocedures for AMI occurred between 1998 and 2008.",
"The authors declare that they have no competing interests.",
"CI made part of the statistical analyses and wrote most of the article; PMV collected\ndata, made part of the statistical analysis and wrote part of the article; FP\nrevised the article for important intellectual content. PMV had full access to the\ndata and is the guarantor of the study. All authors read and approved the final\nmanuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/270/prepub\n",
" Trends in intensive care unit (ICU) utilization for acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008, patients managed in a single hospital (in-house).\nResults are expressed as percentage of patients discharged with a\ndiagnosis of acute myocardial infarction.\nClick here for file\nTrends in the use of drug-eluting and non-drug-eluting stents for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008, patients managed in a single hospital\n(in-house). Results are expressed as percentage of patients discharged\nwith a diagnosis of acute myocardial infarction.\nClick here for file\nTrends in the use of coronary artery bypass graft (CABG) for acute\nmyocardial infarction in Switzerland, overall and by region, for the\nperiod 1998–2008, patients managed in a single hospital\n(in-house). Results are expressed as percentage of patients discharged\nwith a diagnosis of acute myocardial infarction.\nClick here for file\nTrends in seven-day in-hospital mortality rates from acute myocardial\ninfarction in Switzerland, overall and by region, for the period\n1998–2008, patients managed in a single hospital (in-house).\nResults are expressed as percentage of patients discharged with a\ndiagnosis of acute myocardial infarction.\nClick here for file\nTrends in seven-day and overall in-hospital mortality, Switzerland and\nSwiss regions, for period 1998-2008\nClick here for file"
] | [
null,
"methods",
null,
null,
"results",
null,
null,
null,
null,
"discussion",
null,
null,
null,
null,
"conclusions",
null,
null,
null,
"supplementary-material"
] | [
"Acute myocardial infarction",
"Coronary revascularization",
"Epidemiology",
"Switzerland",
"Trends",
"Drug-eluting stent",
"Coronary artery bypass graft",
"Hospital discharge"
] | Background:
Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.
Switzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug
therapies and revascularization interventions. Indeed, management of AMI, namely
regarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].
There are few data on the evolution of AMI management and outcome in Switzerland. A
study published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus
register (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in
invasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if
the findings from this study also apply to non participating centers, and whether
these trends are equally applied in all Swiss regions. Indeed, in Switzerland,
health policies rarely go down to clinical guidelines and, if they exist, they are
decided at the hospital or canton level, with little or no intervention from the
federal bodies. The purpose of this study was to assess the trends in AMI management
and outcome for the whole country and for the main administrative regions, using
data from the Swiss hospital discharge database for the period 1998–2008.
Methods:
Databases and available data Data from the hospital discharge database for years 1998 to 2008 was used. The
database was provided by the Swiss federal office of statistics
(http://www.bfs.admin.ch) and covers 99% of public and private
hospitals within Switzerland [11]. Data providing is compulsory and the information collected includes
gender, age, length of stay, discharge status (main and secondary diagnoses,
vital status) and procedures. Four types of stays could be obtained:
“passing through”, patients who were admitted from another hospital
and transferred to another hospital; “inbound”, patients who were
admitted from another hospital and managed on-site; “outbound”,
patients who were managed on-site and transferred to another hospital and
“in-house”, patients who were admitted and managed without being
transferred to another hospital.
Overall length of stay was indicated in days and length of stay in an intensive
care unit (ICU) in hours. When the length of stay in the ICU was zero, it was
considered as no stay in ICU. Vital status at discharge was indicated as dead or
alive. Overall and seven-day in-hospital all-cause mortalities were computed;
for seven-day mortality, patients who died after seven days were considered as
being alive.
Main and secondary diagnoses at discharge were coded using the International
Classification of Diseases, 10th revision (ICD10) of the World Health
Organization. Acute myocardial infarction was defined as ICD10 code I21X, where
X = any number. The ICD10 coding does not distinguish between STEMI
and non-STEMI. Procedures were coded using the International Classification of
Diseases, 9th Revision, Clinical Modification (ICD9-CM). The
following revascularization procedures (and corresponding IDC9-CM codes) were
assessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)
stent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting
(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and
thrombolysis (3602, 3604 and 991); and 379X for other procedures. All these
procedures were coded as binary (yes/no) variables.
Inclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the
main discharge diagnosis and 2) age ≥18 years. Exclusion criterion
was cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial
infarction (ICD10 code I22), was also excluded, as it was not possible to trace
back to the initial event (and corresponding management) due to the
anonymization of the data.
Population statistics by age were also obtained from the Swiss federal office of
statistics and used to calculate a discharge rate, expressed as the number of
discharges per 100,000 adult (≥18 years) inhabitants.
Data from the hospital discharge database for years 1998 to 2008 was used. The
database was provided by the Swiss federal office of statistics
(http://www.bfs.admin.ch) and covers 99% of public and private
hospitals within Switzerland [11]. Data providing is compulsory and the information collected includes
gender, age, length of stay, discharge status (main and secondary diagnoses,
vital status) and procedures. Four types of stays could be obtained:
“passing through”, patients who were admitted from another hospital
and transferred to another hospital; “inbound”, patients who were
admitted from another hospital and managed on-site; “outbound”,
patients who were managed on-site and transferred to another hospital and
“in-house”, patients who were admitted and managed without being
transferred to another hospital.
Overall length of stay was indicated in days and length of stay in an intensive
care unit (ICU) in hours. When the length of stay in the ICU was zero, it was
considered as no stay in ICU. Vital status at discharge was indicated as dead or
alive. Overall and seven-day in-hospital all-cause mortalities were computed;
for seven-day mortality, patients who died after seven days were considered as
being alive.
Main and secondary diagnoses at discharge were coded using the International
Classification of Diseases, 10th revision (ICD10) of the World Health
Organization. Acute myocardial infarction was defined as ICD10 code I21X, where
X = any number. The ICD10 coding does not distinguish between STEMI
and non-STEMI. Procedures were coded using the International Classification of
Diseases, 9th Revision, Clinical Modification (ICD9-CM). The
following revascularization procedures (and corresponding IDC9-CM codes) were
assessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)
stent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting
(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and
thrombolysis (3602, 3604 and 991); and 379X for other procedures. All these
procedures were coded as binary (yes/no) variables.
Inclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the
main discharge diagnosis and 2) age ≥18 years. Exclusion criterion
was cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial
infarction (ICD10 code I22), was also excluded, as it was not possible to trace
back to the initial event (and corresponding management) due to the
anonymization of the data.
Population statistics by age were also obtained from the Swiss federal office of
statistics and used to calculate a discharge rate, expressed as the number of
discharges per 100,000 adult (≥18 years) inhabitants.
Statistical analysis Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,
Cary, NC, USA). Results were expressed as average ± standard deviation and
as number of subjects and (percentage). Two sets of analyses were performed: the
main analysis for trends in revascularization procedures included all hospital
discharges, a supplementary analysis was carried out to limit the impact of
transfers on significance levels and included only patients who were treated in
one hospital (in-hospital).
Trends in the annual use of revascularisation procedures were assessed by
Cochran-Armitage test for trend and further confirmed by multivariate logistic
regression adjusting for age, gender and type of transfer; when assessing trends
for Switzerland, a further adjustment was performed on region. Trends for
seven-day and overall mortality rates were assessed among inbound and in-house
patients only, as among “passing through” and outbound patients
mortality was zero. Multivariate models assessing trends for seven-day and
overall hospital mortality rates were adjusted for age, gender, ICU stay
(yes/no), hemodynamic assistance (yes/no) and revascularization procedures
(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends
for Switzerland, a further adjustment was performed on region. Results were
expressed as Odds ratio (OR) and (95% confidence interval). Statistical
significance was assessed for p < 0.05.
Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,
Cary, NC, USA). Results were expressed as average ± standard deviation and
as number of subjects and (percentage). Two sets of analyses were performed: the
main analysis for trends in revascularization procedures included all hospital
discharges, a supplementary analysis was carried out to limit the impact of
transfers on significance levels and included only patients who were treated in
one hospital (in-hospital).
Trends in the annual use of revascularisation procedures were assessed by
Cochran-Armitage test for trend and further confirmed by multivariate logistic
regression adjusting for age, gender and type of transfer; when assessing trends
for Switzerland, a further adjustment was performed on region. Trends for
seven-day and overall mortality rates were assessed among inbound and in-house
patients only, as among “passing through” and outbound patients
mortality was zero. Multivariate models assessing trends for seven-day and
overall hospital mortality rates were adjusted for age, gender, ICU stay
(yes/no), hemodynamic assistance (yes/no) and revascularization procedures
(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends
for Switzerland, a further adjustment was performed on region. Results were
expressed as Odds ratio (OR) and (95% confidence interval). Statistical
significance was assessed for p < 0.05.
Databases and available data:
Data from the hospital discharge database for years 1998 to 2008 was used. The
database was provided by the Swiss federal office of statistics
(http://www.bfs.admin.ch) and covers 99% of public and private
hospitals within Switzerland [11]. Data providing is compulsory and the information collected includes
gender, age, length of stay, discharge status (main and secondary diagnoses,
vital status) and procedures. Four types of stays could be obtained:
“passing through”, patients who were admitted from another hospital
and transferred to another hospital; “inbound”, patients who were
admitted from another hospital and managed on-site; “outbound”,
patients who were managed on-site and transferred to another hospital and
“in-house”, patients who were admitted and managed without being
transferred to another hospital.
Overall length of stay was indicated in days and length of stay in an intensive
care unit (ICU) in hours. When the length of stay in the ICU was zero, it was
considered as no stay in ICU. Vital status at discharge was indicated as dead or
alive. Overall and seven-day in-hospital all-cause mortalities were computed;
for seven-day mortality, patients who died after seven days were considered as
being alive.
Main and secondary diagnoses at discharge were coded using the International
Classification of Diseases, 10th revision (ICD10) of the World Health
Organization. Acute myocardial infarction was defined as ICD10 code I21X, where
X = any number. The ICD10 coding does not distinguish between STEMI
and non-STEMI. Procedures were coded using the International Classification of
Diseases, 9th Revision, Clinical Modification (ICD9-CM). The
following revascularization procedures (and corresponding IDC9-CM codes) were
assessed: any percutaneous coronary intervention (360X); bare (non-drug-eluting)
stent (3606); drug-eluting stent (DES: 3607); coronary artery bypass grafting
(CABG: 361X, 362X and 363X); circulatory assistance (376X and 369X) and
thrombolysis (3602, 3604 and 991); and 379X for other procedures. All these
procedures were coded as binary (yes/no) variables.
Inclusion criteria were: 1) Acute myocardial infarction (ICD10 code I21X) as the
main discharge diagnosis and 2) age ≥18 years. Exclusion criterion
was cardiovascular rehabilitation (ICD9-CM code Z500). Subsequent myocardial
infarction (ICD10 code I22), was also excluded, as it was not possible to trace
back to the initial event (and corresponding management) due to the
anonymization of the data.
Population statistics by age were also obtained from the Swiss federal office of
statistics and used to calculate a discharge rate, expressed as the number of
discharges per 100,000 adult (≥18 years) inhabitants.
Statistical analysis:
Statistical analysis was conducted using SAS software version 9.2 (SAS Inc.,
Cary, NC, USA). Results were expressed as average ± standard deviation and
as number of subjects and (percentage). Two sets of analyses were performed: the
main analysis for trends in revascularization procedures included all hospital
discharges, a supplementary analysis was carried out to limit the impact of
transfers on significance levels and included only patients who were treated in
one hospital (in-hospital).
Trends in the annual use of revascularisation procedures were assessed by
Cochran-Armitage test for trend and further confirmed by multivariate logistic
regression adjusting for age, gender and type of transfer; when assessing trends
for Switzerland, a further adjustment was performed on region. Trends for
seven-day and overall mortality rates were assessed among inbound and in-house
patients only, as among “passing through” and outbound patients
mortality was zero. Multivariate models assessing trends for seven-day and
overall hospital mortality rates were adjusted for age, gender, ICU stay
(yes/no), hemodynamic assistance (yes/no) and revascularization procedures
(yes/no code: bare stent, drug eluting stent and CABG); when assessing trends
for Switzerland, a further adjustment was performed on region. Results were
expressed as Odds ratio (OR) and (95% confidence interval). Statistical
significance was assessed for p < 0.05.
Results:
Characteristics of the patients Overall, data from 102,729 hospital discharges from AMI were analyzed. The
characteristics of the patients discharged according to year are summarized in
Table 1. The percentage of women tended to
decrease, while the mean age of patients at discharge increased by one year.
Discharge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to
221.3 in 2008 (Table 1).
Characteristics of patients discharged from hospital with a main
diagnosis of acute myocardial infarction, Switzerland, for period
1998–2008
Results are expressed as percentage or as
mean ± standard deviation. Trends assessed by
Cochran-Armitage test or linear regression: p < 0.001
for gender and age. § defined as the ratio between the number
of discharges and the adult (≥18 years) population,
expressed per 100,000 inhabitants.
The number of discharges from AMI rose considerably from 5530 in 1998 to 13,834
in 2008 (Figures 1, 2, 3 and 4). Most of this increase was
due to between-hospital transfers (inbound, outbound and passing through,
Figures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for
Switzerland). In 2008, over half of hospital discharges for AMI were due to
transfers in Mittelland (52%) and Ticino (68%).
Number of patients discharged with a main diagnosis of acute
myocardial infarction who were managed in a single hospital
(in-house), in Switzerland and by region, 1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were admitted from another hospital and managed
on-site (inbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were managed on-site and transferred to another
hospital (outbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial admitted
from another hospital and transferred to another hospital (passing
through), in Switzerland and by region, 1998–2008.
Overall, data from 102,729 hospital discharges from AMI were analyzed. The
characteristics of the patients discharged according to year are summarized in
Table 1. The percentage of women tended to
decrease, while the mean age of patients at discharge increased by one year.
Discharge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to
221.3 in 2008 (Table 1).
Characteristics of patients discharged from hospital with a main
diagnosis of acute myocardial infarction, Switzerland, for period
1998–2008
Results are expressed as percentage or as
mean ± standard deviation. Trends assessed by
Cochran-Armitage test or linear regression: p < 0.001
for gender and age. § defined as the ratio between the number
of discharges and the adult (≥18 years) population,
expressed per 100,000 inhabitants.
The number of discharges from AMI rose considerably from 5530 in 1998 to 13,834
in 2008 (Figures 1, 2, 3 and 4). Most of this increase was
due to between-hospital transfers (inbound, outbound and passing through,
Figures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for
Switzerland). In 2008, over half of hospital discharges for AMI were due to
transfers in Mittelland (52%) and Ticino (68%).
Number of patients discharged with a main diagnosis of acute
myocardial infarction who were managed in a single hospital
(in-house), in Switzerland and by region, 1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were admitted from another hospital and managed
on-site (inbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were managed on-site and transferred to another
hospital (outbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial admitted
from another hospital and transferred to another hospital (passing
through), in Switzerland and by region, 1998–2008.
Intensive care unit The percentage of hospitalizations with a stay in an Intensive Care Unit
decreased from 38.0% in 1998 to 36.2% in 2008 (p for
trend < 0.001, Figure 5). This trend
was further confirmed after multivariate adjustment for Switzerland. Differing
trends were found according to the region considered: increase in Leman, Eastern
and Central Switzerland; decrease in Northwest, Zurich and Ticino and no change
in Mittelland (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results (Additional file
1: Figure S1).
Trends in intensive care unit (ICU) utilization for acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of hospital
discharges with a diagnosis of acute myocardial infarction.
Multivariate analysis of the trends in revascularisation procedures
and in in-hospital mortality for patients discharged from hospital
with a main diagnosis of acute myocardial infarction, Switzerland
and Swiss regions, for period 1998–2008
Results are expressed as multivariate adjusted Odds ratio and (95%
confidence interval) for a one-year increase. ICU intensive
care unit; CABG coronary artery bypass graft; DNC
model did not converge. Statistical analysis by multivariate
logistic regression. For revascularization procedures, analysis was
conducted adjusting for age (continuous), gender and transfer type
(in-house, inbound, outbound and passing through) for each Swiss
region; for Switzerland, a further adjustment was performed on
region. For in-hospital mortality, analysis was conducted adjusting
for age (continuous), gender, ICU stay (yes/no), hemodynamic
assistance (yes/no) and revascularization procedures (yes/no code:
bare stent, drug eluting stent and CABG) for each Swiss region; for
Switzerland, a further adjustment was performed on region. §,
patients not transferred to another hospital (inboud or in-house);
§§, patients managed in a single hospital (in-house).
The percentage of hospitalizations with a stay in an Intensive Care Unit
decreased from 38.0% in 1998 to 36.2% in 2008 (p for
trend < 0.001, Figure 5). This trend
was further confirmed after multivariate adjustment for Switzerland. Differing
trends were found according to the region considered: increase in Leman, Eastern
and Central Switzerland; decrease in Northwest, Zurich and Ticino and no change
in Mittelland (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results (Additional file
1: Figure S1).
Trends in intensive care unit (ICU) utilization for acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of hospital
discharges with a diagnosis of acute myocardial infarction.
Multivariate analysis of the trends in revascularisation procedures
and in in-hospital mortality for patients discharged from hospital
with a main diagnosis of acute myocardial infarction, Switzerland
and Swiss regions, for period 1998–2008
Results are expressed as multivariate adjusted Odds ratio and (95%
confidence interval) for a one-year increase. ICU intensive
care unit; CABG coronary artery bypass graft; DNC
model did not converge. Statistical analysis by multivariate
logistic regression. For revascularization procedures, analysis was
conducted adjusting for age (continuous), gender and transfer type
(in-house, inbound, outbound and passing through) for each Swiss
region; for Switzerland, a further adjustment was performed on
region. For in-hospital mortality, analysis was conducted adjusting
for age (continuous), gender, ICU stay (yes/no), hemodynamic
assistance (yes/no) and revascularization procedures (yes/no code:
bare stent, drug eluting stent and CABG) for each Swiss region; for
Switzerland, a further adjustment was performed on region. §,
patients not transferred to another hospital (inboud or in-house);
§§, patients managed in a single hospital (in-house).
Revascularisation procedures Percutaneous revascularizations increased sevenfold, from 6.0% of all patients
discharged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in
2008 (p for trend < 0.001, Figure 6).
This trend was further confirmed after multivariate adjustment for Switzerland
and all regions considered (Table 2).
Trends in the use of drug-eluting and non-drug-eluting stents for
acute myocardial infarction in Switzerland, overall and by region,
for the period 1998–2008. Results are expressed as
percentage of hospital discharges with a diagnosis of acute myocardial
infarction.
Bare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,
further decreasing to 16.6% in 2008 (p for trend < 0.001,
Figure 6). This trend was further confirmed after
multivariate adjustment for Switzerland and all Swiss regions considered
(Table 2).
DES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease
to 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment
for Switzerland and all regions concerned, although stronger for Central,
Ticino, Eastern and Zurich than for Leman, Mittelland or Northwest
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 2: Figure S2).
CABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to
3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment
for Switzerland and all Swiss regions, with the exception of Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 3: Figure S3).
Trends in the use of coronary artery bypass graft (CABG) for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008. Results are expressed as percentage of
hospital discharges with a diagnosis of acute myocardial infarction.
Circulatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and
decreased to 1.71% in 2008 (p for trend < 0.001). This trend was
further confirmed after multivariate adjustment for Switzerland and all Swiss
regions, with the exception of Ticino (Table 2).
Finally, no significant trends were found for thrombolysis, which increased from
0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for
trend = 0.09). This trend was further confirmed after multivariate
adjustment for Switzerland. Conversely, differing trends were found according to
the region considered: increase in Leman, Zurich and Eastern Switzerland,
decrease in Northwest and Ticino and no change in Mittelland and Central
Switzerland (Table 2).
Percutaneous revascularizations increased sevenfold, from 6.0% of all patients
discharged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in
2008 (p for trend < 0.001, Figure 6).
This trend was further confirmed after multivariate adjustment for Switzerland
and all regions considered (Table 2).
Trends in the use of drug-eluting and non-drug-eluting stents for
acute myocardial infarction in Switzerland, overall and by region,
for the period 1998–2008. Results are expressed as
percentage of hospital discharges with a diagnosis of acute myocardial
infarction.
Bare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,
further decreasing to 16.6% in 2008 (p for trend < 0.001,
Figure 6). This trend was further confirmed after
multivariate adjustment for Switzerland and all Swiss regions considered
(Table 2).
DES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease
to 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment
for Switzerland and all regions concerned, although stronger for Central,
Ticino, Eastern and Zurich than for Leman, Mittelland or Northwest
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 2: Figure S2).
CABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to
3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment
for Switzerland and all Swiss regions, with the exception of Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 3: Figure S3).
Trends in the use of coronary artery bypass graft (CABG) for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008. Results are expressed as percentage of
hospital discharges with a diagnosis of acute myocardial infarction.
Circulatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and
decreased to 1.71% in 2008 (p for trend < 0.001). This trend was
further confirmed after multivariate adjustment for Switzerland and all Swiss
regions, with the exception of Ticino (Table 2).
Finally, no significant trends were found for thrombolysis, which increased from
0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for
trend = 0.09). This trend was further confirmed after multivariate
adjustment for Switzerland. Conversely, differing trends were found according to
the region considered: increase in Leman, Zurich and Eastern Switzerland,
decrease in Northwest and Ticino and no change in Mittelland and Central
Switzerland (Table 2).
In-hospital mortality Among patients not transferred to another hospital, seven-day in-hospital
mortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for
trend < 0.01, Figure 8), while total
hospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for
trend < 0.01, Figure 9). After
multivariate adjustment on age, gender, ICU and revascularization procedures, no
change in seven-day in-hospital mortality was found for Switzerland and all
Swiss regions (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results, with the exception
of an increase in mortality in Mittelland and Central Switzerland
(Table 2 and Additional file 4: Figure S4).
Trends in seven-day in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
Trends in overall in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
For overall in-hospital death, no significant trend was found for Switzerland,
while differing trends were found for regions: decrease in Eastern; increase in
Mittelland and Central and no change in Leman, Northwest, Zurich and Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results, except that the decrease in
Eastern Switzerland was no longer statistically significant (Table 2).
Among patients not transferred to another hospital, seven-day in-hospital
mortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for
trend < 0.01, Figure 8), while total
hospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for
trend < 0.01, Figure 9). After
multivariate adjustment on age, gender, ICU and revascularization procedures, no
change in seven-day in-hospital mortality was found for Switzerland and all
Swiss regions (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results, with the exception
of an increase in mortality in Mittelland and Central Switzerland
(Table 2 and Additional file 4: Figure S4).
Trends in seven-day in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
Trends in overall in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
For overall in-hospital death, no significant trend was found for Switzerland,
while differing trends were found for regions: decrease in Eastern; increase in
Mittelland and Central and no change in Leman, Northwest, Zurich and Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results, except that the decrease in
Eastern Switzerland was no longer statistically significant (Table 2).
Characteristics of the patients:
Overall, data from 102,729 hospital discharges from AMI were analyzed. The
characteristics of the patients discharged according to year are summarized in
Table 1. The percentage of women tended to
decrease, while the mean age of patients at discharge increased by one year.
Discharge rates more than duplicated, from 98.2/100,000 inhabitants in 1998 to
221.3 in 2008 (Table 1).
Characteristics of patients discharged from hospital with a main
diagnosis of acute myocardial infarction, Switzerland, for period
1998–2008
Results are expressed as percentage or as
mean ± standard deviation. Trends assessed by
Cochran-Armitage test or linear regression: p < 0.001
for gender and age. § defined as the ratio between the number
of discharges and the adult (≥18 years) population,
expressed per 100,000 inhabitants.
The number of discharges from AMI rose considerably from 5530 in 1998 to 13,834
in 2008 (Figures 1, 2, 3 and 4). Most of this increase was
due to between-hospital transfers (inbound, outbound and passing through,
Figures 2, 3 and 4), which increased from 24% in 1998 to 47% in 2008 (for
Switzerland). In 2008, over half of hospital discharges for AMI were due to
transfers in Mittelland (52%) and Ticino (68%).
Number of patients discharged with a main diagnosis of acute
myocardial infarction who were managed in a single hospital
(in-house), in Switzerland and by region, 1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were admitted from another hospital and managed
on-site (inbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial
infarction who were managed on-site and transferred to another
hospital (outbound), in Switzerland and by region,
1998–2008.
Number of patients with a main diagnosis of acute myocardial admitted
from another hospital and transferred to another hospital (passing
through), in Switzerland and by region, 1998–2008.
Intensive care unit:
The percentage of hospitalizations with a stay in an Intensive Care Unit
decreased from 38.0% in 1998 to 36.2% in 2008 (p for
trend < 0.001, Figure 5). This trend
was further confirmed after multivariate adjustment for Switzerland. Differing
trends were found according to the region considered: increase in Leman, Eastern
and Central Switzerland; decrease in Northwest, Zurich and Ticino and no change
in Mittelland (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results (Additional file
1: Figure S1).
Trends in intensive care unit (ICU) utilization for acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of hospital
discharges with a diagnosis of acute myocardial infarction.
Multivariate analysis of the trends in revascularisation procedures
and in in-hospital mortality for patients discharged from hospital
with a main diagnosis of acute myocardial infarction, Switzerland
and Swiss regions, for period 1998–2008
Results are expressed as multivariate adjusted Odds ratio and (95%
confidence interval) for a one-year increase. ICU intensive
care unit; CABG coronary artery bypass graft; DNC
model did not converge. Statistical analysis by multivariate
logistic regression. For revascularization procedures, analysis was
conducted adjusting for age (continuous), gender and transfer type
(in-house, inbound, outbound and passing through) for each Swiss
region; for Switzerland, a further adjustment was performed on
region. For in-hospital mortality, analysis was conducted adjusting
for age (continuous), gender, ICU stay (yes/no), hemodynamic
assistance (yes/no) and revascularization procedures (yes/no code:
bare stent, drug eluting stent and CABG) for each Swiss region; for
Switzerland, a further adjustment was performed on region. §,
patients not transferred to another hospital (inboud or in-house);
§§, patients managed in a single hospital (in-house).
Revascularisation procedures:
Percutaneous revascularizations increased sevenfold, from 6.0% of all patients
discharged with AMI in 1998 to 42.4% in 2006, with a slight decrease to 39.9% in
2008 (p for trend < 0.001, Figure 6).
This trend was further confirmed after multivariate adjustment for Switzerland
and all regions considered (Table 2).
Trends in the use of drug-eluting and non-drug-eluting stents for
acute myocardial infarction in Switzerland, overall and by region,
for the period 1998–2008. Results are expressed as
percentage of hospital discharges with a diagnosis of acute myocardial
infarction.
Bare (non-drug-eluting) stents rose from 1.3% in 1998 to peak at 21.1% in 2003,
further decreasing to 16.6% in 2008 (p for trend < 0.001,
Figure 6). This trend was further confirmed after
multivariate adjustment for Switzerland and all Swiss regions considered
(Table 2).
DES appeared in 2004 and steadily peaked at 24.3% in 2006, with a slight decrease
to 23.5% in 2008 (p for trend < 0.001, Figure 6). This trend was further confirmed after multivariate adjustment
for Switzerland and all regions concerned, although stronger for Central,
Ticino, Eastern and Zurich than for Leman, Mittelland or Northwest
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 2: Figure S2).
CABG increased from 1.0% in 1998 to peak at 3.3% in 2006, further decreasing to
3.0% in 2008 (p for trend < 0.001, Figure 7). This trend was further confirmed after multivariate adjustment
for Switzerland and all Swiss regions, with the exception of Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results (Additional file 3: Figure S3).
Trends in the use of coronary artery bypass graft (CABG) for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008. Results are expressed as percentage of
hospital discharges with a diagnosis of acute myocardial infarction.
Circulatory assistance increased from 0.24% in 1998 to 2.00% in 2007 and
decreased to 1.71% in 2008 (p for trend < 0.001). This trend was
further confirmed after multivariate adjustment for Switzerland and all Swiss
regions, with the exception of Ticino (Table 2).
Finally, no significant trends were found for thrombolysis, which increased from
0.5% in 1998 to 6.0% in 2002 and decreased to 1.9% in 2008 (p for
trend = 0.09). This trend was further confirmed after multivariate
adjustment for Switzerland. Conversely, differing trends were found according to
the region considered: increase in Leman, Zurich and Eastern Switzerland,
decrease in Northwest and Ticino and no change in Mittelland and Central
Switzerland (Table 2).
In-hospital mortality:
Among patients not transferred to another hospital, seven-day in-hospital
mortality decreased from 8.0% in 1998 to 7.0% in 2008 (p for
trend < 0.01, Figure 8), while total
hospital mortality decreased from 11.2% in 1998 to 10.1% in 2008 (p for
trend < 0.01, Figure 9). After
multivariate adjustment on age, gender, ICU and revascularization procedures, no
change in seven-day in-hospital mortality was found for Switzerland and all
Swiss regions (Table 2). Restricting the analysis to
patients managed in a single hospital led to similar results, with the exception
of an increase in mortality in Mittelland and Central Switzerland
(Table 2 and Additional file 4: Figure S4).
Trends in seven-day in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
Trends in overall in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008. Results are expressed as percentage of patients
inbound or in-house discharged with a diagnosis of acute myocardial
infarction.
For overall in-hospital death, no significant trend was found for Switzerland,
while differing trends were found for regions: decrease in Eastern; increase in
Mittelland and Central and no change in Leman, Northwest, Zurich and Ticino
(Table 2). Restricting the analysis to patients
managed in a single hospital led to similar results, except that the decrease in
Eastern Switzerland was no longer statistically significant (Table 2).
Discussion:
Since the end of the MONICA study [12,13], few studies have assessed the management and outcome of AMI in
Switzerland. Our results show an increase in most revascularization procedures,
namely PCI, while CABG tended to stabilize or even to decrease in the most recent
years. Moreover, these trends strongly differ according to region. Overall, the
changes in revascularization procedure in Switzerland follow the ones observed in
other countries, [6,7,14,15]. These findings are also in agreement with a previous study conducted on
19,500 patients of the AMIS Plus registry [8], although the increase in PCI procedures observed [16] was considerably stronger than in ours. This difference is most likely
due to the overrepresentation of PCI procedures among the AMIS Plus registry
hospitals as the Registry includes only selected voluntary teams.
Between 1998 and 2008, the number of hospital discharges for AMI increased over
two-fold in Switzerland. This finding is in contradiction with some previous studies [17,18], but it should be noted that these studies showed declining rates of AMI
rather than number of discharges. Part of this increase might be due to population
aging [2], although the observed increase in population age (from 66.5 years
in 1998 to 67.6 years in 2008) would not lead to a doubling of the number of
hospitalizations for AMI. Further, all analyses were adjusted for age. Another
possibility would be a change in disease coding, but again this is unlikely as
hospital discharge data has been shown to be reliable regarding the diagnosis of AMI [19] and as this study focused on a specific code. It is also possible that
more people are hospitalized for a second or even a third AMI (partly related to the
decrease in case fatality rate of the first AMI, leading to an increased incidence
of 2nd or 3rd AMI). Another possibility is the change in
criteria defining AMI [20], which has been shown to increase the incidence of AMI [21]. The most likely explanation for the considerable increase in the number
of hospital discharges for AMI in Switzerland is the steep rise in hospital
transfers, which accounted for almost half of hospital discharges for AMI in 2008.
The increase in hospital transfers might be due to the limited number of hospitals
capable of performing revascularization procedures, prompting smaller hospitals to
transfer their patients as recommended by European guidelines [22,23]. Still, considerable differences in the percentages of hospital
discharges due to transfers were noted: in 2008 the values ranged from 40% in Leman
to 68% in Ticino. These differences might be due to local policies. As AMI
management is expensive [24], the increase in hospital discharges for AMI would add a considerable
pressure in Swiss health expenditures, which already increased from 9.6% in 1995 to
11.4% of the Swiss GNP in 2009 [25].
Intensive care unit The percentage of hospitalizations with ICU stay remained stable between 1998 and
2008 in Switzerland. This stabilization might be the consequence of improved
management, requiring less ICU admissions before or after a revascularization
intervention. Another likely explanation is the increasing number of transfers,
the patients being stabilized before being transferred to another hospital for
revascularization. Still, considerable differences in the proportion of
hospitalizations with ICU and corresponding trends were found between regions,
suggesting that other factors such as the number of ICU beds available or local
options for AMI management might be at play. Unfortunately there is no available
information regarding the number of ICU beds in each region, so the precise
reasons for the regional differences in ICU admissions for AMI remain to be
further assessed.
The percentage of hospitalizations with ICU stay remained stable between 1998 and
2008 in Switzerland. This stabilization might be the consequence of improved
management, requiring less ICU admissions before or after a revascularization
intervention. Another likely explanation is the increasing number of transfers,
the patients being stabilized before being transferred to another hospital for
revascularization. Still, considerable differences in the proportion of
hospitalizations with ICU and corresponding trends were found between regions,
suggesting that other factors such as the number of ICU beds available or local
options for AMI management might be at play. Unfortunately there is no available
information regarding the number of ICU beds in each region, so the precise
reasons for the regional differences in ICU admissions for AMI remain to be
further assessed.
Revascularisation procedures Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,
a finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several
reports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss
regions were more responsive than others. For instance, in the Leman area DES
use equaled but never exceeded bare stent use, while in South Switzerland
(Ticino) DES represented nearly all stents implanted since 2005. The reasons for
such regional differences are more likely due to local preferences or to local
agreements regarding DES cost than to decisions based on scientific
evidence.
The rate of CABG remained stable at 3% throughout the study period, a finding
also reported in other countries [3,6]. As for stents, very different trends according to the region were
found. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,
starting in 1999 to reach an average rate of 7%, well above the Swiss average.
This trend might partly be explained by the creation in 1999 of new medical
centers with the capacity to carry out these interventions. Conversely, in
Eastern and Central Switzerland, hardly any CABG was performed until 2007,
likely the result of the lack of required infrastructure. Overall, our results
show that CABG rates evolved differently between Swiss regions, the most likely
explanations being the existence of infrastructures and local preferences for
certain types of revascularization procedures.
Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,
a finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several
reports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss
regions were more responsive than others. For instance, in the Leman area DES
use equaled but never exceeded bare stent use, while in South Switzerland
(Ticino) DES represented nearly all stents implanted since 2005. The reasons for
such regional differences are more likely due to local preferences or to local
agreements regarding DES cost than to decisions based on scientific
evidence.
The rate of CABG remained stable at 3% throughout the study period, a finding
also reported in other countries [3,6]. As for stents, very different trends according to the region were
found. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,
starting in 1999 to reach an average rate of 7%, well above the Swiss average.
This trend might partly be explained by the creation in 1999 of new medical
centers with the capacity to carry out these interventions. Conversely, in
Eastern and Central Switzerland, hardly any CABG was performed until 2007,
likely the result of the lack of required infrastructure. Overall, our results
show that CABG rates evolved differently between Swiss regions, the most likely
explanations being the existence of infrastructures and local preferences for
certain types of revascularization procedures.
In-hospital mortality An overall decrease in seven-day in-hospital mortality was found for Switzerland,
a finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,
suggesting that most of the decrease in mortality is due to revascularization
interventions and the improved quality of treatment according to the newest
guidelines [9,10]. Restricting the analysis to patients who survived at least three
days led to similar findings, although some regions presented a slight increase
in mortality (Additional file 5: Table S1). The rising
prevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the
multivariate model as they are not reported systematically in the database.
Similarly, it was not possible to assess whether the patients presented with a
STEMI or a non-STEMI, and which drug treatments were provided during hospital
stay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of
revascularization interventions performed among patients discharged with a main
diagnosis of AMI do not suffice to decrease in-hospital mortality in
Switzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors
associated with in-hospital mortality from AMI in order to optimize patient
management.
An overall decrease in seven-day in-hospital mortality was found for Switzerland,
a finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,
suggesting that most of the decrease in mortality is due to revascularization
interventions and the improved quality of treatment according to the newest
guidelines [9,10]. Restricting the analysis to patients who survived at least three
days led to similar findings, although some regions presented a slight increase
in mortality (Additional file 5: Table S1). The rising
prevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the
multivariate model as they are not reported systematically in the database.
Similarly, it was not possible to assess whether the patients presented with a
STEMI or a non-STEMI, and which drug treatments were provided during hospital
stay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of
revascularization interventions performed among patients discharged with a main
diagnosis of AMI do not suffice to decrease in-hospital mortality in
Switzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors
associated with in-hospital mortality from AMI in order to optimize patient
management.
Study limitations This study has some limitations. Medicines are not included in the hospital
discharge database, thus precluding the assessment of their trends. This absence
might also explain the very low rates for thrombolysis, which are likely due to
the absence of coding rather than a true absence of use. Furthermore, we lack
data to assess whether the changes in thrombolytic therapy are related to
changes in reporting levels or are actually real changes. The ICD10 coding does
not distinguish STEMI from NSTEMI; hence, it is possible that part of the
increase in discharges from AMI during the study period might be related to the
change in the definition of NSTEMI populations due to the generalization of the
use of troponin measurements. Indeed, introduction of troponin measurements led
to the reclassification of many unstable angina patients into NSTEMI patients,
with a 33% increase of the population of NSTEMIs from before to after the
introduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis
led to similar findings, with the exception that the increase in CABG was no
longer significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of
between-hospital transfers and the anonymization of the data (which precluded
the follow-up of the patients) complicated the interpretation of the trends.
Still, restricting the analysis to patients fully managed in a single hospital
led to similar results. It was not possible to assess “true” seven-
and 30-day mortality rates as no follow-up data was available for the patients
discharged. The AMIS registry, which collects vital status data for AMI patients
hospitalized in the participating institutions, will be able to provide such
information [35]. Finally, there is no published information on the validity of
hospital discharge data in Switzerland regarding the diagnosis of AMI. Still,
using the results from an ongoing study (CoLaus), of 159 hospitalizations for
AMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,
a value in agreement with the literature [36,37].
This study has some limitations. Medicines are not included in the hospital
discharge database, thus precluding the assessment of their trends. This absence
might also explain the very low rates for thrombolysis, which are likely due to
the absence of coding rather than a true absence of use. Furthermore, we lack
data to assess whether the changes in thrombolytic therapy are related to
changes in reporting levels or are actually real changes. The ICD10 coding does
not distinguish STEMI from NSTEMI; hence, it is possible that part of the
increase in discharges from AMI during the study period might be related to the
change in the definition of NSTEMI populations due to the generalization of the
use of troponin measurements. Indeed, introduction of troponin measurements led
to the reclassification of many unstable angina patients into NSTEMI patients,
with a 33% increase of the population of NSTEMIs from before to after the
introduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis
led to similar findings, with the exception that the increase in CABG was no
longer significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of
between-hospital transfers and the anonymization of the data (which precluded
the follow-up of the patients) complicated the interpretation of the trends.
Still, restricting the analysis to patients fully managed in a single hospital
led to similar results. It was not possible to assess “true” seven-
and 30-day mortality rates as no follow-up data was available for the patients
discharged. The AMIS registry, which collects vital status data for AMI patients
hospitalized in the participating institutions, will be able to provide such
information [35]. Finally, there is no published information on the validity of
hospital discharge data in Switzerland regarding the diagnosis of AMI. Still,
using the results from an ongoing study (CoLaus), of 159 hospitalizations for
AMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,
a value in agreement with the literature [36,37].
Intensive care unit:
The percentage of hospitalizations with ICU stay remained stable between 1998 and
2008 in Switzerland. This stabilization might be the consequence of improved
management, requiring less ICU admissions before or after a revascularization
intervention. Another likely explanation is the increasing number of transfers,
the patients being stabilized before being transferred to another hospital for
revascularization. Still, considerable differences in the proportion of
hospitalizations with ICU and corresponding trends were found between regions,
suggesting that other factors such as the number of ICU beds available or local
options for AMI management might be at play. Unfortunately there is no available
information regarding the number of ICU beds in each region, so the precise
reasons for the regional differences in ICU admissions for AMI remain to be
further assessed.
Revascularisation procedures:
Stent use increased steeply from slightly over 1% in 1998 to nearly 40% in 2008,
a finding in agreement with the literature [26]. DES appeared in 2004 and rose steadily until 2006, when several
reports questioning their costs [27,28] and long term complications [29,30] were published. These reports slowed down DES use, but some Swiss
regions were more responsive than others. For instance, in the Leman area DES
use equaled but never exceeded bare stent use, while in South Switzerland
(Ticino) DES represented nearly all stents implanted since 2005. The reasons for
such regional differences are more likely due to local preferences or to local
agreements regarding DES cost than to decisions based on scientific
evidence.
The rate of CABG remained stable at 3% throughout the study period, a finding
also reported in other countries [3,6]. As for stents, very different trends according to the region were
found. For instance, South Switzerland (Ticino) showed a steep rise in CABG use,
starting in 1999 to reach an average rate of 7%, well above the Swiss average.
This trend might partly be explained by the creation in 1999 of new medical
centers with the capacity to carry out these interventions. Conversely, in
Eastern and Central Switzerland, hardly any CABG was performed until 2007,
likely the result of the lack of required infrastructure. Overall, our results
show that CABG rates evolved differently between Swiss regions, the most likely
explanations being the existence of infrastructures and local preferences for
certain types of revascularization procedures.
In-hospital mortality:
An overall decrease in seven-day in-hospital mortality was found for Switzerland,
a finding also reported elsewhere [8,16]. This trend was no longer significant after multivariate adjustment,
suggesting that most of the decrease in mortality is due to revascularization
interventions and the improved quality of treatment according to the newest
guidelines [9,10]. Restricting the analysis to patients who survived at least three
days led to similar findings, although some regions presented a slight increase
in mortality (Additional file 5: Table S1). The rising
prevalence of CV risk factors in Switzerland [31] could also contribute to lessen the decrease in AMI mortality [32], but it was not possible to adequately consider them in the
multivariate model as they are not reported systematically in the database.
Similarly, it was not possible to assess whether the patients presented with a
STEMI or a non-STEMI, and which drug treatments were provided during hospital
stay, all factors associated with in-hospital mortality [22,23,33]. Overall, our data suggest that the increasing number of
revascularization interventions performed among patients discharged with a main
diagnosis of AMI do not suffice to decrease in-hospital mortality in
Switzerland, a country presenting the lowest CVD mortality within Europe [1]. Further studies are mandatory to better assess the factors
associated with in-hospital mortality from AMI in order to optimize patient
management.
Study limitations:
This study has some limitations. Medicines are not included in the hospital
discharge database, thus precluding the assessment of their trends. This absence
might also explain the very low rates for thrombolysis, which are likely due to
the absence of coding rather than a true absence of use. Furthermore, we lack
data to assess whether the changes in thrombolytic therapy are related to
changes in reporting levels or are actually real changes. The ICD10 coding does
not distinguish STEMI from NSTEMI; hence, it is possible that part of the
increase in discharges from AMI during the study period might be related to the
change in the definition of NSTEMI populations due to the generalization of the
use of troponin measurements. Indeed, introduction of troponin measurements led
to the reclassification of many unstable angina patients into NSTEMI patients,
with a 33% increase of the population of NSTEMIs from before to after the
introduction of troponin measurements [34]. Still, including unstable angina (ICD10 code I200) in the analysis
led to similar findings, with the exception that the increase in CABG was no
longer significant and even decreased in some regions (Additional file 5: Table S1). The strong increase in the number of
between-hospital transfers and the anonymization of the data (which precluded
the follow-up of the patients) complicated the interpretation of the trends.
Still, restricting the analysis to patients fully managed in a single hospital
led to similar results. It was not possible to assess “true” seven-
and 30-day mortality rates as no follow-up data was available for the patients
discharged. The AMIS registry, which collects vital status data for AMI patients
hospitalized in the participating institutions, will be able to provide such
information [35]. Finally, there is no published information on the validity of
hospital discharge data in Switzerland regarding the diagnosis of AMI. Still,
using the results from an ongoing study (CoLaus), of 159 hospitalizations for
AMI, 145 (91%) were confirmed as such by an independent panel of cardiologists,
a value in agreement with the literature [36,37].
Conclusion:
In Switzerland, a steep rise in hospital discharges and in revascularization
procedures for AMI occurred between 1998 and 2008.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
CI made part of the statistical analyses and wrote most of the article; PMV collected
data, made part of the statistical analysis and wrote part of the article; FP
revised the article for important intellectual content. PMV had full access to the
data and is the guarantor of the study. All authors read and approved the final
manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/13/270/prepub
Supplementary Material:
Trends in intensive care unit (ICU) utilization for acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008, patients managed in a single hospital (in-house).
Results are expressed as percentage of patients discharged with a
diagnosis of acute myocardial infarction.
Click here for file
Trends in the use of drug-eluting and non-drug-eluting stents for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008, patients managed in a single hospital
(in-house). Results are expressed as percentage of patients discharged
with a diagnosis of acute myocardial infarction.
Click here for file
Trends in the use of coronary artery bypass graft (CABG) for acute
myocardial infarction in Switzerland, overall and by region, for the
period 1998–2008, patients managed in a single hospital
(in-house). Results are expressed as percentage of patients discharged
with a diagnosis of acute myocardial infarction.
Click here for file
Trends in seven-day in-hospital mortality rates from acute myocardial
infarction in Switzerland, overall and by region, for the period
1998–2008, patients managed in a single hospital (in-house).
Results are expressed as percentage of patients discharged with a
diagnosis of acute myocardial infarction.
Click here for file
Trends in seven-day and overall in-hospital mortality, Switzerland and
Swiss regions, for period 1998-2008
Click here for file
| Background:
Since the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap.
Methods:
Swiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed.
Results:
Overall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered.
Conclusions:
In Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates. | Background:
Cardiovascular Diseases (CVD) remain one of the major causes of deaths worldwide.
Switzerland has one of the lowest CVD mortality rates within Europe [1] with a sustained downward trend over the last decades [2]. Acute myocardial infarction (AMI) can be treated with various drug
therapies and revascularization interventions. Indeed, management of AMI, namely
regarding revascularization interventions, has evolved over the last decades [3-8], following the continuously renewed guidelines [9,10].
There are few data on the evolution of AMI management and outcome in Switzerland. A
study published in 2006 [8] based on data from 68 medical centers participating in the AMIS Plus
register (http://www.amis-plus.ch/Project.htm) assessed Swiss trends in
invasive treatment and outcome for the period 1997 to 2005. Still, it is unknown if
the findings from this study also apply to non participating centers, and whether
these trends are equally applied in all Swiss regions. Indeed, in Switzerland,
health policies rarely go down to clinical guidelines and, if they exist, they are
decided at the hospital or canton level, with little or no intervention from the
federal bodies. The purpose of this study was to assess the trends in AMI management
and outcome for the whole country and for the main administrative regions, using
data from the Swiss hospital discharge database for the period 1998–2008.
Conclusion:
In Switzerland, a steep rise in hospital discharges and in revascularization
procedures for AMI occurred between 1998 and 2008. | Background:
Since the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap.
Methods:
Swiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed.
Results:
Overall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered.
Conclusions:
In Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates. | 12,726 | 365 | [
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] | [CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY] | [CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY] | [CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY] | [CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY] | [CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY] | [CONTENT] Acute myocardial infarction | Coronary revascularization | Epidemiology | Switzerland | Trends | Drug-eluting stent | Coronary artery bypass graft | Hospital discharge [SUMMARY] | [CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY] | [CONTENT] Aged | Aged, 80 and over | Databases, Factual | Female | Hospital Mortality | Humans | Male | Middle Aged | Myocardial Infarction | Patient Discharge | Switzerland | Treatment Outcome [SUMMARY] | [CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY] | [CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY] | [CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY] | [CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY] | [CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY] | [CONTENT] mortality switzerland swiss | myocardial infarction managed | hospital mortality switzerland | switzerland health policies | infarction switzerland overall [SUMMARY] | [CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY] | [CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY] | [CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY] | [CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY] | [CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY] | [CONTENT] hospital | switzerland | patients | 2008 | 1998 | trends | mortality | myocardial | acute | myocardial infarction [SUMMARY] | [CONTENT] outcome | ami | study | decades | ami management outcome | management | plus | management outcome | amis plus | participating [SUMMARY] | [CONTENT] procedures | hospital | icd10 | length | length stay | discharge | patients | stay | code | age [SUMMARY] | [CONTENT] hospital | 2008 | switzerland | 1998 | trend | acute | myocardial | acute myocardial | patients | figure [SUMMARY] | [CONTENT] rise hospital discharges | revascularization procedures ami | rise hospital discharges revascularization | ami occurred | ami occurred 1998 | ami occurred 1998 2008 | steep rise hospital discharges | discharges revascularization procedures ami | discharges revascularization procedures | discharges revascularization [SUMMARY] | [CONTENT] hospital | patients | switzerland | 2008 | 1998 | ami | mortality | myocardial | trends | myocardial infarction [SUMMARY] | [CONTENT] hospital | patients | switzerland | 2008 | 1998 | ami | mortality | myocardial | trends | myocardial infarction [SUMMARY] | [CONTENT] the late nineties | AMI | Switzerland ||| [SUMMARY] | [CONTENT] Swiss | years 1998 to 2008 ||| AMI | I21 ||| [SUMMARY] | [CONTENT] 102,729 | AMI ||| Intensive Care Unit | 38.0% | 1998 | 36.2% | 2008 | 0.001 ||| 6.0% | 39.9% | 0.001 ||| 1.3% | 16.6% | 0.001 ||| 2004 | 23.5% | 2008 | 0.001 ||| 1.0% to 3.0% | 0.001 ||| 0.2% | 1.7% | 0.001 ||| seven-day | 8.0% to 7.0% | 0.01 | 11.2% | 10.1% ||| ||| [SUMMARY] | [CONTENT] Switzerland | AMI | between 1998 and 2008 ||| [SUMMARY] | [CONTENT] the late nineties | AMI | Switzerland ||| ||| years 1998 to 2008 ||| AMI | I21 ||| ||| 102,729 | AMI ||| Intensive Care Unit | 38.0% | 1998 | 36.2% | 2008 | 0.001 ||| 6.0% | 39.9% | 0.001 ||| 1.3% | 16.6% | 0.001 ||| 2004 | 23.5% | 2008 | 0.001 ||| 1.0% to 3.0% | 0.001 ||| 0.2% | 1.7% | 0.001 ||| seven-day | 8.0% to 7.0% | 0.01 | 11.2% | 10.1% ||| ||| ||| Switzerland | AMI | between 1998 and 2008 ||| [SUMMARY] | [CONTENT] the late nineties | AMI | Switzerland ||| ||| years 1998 to 2008 ||| AMI | I21 ||| ||| 102,729 | AMI ||| Intensive Care Unit | 38.0% | 1998 | 36.2% | 2008 | 0.001 ||| 6.0% | 39.9% | 0.001 ||| 1.3% | 16.6% | 0.001 ||| 2004 | 23.5% | 2008 | 0.001 ||| 1.0% to 3.0% | 0.001 ||| 0.2% | 1.7% | 0.001 ||| seven-day | 8.0% to 7.0% | 0.01 | 11.2% | 10.1% ||| ||| ||| Switzerland | AMI | between 1998 and 2008 ||| [SUMMARY] |
||||||
Clinician preferences for computerised clinical decision support for medications in primary care: a focus group study. | 31039120 | To improve user-centred design efforts and efficiency; there is a need to disseminate information on modern day clinician preferences for technologies such as computerised clinical decision support (CDS). | BACKGROUND | This study included focus groups and clinicians individually describing their ideal CDS. Three focus groups were conducted including prescribing clinicians from a variety of disciplines. Outcome measures included identification of favourable features and unintended consequences of CDS for chronic medication management in primary care. We transcribed recordings, performed thematic qualitative analysis and generated counts when possible. | METHODS | There were 21 participants who identified four categories of beneficial CDS features during the group discussion: non-interruptive alerts, clinically relevant and customisable support, presentation of pertinent clinical information and optimises workflow. Non-interruptive alerts were broadly defined as passive alerts that a user chooses to review, whereas interruptive were active or disruptive alerts that interrupted workflow and one is forced to review before completing a task. The CDS features identified in the individual descriptions were consistent with the focus group discussion, with the exception of non-interruptive alerts. In the individual descriptions, 12 clinicians preferred interruptive CDS compared with seven clinicians describing non-interruptive CDS. | RESULTS | Clinicians identified CDS for chronic medications beneficial when they are clinically relevant and customisable, present pertinent clinical information (eg, labs, vitals) and improve their workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. These features align with literature describing best practices in CDS design and emphasise those features clinicians prioritise, which should be considered when designing CDS for medication management in primary care. These findings highlight the disparity between the current state of CDS design and clinician-stated design features associated with beneficial CDS. | CONCLUSION | [
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] | 7062316 | Introduction | Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.
Clinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12
Unfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.
Understanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design. | Methods | The purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.
Each focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.
The focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.
Focus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.
The major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent. | Results | Twenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).
Clinician characteristics
Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.
Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.
Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.
Features of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)
Clinicians could draw/describe as many features as they wanted, including more than one CDS.
When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.
Features of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)
Clinicians could draw/describe as many features as they wanted, including more than one CDS.
Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.
One clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.
Participants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.
When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.
One clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.
Participants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them. | Open-ended discussion of beneficial features | When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.
One clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.
Participants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them. | [
"What is already known?",
"What does this paper add?",
"Open-ended discussion of beneficial features"
] | [
"There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\nEnd user input in CDS design increases the likelihood of CDS to be successful.",
"Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.",
"Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’."
] | [
null,
null,
null
] | [
"What is already known?",
"What does this paper add?",
"Introduction",
"Methods",
"Results",
"Open-ended discussion of beneficial features",
"Discussion"
] | [
"There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.\nEnd user input in CDS design increases the likelihood of CDS to be successful.",
"Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.\nClinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.",
"Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.\nClinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12\n\nUnfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.\nUnderstanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.",
"The purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.\nEach focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.\nThe focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.\nFocus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.\nThe major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent.",
"Twenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).\nClinician characteristics\n Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\n Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\nWhen asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.\nFeatures of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)\nClinicians could draw/describe as many features as they wanted, including more than one CDS.\n Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.\nWhen asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.\nOne clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.\nParticipants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.",
"Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.\n Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\nClinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.\nClinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.\nDespite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.\nThere was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.\nIn lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.\nAll clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.\n Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\nClinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.\nTo minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.\n Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\nParticipants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.\nFor CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.\n Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.\nClinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.\nClinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.\nClinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.\nClinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.",
"This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care and the need to apply these principles to realise the benefits of CDS, which are pervasive in healthcare, yet do not currently produce consistently positive outcomes. We identified four main categories of CDS features for chronic medication management that clinicians find beneficial: clinically relevant and customisable, presentation of pertinent information and optimisation of workflow. These categories of features align with the best practices in CDS design and highlight aspects that clinicians perceive to be more or less beneficial, which should be prioritised when designing and implementing future CDS for chronic medications in primary care.\nInterestingly, focus group participants expressed strong dislike for interruptive CDS alerts during the open group discussion, which was the most common CDS suggested when individuals were asked to design the ideal CDS. This discrepancy may be due to effects of peer influence during the group discussion or realisation that interruptive alerts were helpful when designed well and that non-interruptive alerts are more likely to be overlooked or ignored. The discussion of whether the alerts should be interruptive or not aligns with literature supporting improved use and effectiveness of interruptive CDS16 22 34 35 and non-interruptive CDS being less likely to be viewed and therefore less likely to be effective.16 22 24 36 37 However, whether an alert is interruptive or not is only one feature of CDS and as the focus group conversation progressed, clinicians discussed other features of CDS that would improve usefulness of an interruptive alert, such as timing of the alert and the ability to customise the alert. These features may have led individual clinicians to favour interruptive alerts if the design incorporated these features. Although not explicitly discussed during the focus groups, the sentiment regarding passive versus active CDS highlights the importance of potentially reserving active CDS for high priority or high-risk patient care situations13 38–40 and carefully designing active CDS such that the other features are optimised to minimise clinician frustrations.\nAlthough there was no consensus in the timing of alerts, the discussion supports efforts to accommodate variations in individual clinician workflow and to personalise CDS to clinical workflows. However, to date, CDS have not been personalised to individual workflows, given the complexity required to account for the highly variable clinical workflows. Future research is needed to evaluate feasibility of such personalisation to workflows. For non-interruptive alerts, personalisation could include designing the CDS such that clinicians can access the information in the alert at a variety of different time points (eg, before entering the patient chart and/or on order entry and/or when signing a note), whereas such an approach with interruptive alerts would increase the frequency of interruptions and augment alert fatigue. Thus, for interruptive alerts, individualised personalisation of the alert timing by the clinician to meet their workflow needs would be the ideal state, but has not been realised yet. The ideal timing of passive or active CDS facilitates clinicians using the CDS without them noticing it, which can be challenging to accomplish in many situations, but nonetheless should be the goal in CDS design. Often personalisation is limited by technical constraints of the CDS software or EHR, which is continually evolving and may be less of a barrier with time and creative solutions.\nTo improve relevance and optimise their workflow, clinicians want CDS to be smarter, synthesising and presenting pertinent patient-specific information, with actionable recommendations that generates automated text in their clinical documentation and patient instructions. These findings are consistent with published recommendations on the ‘grand challenges’ that must be overcome for CDS to reach their potential and positively impact healthcare.39 Summarising pertinent information such as labs, vitals and allergies makes it easier for clinicians to quickly evaluate the appropriateness of a CDS recommendation and actionable links, such as ordering medications or labs, makes it easy to take action immediately. Further, the ability to populate clinical documentation and patient instructions with the plan would decrease duplicate work. Clinicians were specifically interested in auto-population of patient instructions to include titration schedules, timing of lab monitoring and pertinent adverse effects. Although not specifically mentioned in the focus groups, the clinical documentation could include routine monitoring that would assist in planning follow-up care. Some clinicians also expressed interest in presenting medication cost information with CDS recommendations. Although there are efforts to integrate medication costs into EHRs and CDS,41 there is currently no published literature describing the efficacy of such efforts. However, in other situations, such as ordering laboratory tests, clinicians did change ordering behaviour when informed of associated costs,42–45 suggesting the same may be true with medication ordering.\nAnother desired feature of the CDS was flexibility in response options, which is aligned with best practice principles in user-interface design. At times, clinicians felt forced to answer in a way not aligned with their intentions or the clinical scenario and wanted the option to better explain their actions in the form of free text that could be viewed later to trigger recall of their decisions. They also strongly wanted the option to permanently disable or temporarily dismiss CDS to alert at a more opportune time of their choosing. Clinicians who believed CDS would improve usefulness felt the ability to customise the CDS to the clinical scenario and individual workflow was very important. Principles of user-interface design includes ensuring clear and actionable response options,13 but flexibility in options is not well emphasised in the literature and can be critical to support the often nuanced clinical scenarios.\nClinicians had some concerns about unintended consequences, especially related to alert fatigue, mindlessly following CDS and inaccurate or incomplete information being presented by CDS. To avoid these problems, clinicians suggested implementing systematic processes for evaluation of CDS and inclusion of optional information and references. While inclusion of references in CDS are key to building trust, there are many additional strategies recommended to cultivate trust.13 Prioritising CDS recommendations according to specificity, urgency and relevance minimises obtrusiveness and increases trust.13 As CDS becomes increasingly pervasive in healthcare and we drive towards a Learning Health System, provision of a rationale with an assessment of the certainty or quality of the recommendation is important to engender trust.46 Further, as CDS are modified in response to clinician feedback, it is important to monitor for unintended consequences. For example, converting all CDS to be passive or giving clinicians the ability to permanently ‘snooze’ all CDS could result in missed opportunities to optimise evidence-based care for patients.\nThe findings of this study are limited by generalisability, given that participants represent clinicians at a single large academic medical centre using one integrated EHR platform for over 5 years and are exposed routinely to CDS. The results may be less representative of clinicians with less experience using an EHR and CDS, or using a different EHR platform, despite conversations focusing on broad concepts related to CDS that were not specific to an individual EHR. Further, participants represent a convenience sample based on ongoing professional relationships with the study investigators, thus introducing selection bias. However, a notable strength is that participants represent a variety of disciplines across outpatient practice settings and various expertise, with varying degrees of clinical practice responsibilities.\nClinicians characterised CDS for chronic medications as beneficial when it is clinically relevant and customisable, presents pertinent clinical information (eg, labs, vitals) and optimises workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. The design features align with literature describing best practices in CDS design and emphasise features that primary care providers prioritise when using CDS for chronic medication use. Despite awareness of these best practices in CDS design, many CDS are not designed with application of these best practices, which is thought to be one reason for the mixed outcomes of CDS to date.14 19 47 48 This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care. When designing CDS for chronic medications in primary care, developers should consider these user-centred design features and continually re-evaluate CDS design as technical capabilities of CDS and EHRs become more sophisticated."
] | [
null,
null,
"intro",
"methods",
"results",
null,
"discussion"
] | [
"clinical decision support systems",
"electronic prescribing",
"primary health care"
] | What is already known?:
There are best practices in clinical decision support (CDS) design that increase likelihood of CDS to be successful.
End user input in CDS design increases the likelihood of CDS to be successful.
What does this paper add?:
Clinicians identified beneficial design features of CDS for chronic medications that align with best practices in CDS design, including CDS that are clinically relevant and customisable, present pertinent clinical information and optimise workflow.
Clinicians prefer CDS that are not interruptive to their workflow, but may recognise interruptive CDS are more effective when designed well.
Introduction:
Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.
Clinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12
Unfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.
Understanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.
Methods:
The purpose of the focus groups was to inform the optimal design of medication-related CDS for primary care within a large health system that includes academic, suburban, community and underserved settings. The health system cares for an estimated 3.5 million outpatients annually and has used one EHR platform (Epic) for the past 5 years. The Ottawa Decision Support Framework Needs Assessment25 and the grounded theory principle of saturation were used to conduct the focus group.26 To achieve saturation, we conducted three focus groups.27 28 To provide context, CDS for chronic heart failure (HF) medications in primary care was used as a case study during the focus groups. Chronic HF medications were chosen because there is a national need to improve prescribing to align with evidence-based recommendations29 and because these principles can be generalised to management of other chronic diseases in primary care.
Each focus group was planned to include five to 12 participants to facilitate effective discussion, which is aligned with best practices in focus group conduct.30 Eligible participants were prescribing clinicians from a variety of primary care practice settings whose workflow would be impacted by the CDS or who have pertinent clinical expertise (eg, cardiology). Clinical experts were invited to assist with thinking about more challenging clinical needs for the HF case study. The investigators identified participants by ongoing professional relationships and invited clinicians via email to participate based on clinician type (ie, physician, pharmacist, advanced practice provider), practice setting and clinical expertise to ensure broad representation.
The focus groups were 1.5 hours each and followed a moderator guide, led by an investigator (KET) with training and experience in conducting focus groups.30 Each session included five consecutive components: (1) overview of purpose and rationale for focus group and informed consent, (2) brief written clinician demographic questionnaire, (3) open-ended discussion of beneficial CDS features for chronic medication management in primary care, (4) participants describing in writing their ideal CDS for chronic medications on paper and (5) open-ended discussion of potential unintended consequences of the CDS. In the fifth component, individual descriptions of the ideal CDS served to validate the findings of the open discussion and evaluate features that resonated most with individuals, separate from influences from peers that can present in a group environment.
Focus groups were audio recorded and field notes were taken by an investigator (AGV) to identify key issues. Audio recordings were transcribed and clinicians de-identified prior to thematic analysis. Accuracy of the transcriptions was validated by an independent investigator (KET) who reviewed 20% of the transcription and by comparison with field notes. A thematic approach26 31 using ATLAS.ti software (V.7, Scientific Software Development GmbH) was used to analyse the transcripts by one investigator (JAN). The transcriptions were categorised into major themes iteratively using topic coding. Clinician demographics were also coded. Topic coding included inductive general categorisation of text from the transcripts into themes with some connection or pattern, with successive recoding for more specific subcategories.31 Throughout topic coding, interpretation or analytical conclusions were applied to assess the meaning of codes and emergent themes.31 A 20% sample of the coding was reviewed by a second independent investigator (KET) for validation.
The major categories identified in the open discussion of the focus groups were then used to evaluate the individual clinician written descriptions (component five). Characteristics of the individual descriptions were reviewed for consistencies and discrepancies of the categories identified in the open discussion of the focus groups. Counts were generated by tabulating instances of a written description describing categories discussed in the focus group and noting new themes. The written descriptions were reviewed by one clinical pharmacist-investigator (KET) with expertise in chronic medication management and CDS design. The focus group participants provided informed consent.
Results:
Twenty-one clinicians participated in the focus groups, including 11 primary care physicians, four advanced practice providers, four pharmacists and two cardiologists. The individual focus groups consisted of five, six and 10 participants each. Primary care clinician subspecialties included geriatrics, internal medicine and family medicine (table 1).
Clinician characteristics
Open-ended discussion of beneficial features Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.
Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.
Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Key components of individual CDS written descriptions When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.
Features of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)
Clinicians could draw/describe as many features as they wanted, including more than one CDS.
When asked to independently describe their ideal, medication-related CDS, clinicians generally incorporated features discussed during the group discussion (table 2). Most clinicians drew figures of the ideal CDS supplemented with written descriptions. Despite the sentiment of the group discussion, 12 clinicians described their ideal CDS as interruptive compared with seven who described a passive CDS. Of note, some clinicians drew more than one CDS and some expressed wanting both interruptive and passive CDS, whereas some clinicians did not clearly articulate whether the CDS they drew was interruptive or passive. Features most commonly incorporated into the descriptions were: (1) the CDS being patient specific (n=15 clinicians), (2) inclusion of pertinent summary information, including current medications (n=16 clinicians) and diagnostics (n=12 clinicians), (3) actionable links to place orders (n=13 clinicians) and (4) the ability to permanently disable or defer the alert to a later time (n=10 clinicians). Although six clinicians incorporated cost in their descriptions, two articulated they wanted it as optional information they could access on demand instead of having it presented on the CDS user interface.
Features of a medication-related clinical decision support (CDS) described individually by clinicians (N=21 clinicians)
Clinicians could draw/describe as many features as they wanted, including more than one CDS.
Unintended consequences When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.
One clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.
Participants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.
When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.
One clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.
Participants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them.
Open-ended discussion of beneficial features:
Clinicians described four main beneficial features during the open discussion: (1) non-interruptive alerts; (2) clinically relevant and customisable support; (3) summarisation of pertinent clinical information and (4) improving workflow.
Non-interruptive alerts Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinicians overwhelmingly asserted they did not want interruptive or active CDS alerts because they interrupted clinician workflow. The term non-interruptive alerts used by participants was broadly defined as passive alerts that a user chooses to review, whereas interruptive was defined as active or disruptive alerts that interrupted workflow and one is forced to review before completing a task.32 33 Each focus group deliberated on this point for a prolonged period of time compared with discussion of other CDS features. Clinicians repeatedly reported the alerts were “one more thing to get through” to complete a task, reporting interruptive alerts to be a barrier to completing their tasks. One clinician stated ‘…[the alert] not only disrupts your flow but it actually paralyzes you, that I think is the worst of all’.
Clinicians reported ‘alert fatigue’, referred to interruptive alerts as an ‘annoyance’ and stated an overwhelming number of alerts for ‘every patient’. They stated the alerts were not helpful, ‘tell you the obvious’ and often are redundant or irrelevant. Multiple clinicians stated the interruptive alerts were not ‘smart enough’ to recognise that a given task was already complete and sometimes fired multiple times after a task was completed. Some notable ‘irrelevant’ examples included a lactation warning for a 60-year-old patient and an outdated alert regarding foreign travel and exposure to the Ebola virus. Irrelevance was also noted when an alert was out of context with the reason for visit or clinician type.
Despite overwhelmingly negative attitudes towards interruptive alerts, clinicians in one of the three focus groups expressed some positive benefits of interruptive alerts, being careful to emphasise the infrequency of helpfulness. The clinicians reported a reminder alert was ‘sometimes’ helpful and one clinician stated ‘every once in a while I am brainlessly doing something and it pops up and I’m like, oh, oh, that’s helpful’. While not universally endorsed, some participants indicated they liked one interruptive alert for lung cancer screening and found it helpful, because it interrupted workflow at the right time and ‘it does the work for me’, including ability to place the order.
There was no consensus regarding the most appropriate timing of an interruptive alert. Roughly equal numbers of clinicians preferred the CDS to alert at different times such as: (1) the first opening of an encounter; (2) when ordering a medication or reviewing medications, (3) entering the patient’s visit diagnosis or (4) at the end of the encounter. Proponents of alerting on first opening the encounter wanted the information to inform their visit agenda, while opponents stated it was premature. Proponents of alerting on ordering a medication or entering a diagnosis believed it would improve context, whereas opponents expressed this would be too late because ordering tasks are often completed at the conclusion of the visit after a plan was already established. Proponents of alerts appearing at the end of the visit argued the alert serves as a double check while minimising workflow interruptions, whereas opponents stated this was too late. There was also concern that in situations involving medical residents, the attending physician might not see or receive the alert until after the patient was dismissed/discharged.
In lieu of interruptive alerts, clinicians indicated a strong preference for passive CDS. They want ‘a gentle reminder that doesn’t actually interrupt you’, or ‘just sort of there and available’. Many clinicians desired CDS they could access in a way that fits into their workflow. A number of clinicians desired CDS accessible before a patient’s visit and prior to opening an encounter, stating this would be more helpful in planning the visit. Clinicians stated they want summary information in the form of clinical dashboards and summative or snapshot screens. One clinician stated if CDS were ‘…more pro-active, preventative,…something that is not in that moment where you’re trying to just take care of the patient acutely, we could do more outreach and prevention’. Clinicians also expressed favourable views of checklists that could ‘default to fill in the [information] that the system already knew’.
All clinicians from a single focus group asserted that passive alerts would be used more frequently and be more effective than interruptive alerts, whereas clinicians in the other two focus groups felt passive alerts would be less widely seen and therefore less effective. Proponents of interruptive alerts noted they must be properly designed and implemented to be effective and minimise alert fatigue.
Clinically relevant and customisable support Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Clinicians wanted the option to make the CDS smarter, informing the CDS when to alert rather than interruptive CDS alerting all clinicians in the same way for all patients. Clinicians reported CDS might be better accepted if the CDS could be temporarily dismissed. One clinician stated, ‘if the patient is going through an acute thing and you don’t want to address [the interruptive alert] right now, say remind me in ninety days or something like that, I think would be helpful’. Clinicians also looked favourably on CDS with response options allowing them to permanently disable CDS for a specific patient whom they felt the recommendation would never be appropriate. However, clinicians wanted the option to explain why it is not appropriate so they can access these responses in the future as a means of documentation. Clinicians also wanted more flexibility of how to respond to CDS, stating ‘I always want to do something else’, other than the options given to select. Some clinicians suggested having an ‘other’ response option that allowed them to explain and prevent them from feeling forced to respond in a given way.
To minimise instances when the CDS is considered irrelevant, they desired the CDS to be patient specific and provide assistance only in the appropriate context, considering the setting and clinician. Clinicians expressed the desire to tailor the CDS based on the reason for visit. Many indicated they do not want to see a chronic-medication CDS alert for patients who are not assigned to their patient panel or is being seen for an acute reason. In addition, clinicians commented several times that they ‘know the patient better than the computer’, and want CDS to be smarter and patient specific, not disease specific.
Presentation of pertinent clinical information Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Participants wanted the CDS to present pertinent patient-specific information to inform their decision, such as pertinent laboratory results, vital signs, and drug allergy information. Having this information ‘saves you from going to Chart Review and pulling them out’. Participants reported having dates for information such as vitals or labs as a reminder to order new tests, if warranted, and to assess clinically meaningful trends, such as serum creatinine changes for acute kidney injury.
For CDS recommendations that are accepted, clinicians indicated they wanted additional information: information regarding monitoring would serve as a reminder and information on medication costs could improve selection of more affordable medications. Clinicians thought it would be helpful for the CDS to include actual or general costs of medications when making recommendations, which could improve patient adherence and avoid pharmacy requests for less expensive alternatives.
Optimisation of workflow Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Clinicians discussed the role of clinic staff filtering CDS that would otherwise be presented to the clinician. They suggested that clinic staff could handle some of the interruptive CDS while preparing the patient for the clinician’s visit, such as during medication reconciliation. Staff responses to CDS would then inform and streamline the CDS presented to clinicians. One clinician stated clinic staff could do ‘non-physician work that would free up physicians to do some of the higher level patient care management that we just sometimes don’t get to’.
Clinicians also requested easy access to calculations otherwise routinely done manually, such as daily morphine-equivalent doses and 10-year cardiovascular risk estimates. Participants emphasised that information be easy to find. It was recognised that some routine calculations, such as 10-year cardiovascular risk estimates, are available in existing EHRs, but the information was not conveniently located or it was challenging to recall the steps to access them.
Clinicians expressed interest in CDS that would populate text in their clinical documentation or patient instructions to make documentation easier. Clinical documentation and the patient instruction sections of the EHR encounter would automatically be populated to reflect the plan, counselling points and necessary follow-up including labs if they accepted a CDS recommendation to order a new medication for a patient. For example, accepting a CDS recommendation to start spironolactone would lead to standardised text populated in the clinical documentation reflecting the plan to start spironolactone, monitor pertinent labs and include patient instructions. Clinicians specifically expressed interest in CDS that would reduce clinician documentation burden by generating patient instructions, especially for medications requiring titration, with timing of monitoring and common adverse effects.
Clinicians also wanted the ability to take action within the CDS with the ‘least amount of clicks’ to make it easier to follow through with an accepted CDS recommendation. One clinician suggested a CDS recommendation ‘…could just say ‘would you like to order it?’ and you click ‘yes’ and boom, it just happens’ instead of needing to leave the current screen and enter the orders module. One clinician stated ‘if you save the clinician time, you'll always win’.
Discussion:
This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care and the need to apply these principles to realise the benefits of CDS, which are pervasive in healthcare, yet do not currently produce consistently positive outcomes. We identified four main categories of CDS features for chronic medication management that clinicians find beneficial: clinically relevant and customisable, presentation of pertinent information and optimisation of workflow. These categories of features align with the best practices in CDS design and highlight aspects that clinicians perceive to be more or less beneficial, which should be prioritised when designing and implementing future CDS for chronic medications in primary care.
Interestingly, focus group participants expressed strong dislike for interruptive CDS alerts during the open group discussion, which was the most common CDS suggested when individuals were asked to design the ideal CDS. This discrepancy may be due to effects of peer influence during the group discussion or realisation that interruptive alerts were helpful when designed well and that non-interruptive alerts are more likely to be overlooked or ignored. The discussion of whether the alerts should be interruptive or not aligns with literature supporting improved use and effectiveness of interruptive CDS16 22 34 35 and non-interruptive CDS being less likely to be viewed and therefore less likely to be effective.16 22 24 36 37 However, whether an alert is interruptive or not is only one feature of CDS and as the focus group conversation progressed, clinicians discussed other features of CDS that would improve usefulness of an interruptive alert, such as timing of the alert and the ability to customise the alert. These features may have led individual clinicians to favour interruptive alerts if the design incorporated these features. Although not explicitly discussed during the focus groups, the sentiment regarding passive versus active CDS highlights the importance of potentially reserving active CDS for high priority or high-risk patient care situations13 38–40 and carefully designing active CDS such that the other features are optimised to minimise clinician frustrations.
Although there was no consensus in the timing of alerts, the discussion supports efforts to accommodate variations in individual clinician workflow and to personalise CDS to clinical workflows. However, to date, CDS have not been personalised to individual workflows, given the complexity required to account for the highly variable clinical workflows. Future research is needed to evaluate feasibility of such personalisation to workflows. For non-interruptive alerts, personalisation could include designing the CDS such that clinicians can access the information in the alert at a variety of different time points (eg, before entering the patient chart and/or on order entry and/or when signing a note), whereas such an approach with interruptive alerts would increase the frequency of interruptions and augment alert fatigue. Thus, for interruptive alerts, individualised personalisation of the alert timing by the clinician to meet their workflow needs would be the ideal state, but has not been realised yet. The ideal timing of passive or active CDS facilitates clinicians using the CDS without them noticing it, which can be challenging to accomplish in many situations, but nonetheless should be the goal in CDS design. Often personalisation is limited by technical constraints of the CDS software or EHR, which is continually evolving and may be less of a barrier with time and creative solutions.
To improve relevance and optimise their workflow, clinicians want CDS to be smarter, synthesising and presenting pertinent patient-specific information, with actionable recommendations that generates automated text in their clinical documentation and patient instructions. These findings are consistent with published recommendations on the ‘grand challenges’ that must be overcome for CDS to reach their potential and positively impact healthcare.39 Summarising pertinent information such as labs, vitals and allergies makes it easier for clinicians to quickly evaluate the appropriateness of a CDS recommendation and actionable links, such as ordering medications or labs, makes it easy to take action immediately. Further, the ability to populate clinical documentation and patient instructions with the plan would decrease duplicate work. Clinicians were specifically interested in auto-population of patient instructions to include titration schedules, timing of lab monitoring and pertinent adverse effects. Although not specifically mentioned in the focus groups, the clinical documentation could include routine monitoring that would assist in planning follow-up care. Some clinicians also expressed interest in presenting medication cost information with CDS recommendations. Although there are efforts to integrate medication costs into EHRs and CDS,41 there is currently no published literature describing the efficacy of such efforts. However, in other situations, such as ordering laboratory tests, clinicians did change ordering behaviour when informed of associated costs,42–45 suggesting the same may be true with medication ordering.
Another desired feature of the CDS was flexibility in response options, which is aligned with best practice principles in user-interface design. At times, clinicians felt forced to answer in a way not aligned with their intentions or the clinical scenario and wanted the option to better explain their actions in the form of free text that could be viewed later to trigger recall of their decisions. They also strongly wanted the option to permanently disable or temporarily dismiss CDS to alert at a more opportune time of their choosing. Clinicians who believed CDS would improve usefulness felt the ability to customise the CDS to the clinical scenario and individual workflow was very important. Principles of user-interface design includes ensuring clear and actionable response options,13 but flexibility in options is not well emphasised in the literature and can be critical to support the often nuanced clinical scenarios.
Clinicians had some concerns about unintended consequences, especially related to alert fatigue, mindlessly following CDS and inaccurate or incomplete information being presented by CDS. To avoid these problems, clinicians suggested implementing systematic processes for evaluation of CDS and inclusion of optional information and references. While inclusion of references in CDS are key to building trust, there are many additional strategies recommended to cultivate trust.13 Prioritising CDS recommendations according to specificity, urgency and relevance minimises obtrusiveness and increases trust.13 As CDS becomes increasingly pervasive in healthcare and we drive towards a Learning Health System, provision of a rationale with an assessment of the certainty or quality of the recommendation is important to engender trust.46 Further, as CDS are modified in response to clinician feedback, it is important to monitor for unintended consequences. For example, converting all CDS to be passive or giving clinicians the ability to permanently ‘snooze’ all CDS could result in missed opportunities to optimise evidence-based care for patients.
The findings of this study are limited by generalisability, given that participants represent clinicians at a single large academic medical centre using one integrated EHR platform for over 5 years and are exposed routinely to CDS. The results may be less representative of clinicians with less experience using an EHR and CDS, or using a different EHR platform, despite conversations focusing on broad concepts related to CDS that were not specific to an individual EHR. Further, participants represent a convenience sample based on ongoing professional relationships with the study investigators, thus introducing selection bias. However, a notable strength is that participants represent a variety of disciplines across outpatient practice settings and various expertise, with varying degrees of clinical practice responsibilities.
Clinicians characterised CDS for chronic medications as beneficial when it is clinically relevant and customisable, presents pertinent clinical information (eg, labs, vitals) and optimises workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. The design features align with literature describing best practices in CDS design and emphasise features that primary care providers prioritise when using CDS for chronic medication use. Despite awareness of these best practices in CDS design, many CDS are not designed with application of these best practices, which is thought to be one reason for the mixed outcomes of CDS to date.14 19 47 48 This study reinforces the modern day relevance of the best practice principles in CDS design to CDS for chronic medications in primary care. When designing CDS for chronic medications in primary care, developers should consider these user-centred design features and continually re-evaluate CDS design as technical capabilities of CDS and EHRs become more sophisticated.
| Background:
To improve user-centred design efforts and efficiency; there is a need to disseminate information on modern day clinician preferences for technologies such as computerised clinical decision support (CDS).
Methods:
This study included focus groups and clinicians individually describing their ideal CDS. Three focus groups were conducted including prescribing clinicians from a variety of disciplines. Outcome measures included identification of favourable features and unintended consequences of CDS for chronic medication management in primary care. We transcribed recordings, performed thematic qualitative analysis and generated counts when possible.
Results:
There were 21 participants who identified four categories of beneficial CDS features during the group discussion: non-interruptive alerts, clinically relevant and customisable support, presentation of pertinent clinical information and optimises workflow. Non-interruptive alerts were broadly defined as passive alerts that a user chooses to review, whereas interruptive were active or disruptive alerts that interrupted workflow and one is forced to review before completing a task. The CDS features identified in the individual descriptions were consistent with the focus group discussion, with the exception of non-interruptive alerts. In the individual descriptions, 12 clinicians preferred interruptive CDS compared with seven clinicians describing non-interruptive CDS.
Conclusions:
Clinicians identified CDS for chronic medications beneficial when they are clinically relevant and customisable, present pertinent clinical information (eg, labs, vitals) and improve their workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. These features align with literature describing best practices in CDS design and emphasise those features clinicians prioritise, which should be considered when designing CDS for medication management in primary care. These findings highlight the disparity between the current state of CDS design and clinician-stated design features associated with beneficial CDS. | Introduction:
Computerised clinical decision support systems (CDS) are intended to assist clinicians in clinical decision-making and thereby improve quality of healthcare provided.1 In light of the growing body of medical information and the difficulty of clinicians to truly stay up to date, CDS offers a powerful means to intelligently support clinicians in their clinical decision-making. Implementation of CDS across a variety of settings has led to improvements in patient care processes, healthcare costs, use of preventative medicine and adherence to standards of medical practice.2–4 Although CDS are widely used and have led to some positive outcomes, there are also numerous examples of CDS leading to no or negative changes in outcomes.5–8 One key reason for the mixed results is poor clinician adoption.
Clinician engagement or adoption is crucial to the effectiveness of CDS. To be engaged, clinicians must perceive CDS to be useful or beneficial. As stated by the National Academy of Medicine (formerly Institute of Medicine), technologies such as CDS should be designed to make it ‘easy to do the right thing’.9 To improve clinician adoption and the success of CDS, there are established best practice principles for CDS design, which includes tailoring CDS to the preferences of the end user and integration with their workflows.1 10 Incorporation of input from end users regarding CDS content, presentation and functionality is one important step to achieving this goal stated by the National Academy of Medicine and is referred to as user-centred or human-centred design.11 12
Unfortunately, the best practice principles for CDS design are inconsistently followed and there is limited literature describing preimplementation end user perceptions, needs or preferences of CDS for chronic medications in primary care. Several publications describe CDS features that improve end user engagement and outcomes and many of which have formed the basis for the best practice principles in CDS design.13–18 However, these findings are largely based on reviewing publications describing specific features of CDS that were successful or unsuccessful or focused on identification of barriers to using CDS. Further, CDS developers seeking to implement CDS have generally sought end user feedback on clinical content or validation in the form of usability testing following initial CDS design prototyping.19–22 Such input after prototyping introduces potential bias in end-user feedback of what the end product could be. While there are published examples of obtaining end-user input into the design of a paper-based CDS prior to prototyping,23 those results may not apply to electronic CDS workflows. Much of the literature and best practices in CDS design also represents findings from a time period when electronic health records (EHRs) and CDS were new or not a standard part of clinical workflow, which may not be relevant to today when EHRs and CDS are commonplace to clinical workflows.
Understanding the current day preferences and needs of clinicians for CDS targeting chronic medications in primary care and comparison to established best practices in CDS design can be used to optimise design and adoption by end users. This information is critical given inconsistent application of best practices in CDS design and the extensive resources invested in designing CDS despite numerous published examples of CDS that result in suboptimal clinical outcomes. Of CDS implemented, medication-focused CDS is one of the most common types.24 Here, we describe current clinician perceptions regarding beneficial features of CDS for chronic medications in primary care prior to prototyping and implementation as one component of a user-centred CDS design.
Open-ended discussion of beneficial features:
When asked about potential unintended consequences of CDS for a chronic medication, clinician concerns were related to alert fatigue and blindly following the CDS without appropriate knowledge, and/or incomplete or inaccurate information.
One clinician stated, ‘in a lot of ways we are creating clinicians that don’t necessarily know pathophysiology and patient assessment and what they are learning is algorithms for treating patients and best practice alerts that tell them what to do’. This sentiment was not only in reference to clinicians in general but also with particular reference to medical residents. The clinicians acknowledged that uniform acceptance of the CDS recommendations ‘can lead to a lot of harm’, especially if the recommendation was based on inaccurate or incomplete information. It was believed this has a negative effect on their rapport with the patient. To prevent the thoughtless acceptance of CDS recommendations, participants indicated a need for recommendations to include situations where the recommendations should not be accepted and links to supporting evidence.
Participants also reported situations where alert fatigue has led to negative health outcomes. They expressed need for CDS, especially interruptive alerts, to be systematically evaluated for efficacy and end user use to facilitate needed revisions or discontinue them. | Background:
To improve user-centred design efforts and efficiency; there is a need to disseminate information on modern day clinician preferences for technologies such as computerised clinical decision support (CDS).
Methods:
This study included focus groups and clinicians individually describing their ideal CDS. Three focus groups were conducted including prescribing clinicians from a variety of disciplines. Outcome measures included identification of favourable features and unintended consequences of CDS for chronic medication management in primary care. We transcribed recordings, performed thematic qualitative analysis and generated counts when possible.
Results:
There were 21 participants who identified four categories of beneficial CDS features during the group discussion: non-interruptive alerts, clinically relevant and customisable support, presentation of pertinent clinical information and optimises workflow. Non-interruptive alerts were broadly defined as passive alerts that a user chooses to review, whereas interruptive were active or disruptive alerts that interrupted workflow and one is forced to review before completing a task. The CDS features identified in the individual descriptions were consistent with the focus group discussion, with the exception of non-interruptive alerts. In the individual descriptions, 12 clinicians preferred interruptive CDS compared with seven clinicians describing non-interruptive CDS.
Conclusions:
Clinicians identified CDS for chronic medications beneficial when they are clinically relevant and customisable, present pertinent clinical information (eg, labs, vitals) and improve their workflow. Although clinicians preferred passive, non-interruptive alerts, most acknowledged that these may not be widely seen and may be less effective. These features align with literature describing best practices in CDS design and emphasise those features clinicians prioritise, which should be considered when designing CDS for medication management in primary care. These findings highlight the disparity between the current state of CDS design and clinician-stated design features associated with beneficial CDS. | 14,639 | 341 | [
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||||||
Rab11 regulates E-cadherin expression and induces cell transformation in colorectal carcinoma. | 25117932 | In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation. | BACKGROUND | For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy. | METHODS | In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration. | RESULTS | This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment. | CONCLUSIONS | [
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] | 4137074 | Background | Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].
E-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.
As the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].
Vesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.
Colorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study. | Methods | Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.
Under TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.
The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.
Under TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.
Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).
HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).
Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.
The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.
Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.
Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.
Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.
HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.
Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.
Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.
Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.
Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed. | Results | Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.
In the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
In 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).
When E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
No. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.
In the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
In 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).
When E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
No. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view. | Conclusions | Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future. | [
"Background",
"Patients and ethics statements",
"Cell culture and transfection",
"Immunohistochemistry",
"Western blots",
"Trans-well cell migration assay",
"Immunofluorescence microscopy",
"Statistics",
"Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues",
"Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells",
"Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration",
"Rab11 induced colon cell transformation"
] | [
"Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.",
"The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.",
"HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).",
"The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.",
"Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.",
"HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.",
"Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.",
"Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.",
"This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.",
"To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.",
"In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.",
"HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Patients and ethics statements",
"Cell culture and transfection",
"Immunohistochemistry",
"Western blots",
"Trans-well cell migration assay",
"Immunofluorescence microscopy",
"Statistics",
"Results",
"Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues",
"Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells",
"Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration",
"Rab11 induced colon cell transformation",
"Discussion",
"Conclusions"
] | [
"Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].\nE-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.\nAs the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].\nVesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.\nColorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.",
" Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\nThe study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.\n Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\nHT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).\n Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\nThe tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.\n Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\nTissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.\n Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\nHT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.\n Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\nCells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.\n Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.\nResults are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.",
"The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.\nUnder TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.",
"HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).",
"The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.",
"Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.",
"HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.",
"Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.",
"Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.",
" Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\nThis study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\nTo examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\nIn order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\nHT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.",
"This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.\nIn the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\n\nE-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.\nIn 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).\nWhen E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\nNo. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.\n\nE-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients\n\nThe symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.",
"To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.\n\nRab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.",
"In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.\n\nRab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.",
"HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.\n\nRab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.",
"E-cadherin, which functions as an adhesion junction, has been demonstrated to be an important marker in cancer biology, as disassembly of E-cadherin is required for epithelial cell transformation and migration. E-cadherin is bound with most of the β-catenin located at the cytosolic side and also interacts with the actin cytoskeleton. β-catenin is the key component of the Wnt pathway that can be stimulated for cell growth and proliferation [10]. E-cadherin has also been shown to regulate initial cell-cell contacts formation and can further recruit exocyst components to the contacts for the formation of cell surface polarity [24]. Recently, the dark side of E-cadherin has been revealed gradually in pathological study of different cancers. The increased expression of E-cadherin was found to be associated with the formation of epithelium tumors. E-cadherin may play an important role in “collective cell migration” [25] and provide the “anchorage-independent” property of cancer cells [9]. In our study, significant over-expression of E-cadherin was found in colorectal cancer tissues. These findings are consistent with the previous study of Truant et al. [5], who demonstrated that the expression ratio of E-cadherin in reference to the normal adjacent tissue was increased in 57% of primary tumors. In contrast, only 30% of specimens were decreased in terms of the expression of E-cadherin in colorectal carcinoma. Regarding liver metastasis, a significantly higher expression of E-cadherin was found at stage I ~ II than at stage III ~ IV [6]. Therefore, the authors of the study suggested that the role of high expression of E-cadherin in colorectal cancer cells may be protective against widespread metastasis.\nAs Rab11, which functions as a recycling endosome, has been reported to play a role in regulating E-cadherin turnover in vitro, dysregulation may be associated with cancer formation. Our data subsequently demonstrated that the expression of Rab11 was also increased in colorectal cancer tissues. Co-overexpression of E-cadherin and Rab11 was found in 65.5% (74/113) of colorectal carcinomas, which suggests that dysregulation of both molecules might be associated with the occurrence and progression of colorectal carcinoma. The correlations of expressions of E-cadherin and Rab11 with stages of colorectal carcinoma were not statistically significant. However, the number of E-cadherin and Rab11 co-expressing cases was decreased in advanced stage cancers (81.8% in stage I, but 50% in stage IV). As we know, pathological progression from early adenomatous proliferation through adenomatous polyp, high grade dysplasia and ultimately, invasive colorectal carcinoma to metastasis occurs as a continuum. This progression coincides with the accumulation of multiple genetic alterations during neoplastic progression as originally described by Fearon and Volgelstein [26]. The regulatory alteration of E-cadherin and Rab11 may vary dynamically in the multistep model of progression of colorectal carcinoma in different stages.\nE-cadherin may participate in tumor progression through its associated cellular mechanisms. In epithelial cells, β-catenin acts as a linker between transmembrane E-cadherin and cytosolic actin fibers and forms a junctional adhesion complex. β-catenin is either stable connected to E-cadherin or translocated into the nucleus and binds to Lef/Tcf transcription factor upon stimulation for cell proliferation. Free cytosol β-catenin is degraded through ubiquitin-mediated degradation. Therefore, dynamic regulation of membrane E-cadherin and Rab11 may be necessary for cell proliferation and tumor growth. Rab11 was suggested to play a role in E-cadherin recycling and enhance membrane E-cadherin dynamics, which may be involved in cell signaling for cancer cell growth.\nThe in vitro experiments used GFP-Rab11 plasmid overexpressed in HT-29 cells induced cell transformation and migration. Intense staining of E-cadherin and Rab11 were also observed in infiltrated tumor nest cells. Actin dynamics are required for cell motility, and it has also been demonstrated that Rab11 interacts with RTK down-stream target Rac1 and controls moesin activity to regulate the endocytic cycle and actin cytoskeleton in cell migration and collective cell migration [27–29]. Rac1 has been shown to regulate actin nucleation via neural WASP (N-WASP) and the down-stream Arp2/3 complex [30]. A recent study has also shown that Rac1 activation is involved in twist1-induced cell transformation and migration [31]. Taken together, Rab11 plays a role not just in E-cadherin turnover but also improves the cytoskeleton reorganization for cell migration.\nDiscovering markers for cancer progression is important; however, the formation of cancer is multi-stepped, and specific protein expressions are restricted and responsible for different steps of EMT. Cell migration is an integrated process that requires continuous, coordinated formation and disassembly of adhesions. These processes are complex and require regulated interaction of numerous transcription factors such as snail/slug or twist, and activation of specific signaling pathways [32–35]. In this study, the expression of Rab11 was shown to be associated with cancer formation, and the expression of Rab11 also induced cell transformation, which is associated with cell motility. Moreover, Rab 11 could upregulate the expression of E-cadherin. Thus, Rab11 may be a useful maker together with E-cadherin for the diagnosis of colorectal cancer progression. However, the roles of other transcription factors in this mechanism have not been elucidated and require further investigation.\nIn the report of Anastasiadis et al. [9], several possible mechanisms were suggested to explain the positive role of E-cadherin in tumor progression. First, E-cadherin may cross-talk with EGFR signaling. Second, increased E-cadherin expression may up-regulate the expression of anti-apoptotic proteins, such as Bcl-2 and Bcl-xL. Third, E-cadherin may trigger the activation of the PI3K/AKT pathway through p85. In addition, extracellular N-terminal E-cadherin ectodomain “shedding” could be a role in tumor-promoting activities [9]. Our results showing that Rab11 up-regulated E-cadherin to induce the transformation of cancer cells might be another mechanism of alteration of neoplastic progression by E-cadherin.",
"Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future."
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] | [
"Rab11",
"E-cadherin",
"Colorectal carcinoma",
"Vesicle recycling",
"Epithelial mesenchymal transition"
] | Background:
Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].
E-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.
As the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].
Vesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.
Colorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.
Methods:
Patients and ethics statements The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.
Under TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.
The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.
Under TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.
Cell culture and transfection HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).
HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).
Immunohistochemistry The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.
The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.
Western blots Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.
Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.
Trans-well cell migration assay HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.
HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.
Immunofluorescence microscopy Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.
Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.
Statistics Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.
Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.
Patients and ethics statements:
The study group consisted of 113 consecutive patients (age range, 24–93 years old, median age, 59 years old, 65 male, 48 female) who had undergone resection for localized colorectal cancer from April 1997 to December 2003 at Ching-Cheng General Hospital, Taiwan. The protocol was reviewed and approved by the Ching-Cheng General Hospital Institutional Review Board (HT110018). Written informed consent was obtained from all patients. Archival paraffin-embedded samples were used to build up tissue microarray blocks in the Department of Medical Technology of Yuanpei University in 2008. Patients with inflammatory disease, infection, bowel obstruction or perforation were excluded. Tumors were located in the ascending colon in 21 patients (19%), transverse colon in 6 patients (5%), descending colon in 5 patients (4%), sigmoid colon in 26 patients (23%) and rectum in 55 patients (49%). All primary cancerous tissues were excised.
Under TNM (AJCC, 7th ed.) classification, 11 patients had stage I disease, 42 patients had stage II disease, 52 patients had stage III disease and 8 patients had stage IV disease. Colorectal carcinoma specimens and uninvolved mucosa specimens were obtained during surgery. All protein expression assessments for this study were carried out without knowledge of the pathological data.
Cell culture and transfection:
HT-29 and SW 480 colon cells (ATCC, VA, USA) were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin and streptomycin (GIBCO-BRL, Gaithersberg, MD, USA) and kept in an incubator under 5% CO2 at 37°C. For transfection, cells were grown on 24-well plates in normal growth medium without antibiotics, and Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) was used for GFP-tagged Rab11 wild-type, dominant negative (DN) mutant (Addgene, MA, USA) and Rab11 shRNA plasmid (RANi core, Academia Sinica, Taiwan) transfection. Cells were analyzed 24 hr post-transfection, and the efficacy of transfection was confirmed by immunoblot analysis of cell lysates using a rabbit anti-GFP antibody (abcam, MA, USA).
Immunohistochemistry:
The tissue specimens were first fixed in 4% paraformaldehyde for 2 hrs. After dehydration, specimens were then embedded in paraffin blocks. 5-μm-thick paraffin sections were cut and deparaffinized in xylene substitute and rehydrated in graded alcohols and distilled water. Antigen retrieval was achieved by heating the samples without boiling in 0.01 M citrate buffer, pH 6.0, with 0.1% tween 20. This treatment was conducted twice for 10 min. The sections were washed in double distilled water (ddH2O). The endogenous peroxide was blocked by 0.3% hydrogen peroxide in methanol for 10 min. The sections were then incubated with E-cadherin (1:150) (BD Biosciences, USA) or Rab11 (1:80) antibodies (Cell signaling technology, MA, USA) at room temperature for 1 hr. A histostain-SP DAB kit (Invitrogen) was then used to reveal the primary antibody; the secondary antibody (reagent 1B in DAB kit) was incubated with the sections for 10 min. After washing in ddH2O thrice for 2 min, the sections were then incubated with streptavidin-peroxidase conjugate (reagent 2 in DAB kit) for 10 min. After washing, the final staining was performed in diaminobenzidine tetrahydrochloride (DAB) solution (reagent 3A-3C in 1 ml ddH2O) for 5 min. The nuclei were counterstained with Mayer’s hematoxylin (reagent 4 in DAB kit) for 3 min. After washing with ddH2O, the slides were then transferred through an ascending ethanol series (95%, 100%) and xylene substitute before mounting. The scoring used for immunohistochemistry was the “I” index [6], the equation for which is I = 0*f0 + 1*f1 + 2*f2 + 3*f3, where f0-f3 are the fractions of the cells showing a defined level of staining intensity (from 0–3); the numbers 0–3 represent the following: “0” negative, no detectable staining, “1” weak, but still detectable staining, “2” moderate, clearly positive but still weak; and “3” heavy and intense staining.
Western blots:
Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.
Trans-well cell migration assay:
HT-29 cells were transfected with GFP-Rab11 or Rab11 shRNA. After 48 hours, cells were trypsinized into trans-well insert (BD Biosciences) for cell migration assay. Transfected cells were transferred to the upper chamber of the trans-well insert that with 8 μm pore size and containing serum-free medium. Cells were allowed to migrate for 12 hours toward the bottom chamber which was filled with normal serum medium. Cells remaining on the upper membrane were removed by cotton swab. The migrated cells on the bottom side were fixed and stained with DAPI nuclear dye. The migrated cells were then revealed by fluorescence microscope and counted for quantification.
Immunofluorescence microscopy:
Cells grown on glass coverslips were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton-X 100. For the transfection experiment, cells were first grown on cover slips for 24 hrs and then transfected with GFP-tagged Rab11 wild-type or dominant negative plasmid for an additional 24 hrs. The fixed cells were incubated with mouse anti-E-cadherin antibody (1:100 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr and then incubated with Cy3 conjugated anti-mouse secondary antibodies (1:200 dilution in PBS/0.1% Triton-100/3% BSA) at room temperature for 1 hr. Coverslips were mounted with Gel Mount aqueous mounting medium (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss LSM 510 META confocal microscope with a 63× objective (1.4 oil). To analyze the cell morphology changes for transformation, cells were scanned by a laser confocal microscope with z-sections for 3D image construction of the side view. The transfected cells chosen for scanning were either localized inside the cell colony or on the margin of the island.
Statistics:
Results are expressed as mean ± standard deviation. Chi-squared tests were used to compare categorical variables. The Student t-test was used to compare continuous variables. Differences at the p <0.05 level were considered statistically significant. For cell culture experiments, at least three independent experiments were performed.
Results:
Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.
In the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
In 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).
When E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
No. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.
In the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
In 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).
When E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
No. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 induced colon cell transformation HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Elevated E-cadherin and Rab11 expressions were revealed in colon tumor tissues:
This study first examined the E-cadherin and Rab11 expression patterns in nine colon carcinoma patients. Tumor tissue and non-tumor tissue including the distal and proximal ends of colon tissues from the same patient were collected. Tissues were processed for immunohistochemistry (IHC) with antibodies against E-cadherin and Rab11.
In the IHC results, the mucosa cells of non-tumor tissue, which was indicated to be a normal tissue, exhibited the basal level expression of E-cadherin in most of the cell membrane; weak expression of Rab11 was detected in the epithelial cytoplasm. In colon tumor tissue, serious proliferation of the cancer cells was observed, and E-cadherin was found to be intensively expressed in the cell membrane; the Rab11 expression was also found to be increased in cancerous cells (Figure 1a). The intensities of E-cadherin and Rab11 expressions in tissues were scored and quantified using the “I” index as described in Methods, and the results showed that the expressions of E-cadherin and Rab11 in tumor tissues were significantly higher than in normal tissues in the preliminary 9 cases (Figure 1b). Tissue proteins were also extracted for Western blot analysis of E-cadherin and Rab11 expressions. Samples of one of the patients, demonstrated in Figure 1c, showed that E-cadherin and Rab11 were both highly expressed in tumor tissue. By Western blot analysis, the expression levels of E-cadherin and Rab11 were quantified in the initial 9 patients (Figure 1d). By immunocytochemistry, infiltrated tumor nests were also found in the stroma of colon tumor tissue, and E-cadherin and Rab11 were stained intensively in these infiltrated tumor cells (Figure 1e). These data suggested that E-cadherin and Rab11 might play important roles in colon tumor cell transformation and migration based on pathological evidence. In order to examine the relationship between E-cadherin and Rab11 expressions in colorectal tumors with statistical significance, tissue array chips were created from 113 patients and IHC staining with E-cadherin and Rab11 antibodies was performed. The quantified results showed that the expressions of E-cadherin and Rab11 (Figure 1f) were both higher in tumor parts than in non-tumor parts. The mean scores of E-cadherin in the tumor and normal mucosa were 1.41 ± 0.06 and 1.08 ± 0.06, respectively, which were significantly different (p < 0.05). In addition, the mean scores of Rab11 in the tumor and normal mucosa were 0.51 ± 0.05 and 0.18 ± 0.02, which were also significantly different between the two groups (p < 0.05).Figure 1
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
E-cadherin and Rab11 expressions in colon tumor tissues. (a) Colon tumor tissue and non-tumor (indicated to be normal) tissue samples from the same patient were processed for immunohistochemistry with antibodies against E-cadherin or Rab11; micrographs were taken using a Zeiss light microscope. Scale bar = 50 μm. (b) E-cadherin and Rab11 expressions in tissues from 9 patients were quantified by “I” index scoring as described in Methods. (c) Protein extracts from colon tumor tissue and non-tumor tissue were subjected to SDS-PAGE and Western blot analysis to determine the expression levels of E-cadherin and Rab11. GAPDH was used as the internal loading control. (d) Quantification of E-cadherin and Rab11 protein expressions in 9 patients by Western blot assay. Value = mean ± SD, * p < 0.05. (e) Immunohistochemistry study of colon tumor tissue showed the E-cadherin and Rab11 expressions in colon tumor infiltrated cells. The asterisk indicates the tumor region, arrows indicate the tumor infiltrated cell nest, and the arrowhead indicates a disarranged gland-like structure. Scale bar = 50 μm. (f) Colorectal tumor and non-tumor (indicated to be normal) tissue paraffin sections from 113 patients were mounted on glass slides as a tissue microarray. E-cadherin and Rab11 were detected by immunohistochemistry. The expressions of E-cadherin and Rab11 were scored and quantified by the “I” index as described in Methods. Value = mean ± SD, * p < 0.05.
In 84 patients (74.3%), the E-cadherin expression was higher in tumor tissues than in non-tumor tissues. The specimens of stage IV disease had the largest percentage of over-expression (87.5% versus 12.5%). The Rab11 expression was higher in tumor tissues than in non-tumor tissues in 100 cases (88.5%), especially specimens of stage III tumors, which showed the largest percentage of over-expression of Rab11 (94.2% versus 5.8%).
When E-cadherin and Rab11 were combined together for observation, over-expression of both proteins was presented in 74 patients (65.5%). Solitary over-expression of Rab11 was found in 26 patients (23.0%) and solitary over-expression of E-cadherin was found in 10 patients (8.9%). Only 3 patients (2.7%) lacked any over-expression of the two proteins. Furthermore, all of these patients were classed as stage I or II. However, there was no significant difference between the stages with different expressions of E-cadherin or Rab11 (p = 0.09). These results suggested that the elevation of E-cadherin or Rab11 plays an important role in colorectal carcinoma formation regardless of stage (Table 1).Table 1
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
No. of cases, (%)E-cadherin + Rab11+E-cadherin - Rab11 +E-cadherin + Rab11-E-cadherin - Rab11 -Stage I9/11(81.82%)1/11(9.09%)0/11(0%)1/11(9.09%)Stage II23/42 (54.76%)13/42 (30.95%)4/42 (9.52%)2/42 (4.76%)Stage III38/52 (73.08%)11/52 (21.15%)3/52 (5.77%)0/52 (0%)Stage IV4/8 (50.00%)1/8 (12.50%)3/8 (37.50%)0/8 (0%)Total patients74/113 (65.49%)26/113 (23.01%)10/113 (8.85%)3/113 (2.65%)The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
E-cadherin and Rab11 expression patterns in 113 colorectal carcinoma patients
The symbols (+) and (-) indicate the expression level according to tissue scoring in the tumor part being higher or lower as compared with normal tissue.
Proportional expressions of E-cadherin and Rab11 proteins were associated with epithelium morphology in cultured HT-29 colon cells:
To examine the functions of E-cadherin and Rab11 in vitro, colon cell line HT-29 was used in this study. HT-29 cells have been demonstrated to be an ideal cell model for cell differentiation [23]. The phenotype of HT-29 was first examined and compared with another colon cell line, SW480. Cells grown on coverslips were fixed, and the E-cadherin expression was analyzed by immunofluorescent microscopy. An organized membrane E-cadherin staining pattern was revealed in HT-29 cells; however, the distribution of E-cadherin was shown to be diffused in SW480 cells (Figure 2a). The Western blot results showed greater Rab11 and E-cadherin protein expressions in HT-29 cells than in SW480 cells (Figure 2b). The results suggested that the proportional expression levels of junctional adhesion protein E-cadherin and recycling endosome protein Rab11 are associated with colon cell morphology.Figure 2
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 and E-cadherin expressions in cultured colon cells. (a) HT-29 and SW480 colon cells were grown on coverslips, cells were fixed and stained with E-cadherin antibody, and E-cadherin was then revealed by Cy2-conjugated secondary antibody and observed by confocal microscopy. (b) HT-29 and SW480 cell lysates were collected and subjected to SDS-page. E-cadherin and Rab11 were detected by Western blot, and GAPDH was used as the internal loading control.
Rab11 overexpression in colon cells up-regulated E-cadherin expression and induced membrane protrusion and cell migration:
In order to determine the effects of Rab11 in colon cell morphogenesis, GFP-tagged Rab11 plasmid was overexpressed in HT-29 colon cells. Western blot results showed that the E-cadherin expression was up-regulated in Rab11-overexpressing cells (Figure 3a, b). Rab11 overexpression also promoted cell migration significantly (Figure 3c, p < 0.01). When cells were treated with shRNA that targets Rab11, the depletion of Rab11 expression suppressed E-cadherin expression (3d, 3e), and inhibited cell migration (Figure 3f, p < 0.001). HT-29 cells transfected with GFP-tagged wild-type Rab11 plasmid were stained with E-cadherin antibody and analyzed by immunofluorescent microscopy. The results showed that GFP-Rab11 was distributed in the cell leading edge and had induced the cell membrane protrusion. When the Rab11-overexpressing cell was localized at the margins of the cell colony and started to migrate out of the colony, the expression of E-cadherin in the cell membrane was shown to be decreased (Figure 3g). When the GFP-Rab11-overexpressing cell was migrating towards the center of the cell colony, the transfected cells still showed Rab11-induced membrane protrusion; however, E-cadherin was stained intensively in the cell-cell contacts (Figure 3h). These results demonstrated that the overexpression of Rab11 induced HT-29 membrane protrusion and resulted in two distinct E-cadherin expression patterns depending on the location of the cells: E-cadherin was down-regulated when the cell was migrating out of the cell colony, but E-cadherin was strengthened on the cell membrane when the cell was migrating in the cell colony between neighbor cells.Figure 3
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 overexpression induced E-cadherin expression and cell migration in HT-29 cells. HT-29 cells were transfected with GFP-tagged Rab11 plasmid (a-c) or plasmid constructed with Rab11 shRNA (d-f). After 24 hr, cell lysates were collected and subjected to Western blot analysis for GFP-Rab11, E-cadherin and Rab11 expressions using anti-GFP, anti-E-cadherin and anti-Rab11 antibodies, respectively. GAPDH was used as the internal loading control. Quantifications of E-cadherin expression in GFP-Rab11 overexpressed cells (b) and in Rab11 knockdown cells (e) are shown from at least three independent experiments. Value = mean ± SD, * p < 0.05. Trans-well migration assay for Rab11 overexpression (c) and knockdown cells (f) were quantified. Value = mean ± SD were from three independent experiments, ** p < 0.01; *** p < 0.001. HT-29 cells grown on glass coverslips were transfected with GFP-Rab11; cells were then fixed and stained with anti-E-cadherin antibody, followed by staining with Cy3 conjugated secondary antibody. (g) GFP-Rab11-overexpressing cell migration out of the cell colony or (h) migration towards the center of the cell colony was observed by confocal microscopy. Arrowheads indicate cell membrane protrusion.
Rab11 induced colon cell transformation:
HT-29 cells transfected with Rab11 as shown in Figure 3f were further analyzed by a confocal microscope with z-sections. A 3D image was composed from the z-section images. The side view image showed that the Rab11 overexpressed cells (Figure 4a) induced cell transformation, resulting in cell migration; the Rab11 overexpressed cells migrated beneath a neighbor cell (see 3D side view). However, when cells were transfected with GFP-tagged dominant negative (DN) Rab11, the GFP-Rab11 DN was evenly distributed in the cytosol and did not induce cell transformation and migration (Figure 4b). This result indicated that overexpression of Rab11 induced HT-29 cell transformation, which was dependent on Rab11 activity.Figure 4
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Rab11 overexpression promoted cell transformation. (a) GFP-Rab11 wild type and (b) GFP-Rab11 dominant negative over-expressing HT-29 cells were stained with E-cadherin antibody and analyzed by a confocal microscope as described in Figure 3h. The Z-section scanning images in positions A and B (as indicated in the diagram above) are shown, and Z-stacked images were composed for a 3D side view.
Discussion:
E-cadherin, which functions as an adhesion junction, has been demonstrated to be an important marker in cancer biology, as disassembly of E-cadherin is required for epithelial cell transformation and migration. E-cadherin is bound with most of the β-catenin located at the cytosolic side and also interacts with the actin cytoskeleton. β-catenin is the key component of the Wnt pathway that can be stimulated for cell growth and proliferation [10]. E-cadherin has also been shown to regulate initial cell-cell contacts formation and can further recruit exocyst components to the contacts for the formation of cell surface polarity [24]. Recently, the dark side of E-cadherin has been revealed gradually in pathological study of different cancers. The increased expression of E-cadherin was found to be associated with the formation of epithelium tumors. E-cadherin may play an important role in “collective cell migration” [25] and provide the “anchorage-independent” property of cancer cells [9]. In our study, significant over-expression of E-cadherin was found in colorectal cancer tissues. These findings are consistent with the previous study of Truant et al. [5], who demonstrated that the expression ratio of E-cadherin in reference to the normal adjacent tissue was increased in 57% of primary tumors. In contrast, only 30% of specimens were decreased in terms of the expression of E-cadherin in colorectal carcinoma. Regarding liver metastasis, a significantly higher expression of E-cadherin was found at stage I ~ II than at stage III ~ IV [6]. Therefore, the authors of the study suggested that the role of high expression of E-cadherin in colorectal cancer cells may be protective against widespread metastasis.
As Rab11, which functions as a recycling endosome, has been reported to play a role in regulating E-cadherin turnover in vitro, dysregulation may be associated with cancer formation. Our data subsequently demonstrated that the expression of Rab11 was also increased in colorectal cancer tissues. Co-overexpression of E-cadherin and Rab11 was found in 65.5% (74/113) of colorectal carcinomas, which suggests that dysregulation of both molecules might be associated with the occurrence and progression of colorectal carcinoma. The correlations of expressions of E-cadherin and Rab11 with stages of colorectal carcinoma were not statistically significant. However, the number of E-cadherin and Rab11 co-expressing cases was decreased in advanced stage cancers (81.8% in stage I, but 50% in stage IV). As we know, pathological progression from early adenomatous proliferation through adenomatous polyp, high grade dysplasia and ultimately, invasive colorectal carcinoma to metastasis occurs as a continuum. This progression coincides with the accumulation of multiple genetic alterations during neoplastic progression as originally described by Fearon and Volgelstein [26]. The regulatory alteration of E-cadherin and Rab11 may vary dynamically in the multistep model of progression of colorectal carcinoma in different stages.
E-cadherin may participate in tumor progression through its associated cellular mechanisms. In epithelial cells, β-catenin acts as a linker between transmembrane E-cadherin and cytosolic actin fibers and forms a junctional adhesion complex. β-catenin is either stable connected to E-cadherin or translocated into the nucleus and binds to Lef/Tcf transcription factor upon stimulation for cell proliferation. Free cytosol β-catenin is degraded through ubiquitin-mediated degradation. Therefore, dynamic regulation of membrane E-cadherin and Rab11 may be necessary for cell proliferation and tumor growth. Rab11 was suggested to play a role in E-cadherin recycling and enhance membrane E-cadherin dynamics, which may be involved in cell signaling for cancer cell growth.
The in vitro experiments used GFP-Rab11 plasmid overexpressed in HT-29 cells induced cell transformation and migration. Intense staining of E-cadherin and Rab11 were also observed in infiltrated tumor nest cells. Actin dynamics are required for cell motility, and it has also been demonstrated that Rab11 interacts with RTK down-stream target Rac1 and controls moesin activity to regulate the endocytic cycle and actin cytoskeleton in cell migration and collective cell migration [27–29]. Rac1 has been shown to regulate actin nucleation via neural WASP (N-WASP) and the down-stream Arp2/3 complex [30]. A recent study has also shown that Rac1 activation is involved in twist1-induced cell transformation and migration [31]. Taken together, Rab11 plays a role not just in E-cadherin turnover but also improves the cytoskeleton reorganization for cell migration.
Discovering markers for cancer progression is important; however, the formation of cancer is multi-stepped, and specific protein expressions are restricted and responsible for different steps of EMT. Cell migration is an integrated process that requires continuous, coordinated formation and disassembly of adhesions. These processes are complex and require regulated interaction of numerous transcription factors such as snail/slug or twist, and activation of specific signaling pathways [32–35]. In this study, the expression of Rab11 was shown to be associated with cancer formation, and the expression of Rab11 also induced cell transformation, which is associated with cell motility. Moreover, Rab 11 could upregulate the expression of E-cadherin. Thus, Rab11 may be a useful maker together with E-cadherin for the diagnosis of colorectal cancer progression. However, the roles of other transcription factors in this mechanism have not been elucidated and require further investigation.
In the report of Anastasiadis et al. [9], several possible mechanisms were suggested to explain the positive role of E-cadherin in tumor progression. First, E-cadherin may cross-talk with EGFR signaling. Second, increased E-cadherin expression may up-regulate the expression of anti-apoptotic proteins, such as Bcl-2 and Bcl-xL. Third, E-cadherin may trigger the activation of the PI3K/AKT pathway through p85. In addition, extracellular N-terminal E-cadherin ectodomain “shedding” could be a role in tumor-promoting activities [9]. Our results showing that Rab11 up-regulated E-cadherin to induce the transformation of cancer cells might be another mechanism of alteration of neoplastic progression by E-cadherin.
Conclusions:
Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future.
| Background:
In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation.
Methods:
For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy.
Results:
In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration.
Conclusions:
This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment. | Background:
Most tumors are epithelial based cell types. Epithelial Mesenchymal Transition (EMT) is thought to be a marker of tumor progression and metastasis. Normal epithelial cells express cadherin, catenin and other junctional adhesion proteins in the areas of cell-cell contacts; however, tumor cells that express mesenchymal markers have a greater tendency to be invasive and metastasize [1].
E-cadhrin has been considered to be a “tumor suppressor” marker, as the breakdown of cell-cell contacts promotes cell transformation and further migration. However, recent evidence demonstrated a promoting role of high expression of E-cadherin in aspects of tumor progression. An unexpected high expression of E-cadherin in tumor progression was observed in aggressive brain tumor [2] and in inflammatory breast carcinoma; E-cadherin was identified as being involved in the pathogenesis of advanced breast carcinoma [3, 4]. It has also been demonstrated in clinical studies that the E-cadherin and β-catenin mRNA levels were increased in colon cancer progression and in liver metastasis [5]. E-cadherin protein expression and localization have also been found to be increased in primary colorectal tumors [6]. However, the related biological meaning and the underlying cellular mechanism are still under investigation.
As the process of metastasis involves transformation of epithelial cells between EMT and MET, the expression of E-cadherin is regulated dynamically and does not just act in the role of tumor suppressor [7]. Recent reports have also pointed towards an alternative role of E-cadherin in carcinogenesis, which suggests that it may not just be that of a “sticky” molecular complex in between cells – the dysregulated over-expression of E-cadherin may participate in tumor progression through its associated cellular mechanisms [8–10]. In epithelial cells, cadherins and catenins form strong cell-cell contacts and are also dependent on vesicle-mediated intracellular transport. Continual trafficking of E-cadherin to form the cell junction is essential for morphogenesis [11, 12]. Increases of E-cadherin endocytosis and recycling have been shown to be correlated with cancer progression [13, 14].
Vesicles transport mediated through the endocytic system including endocytosis and recycling is controlled primarily by small GTPases of the Rab family [15]. Different Rab proteins are localized in cellular compartments and regulate distinct vesicles and endosome transport routes. It has been demonstrated that Rab proteins were associated with cancer metastasis [16–18]. Rab11 has been shown to function in recycling endosome movement to the membrane and regulate epithelial cell polarity [19, 20], and also been demonstrated to be related to hypoxia-stimulated cell invasion in breast carcinoma [21]. Hence, dysregulation of the expressions of Rab proteins may be an important component of human carcinogenesis, and a recent study also illustrated that Rab11-mediated recycling endosome is required for E-cadherin trafficking during epithelial morphogenesis. Active Rab11 can carry E-cadherin to the cell-cell contacts; however, the Rab11 inactive form fails to regulate recycling endosome for E-cadherin membrane targeting [22]. Although in vitro studies have demonstrated that Rab11 can regulate E-cadherin membrane targeting, its role in cancer cell transformation is still not clear, and the relationship with the tumor suppressor role of E-cadherin is still controversial.
Colorectal cancer is one of the major causes of death worldwide, and the E-cadherin expression dynamics may be critical in colorectal tumor progression. Thus, we speculated that Rab11-mediated E-cadherin turnover is an important mechanism in colorectal tumor formation. In this study, the expressions of E-cadherin and Rab11 were examined pathologically in colorectal tumor specimens, and Rab11 was also over-expressed in cultured colon cells for in vitro transformation study.
Conclusions:
Our data for the first time demonstrated that Rab11 regulated E-cadherin expression and promoted colon cancer cell transformation. Overexpression of both E-cadherin and Rab11 was also detected in the majority of colorectal carcinoma samples. Regulation of the dual protein motifs might facilitate targeting of the progression of colorectal carcinoma in the future. | Background:
In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation.
Methods:
For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy.
Results:
In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration.
Conclusions:
This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment. | 15,522 | 329 | [
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] | [CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY] | [CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY] | [CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY] | [CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY] | [CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY] | [CONTENT] Rab11 | E-cadherin | Colorectal carcinoma | Vesicle recycling | Epithelial mesenchymal transition [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Cadherins | Cell Line, Tumor | Cell Transformation, Neoplastic | Colorectal Neoplasms | Epithelial-Mesenchymal Transition | Female | Gene Expression Regulation, Neoplastic | HT29 Cells | Humans | Male | Middle Aged | rab GTP-Binding Proteins [SUMMARY] | [CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY] | [CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY] | [CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY] | [CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY] | [CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY] | [CONTENT] cadherin tumor progression | cadherin trafficking epithelial | role cadherin carcinogenesis | epithelium tumors cadherin | metastasis cadherin protein [SUMMARY] | [CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY] | [CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY] | [CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY] | [CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY] | [CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY] | [CONTENT] rab11 | cadherin | cell | cells | tumor | cadherin rab11 | expression | colon | patients | tissue [SUMMARY] | [CONTENT] tumor | cadherin | progression | cell | epithelial | tumor progression | role | recycling | metastasis | regulate [SUMMARY] | [CONTENT] patients | usa | cells | min | transfection | mm | dab | reagent | incubated | sections [SUMMARY] | [CONTENT] rab11 | cadherin | tumor | cadherin rab11 | cells | cell | tissue | expression | figure | expressions [SUMMARY] | [CONTENT] colorectal carcinoma | colorectal | carcinoma | promoted colon cancer cell | regulation dual | targeting progression | regulated cadherin expression promoted | targeting progression colorectal | targeting progression colorectal carcinoma | overexpression cadherin rab11 detected [SUMMARY] | [CONTENT] cadherin | rab11 | cells | cell | tumor | patients | cadherin rab11 | expression | colon | gfp [SUMMARY] | [CONTENT] cadherin | rab11 | cells | cell | tumor | patients | cadherin rab11 | expression | colon | gfp [SUMMARY] | [CONTENT] EMT ||| ||| [SUMMARY] | [CONTENT] 113 ||| GFP [SUMMARY] | [CONTENT] 1.41 ± | 0.06 | 1.08 ± | 0.06 ||| 0.05 ||| 0.51 ± | 0.05 | 0.18 | 0.02 | 0.05 ||| 74 | 66.5% ||| GFP | HT-29 [SUMMARY] | [CONTENT] ||| [SUMMARY] | [CONTENT] EMT ||| ||| ||| 113 ||| GFP ||| ||| 1.41 ± | 0.06 | 1.08 ± | 0.06 | 0.05 ||| 0.51 ± | 0.05 | 0.18 | 0.02 | 0.05 ||| 74 | 66.5% ||| GFP | HT-29 ||| ||| [SUMMARY] | [CONTENT] EMT ||| ||| ||| 113 ||| GFP ||| ||| 1.41 ± | 0.06 | 1.08 ± | 0.06 | 0.05 ||| 0.51 ± | 0.05 | 0.18 | 0.02 | 0.05 ||| 74 | 66.5% ||| GFP | HT-29 ||| ||| [SUMMARY] |
||||||
Reexamining treatment of high-grade T1 bladder cancer according to depth of lamina propria invasion: a prospective trial of 200 patients. | 25535728 | Management of high-grade T1 (HGT1) bladder cancer represents a major challenge. We studied a treatment strategy according to substaging by depth of lamina propria invasion. | BACKGROUND | In this prospective observational cohort study, patients received initial transurethral resection (TUR), mitomycin-C, and BCG. Subjects with shallower lamina propria invasion (HGT1a) were followed without further surgery, whereas subjects with HGT1b received a second TUR. Association of clinical and histological features with outcomes (primary: progression; secondary: recurrence and cancer-specific survival) was assessed using Cox regression. | METHODS | Median age was 71 years; 89.5% were males, with 89 (44.5%) cases T1a and 111 (55.5%) T1b. At median follow-up of 71 months, disease progression was observed in 31 (15.5%) and in univariate analysis, substaging, carcinoma in situ, tumour size, and tumour pattern predicted progression. On multivariate analysis only substaging, associated carcinoma in situ, and tumour size remained significant for progression. | RESULTS | In HGT1 bladder cancer, the strategy of performing a second TUR only in T1b cases results in a global low progression rate of 15.5%. Tumours deeply invading the lamina propria (HGT1b) showed a three-fold increase in risk of progression. Substaging should be routinely evaluated, with HGT1b cases being thoroughly evaluated for cystectomy. Inclusion in the TNM system should also be carefully considered. | CONCLUSIONS | [
"Adult",
"Aged",
"Aged, 80 and over",
"Carcinoma in Situ",
"Carcinoma, Squamous Cell",
"Cohort Studies",
"Cystectomy",
"Female",
"Humans",
"Male",
"Middle Aged",
"Mucous Membrane",
"Neoplasm Grading",
"Neoplasm Invasiveness",
"Neoplasm Recurrence, Local",
"Reoperation",
"Urinary Bladder Neoplasms",
"Urinary Tract"
] | 4453654 | null | null | null | null | Results | Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.
Recurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.
The association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.
Progression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.
The low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.
Finally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest. | Conclusions | With this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy. | [] | [] | [] | [
"Materials and methods",
"Results",
"Discussion",
"Conclusions"
] | [
"Trial design and inclusion of this prospective observational cohort study began in April 2005. Since then, patients diagnosed at initial TUR with HGT1 BC were offered entry into this protocol. Transurethral resection BT included complete resection of all visible tumours, with a separate base biopsy. Cold cup biopsies to detect CIS were taken, depending on surgeon preference, in a standardised manner or according to EAU Guidelines (abnormal urothelium, non-papillary tumour appearance, or positive cytology) (Babjuk et al, 2013). All patients received a postoperative dose of intravesical mitomycin-C.\nTumours were graded according to the 2004 WHO system (2006) after pathological assessment of the total specimen. A two-tier system (Cottrell et al, 2007) was used to assess the depth of LP invasion: T1a when tumour involved the subepithelial connective tissue superficial to muscularis mucosae (MM); T1b when tumour was found at the level of or beyond MM. When MM was not identifiable, thick-walled blood vessels deep in LP served as a landmark, or alternatively location of MM in tumour-free tissue of the same TUR was used for reference. Only initial HGT1 tumours with a visible, clearly identifiable and disease-free muscularis propria were included in this study. Patients with LP invasion (T1) but without muscularis propria in the specimen represent ∼10% of all T1 in our centre. These cases, identified as Tx, did not fulfill the requirements to enter this protocol and, following guidelines, directly underwent a repeat resection.\nThis protocol was approved by the Hospital Ethics Committee and registered at clinicaltrials.gov (NCT02113501), and last evaluation of patients was performed in February 2014. Enrolled subjects received six weekly instillations of 2–8 × 108 UFC BCG-Tice (OncoTICE, Schering-Plough Canada Inc., Kirkland, QC, Canada) and, at 3 months after initial TUR, underwent either a second TUR post-BCG (T1b) or a cystoscopy plus cytology (T1a). Follow-up included BCG maintenance; the strategy is summarised in Figure 1 and described in detail in our previous report (Orsola et al, 2010). Baseline characteristics were compared between the T1a and T1b groups using Fisher's exact test.\nThe primary objective was the assessment of progression, recurrence, and CSS. Progression was defined as occurrence of an invasive tumour at post-BCG TUR, the development of muscle invasive or more advanced stage carcinoma, distant metastasis, or death from BC. Time to progression was defined as time from diagnosis of BC to the date when disease progression was observed, or censored on the last known date alive without progression. Recurrence was defined as the first evidence of any NMIBC detected on follow-up. Time to recurrence was defined as time from diagnosis of BC to the date when disease recurrence was observed, or censored at the last known date alive without disease recurrence, or at date of progression. Cancer-specific survival was defined as death from the same cancer or related causes.\nThe secondary objective was the assessment of prognostic factors. Eleven clinical and pathological variables were prospectively documented to evaluate their capacity to predict events. Kaplan–Meier analysis was used to summarise median time to recurrence and time to progression. Association of time to recurrence, time to progression, and CSS with the variables of interest was assessed using Cox regression models in univariate and multivariable models, reporting hazard ratios (HRs) and 95% CI.",
"Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.\nRecurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.\nThe association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.\nProgression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.\nThe low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.\nFinally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest.",
"In HGT1 BC, the proposed strategy of deferring a second TUR until after induction of BCG and doing so only in T1b cases, results in a global low progression rate of 15.5%. Cases in which tumours invaded the MM (T1b) had a three-fold increased risk of progression compared with those without MM invasion (T1a), even though the latter group was treated more conservatively. Our report goes on to confirm the role of CIS, tumour size, and tumour pattern as prognostic factors for worse outcomes in this group of high-risk non-muscle invasive BC.\nTo the best of our knowledge, this is the first report addressing the role of substaging HGT1 BC prospectively to guide therapeutic management. This novel strategy, together with the relatively large number of cases of initial HGT1, the long follow-up and the consistent treatment approach are the main strengths of our series. In addition, the evaluation of depth of tumour invasion was performed prospectively after TUR as part of the routine pathological report, whereas most reports on HGT1 are retrospective and observational. We cannot rule out bias due to patient selection since cases with recurrent tumours and those with no muscularis propria identified were not included; our series might also include some G2 cases (WHO 1974 classification) under the broader definition of HG BC. On the other hand, tumours that could have been understaged (early progressors or positive second TUR) were not excluded from the analysis in the understanding that this represents a more generalisable real-world practice, but might also be seen as a bias. Finally, our results are limited by the fact that the study was performed in a non-randomised manner. This limitation is widespread in the high-risk BC literature as most reports have retrospective observational designs, with few randomised studies (Martin-Doyle et al, 2014). Given the public nature of our health-care system, we chose to differentially allocate patients to treatment, using a more aggressive approach for T1b tumours, aiming to optimise resources.\nThe role of substaging has been well established throughout the past 20 years, with over 2500 published cases substaged according to invasion of the MM (Rouprêt et al, 2013). The relevance of this factor has been recently confirmed in the largest meta-analysis ever conducted on prognostic factors in HGT1 (Martin-Doyle et al, 2014). Because the evaluation of the depth of invasion is subject to interobserver and intraobserver variability, this parameter has been criticised for the last two decades, but there is growing evidence on the reproducibility of this evaluation. In a study specifically addressing this issue, Cottrell et al (2007) elegantly showed a high concordance rate among general pathologists after guidance based only in a presentation of the staging systems as described by the SIGN guideline. In their report, the median K concordance rate was 0.623, which is higher than that attained for Gleason score among pathologists (Bottke et al, 2013). In a more recent report, Rouprêt et al (2013) showed a reclassification rate for substaging of only 16% after central review of 587 HGT1 BC tumour sets from a multicentre data set. Other methods to substage HGT1 have been described using either millimetric evaluation of the depth of invasion (Cheng et al, 1999; Chang et al, 2012), measuring the size of the infiltrative tumour area as ⩽ or > 1 high-power field (Cheng et al, 1999; Bertz et al, 2011) or describing the extent of invasion (microinvasive or extensive) (van der Aa et al, 2005; van Rhijn et al, 2012). These alternative systems, however, have all been studied retrospectively in single data sets of patients and have not yet been externally validated (Martin-Doyle et al, 2014). A head-to-head comparison among these approaches in a large data set unfortunately is unavailable to date. Nonetheless, the fact that substaging remained a robust prognostic factor in our series (three-fold increase risk of progression in the adjusted multivariable model, and five-fold increase in the univariate model), even though patients with HGT1a underwent a more conservative approach demonstrates the magnitude of negative impact associated with depth of invasion.\nWe report a 15.5% progression rate, which is lower than the recently published pooled results of ∼20% progression rate for this high-risk group of tumours (van den Bosch and Alfred Witjes, 2011; Gontero et al, 2014; Martin-Doyle et al, 2014). Progression rates for high-risk BC have typically been considered to be over 30% and up to 50%, but more current data support that approximately one in five cases progress and will typically do so within 48 months of initial diagnosis (van den Bosch and Alfred Witjes, 2011). Our approach of proceeding with the second TUR after the 6-week induction of BCG (Orsola et al, 2010) was also used by Denzinger et al (2007) with an overall progression rate of 41% in 132 patients. We contend that the better outcome in our series might be attributable to a complete, deep, and radical initial TUR together with the exclusion of cases without clear muscularis propria in the TURBT specimen. It is possible the use of BCG (induction and maintenance) and the second TUR have also contributed an added therapeutic benefit. The combined influence of all these factors might explain the low rate of understaging we found in the second TUR (9.9%) as well as the low rate of positive findings at 3 months (Orsola et al, 2010), but the limitations mentioned previously should also be taken into account. Mitomycin-C might have only played a minor role given that no impact on progression has been demonstrated with this agent in all randomised published trials. Also the proposed approach in delaying the second TUR makes it impossible to accurately differentiate between true understaging and early invasive recurrence.\nOur study goes on to corroborate previous observations that CIS is a strong prognostic factor with a two-fold increase in risk of progression (Sylvester et al, 2006). In our report, the impact was even stronger with a 0.23 HR favouring lack of CIS. Interestingly, we observed a high rate of detection in those patients who, according to surgeon preference, underwent multiple random bladder biopsies. This adds to the debate of whether routine bladder biopsies, which we have previously defended (Orsola et al, 2010), should be standardised at the time of initial BC TUR (Millan-Rodriguez et al, 2000). The usefulness of random bladder biopsies in the broad population of NMIBC is controversial (Kiemeney et al, 1994), but our findings suggest that a high positive yield is obtained in HGT1 cases. Alternatively, the diagnosis of CIS is based on its detection in either peritumoral areas of the specimen or if suspicious areas are biopsied. This approach, currently recommended by guidelines (American Urological Association, 2007; Babjuk et al, 2013), is highly limited by interpretation and likely leads to underdetection of true CIS, even though fluorescence-guided TURBT might improve identification of suspicious areas of CIS (Witjes et al, 2014). Finally, the findings of our study suggest that larger tumours and those with a solid pattern are associated with a worse outcome in HGT1, are consistent with the relevance of these parameters in the broader NMIBC population. Combining these factors we observed that patients with T1b tumours that were larger than 3 cm and associated with CIS were 4.8 times more likely to progress than the rest.\nWe failed to demonstrate a worse prognosis for LVI and for females in any of the outcomes. The incidence of LVI was very low (under 5%) and, because its effects have been noted to correlate with depth of LP invasion, it is unclear whether LVI independently predicts outcome, or whether its detrimental impact is already captured by pathologic information on substaging (Martin-Doyle et al, 2014). Regarding gender, large population-based studies may be required to confirm a worse prognosis of females that has been suggested previously (Palou et al, 2012).\nIn summary, our report proposes a novel approach for HGT1 BC based on the selection of patients who will most benefit from a second TUR, specifically those with deep invasion of the MM (T1b). The main clinical implication of separately identifying almost half of HGT1 cases with T1a substaging as having a low (4%) progression rate is that they can be spared a second endoscopic procedure with no harm. More accurate risk stratification incorporating depth of invasion, associated CIS, and tumour size would enable improved resource allocation for reTUR. It would also help identify optimal candidates for prompt cystectomy, namely T1b tumours larger than 3 cm and with associated CIS. If the one in five HGT1 patients who will eventually develop a muscle invasive BC could be identified after diagnostic TUR, then cystectomy could be offered more selectively. Also, overtreatment and the morbidity and anxiety of undergoing additional procedures could be spared in patients with more indolent prognosis. Our results further support the notion that the ‘rule of thirds for HGT1' (i.e., one-third will never recur, one-third will require deferred cystectomy, and one-third will die of disease) is currently outdated.\nGiven the low impact of available scoring systems and biological markers (van Rhijn et al, 2012), and in view of this data, depth of invasion might be considered as the strongest prognostic factor for progression in HGT1. In addition, recent evidence suggests that histopathological classification into pTa, pT1a, and pT1b can be translated into a molecular signature, reflecting progressive dysregulation to more invasive stages (Descotes et al, 2013). Based on the prognostic impact of T1 substaging in the present and previous studies, we believe there is an argument for evolution of the current TNM classification. Together with tumour size and the detection of CIS an updated classification would help to identify the patients most suitable for cystectomy. Randomised control trials to evaluate the positive impact of this subclassification of tumours would also be beneficial.",
"With this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy."
] | [
"materials|methods",
"results",
"discussion",
"conclusions"
] | [
"high risk bladder cancer",
"HGT1",
"substaging",
"reTUR",
"prognosis"
] | Materials and methods:
Trial design and inclusion of this prospective observational cohort study began in April 2005. Since then, patients diagnosed at initial TUR with HGT1 BC were offered entry into this protocol. Transurethral resection BT included complete resection of all visible tumours, with a separate base biopsy. Cold cup biopsies to detect CIS were taken, depending on surgeon preference, in a standardised manner or according to EAU Guidelines (abnormal urothelium, non-papillary tumour appearance, or positive cytology) (Babjuk et al, 2013). All patients received a postoperative dose of intravesical mitomycin-C.
Tumours were graded according to the 2004 WHO system (2006) after pathological assessment of the total specimen. A two-tier system (Cottrell et al, 2007) was used to assess the depth of LP invasion: T1a when tumour involved the subepithelial connective tissue superficial to muscularis mucosae (MM); T1b when tumour was found at the level of or beyond MM. When MM was not identifiable, thick-walled blood vessels deep in LP served as a landmark, or alternatively location of MM in tumour-free tissue of the same TUR was used for reference. Only initial HGT1 tumours with a visible, clearly identifiable and disease-free muscularis propria were included in this study. Patients with LP invasion (T1) but without muscularis propria in the specimen represent ∼10% of all T1 in our centre. These cases, identified as Tx, did not fulfill the requirements to enter this protocol and, following guidelines, directly underwent a repeat resection.
This protocol was approved by the Hospital Ethics Committee and registered at clinicaltrials.gov (NCT02113501), and last evaluation of patients was performed in February 2014. Enrolled subjects received six weekly instillations of 2–8 × 108 UFC BCG-Tice (OncoTICE, Schering-Plough Canada Inc., Kirkland, QC, Canada) and, at 3 months after initial TUR, underwent either a second TUR post-BCG (T1b) or a cystoscopy plus cytology (T1a). Follow-up included BCG maintenance; the strategy is summarised in Figure 1 and described in detail in our previous report (Orsola et al, 2010). Baseline characteristics were compared between the T1a and T1b groups using Fisher's exact test.
The primary objective was the assessment of progression, recurrence, and CSS. Progression was defined as occurrence of an invasive tumour at post-BCG TUR, the development of muscle invasive or more advanced stage carcinoma, distant metastasis, or death from BC. Time to progression was defined as time from diagnosis of BC to the date when disease progression was observed, or censored on the last known date alive without progression. Recurrence was defined as the first evidence of any NMIBC detected on follow-up. Time to recurrence was defined as time from diagnosis of BC to the date when disease recurrence was observed, or censored at the last known date alive without disease recurrence, or at date of progression. Cancer-specific survival was defined as death from the same cancer or related causes.
The secondary objective was the assessment of prognostic factors. Eleven clinical and pathological variables were prospectively documented to evaluate their capacity to predict events. Kaplan–Meier analysis was used to summarise median time to recurrence and time to progression. Association of time to recurrence, time to progression, and CSS with the variables of interest was assessed using Cox regression models in univariate and multivariable models, reporting hazard ratios (HRs) and 95% CI.
Results:
Two hundred primary urothelial HGT1 cases have been identified at TUR and have entered the present protocol. Median age at diagnosis was 71 years; 89.5% were males, with 89 (44.5%) cases being T1a and 111 (55.5%) T1b. Patients and tumour characteristics are shown in Table 1 classified by substaging. The median follow-up period was 71 months (range: 5–107 months). The second TUR (performed only in T1b) was negative in 67.5% of cases and positive in the remaining 32.4% (11 invasive and 25 non-invasive; primary histology being persistent HGT1). Tolerance and patient dropout of BCG was not documented specifically within the study group, but for the broader population of high-risk BC at our hospital ∼30% do not complete the first year.
Recurrence was observed in 57 (28.5%) patients and disease progression in 31 (15.5%). In total, 46 patients died (13 of BC, 14 of other cancers, 18 of other causes, and one of unknown cause) and 18 underwent cystectomy (2 pT0, 1 pT1, 4 pTIS, 3 pT2, 4 pT3, and 3 pT4; one cystectomy at another centre; and 3 presented with positive lymph nodes). Ten potential candidates for cystectomy received palliative treatment because they were considered ineligible due to high surgical and/or anaesthetic risk.
The association of relevant clinical and pathological variables with time to progression and recurrence is presented in Table 2. Median time to recurrence was not reached; 2- and 5-year recurrence-free rates are summarised. Recurrence rate was not significantly different among HGT1a (21 cases, 22%) and HGT1b (36 cases, 34.6%), with only age at diagnosis remaining significant on multivariate analysis. Table 3a and b summarise univariate and multivariate analysis for recurrence.
Progression rate demonstrated a significant difference based on substaging, with HGT1b tumours showing an increased risk of progression (5 T1a (5.6%) vs 26 T1b (23.6%)). Median time to progression was not reached; the 2- and 5-year progression-free rates are summarised in Table 4a. Variables significant in univariate analysis together with lymphovascular invasion (LVI) and gender (significant in previous publications, but not in the current univariate analysis) were evaluated in a multivariable model for time to progression (Table 4b). Substaging, CIS, and tumour size remained significant in this model. Figure 2 illustrates the Kaplan–Meier curves for progression in relation to substaging, tumour pattern, tumour size, and associated CIS.
The low number of deaths due to BC precluded analysis of predictors of CSS. Five-year CSS was 99% for T1a and 95% for T1b. Of the 13 patients who died due to BC (3 of local progression (no cystectomy) and 10 from metastatic BC, 6 of which had undergone cystectomy) all except one were originally T1b.
Finally, the association of TIS and tumour size in deeply invasive cases (T1b) gave an HR for progression of 4.81 with 95% CI (2.07, 11.20) (P=0.0003) for patients with T1b+CIS+>3 cm compared with the rest.
Discussion:
In HGT1 BC, the proposed strategy of deferring a second TUR until after induction of BCG and doing so only in T1b cases, results in a global low progression rate of 15.5%. Cases in which tumours invaded the MM (T1b) had a three-fold increased risk of progression compared with those without MM invasion (T1a), even though the latter group was treated more conservatively. Our report goes on to confirm the role of CIS, tumour size, and tumour pattern as prognostic factors for worse outcomes in this group of high-risk non-muscle invasive BC.
To the best of our knowledge, this is the first report addressing the role of substaging HGT1 BC prospectively to guide therapeutic management. This novel strategy, together with the relatively large number of cases of initial HGT1, the long follow-up and the consistent treatment approach are the main strengths of our series. In addition, the evaluation of depth of tumour invasion was performed prospectively after TUR as part of the routine pathological report, whereas most reports on HGT1 are retrospective and observational. We cannot rule out bias due to patient selection since cases with recurrent tumours and those with no muscularis propria identified were not included; our series might also include some G2 cases (WHO 1974 classification) under the broader definition of HG BC. On the other hand, tumours that could have been understaged (early progressors or positive second TUR) were not excluded from the analysis in the understanding that this represents a more generalisable real-world practice, but might also be seen as a bias. Finally, our results are limited by the fact that the study was performed in a non-randomised manner. This limitation is widespread in the high-risk BC literature as most reports have retrospective observational designs, with few randomised studies (Martin-Doyle et al, 2014). Given the public nature of our health-care system, we chose to differentially allocate patients to treatment, using a more aggressive approach for T1b tumours, aiming to optimise resources.
The role of substaging has been well established throughout the past 20 years, with over 2500 published cases substaged according to invasion of the MM (Rouprêt et al, 2013). The relevance of this factor has been recently confirmed in the largest meta-analysis ever conducted on prognostic factors in HGT1 (Martin-Doyle et al, 2014). Because the evaluation of the depth of invasion is subject to interobserver and intraobserver variability, this parameter has been criticised for the last two decades, but there is growing evidence on the reproducibility of this evaluation. In a study specifically addressing this issue, Cottrell et al (2007) elegantly showed a high concordance rate among general pathologists after guidance based only in a presentation of the staging systems as described by the SIGN guideline. In their report, the median K concordance rate was 0.623, which is higher than that attained for Gleason score among pathologists (Bottke et al, 2013). In a more recent report, Rouprêt et al (2013) showed a reclassification rate for substaging of only 16% after central review of 587 HGT1 BC tumour sets from a multicentre data set. Other methods to substage HGT1 have been described using either millimetric evaluation of the depth of invasion (Cheng et al, 1999; Chang et al, 2012), measuring the size of the infiltrative tumour area as ⩽ or > 1 high-power field (Cheng et al, 1999; Bertz et al, 2011) or describing the extent of invasion (microinvasive or extensive) (van der Aa et al, 2005; van Rhijn et al, 2012). These alternative systems, however, have all been studied retrospectively in single data sets of patients and have not yet been externally validated (Martin-Doyle et al, 2014). A head-to-head comparison among these approaches in a large data set unfortunately is unavailable to date. Nonetheless, the fact that substaging remained a robust prognostic factor in our series (three-fold increase risk of progression in the adjusted multivariable model, and five-fold increase in the univariate model), even though patients with HGT1a underwent a more conservative approach demonstrates the magnitude of negative impact associated with depth of invasion.
We report a 15.5% progression rate, which is lower than the recently published pooled results of ∼20% progression rate for this high-risk group of tumours (van den Bosch and Alfred Witjes, 2011; Gontero et al, 2014; Martin-Doyle et al, 2014). Progression rates for high-risk BC have typically been considered to be over 30% and up to 50%, but more current data support that approximately one in five cases progress and will typically do so within 48 months of initial diagnosis (van den Bosch and Alfred Witjes, 2011). Our approach of proceeding with the second TUR after the 6-week induction of BCG (Orsola et al, 2010) was also used by Denzinger et al (2007) with an overall progression rate of 41% in 132 patients. We contend that the better outcome in our series might be attributable to a complete, deep, and radical initial TUR together with the exclusion of cases without clear muscularis propria in the TURBT specimen. It is possible the use of BCG (induction and maintenance) and the second TUR have also contributed an added therapeutic benefit. The combined influence of all these factors might explain the low rate of understaging we found in the second TUR (9.9%) as well as the low rate of positive findings at 3 months (Orsola et al, 2010), but the limitations mentioned previously should also be taken into account. Mitomycin-C might have only played a minor role given that no impact on progression has been demonstrated with this agent in all randomised published trials. Also the proposed approach in delaying the second TUR makes it impossible to accurately differentiate between true understaging and early invasive recurrence.
Our study goes on to corroborate previous observations that CIS is a strong prognostic factor with a two-fold increase in risk of progression (Sylvester et al, 2006). In our report, the impact was even stronger with a 0.23 HR favouring lack of CIS. Interestingly, we observed a high rate of detection in those patients who, according to surgeon preference, underwent multiple random bladder biopsies. This adds to the debate of whether routine bladder biopsies, which we have previously defended (Orsola et al, 2010), should be standardised at the time of initial BC TUR (Millan-Rodriguez et al, 2000). The usefulness of random bladder biopsies in the broad population of NMIBC is controversial (Kiemeney et al, 1994), but our findings suggest that a high positive yield is obtained in HGT1 cases. Alternatively, the diagnosis of CIS is based on its detection in either peritumoral areas of the specimen or if suspicious areas are biopsied. This approach, currently recommended by guidelines (American Urological Association, 2007; Babjuk et al, 2013), is highly limited by interpretation and likely leads to underdetection of true CIS, even though fluorescence-guided TURBT might improve identification of suspicious areas of CIS (Witjes et al, 2014). Finally, the findings of our study suggest that larger tumours and those with a solid pattern are associated with a worse outcome in HGT1, are consistent with the relevance of these parameters in the broader NMIBC population. Combining these factors we observed that patients with T1b tumours that were larger than 3 cm and associated with CIS were 4.8 times more likely to progress than the rest.
We failed to demonstrate a worse prognosis for LVI and for females in any of the outcomes. The incidence of LVI was very low (under 5%) and, because its effects have been noted to correlate with depth of LP invasion, it is unclear whether LVI independently predicts outcome, or whether its detrimental impact is already captured by pathologic information on substaging (Martin-Doyle et al, 2014). Regarding gender, large population-based studies may be required to confirm a worse prognosis of females that has been suggested previously (Palou et al, 2012).
In summary, our report proposes a novel approach for HGT1 BC based on the selection of patients who will most benefit from a second TUR, specifically those with deep invasion of the MM (T1b). The main clinical implication of separately identifying almost half of HGT1 cases with T1a substaging as having a low (4%) progression rate is that they can be spared a second endoscopic procedure with no harm. More accurate risk stratification incorporating depth of invasion, associated CIS, and tumour size would enable improved resource allocation for reTUR. It would also help identify optimal candidates for prompt cystectomy, namely T1b tumours larger than 3 cm and with associated CIS. If the one in five HGT1 patients who will eventually develop a muscle invasive BC could be identified after diagnostic TUR, then cystectomy could be offered more selectively. Also, overtreatment and the morbidity and anxiety of undergoing additional procedures could be spared in patients with more indolent prognosis. Our results further support the notion that the ‘rule of thirds for HGT1' (i.e., one-third will never recur, one-third will require deferred cystectomy, and one-third will die of disease) is currently outdated.
Given the low impact of available scoring systems and biological markers (van Rhijn et al, 2012), and in view of this data, depth of invasion might be considered as the strongest prognostic factor for progression in HGT1. In addition, recent evidence suggests that histopathological classification into pTa, pT1a, and pT1b can be translated into a molecular signature, reflecting progressive dysregulation to more invasive stages (Descotes et al, 2013). Based on the prognostic impact of T1 substaging in the present and previous studies, we believe there is an argument for evolution of the current TNM classification. Together with tumour size and the detection of CIS an updated classification would help to identify the patients most suitable for cystectomy. Randomised control trials to evaluate the positive impact of this subclassification of tumours would also be beneficial.
Conclusions:
With this contemporary approach for the management of HGT1 BC, in which the strategy of complete initial TUR+MitC+BCG is only followed by a second TUR in T1b cases, low recurrence and progression rates (28.5% and 15.5%, respectively) are achieved. Together with LP substaging (T1a vs T1b), tumour size and associated CIS are also prognostic factors for progression, which should be contemplated in future strategies and aid in the selection of patients for cystectomy.
| Background:
Management of high-grade T1 (HGT1) bladder cancer represents a major challenge. We studied a treatment strategy according to substaging by depth of lamina propria invasion.
Methods:
In this prospective observational cohort study, patients received initial transurethral resection (TUR), mitomycin-C, and BCG. Subjects with shallower lamina propria invasion (HGT1a) were followed without further surgery, whereas subjects with HGT1b received a second TUR. Association of clinical and histological features with outcomes (primary: progression; secondary: recurrence and cancer-specific survival) was assessed using Cox regression.
Results:
Median age was 71 years; 89.5% were males, with 89 (44.5%) cases T1a and 111 (55.5%) T1b. At median follow-up of 71 months, disease progression was observed in 31 (15.5%) and in univariate analysis, substaging, carcinoma in situ, tumour size, and tumour pattern predicted progression. On multivariate analysis only substaging, associated carcinoma in situ, and tumour size remained significant for progression.
Conclusions:
In HGT1 bladder cancer, the strategy of performing a second TUR only in T1b cases results in a global low progression rate of 15.5%. Tumours deeply invading the lamina propria (HGT1b) showed a three-fold increase in risk of progression. Substaging should be routinely evaluated, with HGT1b cases being thoroughly evaluated for cystectomy. Inclusion in the TNM system should also be carefully considered. | null | null | 3,299 | 280 | [] | 4 | [
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] | null | null | null | null | null | [CONTENT] high risk bladder cancer | HGT1 | substaging | reTUR | prognosis [SUMMARY] | [CONTENT] high risk bladder cancer | HGT1 | substaging | reTUR | prognosis [SUMMARY] | [CONTENT] high risk bladder cancer | HGT1 | substaging | reTUR | prognosis [SUMMARY] | null | null | null | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma in Situ | Carcinoma, Squamous Cell | Cohort Studies | Cystectomy | Female | Humans | Male | Middle Aged | Mucous Membrane | Neoplasm Grading | Neoplasm Invasiveness | Neoplasm Recurrence, Local | Reoperation | Urinary Bladder Neoplasms | Urinary Tract [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma in Situ | Carcinoma, Squamous Cell | Cohort Studies | Cystectomy | Female | Humans | Male | Middle Aged | Mucous Membrane | Neoplasm Grading | Neoplasm Invasiveness | Neoplasm Recurrence, Local | Reoperation | Urinary Bladder Neoplasms | Urinary Tract [SUMMARY] | [CONTENT] Adult | Aged | Aged, 80 and over | Carcinoma in Situ | Carcinoma, Squamous Cell | Cohort Studies | Cystectomy | Female | Humans | Male | Middle Aged | Mucous Membrane | Neoplasm Grading | Neoplasm Invasiveness | Neoplasm Recurrence, Local | Reoperation | Urinary Bladder Neoplasms | Urinary Tract [SUMMARY] | null | null | null | [CONTENT] resection visible tumours | substaging cis tumour | cytology t1a follow | intravesical mitomycin | dose intravesical mitomycin [SUMMARY] | [CONTENT] resection visible tumours | substaging cis tumour | cytology t1a follow | intravesical mitomycin | dose intravesical mitomycin [SUMMARY] | [CONTENT] resection visible tumours | substaging cis tumour | cytology t1a follow | intravesical mitomycin | dose intravesical mitomycin [SUMMARY] | null | null | null | [CONTENT] progression | patients | bc | tur | hgt1 | cases | t1b | tumour | cis | recurrence [SUMMARY] | [CONTENT] progression | patients | bc | tur | hgt1 | cases | t1b | tumour | cis | recurrence [SUMMARY] | [CONTENT] progression | patients | bc | tur | hgt1 | cases | t1b | tumour | cis | recurrence [SUMMARY] | null | null | null | [CONTENT] progression | table | significant | year | t1b | cystectomy | analysis | cases | recurrence | bc [SUMMARY] | [CONTENT] tur | t1b | progression | strategies | strategies aid selection patients | initial tur mitc | t1a vs t1b | t1a vs t1b tumour | strategy complete initial tur | strategy complete initial [SUMMARY] | [CONTENT] progression | t1b | tur | bc | patients | tumour | recurrence | cases | time | hgt1 [SUMMARY] | null | null | null | [CONTENT] 71 years | 89.5% | 89 | 44.5% | 111 | 55.5% | T1b ||| 71 months | 31 | 15.5% ||| [SUMMARY] | [CONTENT] second | T1b | 15.5% ||| three-fold ||| ||| TNM [SUMMARY] | [CONTENT] ||| ||| BCG ||| second ||| ||| ||| 71 years | 89.5% | 89 | 44.5% | 111 | 55.5% | T1b ||| 71 months | 31 | 15.5% ||| ||| second | T1b | 15.5% ||| three-fold ||| ||| TNM [SUMMARY] | null |
|||
Current manufactured cigarette smoking and roll-your-own cigarette smoking in Thailand: findings from the 2009 Global Adult Tobacco Survey. | 23530750 | Current smoking prevalence in Thailand decreased from 1991 to 2004 and since that time the prevalence has remained flat. It has been suggested that one of the reasons that the prevalence of current smoking in Thailand has stopped decreasing is due to the use of RYO cigarettes. The aim of this study was to examine characteristics of users of manufactured and RYO cigarettes and dual users in Thailand, in order to determine whether there are differences in the characteristics of users of the different products. | BACKGROUND | The 2009 Global Adult Tobacco Survey (GATS Thailand) provides detailed information on current smoking patterns. GATS Thailand used a nationally and regionally representative probability sample of 20,566 adults (ages 15 years and above) who were chosen through stratified three-stage cluster sampling and then interviewed face-to-face. | METHODS | The prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only. 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokers were more likely to live in rural areas. Smokers of manufactured cigarettes appeared to be more knowledgeable about the health risks of tobacco use. However, the difference was confounded with age and education; when demographic variables were controlled, the knowledge differences no longer remained. Smokers of manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appeared to be more addicted than the other two groups as measured by time to first cigarette. | RESULTS | There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. | CONCLUSIONS | [
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] | 3621680 | Background | In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].
Sangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.
One of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.
1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?
2. Are there differences in current use by age, by geographic location, by socio-economic status?
3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users? | Methods | From 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.
The target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.
All interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.
The interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.
The questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.
Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.
Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.
Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.
The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded. | Results | A total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.
Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).
Prevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics
1 Smoked manufactured cigarettes only.
2 Smoked roll-your-own cigarettes only.
3 None or less than primary school.
* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.
The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).
Prevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics
1 Smoked manufactured cigarettes only.
2 Smoked roll-your-own cigarettes only.
3 None or less than primary school.
* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.
Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.
Prevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region
Table 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.
Multivariate analysis of type of smokers with demographic variables
*p-value of the test for importance of the factor.
There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.
Prevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region
Table 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.
Multivariate analysis of type of smokers with demographic variables
*p-value of the test for importance of the factor.
Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.
Percentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases
*The number of knowledge questions correctly answered.
Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.
Percentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases
*The number of knowledge questions correctly answered.
Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).
Percentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
As shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).
Percentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).
Percentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
As shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).
Percentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers. | Conclusions | There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered. | [
"Background",
"Definition of variables",
"Analysis",
"Prevalence of smoking of tobacco products",
"Demographic variables",
"Knowledge",
"Addiction and quitting",
"Competing interest",
"Authors’ contributions",
"Pre-publication history"
] | [
"In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?",
"Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.",
"The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.",
"The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.",
"There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.",
"Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.",
"Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.",
"All authors declare to have no competing interest.",
"SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Definition of variables",
"Analysis",
"Results",
"Prevalence of smoking of tobacco products",
"Demographic variables",
"Knowledge",
"Addiction and quitting",
"Discussion",
"Conclusions",
"Competing interest",
"Authors’ contributions",
"Pre-publication history"
] | [
"In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].\nSangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.\nOne of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.\n1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?\n2. Are there differences in current use by age, by geographic location, by socio-economic status?\n3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?",
"From 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.\nThe target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.\nAll interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.\nThe interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.\nThe questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.\n Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\nUsed as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.\n Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.\nThe data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.",
"Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.",
"The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.",
"A total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.\n Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\nThe prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.\n Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\nThere were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.\n Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\nTable 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.\n Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nTable 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.",
"The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).\nPrevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics\n1 Smoked manufactured cigarettes only.\n2 Smoked roll-your-own cigarettes only.\n3 None or less than primary school.\n* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.",
"There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.\nPrevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region\nTable 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.\nMultivariate analysis of type of smokers with demographic variables\n*p-value of the test for importance of the factor.",
"Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.\nPercentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases\n*The number of knowledge questions correctly answered.",
"Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).\nPercentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.\nAs shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).\nPercentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics\n* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.\n** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.",
"In 2009, we found that the prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only and 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokes were also more likely to live in rural areas. When the demographic variables were controlled, there were no differences between the three groups of smoking either in the percentage who answered all seven questions correctly or in total knowledge score. Respondents who smoked only manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appear to be more addicted than the other two groups as measured by time to first cigarette. These analyses provide recent estimates than have been reported and present information on knowledge and addiction that has not been reported in earlier work.\nWhile comparisons with other surveys such as that National Cigarette Smoking and Alcohol Drinking Survey and the ITC survey can be made, caution should be exercised in that sampling and question wording differed. However, the findings from this study closely mirror those from Young et al. [6] who found that any RYO use was associated with living in rural areas, older average age, lower level of education, male gender, not being in paid work, slightly lower consumption of cigarettes, higher social acceptability of smoking, and positive attitudes toward tobacco regulation.\nThe proportion of RYO cigarette smokers is higher in Thailand than in its neighbors, such Vietnam (1.1%) and China (2.3%) [11,12]. As Young et al. [6] noted there are many factors that may play a role in differences in prevalence in different countries including cultural, legislative and economic factors.\nOne important factor in stimulating the decrease in the prevalence of current smoking in Thailand can be found in the increase in the taxation of manufactured cigarettes [13,14]; the excise tax for manufactured cigarettes has increased from 55% in 1992 to 85% in 2009 [13]. However, the taxation rate for blended shredded tobacco, which is used in RYO cigarettes, has remained as low as 500 Thai Baht (approximately 16.5 U.S. dollars, per kilogram since October 13, 1999), leading to the possibility that some smokers of manufactured cigarettes may have started smoking more RYO cigarettes and reduced or eliminated their smoking of manufactured cigarettes.\nSince there were differences noted in rural areas and for those with lower education and income, it may be that more focused education programs should be developed focusing on RYO smoking. Enhancement of health education programs with information on the harmful effects of both types of cigarettes may provide much needed information for smokers.\nThere are some limitations that should be noted about this survey. Questions on reasons for use of RYO were not included and, therefore, the explanations about economic and cultural facts are speculative. Given that smoking among women does not appear to be acceptable, the estimate of prevalence for women may be subject to some misreporting. Some of the strengths include the rigorous procedures for sampling and interviewing used in GATS Thailand and the high response rate.\nThailand has implemented comprehensive tobacco control interventions, including educational campaigns in the media, legislation to ban smoking in public places, increases in the cigarette excise tax, bans on advertising, and the use of a pictorial warning labels on manufactured cigarettes. Taken together, these legislative measures may have changed social norms and perceptions about tobacco use in Thailand. Meanwhile, lack of comparable policies in the control of RYO cigarettes may be playing a role in the overall prevalence of smoking in Thailand, diluting the success of existing programs.",
"There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered.",
"All authors declare to have no competing interest.",
"SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/277/prepub\n"
] | [
null,
"methods",
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null
] | [
"Thailand",
"Manufactured cigarettes",
"Roll-your-own cigarettes (RYO)",
"Prevalence of current smoking"
] | Background:
In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].
Sangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.
One of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.
1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?
2. Are there differences in current use by age, by geographic location, by socio-economic status?
3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?
Methods:
From 2008 to 2010, 14 countries, most of them being either low or middle income with a heavy burden of tobacco use and highly populated, conducted the Global Adult Tobacco Survey (GATS) to monitor tobacco use in their countries and the changes driven by policies enacted in those nations [7]. Selected as one of the 14 countries, Thailand conducted GATS in 2009 [8,9]. To ensure useful comparisons across the 14 countries, the survey followed the standard GATS protocol.
The target population was noninstitutionalized residents, in Thailand, aged 15 years or above (defined as adults in the current study), and the sample was a nationally and regionally representative probability sample. A stratified three-stage cluster sampling design was used with the strata being five geographic regions: the Bangkok metropolitan area and the North, Northeast, Central, and South regions of Thailand which were further stratified into urban and rural areas. The sampling frame used was supplied by the National Statistical Office (NSO), derived from Thailand’s National Population and Housing Census 2000 [10]. Primary sampling units (PSUs) were blocks in urban areas and villages in rural areas and were randomly selected, in the first stage, using selection probability proportional to size sampling. In the second stage, 16 and 28 households were randomly chosen from the previously selected urban or rural PSUs, respectively, using simple systematic sampling. In the third stage, a face-to-face screening interview was used at each randomly selected household, in which a list of all eligible individuals in the household was drawn up and one person from the list was randomly selected to participate in the interview, using an algorithm of simple random sampling on the handheld device designed for data collection. If the respondent was not available, the interviewer would schedule another appointment to interview. Three attempts were made before the individual was considered a nonrespondent. If a residence was found to be empty, it was declared to be ineligible; if a selected respondent refused to participate, the individual was considered to be a nonrespondent.
All interviewees were informed that they could stop the survey and the response was confidential. Interviewers were selected by provincial statistical offices (PSO) and were only responsible for their own province, with supervisors from corresponding PSOs. Both interviewers and supervisors had experience on national health related surveys fieldwork. All of them had at least a Bachelor’s degree in education. There were 2 interviewers/supervisors - training sessions using the same trainers with technical support from CDC and WHO experts.
The interview was conducted in the selected household; while other members of the household could be present, they were instructed to remain silent during the interview. No proxy responding was allowed and no incentives were provided for participation. For the questionnaire interview (average duration was 19.1 minutes, SD = 7.6 minutes), the trained interviewers used personal digital assistants (PDAs) to collect data. The fieldwork started on February 1, 2009, and was completed on May 31 of the same year.
The questionnaire included information on demographics (age, gender, education, income); use of smoking tobacco and of smokeless tobacco; cessation attempts and interest in quitting; exposure to secondhand smoke; purchase of cigarettes during the last 30 days; exposure to media that provided information on smoking and health effects; questions about pictorial warning labels on manufactured cigarette packs, and knowledge, attitudes, and perceptions about tobacco use. Some questions were added by the Thailand GATS working group and reviewed by the Thailand GATS expert committee [9]. GATS protocols were approved by the Ethics Committee of the Faculty of Public Health, Mahidol University.
Definition of variables Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.
Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.
Analysis The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.
The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.
Definition of variables:
Used as an indicator of socioeconomic status, total personal income was grouped as low (less than 4,780 baht or less than 140.6 USD per month), middle (4,780 to 7,000 baht or 140.6 to 205.9 USD per month), and high (7,001 baht or higher or 206.0 USD or higher per month). Education was grouped into none or less than primary school, primary school, secondary school and university and above. RYO cigarettes, also known as hand-rolled cigarettes are referred to the cigarettes made by hand using shredded tobacco and papers. Current smoking was defined as responding yes to a question about smoking daily or less than daily. Whether smoking can cause seven well proved diseases, including stroke, heart stack, lung cancer, mouth cancer, larynx cancer, impotence, emphysema, was used as information to assess the extent of participants knowledge of health effects. A knowledge score, defined as the total number of the seven knowledge items answered correctly, was used as a summary measure of knowledge.
Analysis:
The data were reviewed for inconsistencies and out of range responses, edited, and weighted, using the complex survey analysis module of SPSS Version 18. Weights were computed as a product of three components: base weight, which was an inverse of the final probability of selection, adjustment for nonresponse at both the household and individual level, and post-stratification adjustment based on residence (urban or rural), age, and gender from the 2008 population projection for Thailand. A two sample test was used for pair comparison of prevalence among different group of users at statistically significant level of p < 0.05. Analyses of knowledge, addiction, and quitting, as well as multivariate analysis, were carried out for men only, because the levels of use of the tobacco products among females were too low for reliable analyses. Multivariate analyses, taking the complex sample design into account, used logistic regression models to see if the type of cigarettes smoked (manufactured cigarette only, RYO only and dual use) was associated with age, education, income, residence, and region. These variables were selected because of previous studies that have shown these variables to be related to tobacco use and the use of RYO. In the multivariate models, we determined the relationship of one variable while controlling for the effects of the others. The test for importance of a factor was carried out by a comparison of the full model with all factors and a reduced model where the factor of interest was excluded.
Results:
A total of 20,566 people aged 15 and over were surveyed in Bangkok and in the four regions of Thailand: North, Northeast, Central and South. The household-level response rate was 97.9%, the individual-level response rate was 96.2%, and the overall response rate, a product of household response rate and individual response rate, was 94.2%. Each of the four non-Bangkok regions had high response rates, with at least 95.1% overall, at least 98.4% at the household level, and at least 96.2% at the individual level. The response rate in the Bangkok metropolitan area was 94.6% at the household level, 90.0% at the individual level, and 85.1% overall.
Prevalence of smoking of tobacco products The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).
Prevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics
1 Smoked manufactured cigarettes only.
2 Smoked roll-your-own cigarettes only.
3 None or less than primary school.
* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.
The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).
Prevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics
1 Smoked manufactured cigarettes only.
2 Smoked roll-your-own cigarettes only.
3 None or less than primary school.
* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.
Demographic variables There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.
Prevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region
Table 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.
Multivariate analysis of type of smokers with demographic variables
*p-value of the test for importance of the factor.
There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.
Prevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region
Table 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.
Multivariate analysis of type of smokers with demographic variables
*p-value of the test for importance of the factor.
Knowledge Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.
Percentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases
*The number of knowledge questions correctly answered.
Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.
Percentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases
*The number of knowledge questions correctly answered.
Addiction and quitting Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).
Percentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
As shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).
Percentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).
Percentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
As shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).
Percentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
Prevalence of smoking of tobacco products:
The prevalence of current smoking for all tobacco products (data not shown) was 45.6% among men and 3.1% among women. Although a variety of tobacco products were used including smokeless tobacco, pipes, cigars, and water pipes, 86.3% of all current tobacco users predominantly used two major products, manufactured cigarettes (54.9% of users) and RYO cigarettes (51.6% of users), and 20.2% of users smoked both (data not shown). Among smokers of any tobacco products, 40.4% of them smoked manufactured cigarettes only, 36.6% of them smoked RYO cigarettes only, and 23.4 of them were dual users (40.5%, 34.8%, and 24.7% respectively for men, 35.7%, 60.7%, and 3.6% respectively for women). As shown in Table 1, at a population level, 18.4% of men and 1.0% of women were current smokers of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current smokers of RYO cigarettes only. For men, 11.2% used both manufactured and RYO. Among women, only 0.1% used both products. Among the dual users, 33.7% of men and 63.0% of women smoked more RYO cigarettes than did manufactured cigarettes (Table 1).
Prevalence (in percent and 95%CI ) of current smoking by classification of product and demographic characteristics
1 Smoked manufactured cigarettes only.
2 Smoked roll-your-own cigarettes only.
3 None or less than primary school.
* Percentage of dual users who smoked more roll-your-own cigarettes than manufactured cigarettes.
Demographic variables:
There were differences in product use by education and income levels (Table 1). Among men who used manufactured cigarettes only, the prevalence of use was highest in those aged 25–44 years (23.3%). Among men who used RYO cigarettes only, the highest prevalence was found in those aged 65 years or above (28.6%) for men, with the second highest prevalence group being those aged 45–64 (21.5%). Among women, the highest use of RYO cigarettes only was among those aged 45 or above. Dual use for men occurred most often in those under age 45. For both genders, adults with lower education levels or with lower incomes were more likely to smoke RYO cigarettes only or use both products than were those with university education (p < .001 for men and women) or high income (p < .001 for men and women). In the case of manufactured cigarettes, both men and women with middle or high income or secondary school or university level of education were more likely to smoke these products than those with low income (p < .001 for men and women) and those with primary school or less level of education (p < .001 for men and women). Across regions (Table 2), RYO cigarettes were more likely to be smoked in rural than in urban areas (p < .001), particularly in the South.
Prevalence (in percent and 95% CI) of men’s current smoking by type of product, residence and region
Table 3 shows the multivariate analysis of pairwise comparison of three categories of smokers: manufactured cigarette only, RYO cigarette only, and dual users, with demographic variables, age, education, income, residence, and region. In the comparison of smokers between RYO cigarette only and manufactured cigarette only, all five factors studied were statistically significant. Older age, lower education level, lower income, rural residence, and living in the South region were associated with being a RYO smoker. Similarly, in the comparison of smokers between RYO cigarette only and the dual users, older age, lower education, lower income, and living in the Central and North region, were associated with being a RYO smoker, while rural residence was not. Finally, in the comparison between the dual users and manufactured cigarette only, lower education, lower income, rural residence, living in the South region were associated with being a dual user, while age was not a significant factor.
Multivariate analysis of type of smokers with demographic variables
*p-value of the test for importance of the factor.
Knowledge:
Table 4 presents the results of the univariate analyses. The percent of males who answered all 7 knowledge questions correctly was calculated, with 57.7% who smoked manufactured cigarettes only and 49.0% who smoked RYO (p = 0.003). There was no difference between the dual users and those who used manufactured cigarettes only. After controlling for age, education, income, residence, and region, we did not find statistically significant differences between RYO only and manufactured cigarette only smokers (p = .194). A similar pattern of results was found when the knowledge scores were examined. In the univariate analysis, those who smoked manufactured cigarettes only had the higher average knowledge score than those who used RYO only (6.1 vs. 5.5, p < .001). After controlling for age, education, income, residence, and region, the p-value became 0.11.
Percentage (95% CI) of men’s daily smokers who have correct knowledge of smoking causing diseases
*The number of knowledge questions correctly answered.
Addiction and quitting:
Table 5 presents the results of analyses of addition. Time to the first smoking of a cigarette after waking can be used as an indicator of nicotine dependence. Overall, a higher percentage of smokers of RYO cigarettes were significantly more likely to report smoking their first cigarette within 30 minutes after waking up than smokers of manufactured cigarettes (64.3% vs. 57.6%, p = .018).
Percentage (95% CI) of men daily smokers who smoke within 30 minutes of awakening by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
As shown in Table 6, among men, smokers of manufactured cigarettes were more likely to report plans to quit within the next month than were men who RYO cigarettes (7.8% vs. 4.4% and 4.2% for dual users p < 0.001), and overall men who smoked RYO were more likely to report not being interested in quitting than were those who smoked manufactured cigarettes. A multivariate analysis (not shown) adjusting for age, education, income, residence and region showed the same pattern (p = .005).
Percentage (95% CI) of men daily smokers who intent to quit smoking by demographic characteristics
* p-value < 0.05 of the test for difference between roll-your-own cigarette only smokers and manufactured cigarette smokers.
** p-value < 0.05 of the test for difference between dual users and manufactured cigarette smokers.
Discussion:
In 2009, we found that the prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only and 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokes were also more likely to live in rural areas. When the demographic variables were controlled, there were no differences between the three groups of smoking either in the percentage who answered all seven questions correctly or in total knowledge score. Respondents who smoked only manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appear to be more addicted than the other two groups as measured by time to first cigarette. These analyses provide recent estimates than have been reported and present information on knowledge and addiction that has not been reported in earlier work.
While comparisons with other surveys such as that National Cigarette Smoking and Alcohol Drinking Survey and the ITC survey can be made, caution should be exercised in that sampling and question wording differed. However, the findings from this study closely mirror those from Young et al. [6] who found that any RYO use was associated with living in rural areas, older average age, lower level of education, male gender, not being in paid work, slightly lower consumption of cigarettes, higher social acceptability of smoking, and positive attitudes toward tobacco regulation.
The proportion of RYO cigarette smokers is higher in Thailand than in its neighbors, such Vietnam (1.1%) and China (2.3%) [11,12]. As Young et al. [6] noted there are many factors that may play a role in differences in prevalence in different countries including cultural, legislative and economic factors.
One important factor in stimulating the decrease in the prevalence of current smoking in Thailand can be found in the increase in the taxation of manufactured cigarettes [13,14]; the excise tax for manufactured cigarettes has increased from 55% in 1992 to 85% in 2009 [13]. However, the taxation rate for blended shredded tobacco, which is used in RYO cigarettes, has remained as low as 500 Thai Baht (approximately 16.5 U.S. dollars, per kilogram since October 13, 1999), leading to the possibility that some smokers of manufactured cigarettes may have started smoking more RYO cigarettes and reduced or eliminated their smoking of manufactured cigarettes.
Since there were differences noted in rural areas and for those with lower education and income, it may be that more focused education programs should be developed focusing on RYO smoking. Enhancement of health education programs with information on the harmful effects of both types of cigarettes may provide much needed information for smokers.
There are some limitations that should be noted about this survey. Questions on reasons for use of RYO were not included and, therefore, the explanations about economic and cultural facts are speculative. Given that smoking among women does not appear to be acceptable, the estimate of prevalence for women may be subject to some misreporting. Some of the strengths include the rigorous procedures for sampling and interviewing used in GATS Thailand and the high response rate.
Thailand has implemented comprehensive tobacco control interventions, including educational campaigns in the media, legislation to ban smoking in public places, increases in the cigarette excise tax, bans on advertising, and the use of a pictorial warning labels on manufactured cigarettes. Taken together, these legislative measures may have changed social norms and perceptions about tobacco use in Thailand. Meanwhile, lack of comparable policies in the control of RYO cigarettes may be playing a role in the overall prevalence of smoking in Thailand, diluting the success of existing programs.
Conclusions:
There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered.
Competing interest:
All authors declare to have no competing interest.
Authors’ contributions:
SB and LT supervised the GATS Thailand and critically commented the manuscript. JH provided technical support for the GATS Thailand, analyzed the data and drafted the manuscript. MK, CT and AL contributed to the GATS Thailand operation. HP contributed to the sample design. LA contributed to the data analysis. SA critically reviewed the manuscript. All authors read and approved the final version of the manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/13/277/prepub
| Background:
Current smoking prevalence in Thailand decreased from 1991 to 2004 and since that time the prevalence has remained flat. It has been suggested that one of the reasons that the prevalence of current smoking in Thailand has stopped decreasing is due to the use of RYO cigarettes. The aim of this study was to examine characteristics of users of manufactured and RYO cigarettes and dual users in Thailand, in order to determine whether there are differences in the characteristics of users of the different products.
Methods:
The 2009 Global Adult Tobacco Survey (GATS Thailand) provides detailed information on current smoking patterns. GATS Thailand used a nationally and regionally representative probability sample of 20,566 adults (ages 15 years and above) who were chosen through stratified three-stage cluster sampling and then interviewed face-to-face.
Results:
The prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only. 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokers were more likely to live in rural areas. Smokers of manufactured cigarettes appeared to be more knowledgeable about the health risks of tobacco use. However, the difference was confounded with age and education; when demographic variables were controlled, the knowledge differences no longer remained. Smokers of manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appeared to be more addicted than the other two groups as measured by time to first cigarette.
Conclusions:
There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. | Background:
In Thailand, more than 20 years of tobacco control regulations and laws [1,2], may have contributed, at least in part to decreasing the prevalence of smoking and exposure to secondhand smoke [3]. There has been a reduction in the prevalence of current smoking among men from 59.3% in 1991 to 41.7% in 2007 [3], while the prevalence among women has continued to remain low. However, there does not appear to have been any substantial decrease in prevalence since that time; current smoking among male adults in 2009 still remains relatively high at 40.5% [4].
Sangthong et al. [5] reported that the prevalence of current smoking in Thailand decreased from 1991 to 2004 and subsequently has remained flat. During this period, the rate of decline in the prevalence of manufactured cigarette smoking decreased later in the period. However, the prevalence of roll-your-own (RYO) cigarette smoking has remained flat since 1996. Young et al. [6] compared cigarette smokers in Thailand and in Malaysia in 2005, and found that many factors may play a role in tobacco use including taxation, and socio-cultural and economic factors, which may not act independently. “Economic motives are obviously extremely important, because RYO cigarettes are typically less expensive than FM (factory manufactured, added by the current authors) cigarettes and serve as a “discount” option for smokers” (Young et al. [6]). They also reported that any RYO use was associated with living in rural areas, older average age, lower level of education, being male, not working for pay, greater social acceptability of smoking, and attitudes toward tobacco regulation.
One of the purposes of this study was to provide an update of the information presented by Young et al. [6]. The stability of the findings is important given differences in the time period, sampling strategies and question wording. We were interested in determining whether similar patterns related to the demographic variables emerged after 4 years.
1. What is the prevalence of current tobacco use for males and females by type of cigarettes (manufactured vs RYO)? Do many people use both types of products (dual use)?
2. Are there differences in current use by age, by geographic location, by socio-economic status?
3. Are there differences in knowledge, addiction, and in desire to quit among different groups of users?
Conclusions:
There are demographic differences among the three groups of male tobacco users. RYO smokers are older and more likely to live in rural areas. There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. In addition, adoption of tobacco control measures for RYO products similar to those for manufactured cigarettes could be considered. | Background:
Current smoking prevalence in Thailand decreased from 1991 to 2004 and since that time the prevalence has remained flat. It has been suggested that one of the reasons that the prevalence of current smoking in Thailand has stopped decreasing is due to the use of RYO cigarettes. The aim of this study was to examine characteristics of users of manufactured and RYO cigarettes and dual users in Thailand, in order to determine whether there are differences in the characteristics of users of the different products.
Methods:
The 2009 Global Adult Tobacco Survey (GATS Thailand) provides detailed information on current smoking patterns. GATS Thailand used a nationally and regionally representative probability sample of 20,566 adults (ages 15 years and above) who were chosen through stratified three-stage cluster sampling and then interviewed face-to-face.
Results:
The prevalence of current smoking among Thai adults was 45.6% for men and 3.1% for women. In all, 18.4% of men and 1.0% of women were current users of manufactured cigarettes only, while 15.8% of men and 1.7% of women were current users of RYO cigarettes only. 11.2% of men and 0.1% of women used both RYO and manufactured cigarettes. Users of manufactured cigarettes were younger and users of RYO were older. RYO smokers were more likely to live in rural areas. Smokers of manufactured cigarettes appeared to be more knowledgeable about the health risks of tobacco use. However, the difference was confounded with age and education; when demographic variables were controlled, the knowledge differences no longer remained. Smokers of manufactured cigarettes were more likely than dual users and those who used only RYO to report that they were planning on quitting in the next month. Users of RYO only appeared to be more addicted than the other two groups as measured by time to first cigarette.
Conclusions:
There appears to be a need for product targeted cessation and prevention efforts that are directed toward specific population subgroups in Thailand and include information on manufactured and RYO cigarettes. | 7,795 | 381 | [
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] | [CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY] | [CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY] | [CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY] | [CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY] | [CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY] | [CONTENT] Thailand | Manufactured cigarettes | Roll-your-own cigarettes (RYO) | Prevalence of current smoking [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Aged | China | Female | Health Knowledge, Attitudes, Practice | Health Surveys | Humans | Male | Middle Aged | Multivariate Analysis | Population Surveillance | Prevalence | Product Packaging | Residence Characteristics | Sex Distribution | Smoking | Smoking Cessation | Socioeconomic Factors | Thailand | Tobacco Products [SUMMARY] | [CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY] | [CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY] | [CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY] | [CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY] | [CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY] | [CONTENT] smoking thailand diluting | smoking thai adults | smokers thailand malaysia | smoking thailand decreased | tobacco use thailand [SUMMARY] | [CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY] | [CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY] | [CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY] | [CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY] | [CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY] | [CONTENT] cigarettes | ryo | manufactured | men | smokers | cigarette | users | smoking | manufactured cigarettes | education [SUMMARY] | [CONTENT] prevalence | use | smoking | current | period | economic | young | prevalence current | tobacco | decreased [SUMMARY] | [CONTENT] selected | use | tobacco | interview | knowledge | cancer | usd | sample | sampling | household [SUMMARY] | [CONTENT] cigarettes | men | smokers | manufactured | ryo | women | manufactured cigarettes | cigarette | smoked | users [SUMMARY] | [CONTENT] ryo | tobacco | ryo cigarettes addition adoption | population subgroups thailand | manufactured ryo cigarettes | thailand include information | thailand include | targeted cessation prevention efforts | targeted cessation prevention | targeted cessation [SUMMARY] | [CONTENT] cigarettes | manufactured | ryo | smokers | men | manufactured cigarettes | smoking | cigarette | use | women [SUMMARY] | [CONTENT] cigarettes | manufactured | ryo | smokers | men | manufactured cigarettes | smoking | cigarette | use | women [SUMMARY] | [CONTENT] Thailand | 1991 | 2004 ||| one | Thailand | RYO ||| RYO | Thailand [SUMMARY] | [CONTENT] 2009 | Global Adult Tobacco Survey | Thailand ||| 20,566 | (ages 15 years | three [SUMMARY] | [CONTENT] Thai | 45.6% | 3.1% ||| 18.4% | 1.0% | 15.8% | 1.7% | RYO ||| 11.2% | 0.1% | RYO ||| RYO ||| RYO ||| ||| ||| RYO | the next month ||| RYO | two | first [SUMMARY] | [CONTENT] Thailand | RYO [SUMMARY] | [CONTENT] Thailand | 1991 | 2004 ||| one | Thailand | RYO ||| RYO | Thailand ||| 2009 | Global Adult Tobacco Survey | Thailand ||| 20,566 | (ages 15 years | three ||| ||| Thai | 45.6% | 3.1% ||| 18.4% | 1.0% | 15.8% | 1.7% | RYO ||| 11.2% | 0.1% | RYO ||| RYO ||| RYO ||| ||| ||| RYO | the next month ||| RYO | two | first ||| Thailand | RYO [SUMMARY] | [CONTENT] Thailand | 1991 | 2004 ||| one | Thailand | RYO ||| RYO | Thailand ||| 2009 | Global Adult Tobacco Survey | Thailand ||| 20,566 | (ages 15 years | three ||| ||| Thai | 45.6% | 3.1% ||| 18.4% | 1.0% | 15.8% | 1.7% | RYO ||| 11.2% | 0.1% | RYO ||| RYO ||| RYO ||| ||| ||| RYO | the next month ||| RYO | two | first ||| Thailand | RYO [SUMMARY] |
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Clinical Profiles of Asians with NAFLD: A Systematic Review and Meta-Analysis. | 34942625 | NAFLD is increasingly prevalent in Asia, where people suffer more metabolic comorbidities at a lower body mass index (BMI), suggesting potential differences in their clinical profile. Therefore, we attempted to characterize the clinical profile of Asians with NAFLD via a meta-analytic approach. | INTRODUCTION | We searched PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019. Two authors independently reviewed and selected 104 articles (2,247,754 persons) that identified NAFLD in Asians and reported relevant data, especially BMI and ALT, and excluded individuals with other liver disease and excessive alcohol consumption. Individual patient-level data were obtained from seven cohorts in Asia to complement meta-analyzed data. | METHODS | Overall, the mean age was 52.07 (95% CI: 51.28-52.85) years, with those from Southeast Asia (42.66, 95% CI: 32.23-53.11) being significantly younger. The mean BMI was 26.2 kg/m2, higher in moderate-severe versus mild hepatic steatosis (28.3 vs. 25.7) patients and NFS ≥ -1.455 versus <-1.455 (27.09 vs. 26.02), with 34% having nonobese NAFLD. The mean ALT was 31.74 U/L, higher in NFS < -1.455 versus ≥-1.455 (33.74 vs. 27.83), though no differences were found by obesity or steatosis severity. The majority of males (85.7%) and females (60.7%) had normal to minimally elevated ALT (1-1.5 × 95% ULN). Individual patient-level data analysis (N = 7,668) demonstrated similar results. | RESULTS | About one-third of Asians with NAFLD were nonobese, and the majority did not have markedly elevated ALT. Therefore, abnormal ALT or BMI is not recommended as a criterion for NAFLD screening in this population. Additionally, there were significant differences in the clinical profiles of NAFLD among the different regions of Asia. | CONCLUSION | [
"Male",
"Female",
"Humans",
"Middle Aged",
"Non-alcoholic Fatty Liver Disease",
"Body Mass Index",
"Obesity",
"Comorbidity"
] | 9808705 | Introduction | Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.
Through a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results. | Methods | Search Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).
This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).
Systemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).
Baseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].
The quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.
Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).
Baseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].
The quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.
Individual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.
In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.
Statistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).
We determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].
Heterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.
All meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).
We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).
We determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].
Heterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.
All meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA). | Results | Study-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).
There was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).
We also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).
For subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).
For analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).
In analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.
Online supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).
As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).
There was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).
We also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).
For subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).
For analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).
In analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.
Online supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).
Individual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.
About half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).
When comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).
Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.
About half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).
When comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5). | Conclusion | In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed. | [
"Search Strategy",
"Systemic Literature Review, Data Extraction, and Quality Assessment",
"Individual Patient Data",
"Statistical Analysis",
"Study-Level Analysis",
"Individual Patient Data Analysis",
"Statement of Ethics",
"Funding Sources",
"Author Contributions"
] | [
"This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).",
"Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.",
"In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.",
"We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).",
"As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).",
"Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).",
"The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).",
"There is no funding to disclose.",
"All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Search Strategy",
"Systemic Literature Review, Data Extraction, and Quality Assessment",
"Individual Patient Data",
"Statistical Analysis",
"Results",
"Study-Level Analysis",
"Individual Patient Data Analysis",
"Discussion",
"Conclusion",
"Statement of Ethics",
"Conflict of Interest Statement",
"Funding Sources",
"Author Contributions",
"Data Availability Statement",
"Supplementary Material"
] | [
"Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.\nThrough a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.",
"Search Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nThis study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).\nSystemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nTwo authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.\nIndividual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nIn addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.\nStatistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).\nWe calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).",
"This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).",
"Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).\nBaseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].\nThe quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.",
"In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.",
"We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).\nWe determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].\nHeterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.\nAll meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).",
"Study-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nAs described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).\nIndividual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).\nOverall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).",
"As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).\nThere was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).\nWe also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).\nFor subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).\nFor analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).\nIn analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.\nOnline supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).",
"Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.\nAbout half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).\nWhen comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).",
"Our study found that patients with NAFLD in Asia were approximately 52 years old, overweight by Asian cutoffs, about 34% nonobese, with slightly elevated ALT and total cholesterol levels, around 17% diabetic, and 85% with NFS < −1.455 indicating a very low probability of having advanced fibrosis. Males tended to be approximately 4 years younger than females, but their BMIs were similar. As with other populations, neither the absence of obesity nor an elevated ALT should be used to guide the screening or diagnosis of NAFLD for Asians [2, 17, 18].\nWe also found different clinical phenotypes of NAFLD among the various Asian subregions. Those with NAFLD in Southeast Asia were significantly younger, with mean age only 43 years, suggesting that the possibility of NAFLD should not be excluded in young patients. Those from the Western Pacific were older by 10 years and 5 years than those from Southeast Asia or the Eastern Mediterranean, respectively. Those from the Eastern Mediterranean had the highest BMIs though there was no statistical difference among regions. However, all three regions demonstrated only slightly elevated ALT levels without regional differences, again showing that ALT levels should not be a criterion for detecting the presence of NAFLD. Notably, ALT levels were also lower in subgroups with higher likelihood for advanced fibrosis, which is likely due to the increase in the AST/ALT ratio that is part of the NFS, and AST/ALT ratio has been shown to correlate with liver fibrosis in chronic liver disease [19].\nNotably, when we reviewed the characteristics of patients by classification of their hepatic steatosis as mild, moderate, and severe by ultrasound, we found those with severe steatosis on average had BMIs that were 14% greater than those with mild hepatic steatosis and 7% greater than those with moderate steatosis. In addition, those with severe steatosis demonstrated average ALT levels that were increased at 1.5–2 × 95% ULN ALT and were 31% higher than those with mild steatosis. Such a finding suggests that if an elevated ALT level is found in one suspected of having NAFLD, that patient may in fact have severe steatosis and require further assessment.\nTo strengthen our meta-analysis findings, we used individual patient-level data from 7,668 subjects in seven Asian cities in three countries and found that overall the trends of differences between males and females, obese and nonobese, and those with NFS <−1.455 or ≥−1.455 that we reported in our meta-analysis were similar to those in the analyzed individual patient-level data. Of particular interest are our findings on the percent of NAFLD patients with fibrosis, since fibrosis is the single most important predictor of death for those with NAFLD [20, 21]. Since we had individual data, we were able to calculate both the NFS and FIB-4 scores and found similar findings to our meta-analysis in that between 66% and 71% (respectively) fell into the low probability of having advanced fibrosis, while 3.4% and 4.2% (respectively) had a high probability of having advanced fibrosis or cirrhosis. In addition, we found that older, female, obese, and diabetic were more likely to have advanced fibrosis. Therefore, although the percentage of patients with advanced fibrosis is low, the large number of patients with NAFLD amplifies the number of patients negatively affected, especially as the NAFLD burden continues to grow in Asia [22, 23]. Consequently, efforts must continue to raise awareness about NAFLD and its risk factors as well as its management, which includes weight loss through diet and exercise as well as management of patients' cardiometabolic risk factors [24, 25]. Furthermore, efforts must be made to raise awareness about the negative effects of NAFLD among older women as seen in this study and in light of a recent study that found that NASH is now the number one indicator for liver transplant among women [26].\nThis study has its strengths. First, the number of studies included is large, which provided a study population of over 2 million for many of the analyses. As a result, the majority of our reported findings have a good level of precision as noted in our relatively tight 95% confidence intervals. In addition, the quality of our included studies overall was high. Besides published data, we also obtained individual patient data from seven centers for additional patient-level data. We also recognize that there are several limitations. One was the lack of data to determine the NFS >0.676 for the meta-analysis. We are aware that a score between −1.455 and 0.676 is in the indeterminate zone, where certainty of the presence or absence of advanced fibrosis is not available, but we were able to determine the level of fibrosis with data from our patient-level data, which indicate that the vast majority of patients are at low risk for having advanced fibrosis. Another limitation is that the severity of steatosis was determined by ultrasound which may have interobserver variability. To account for this limitation, we included analyses of mild versus moderate versus severe steatosis as well as mild versus moderate-severe steatosis and found that the trends presented were similar. Third, a few of our subanalyses were from only a few studies; however, these studies provided a large number of patients such that our confidence in our reported findings is relatively high, especially as the majority of our findings were replicated using individual data. Lastly, for both our study level and individual patient-level data analyses, the majority of the data were from the Western Pacific region, so our data may be most representative of the Western Pacific region, and thus additional studies are needed for other regions of Asia.",
"In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed.",
"The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).",
"D.Q.H. has received grant support from Exxon Mobil-NUS Research Fellowship for Clinicians and Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship. J.F. is on the advisory board/consulting for Gilead. R.C.C. has received grant support from Gilead. T.K. has received grant support and personal speaker fees from Gilead and AbbVie. M.Y. is on the advisory board/consulting for AbbVie, Arbutus Biopharma, Assembly Biosciences, Bristol Myers Squibb, Dicerna Pharmaceuticals, GlaxoSmithKline, Gilead Sciences, Janssen, Merck Sharp and Dohme, ClearB Therapeutics, and Springbank Pharmaceuticals. H.T. has received grant support from AbbVie, Merck Sharp and Dohme, and Bayer. M.H.N. has received grant support from Pfizer, Gilead, and Enanta and is on the advisory board/consulting for Intercept and Gilead. Other authors have no disclosures.",
"There is no funding to disclose.",
"All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision.",
"The majority of data generated or analyzed during this study are included in this article and its online supplementary files. Further enquiries can be directed to the corresponding author.",
"Supplementary data\nClick here for additional data file."
] | [
"intro",
"methods",
null,
null,
null,
null,
"results",
null,
null,
"discussion",
"conclusions",
null,
"COI-statement",
null,
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"data-availability",
"supplementary-material"
] | [
"Nonalcoholic fatty liver disease",
"Epidemiology",
"Liver function tests",
"Obesity",
"Fibrosis"
] | Introduction:
Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.
Through a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.
Methods:
Search Strategy This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).
This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).
Systemic Literature Review, Data Extraction, and Quality Assessment Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).
Baseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].
The quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.
Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).
Baseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].
The quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.
Individual Patient Data In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.
In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.
Statistical Analysis We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).
We determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].
Heterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.
All meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).
We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).
We determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].
Heterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.
All meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).
Search Strategy:
This study was carried out using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and was not registered (online suppl. File; for all online suppl. material, see www.karger.com/doi/10.1159/000521662). The search was conducted using PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019, and details of the search strategy were previously published and provided in the online supplementary material. We included studies that provided data for specific characteristics of NAFLD patients, especially BMI and ALT. Excluded studies included individuals with other liver diseases (e.g., viral hepatitis and autoimmune hepatitis) or excessive alcoholic consumption (>30 g/day for men and >20 g/day for women).
Systemic Literature Review, Data Extraction, and Quality Assessment:
Two authors independently reviewed, selected, and extracted relevant data using a standardized form. Studies using duplicate cohorts were excluded. Discordance between the two reviewers was resolved by discussion between the two reviewers and/or by consultation with a senior investigator (M.H.N.).
Baseline characteristics recorded included age, BMI, ALT, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, and triglycerides. We also collected data on diabetes mellitus, degree of hepatic steatosis, and fibrosis scores when available. Characteristics of NAFLD patients were further categorized by sex, BMI, severity of NAFLD, or presence of fibrosis. Obesity in Asians was defined as BMI ≥25 kg/m2 as this was the cutoff most often used in the studies included in this meta-analysis. For the purpose of this study, we defined severity of hepatic steatosis using the following ultrasound criteria, since ultrasound is the most commonly used diagnostic method for NAFLD [7]. Mild steatosis was defined as a mild increase in liver echogenicity with normal or slightly increased visualization of the diaphragm and intrahepatic vessel borders, moderate steatosis as moderate increase in liver echogenicity with decreased visualization of the diaphragm and intrahepatic vessel borders, and severe steatosis as gross increase in liver echogenicity with poor visualization of the diaphragm, intrahepatic vessel borders, and posterior section of the right lobe. We assessed for the presence of fibrosis by NAFLD fibrosis score (NFS) and Fibrosis-4 (FIB-4) index [8, 9]. We used NFS < −1.455 to define the absence of advanced fibrosis (negative predictive value = 93%) and NFS > 0.676 as a cutoff for advanced fibrosis. We used FIB-4 ≥ 2.67 as the cutoff for advanced fibrosis, since it has been shown to have a highly positive predictive value for advanced fibrosis among NAFLD patients [10, 11].
The quality assessment tool was based on the Newcastle-Ottawa scale (NOS) to evaluate the risk of bias and methodological quality of the included studies [12]. Each article was evaluated for its representativeness, comparability, and outcome. Studies with 7 or more stars were considered to have low risk of bias, 4–6 stars as moderate risk of bias, and 3 or less stars as high risk of bias.
Individual Patient Data:
In addition to study-level data, we obtained individual patient-level data from seven centers for additional analysis (Taichung Veterans General Hospital, Taichung, Taiwan; University of Hong Kong, Hong Kong; Kumamoto University, Kumamoto, Japan; Ogaki Municipal Hospital, Ogaki, Japan; Eguchi Hospital, Saga, Japan; Kawamura Clinic Health Center, Hiroshima, Japan; and Kochi Medical School Hospital, Kochi, Japan). All patients were diagnosed with NAFLD by ultrasound. The data derived from Kumamoto University were obtained from a health screening program performed by the Japanese Red Cross Kumamoto Health Center, Kumamoto (May 2003–April 2012) [13]. The data derived from Ogaki Municipal Hospital were obtained from clinic records of consecutive patients who presented for either health screening or a medical visit (March 2010–September 2015). The data derived from Saga Prefecture (Eguchi Hospital Health Center from 2009 to 2010), Kawamura Clinic Health Center, Hiroshima (April 2009–March 2010), and Kochi Medical School Hospital, Kochi (April 2009–March 2010) were all obtained from general health checkup clinics. The data derived from Taichung Veterans General Hospital were collected from the Medical Screening Center (January–December 2009) [14]. The data derived from the University of Hong Kong were obtained from blood donors from the Hong Kong Red Cross Blood Transfusion Center and volunteers from the general population (August 2010–March 2012) [15]. The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional review board at each study center that provided individual patient-level data.
Statistical Analysis:
We calculated the pooled estimates for mean values of characteristics of NAFLD patients using a random-effects model. For studies reporting only median values, interquartile range values were used to calculate the mean and standard deviation based on the assumption that the distribution of data was symmetrical with large sample size and with other liver diseases excluded. We performed subanalysis for the following populations: male, female, obese, nonobese, Western Pacific, Southeast Asian, Eastern Mediterranean, mild steatosis, moderate steatosis, severe steatosis, moderate to severe steatosis, NFS ≥ −1.455, and NFS < −1.455 (as data were generally reported using these cutoffs in the studies included in this meta-analysis).
We determined the ALT distribution among males and females with NAFLD using a bootstrap model with 10,000 resamples from the included study cohorts. Based on the corresponding mean and standard deviations of ALT values in the included studies, we estimated the proportion of patients with ALT <95% upper limit of normal (ULN), ALT (defined as 30 and 19 U/L in males and females, respectively), 1–1.5 × 95% ULN ALT (30–45 and 19.1–28.5 U/L), 1.5–2 × 95% ULN ALT (45–60 and 28.5–38 U/L), and >2 × 95% ULN ALT (60 and 38 U/L) [16].
Heterogeneity was assessed using I2 statistic, and estimates with I2 ≥50% and p value of <0.05 in Q-statistic were considered to have moderate to severe heterogeneity. We used Egger's test to evaluate for publication bias.
All meta-analyses were conducted using the meta-analysis package in R statistical software (version 3.5.1). Bootstrap modeling was performed with SAS (version 9.4; Cary, NC, USA).
Results:
Study-Level Analysis As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).
There was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).
We also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).
For subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).
For analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).
In analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.
Online supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).
As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).
There was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).
We also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).
For subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).
For analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).
In analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.
Online supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).
Individual Patient Data Analysis Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.
About half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).
When comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).
Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.
About half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).
When comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).
Study-Level Analysis:
As described in Figure 1, 4,995 citations were initially identified with our search strategy where 104 articles (2,247,754 participants) met study criteria and were included in study analysis. Of these, 103 studies (n = 2,245,083 participants) provided BMI data, and 87 studies (n = 2,204,503 participants) provided ALT data for participants with NAFLD. Seven studies used either CT, MRI, controlled attenuation parameter, or the fatty liver index to diagnose NAFLD while the remaining studies were diagnosed by ultrasound. All were from January 1, 2000, to January 17, 2019 (online suppl. Table 1). Overall, the pooled mean age of NAFLD participants was 52.07 (95% CI: 51.28–52.85) years, the mean BMI was 26.2 kg/m2 (95% CI: 25.94–26.46), the mean ALT was 31.74 U/L (95% CI: 30.75–32.72), and the prevalence of diabetes was 16.7% (95% CI: 13.6–20.1) (Table 1a).
There was no difference between males or females in the pooled mean age (49.33 vs.53.43, p = 0.07) or BMI (26.01 vs. 26.73, p = 0.36), though males had a higher ALT level (37.91 vs. 27.84, p < 0.001) (Table 1b). While 74% of males compared to 53% of females had ALT levels between 1 and 1.5 × 95% ULN, more females than males (37% vs. 12%) had a higher degree of ALT elevation 1.5–2 × 95% ULN (shown in Fig. 2a).
We also found that 66% of NAFLD participants were considered obese while 34% were nonobese (Table 1c). Obese and nonobese patients had similar mean age (p = 0.78), but the obese group had a higher mean ALT level and a lower mean HDL (p = 0.01 for both) (Table 1c).
For subanalysis by region, there were 94 studies (n = 2,244,590) from the Western Pacific, 5 studies (n = 1,981) from Southeast Asia, and 5 studies (n = 1,183) from the Eastern Mediterranean (Table 1d). The ages of those from Southeast Asia (42.66, 95% CI: 32.23–53.11) were significantly younger than those from the Western Pacific (52.80, 95% CI: 51.85–53.75) or the Eastern Mediterranean regions (47.83, 95% CI: 46.62–49.04, p < 0.001), but there was no significant difference in the pooled mean ALT or AST levels among the regions (p = 0.79 and 0.16, respectively) (Table 1d).
For analysis of steatosis severity, 4 studies provided data for mild, moderate, and severe steatosis and 6 for mild versus moderate-severe steatosis. In total, 6,262 patients with mild steatosis, 1,576 patients with moderate steatosis, 543 patients with severe steatosis, and 3,952 patients with moderate-severe steatosis were included in this analysis (Table 2). NAFLD participants with severe steatosis had a significantly higher pooled mean BMI (29.89, 95% CI: 27.94–31.84) compared to those with moderate (27.85, 95% CI: 26.66–29.06) and mild steatosis (25.71, 95% CI: 24.90–26.53, p < 0.001), as well as a significantly higher pooled mean ALT level (50.34 vs. 49.61 vs. 34.62, respectively, p = 0.01) (Table 2a; online suppl. Table 2). We found similar results in analysis comparing NAFLD participants with moderate to severe steatosis to those with mild steatosis (Table 2b).
In analysis of fibrosis, there were three studies with 16,729 patients with NFS <−1.455 and 3,008 patients with NFS ≥ −1.455. Those with an NFS <−1.455 had a significantly lower BMI (26.02, 95% CI: 25.66–26.40) compared to those with an NFS >−1.455 (27.09, 95% CI: 26.36–27.83, p < 0.001), but a higher pooled mean ALT level (33.74 vs. 27.83, respectively, p = 0.01) (Table 2c). As mentioned above, most studies provided NFS data using cutoff of −1.455 but not for the 0.676 cutoff.
Online supplementary Tables 3a–g and 4a–e provide the quality assessment of the studies included in the overall and subanalyses with overall scores of 7.45 and 8.83 indicating that the majority of included studies had a low risk of bias and used sound methodology in their respective studies, respectively (online suppl. Tables 3–4). However, heterogeneity was considerable for the majority of studies included with I2 > 50 for most analyses and >70 for subanalyses for liver steatosis and liver fibrosis. There was no significant publication bias found using Egger's test for the analysis of ALT in the overall population (p = 0.15).
Individual Patient Data Analysis:
Overall (n = 7,668), the mean age was 55.1 (±13) years, the mean BMI was 25.5 (±3.4) kg/m2, the median ALT was 29 (range 20–43) U/L, 26.3% were diabetic, 4.2% had FIB-4 ≥ 3.25, and 3.4% had NFS > 0.676 (Table 3a). Females (n = 3,169) were significantly older than males (n = 4,499) (57.3 ± 12.5 vs. 53.5 ± 13.1, p < 0.001), had significantly lower median ALT levels (25 vs. 31, p < 0.001), were significantly less likely to have FIB-4 ≤ 1.45 (68.2% vs. 73.5%), and were more likely to have FIB-4 ≥ 2.67 (6.9% vs. 5.8%, p < 0.001) and FIB-4 ≥ 3.25 (4.5% vs. 4%, p < 0.001) (Table 3a). However, ALT levels were low for both males and females (shown in Fig. 2b), as 46% of males and 32% of females had ALT levels <95% ULN.
About half (48.5%) were obese, and obese NAFLD participants were younger (p < 0.001), more likely male (p = 0.03), and had a higher median ALT (p < 0.001), low-density lipoprotein (p = 0.02), and a lower HDL (p < 0.001) (Table 3b). Obese patients were also more likely to have lower FIB-4 (p < 0.001).
When comparing participants with high versus low FIB-4, high FIB-4 participants using the FIB-4 cutoff of 2.67 were about 10 years older (67.4 ± 10.7 years vs. 56.1 ± 12.8 years, p < 0.001), had higher BMIs (26 ± 3.8 vs. 25.5 ± 3.5 kg/m2, p = 0.01), higher ALT levels (34 vs. 26 U/L, p < 0.001), and a greater proportion of diabetes (36.7% vs. 24.2%, p < 0.001). We found similar findings for those with an FIB-4 of 3.25 or above (online suppl. Table 5).
Discussion:
Our study found that patients with NAFLD in Asia were approximately 52 years old, overweight by Asian cutoffs, about 34% nonobese, with slightly elevated ALT and total cholesterol levels, around 17% diabetic, and 85% with NFS < −1.455 indicating a very low probability of having advanced fibrosis. Males tended to be approximately 4 years younger than females, but their BMIs were similar. As with other populations, neither the absence of obesity nor an elevated ALT should be used to guide the screening or diagnosis of NAFLD for Asians [2, 17, 18].
We also found different clinical phenotypes of NAFLD among the various Asian subregions. Those with NAFLD in Southeast Asia were significantly younger, with mean age only 43 years, suggesting that the possibility of NAFLD should not be excluded in young patients. Those from the Western Pacific were older by 10 years and 5 years than those from Southeast Asia or the Eastern Mediterranean, respectively. Those from the Eastern Mediterranean had the highest BMIs though there was no statistical difference among regions. However, all three regions demonstrated only slightly elevated ALT levels without regional differences, again showing that ALT levels should not be a criterion for detecting the presence of NAFLD. Notably, ALT levels were also lower in subgroups with higher likelihood for advanced fibrosis, which is likely due to the increase in the AST/ALT ratio that is part of the NFS, and AST/ALT ratio has been shown to correlate with liver fibrosis in chronic liver disease [19].
Notably, when we reviewed the characteristics of patients by classification of their hepatic steatosis as mild, moderate, and severe by ultrasound, we found those with severe steatosis on average had BMIs that were 14% greater than those with mild hepatic steatosis and 7% greater than those with moderate steatosis. In addition, those with severe steatosis demonstrated average ALT levels that were increased at 1.5–2 × 95% ULN ALT and were 31% higher than those with mild steatosis. Such a finding suggests that if an elevated ALT level is found in one suspected of having NAFLD, that patient may in fact have severe steatosis and require further assessment.
To strengthen our meta-analysis findings, we used individual patient-level data from 7,668 subjects in seven Asian cities in three countries and found that overall the trends of differences between males and females, obese and nonobese, and those with NFS <−1.455 or ≥−1.455 that we reported in our meta-analysis were similar to those in the analyzed individual patient-level data. Of particular interest are our findings on the percent of NAFLD patients with fibrosis, since fibrosis is the single most important predictor of death for those with NAFLD [20, 21]. Since we had individual data, we were able to calculate both the NFS and FIB-4 scores and found similar findings to our meta-analysis in that between 66% and 71% (respectively) fell into the low probability of having advanced fibrosis, while 3.4% and 4.2% (respectively) had a high probability of having advanced fibrosis or cirrhosis. In addition, we found that older, female, obese, and diabetic were more likely to have advanced fibrosis. Therefore, although the percentage of patients with advanced fibrosis is low, the large number of patients with NAFLD amplifies the number of patients negatively affected, especially as the NAFLD burden continues to grow in Asia [22, 23]. Consequently, efforts must continue to raise awareness about NAFLD and its risk factors as well as its management, which includes weight loss through diet and exercise as well as management of patients' cardiometabolic risk factors [24, 25]. Furthermore, efforts must be made to raise awareness about the negative effects of NAFLD among older women as seen in this study and in light of a recent study that found that NASH is now the number one indicator for liver transplant among women [26].
This study has its strengths. First, the number of studies included is large, which provided a study population of over 2 million for many of the analyses. As a result, the majority of our reported findings have a good level of precision as noted in our relatively tight 95% confidence intervals. In addition, the quality of our included studies overall was high. Besides published data, we also obtained individual patient data from seven centers for additional patient-level data. We also recognize that there are several limitations. One was the lack of data to determine the NFS >0.676 for the meta-analysis. We are aware that a score between −1.455 and 0.676 is in the indeterminate zone, where certainty of the presence or absence of advanced fibrosis is not available, but we were able to determine the level of fibrosis with data from our patient-level data, which indicate that the vast majority of patients are at low risk for having advanced fibrosis. Another limitation is that the severity of steatosis was determined by ultrasound which may have interobserver variability. To account for this limitation, we included analyses of mild versus moderate versus severe steatosis as well as mild versus moderate-severe steatosis and found that the trends presented were similar. Third, a few of our subanalyses were from only a few studies; however, these studies provided a large number of patients such that our confidence in our reported findings is relatively high, especially as the majority of our findings were replicated using individual data. Lastly, for both our study level and individual patient-level data analyses, the majority of the data were from the Western Pacific region, so our data may be most representative of the Western Pacific region, and thus additional studies are needed for other regions of Asia.
Conclusion:
In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed.
Statement of Ethics:
The study was performed in accordance with the World Medical Association Declaration of Helsinki. The individual patient-level database was approved by each study center that provided individual patient-level data, received written informed consent, and the study protocol was approved by the Institutional Review Board at their respective institution (Approval No. CF14040 from the Ethics Committee of the Taichung Veterans General Hospital; No. UW-10-325 from the Ethics Committee/IRB of the University of Hong Kong and Hospital Authority Western Cluster; No. 169 from Faculty of Life Sciences, Kumamoto University; No. 20190328-7 from Ogaki Municipal Hospital; No. 2011-06-04 from Saga University Hospital).
Conflict of Interest Statement:
D.Q.H. has received grant support from Exxon Mobil-NUS Research Fellowship for Clinicians and Singapore Ministry of Health's National Medical Research Council under its NMRC Research Training Fellowship. J.F. is on the advisory board/consulting for Gilead. R.C.C. has received grant support from Gilead. T.K. has received grant support and personal speaker fees from Gilead and AbbVie. M.Y. is on the advisory board/consulting for AbbVie, Arbutus Biopharma, Assembly Biosciences, Bristol Myers Squibb, Dicerna Pharmaceuticals, GlaxoSmithKline, Gilead Sciences, Janssen, Merck Sharp and Dohme, ClearB Therapeutics, and Springbank Pharmaceuticals. H.T. has received grant support from AbbVie, Merck Sharp and Dohme, and Bayer. M.H.N. has received grant support from Pfizer, Gilead, and Enanta and is on the advisory board/consulting for Intercept and Gilead. Other authors have no disclosures.
Funding Sources:
There is no funding to disclose.
Author Contributions:
All authors participated in data acquisition, data interpretation, and review/revision of the manuscript. L.Y.K.: study design, data analysis, and manuscript drafting. D.Q.H.: study design. Y.H.Y.: study design. S.B.: data analysis. L.H.: manuscript drafting. M.H.N.: study concept and design, data analysis, manuscript drafting, and study supervision.
Data Availability Statement:
The majority of data generated or analyzed during this study are included in this article and its online supplementary files. Further enquiries can be directed to the corresponding author.
Supplementary Material:
Supplementary data
Click here for additional data file.
| Background:
NAFLD is increasingly prevalent in Asia, where people suffer more metabolic comorbidities at a lower body mass index (BMI), suggesting potential differences in their clinical profile. Therefore, we attempted to characterize the clinical profile of Asians with NAFLD via a meta-analytic approach.
Methods:
We searched PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019. Two authors independently reviewed and selected 104 articles (2,247,754 persons) that identified NAFLD in Asians and reported relevant data, especially BMI and ALT, and excluded individuals with other liver disease and excessive alcohol consumption. Individual patient-level data were obtained from seven cohorts in Asia to complement meta-analyzed data.
Results:
Overall, the mean age was 52.07 (95% CI: 51.28-52.85) years, with those from Southeast Asia (42.66, 95% CI: 32.23-53.11) being significantly younger. The mean BMI was 26.2 kg/m2, higher in moderate-severe versus mild hepatic steatosis (28.3 vs. 25.7) patients and NFS ≥ -1.455 versus <-1.455 (27.09 vs. 26.02), with 34% having nonobese NAFLD. The mean ALT was 31.74 U/L, higher in NFS < -1.455 versus ≥-1.455 (33.74 vs. 27.83), though no differences were found by obesity or steatosis severity. The majority of males (85.7%) and females (60.7%) had normal to minimally elevated ALT (1-1.5 × 95% ULN). Individual patient-level data analysis (N = 7,668) demonstrated similar results.
Conclusions:
About one-third of Asians with NAFLD were nonobese, and the majority did not have markedly elevated ALT. Therefore, abnormal ALT or BMI is not recommended as a criterion for NAFLD screening in this population. Additionally, there were significant differences in the clinical profiles of NAFLD among the different regions of Asia. | Introduction:
Nonalcoholic fatty liver disease (NAFLD) is considered a metabolic disorder of the liver associated with increased body mass index (BMI) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and death [1]. In recent decades, NAFLD has also become more prevalent in Asia [2]. In fact, the overall prevalence of NAFLD in Asia is about 30%, closely tracking that of the West [1]. However, obesity is defined at a lower BMI for Asians, as the risk of metabolic comorbidities is known to rise at these lower BMI cutoffs [3]. In addition, ALT thresholds for normal ALT may also be lower in Asians [4, 5]. Therefore, it is important to profile the Asian NAFLD population to provide ethnic and region-specific data to inform local practice and public health policy. This is particularly relevant as a recent population-based study from the USA reported very poor liver disease awareness in people affected by NAFLD in general and especially among the Asian group [6]. This disparity could be due to underdiagnosis by care providers, as well as a misperception of being well by patients because of a lack of disease knowledge specific to Asian patients with NAFLD.
Through a systematic review and meta-analytic approach, we characterized the demographic, anthropometric, and laboratory characteristics, as well as the severity of hepatic steatosis and fibrosis in Asians with NAFLD, with a focus on BMI and ALT distributions. We also obtained individual patient-level data to complement our meta-analyzed results.
Conclusion:
In Asia, using a BMI of 25 or greater as an indicator of obesity, 66% of the studied population with NAFLD were obese, while 34% were nonobese. ALT levels were not found to be elevated except in cases with combined moderate-severe as well as severe steatosis, with the majority of both males and females presenting with ALT within 1–1.5 × 95% ULN or lower, suggesting that ALT levels should not be used as a screening or diagnostic criterion for the presence of NAFLD. Among the three regions, Southeast Asians were significantly younger, Western Pacific Asians had the lowest BMIs, and Eastern Mediterranean Asians had the highest BMIs. Additional efforts to raise awareness about NAFLD among Asians are needed. | Background:
NAFLD is increasingly prevalent in Asia, where people suffer more metabolic comorbidities at a lower body mass index (BMI), suggesting potential differences in their clinical profile. Therefore, we attempted to characterize the clinical profile of Asians with NAFLD via a meta-analytic approach.
Methods:
We searched PubMed, EMBASE, and Cochrane databases from January 1, 2000, to January 17, 2019. Two authors independently reviewed and selected 104 articles (2,247,754 persons) that identified NAFLD in Asians and reported relevant data, especially BMI and ALT, and excluded individuals with other liver disease and excessive alcohol consumption. Individual patient-level data were obtained from seven cohorts in Asia to complement meta-analyzed data.
Results:
Overall, the mean age was 52.07 (95% CI: 51.28-52.85) years, with those from Southeast Asia (42.66, 95% CI: 32.23-53.11) being significantly younger. The mean BMI was 26.2 kg/m2, higher in moderate-severe versus mild hepatic steatosis (28.3 vs. 25.7) patients and NFS ≥ -1.455 versus <-1.455 (27.09 vs. 26.02), with 34% having nonobese NAFLD. The mean ALT was 31.74 U/L, higher in NFS < -1.455 versus ≥-1.455 (33.74 vs. 27.83), though no differences were found by obesity or steatosis severity. The majority of males (85.7%) and females (60.7%) had normal to minimally elevated ALT (1-1.5 × 95% ULN). Individual patient-level data analysis (N = 7,668) demonstrated similar results.
Conclusions:
About one-third of Asians with NAFLD were nonobese, and the majority did not have markedly elevated ALT. Therefore, abnormal ALT or BMI is not recommended as a criterion for NAFLD screening in this population. Additionally, there were significant differences in the clinical profiles of NAFLD among the different regions of Asia. | 9,611 | 364 | [
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Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY] | [CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY] | [CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY] | [CONTENT] Male | Female | Humans | Middle Aged | Non-alcoholic Fatty Liver Disease | Body Mass Index | Obesity | Comorbidity [SUMMARY] | [CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY] | [CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY] | [CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY] | [CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY] | [CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY] | [CONTENT] fatty liver disease | liver disease nafld | specific asian patients | bmi asians risk | obesity asians defined [SUMMARY] | [CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY] | [CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY] | [CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY] | [CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY] | [CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY] | [CONTENT] alt | data | steatosis | studies | nafld | 95 | patients | mean | table | vs [SUMMARY] | [CONTENT] nafld | disease | asian | bmi | metabolic | nonalcoholic | asians | lower | liver disease | recent [SUMMARY] | [CONTENT] hospital | center | data | fibrosis | studies | health | steatosis | japan | data derived | derived [SUMMARY] | [CONTENT] vs | table | 001 | ci | 95 ci | participants | 26 | 95 | studies | mean [SUMMARY] | [CONTENT] asians | bmis | alt | nafld | levels | alt levels | severe | regions southeast asians | asians significantly younger western | alt levels screening [SUMMARY] | [CONTENT] data | alt | nafld | studies | steatosis | vs | 001 | funding | disclose | funding disclose [SUMMARY] | [CONTENT] data | alt | nafld | studies | steatosis | vs | 001 | funding | disclose | funding disclose [SUMMARY] | [CONTENT] NAFLD | Asia | BMI ||| Asians | NAFLD [SUMMARY] | [CONTENT] PubMed | EMBASE | Cochrane | January 1, 2000 | January 17, 2019 ||| Two | 104 | 2,247,754 | Asians | BMI | ALT ||| seven | Asia [SUMMARY] | [CONTENT] 52.07 | 95% | CI | 51.28-52.85 | years | Southeast Asia | 42.66 | 95% | CI | 32.23-53.11 ||| BMI | 26.2 kg | m2 | 28.3 | 25.7 | NFS ≥ | 27.09 | 26.02 | 34% | nonobese NAFLD ||| ALT | 31.74 | U/L | NFS | 33.74 | 27.83 ||| 85.7% | 60.7% | ALT | 1-1.5 | 95% | ULN ||| 7,668 [SUMMARY] | [CONTENT] About one-third | Asians | NAFLD | ALT ||| ALT | BMI | NAFLD ||| NAFLD | Asia [SUMMARY] | [CONTENT] NAFLD | Asia | BMI ||| Asians | NAFLD ||| PubMed | EMBASE | Cochrane | January 1, 2000 | January 17, 2019 ||| Two | 104 | 2,247,754 | Asians | BMI | ALT ||| seven | Asia ||| 52.07 | 95% | CI | 51.28-52.85 | years | Southeast Asia | 42.66 | 95% | CI | 32.23-53.11 ||| BMI | 26.2 kg | m2 | 28.3 | 25.7 | NFS ≥ | 27.09 | 26.02 | 34% | nonobese NAFLD ||| ALT | 31.74 | U/L | NFS | 33.74 | 27.83 ||| 85.7% | 60.7% | ALT | 1-1.5 | 95% | ULN ||| 7,668 ||| About one-third | Asians | NAFLD | ALT ||| ALT | BMI | NAFLD ||| NAFLD | Asia [SUMMARY] | [CONTENT] NAFLD | Asia | BMI ||| Asians | NAFLD ||| PubMed | EMBASE | Cochrane | January 1, 2000 | January 17, 2019 ||| Two | 104 | 2,247,754 | Asians | BMI | ALT ||| seven | Asia ||| 52.07 | 95% | CI | 51.28-52.85 | years | Southeast Asia | 42.66 | 95% | CI | 32.23-53.11 ||| BMI | 26.2 kg | m2 | 28.3 | 25.7 | NFS ≥ | 27.09 | 26.02 | 34% | nonobese NAFLD ||| ALT | 31.74 | U/L | NFS | 33.74 | 27.83 ||| 85.7% | 60.7% | ALT | 1-1.5 | 95% | ULN ||| 7,668 ||| About one-third | Asians | NAFLD | ALT ||| ALT | BMI | NAFLD ||| NAFLD | Asia [SUMMARY] |
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Reliability and Validity of the Tinnitus Handicap Inventory: A Clinical Study of Questionnaires. | 36349675 | The aim of this study is to observe the application of the Chinese version of Tinnitus Handicap Inventory-China in Tinnitus patients and verify its reliability and validity. | BACKGROUND | About 1129 patients with tinnitus as the first complaint were selected as subjects. The patients were randomly divided into 2 groups: exploration group (n = 565), whose data were analyzed with reliability analysis method using Statistical Package for the Social Sciences software 19.0, validation group (n = 564), whose data were analyzed with validity analysis method using AMOS21.0. | METHODS | (1) Reliability test: The Cronbach's α coefficients of the Tinnitus Handicap Inventory-China scale in both groups were 0.94, among which, the Cronbach's α coefficients of functional factor (F), emotion factor (E), and catastrophic factor (C) in group E were 0.87, 0.90, and 0.78, respectively. The half-reliability of the 2 components is 0.87. The correlation coefficient between items and the scale in group E and group V is 0.36-0.78 and 0.33-0.77, respectively. (2) Content validity: The Kaiser-Meyer-Olkin value of group E is 0.96, a total of 4 common factors were extracted, and the cumulative interpretation rate is 57.844%. The number of factors with load less than 0.4 on the 4 common factors is only 1 (F24), suggesting that this factor had little significance; the number of factors with load more than 0.4 on the 2 common factors is 8 (F1, E6, F9, C11, F15, E21, E22, and C23), suggesting that patients had different understandings of these 8 questions. (3) Structural validity: The root mean square error of approximation value of the AMOS structural model in group V is 0.065, and the root mean square residual value is 0.114, indicating low fitness; the NC value is 3.353, indicating good fitness of the scale, but it still needed to be simplified. | RESULTS | The Chinese version of Tinnitus Handicap Inventory-China has a high reliability when applied in China, but the content validity and structure validity are not high, and the clinical practicability needs to be improved. | CONCLUSION | [
"Humans",
"Tinnitus",
"Reproducibility of Results",
"Surveys and Questionnaires",
"China",
"Disease Progression",
"Psychometrics"
] | 9682740 | Introduction | Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.
Data from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3
Because of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12 | Methods | Ethical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.
The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.
Subjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent.
The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent.
Study Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).
Reliability analysis
Statistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.
Validity analysis
Exploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.
The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).
Reliability analysis
Statistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.
Validity analysis
Exploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations. | Results | Tinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.
Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.
Reliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.
The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.
Exploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).
The coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.
The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).
The coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.
Confirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.
Researchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.
Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.
Researchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.
Statistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755.
Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755.
Reliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.
The split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.
After analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.
A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.
The split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.
After analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.
Validity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Confirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Confirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model. | Conclusions | The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study. | [
"Main Points",
"Ethical Review",
"Subjects",
"Study Design and Statistical Analysis",
"Tinnitus Handicap Inventory Data",
"Reliability Analysis: Reliability Detection",
"Exploratory Factor Analysis: Content Validity",
"Confirmatory Factor Analysis: Structural Validity",
"Statistical Analysis",
"Reliability Analysis",
"Validity Analysis",
"Exploratory Factor Analysis",
"Confirmatory Factor Analysis"
] | [
"The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.",
"The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.",
"The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. ",
"The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.",
"Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.",
"The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.",
"The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.",
"Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.",
"Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. ",
"A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.",
"Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.",
"Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.",
"Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Main Points",
"Introduction",
"Methods",
"Ethical Review",
"Subjects",
"Study Design and Statistical Analysis",
"Results",
"Tinnitus Handicap Inventory Data",
"Reliability Analysis: Reliability Detection",
"Exploratory Factor Analysis: Content Validity",
"Confirmatory Factor Analysis: Structural Validity",
"Statistical Analysis",
"Reliability Analysis",
"Validity Analysis",
"Exploratory Factor Analysis",
"Confirmatory Factor Analysis",
"Discussion",
"Conclusions"
] | [
"The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.\nThe factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23. \nThe content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.",
"Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.\nData from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3\nBecause of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12",
"Ethical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nThe participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.\nSubjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nThe Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. \nStudy Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.\nThe patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.",
"The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.",
"The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent. ",
"The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).\n\nReliability analysis\n\nStatistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.\n\nValidity analysis\n\nExploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.",
"Tinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nFrequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.\nReliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nThe Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.\nExploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nThe KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.\nConfirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nUsing the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.\nStatistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nFrequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. \nReliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nA scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.\nValidity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nExploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.",
"Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.",
"The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.",
"The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).\nThe coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.",
"Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.\nResearchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.",
"Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755. ",
"A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.\nThe split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.\nAfter analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.",
"Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nExploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.\nConfirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.\nConfirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.",
"Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.\nThere is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.\nThe loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.",
"Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters. \nAccording to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.\nThe latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.\nThe model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.\nThere are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.",
"The linguistic validation of the Chinese THI from the original THI consisted of 3 phases: (1) The scale was translated and tested by Qiulan Shi14 in 2007. After the full text was translated into English, 3 professionals who were proficient in English and familiar with the scale independently translated it into Chinese and then 1 English professional with no knowledge of the scale and 1 doctor translated it back into Chinese and compared both scales to ensure the Chinese version of the scale was equivalent to the original scale. (2) A cultural adjustment of the scale was then performed. As the original scale was established under a specific cultural background,13 it was discussed among tinnitus patients, otologists, nursing staff, and professionals engaged in quality-of-life research, and the questions were determined to be readily understood by Chinese patients with different education levels. (3) The validation was then finalized, according to the reference method.4\nPrevious studies have reported that the THI performs comparably well in several countries and languages.7,9,15-21 The findings of our study suggest that the Chinese THI has good internal consistency reliability for both the total scale (Cronbach’s alpha = 0.94) and the 3 subscales-functional (Cronbach’s alpha = 0.87), emotional (Cronbach’s alpha = 0.90), and catastrophic (Cronbach’s alpha = 0.78-0.76). The internal consistency reliability of the Chinese THI is similar to the validation of the scale in other languages. For example, Cronbach’s alpha is 0.930 for the United States, Danish, German, Dutch, Lithuanian, and Hebrew THI scales; and it is 0.79-0.95 for the Korean THI, 0.72-0.94 for the Chinese THI (Cantonese), 0.91 for the Italian THI, and 0.94 for the Portuguese THI (Lithuanian). Cronbach’s alpha coefficients for the functional, emotional, and catastrophic subscales corresponded well with those of the original THI version. Our study found that the mean score of the question “Do you feel that your tinnitus problem has placed stress on your relationships with members of your family and friends?” (17E) is 0.6, which is the minimum of all items and consistent with previous Lithuanian research.8 Therefore, Tinnitus appears to have a little emotional impact on family and friends.\nThe THI is widely used in clinical practice and research as a 3-dimensional measure of tinnitus severity. Despite its extensive use, its factor structure remains unclear. Furthermore, the THI can be considered a reliable measure only if both Cronbach’s alpha coefficient and classical test theory are used. Recently, Gos22 analyzed 1115 patients with tinnitus using the more modern and robust item response theory, finding that this bifactor model had the best fit (RMSEA = 0.055; CFI = 0.976; and SRMR = 0.040) and identifying 1 strong general factor and several weak specific factors. Further, Mokken scaling generated a reliable unidimensional scale (Loevinger’s H = 0.463). To refine the THI, these authors proposed that 5 items have to be removed. Their findings support the use of the THI to evaluate tinnitus severity as a reliable, unidimensional scale. However, clinicians and researchers should rely only on its overall score, which reflects global tinnitus severity. Our study confirmed the utility of the 3-factor structure of the THI, which is consistent with an earlier German study of 373 patients with tinnitus.23 On the bases of several combined criteria, we propose that certain THI items be removed to refine the scale, which is consistent with Elżbieta’s study in which THI2, THI8, THI13, THI19, and THI24 were removed. Newman et al24 proposed a shorter version of the THI, consisting of only 10 items, which were selected on the bases of just 3 criteria: a high item/total correlation, the representativeness of the 3 content domains, and face validity. We find that such criteria are insufficient and propose refining the THI instrument by removing just those items with a low degree of fitness. We believe that short-form questionnaires are essential in busy clinical practices and extensive research protocols.\nAdditionally, Kennedy et al25 noted that the THI, compared to other tinnitus-related questionnaires, contains a disproportionately large number of items related to the psychological/emotional aspects of tinnitus. The results of our study also suggest that tinnitus severity, as measured by the Chinese THI, captures mainly the emotional aspects of tinnitus, which have a high Cronbach’s coefficient for the emotional subscale (0.90) in both the E and V groups as shown in Table 2.\nIt should be noted that the THI uses a 3-label nominal or category scale. Many psychophysicists would argue that this makes it inappropriate to average across scores and users. Stevens,26 often referred to as the father of human psychophysics, summarized the different scales (nominal, ordinal, interval, and ratio) and noted their appropriate use and limitations. Numerical scales are appropriate for equal differences and equal ratios. This does not apply to ordinal scales. The sensitivity of the THI has been challenged.27 Other considerations for tinnitus trials designs were proposed by professor Tyler.27,28 He created a questionnaire (developed on emotions, hearing, sleep, and concentration) focused on the primary activities impaired by tinnitus and therefore more sensitive to treatments and the questions and found that the tinnitus primary function questionnaire is valid, reliable, and sensitive and can be used to determine.29 Many scales attempt to quantify the assessment of “quality of life”. However, we believe that understanding the quality of life is complex, and many widely used questionnaires do not capture the broad range of factors that we believe are important. Many do not include questions about communicating. Professor Tyler also developed a preliminary questionnaire designed to measure “The Meaning of Life” from a broader perspective in 2020 year. Four factors were prominent in this initial sample, which called (1) friendship and positive outlook, (2) physical health, (3) hearing and mental health, and (4) satisfaction with life. Participants with tinnitus reported more trouble sleeping than participants with cochlear implants, whereas both groups had lower scores on hearing. Older patients reported more difficulty with remembering things but were more satisfied with their financial situation.30 Therefore, based on the complexity and particularity of tinnitus disease, the evaluation of tinnitus is also complex, and the scale is widely used rather than limited.",
"The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study."
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] | [
"Tinnitus",
"Tinnitus Handicap Inventory",
"reliability",
"questionnaire"
] | Main Points:
The Chinese version of Tinnitus Handicap Inventory-China (THI-CN) has a high reliability when applied in China.
The factor F24 had little significance in THI-CN; and the patients had different understandings of 8 questions including F1, E6, F9, C11, F15, E21, E22, and C23.
The content validity and structure validity of THI-CN are not high, and the clinical practicability needs to be improved.
Introduction:
Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.
Data from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3
Because of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12
Methods:
Ethical Review The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.
The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.
Subjects The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent.
The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent.
Study Design and Statistical Analysis The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).
Reliability analysis
Statistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.
Validity analysis
Exploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.
The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).
Reliability analysis
Statistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.
Validity analysis
Exploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.
Ethical Review:
The participants in this study had the right to withdraw from the study at any time. The subjects were informed about the purpose, nature, potential benefits, and risks of the study, and all provided written informed consent. This clinical trial originated from Shanghai Municipal Hospital, Shenkang Hospital Development Center (SHDC12014125), and was approved by the Ethics Committee of Yueyang Hospital, which is affiliated with the Shanghai University.
Subjects:
The Chinese THI was administered to 1129 patients (between the ages of 14 and 88 years; average: 50.17 ± 14.606 years) who reported chronic tinnitus as their primary complaint or as their secondary complaint to hearing loss. These patients were seen in the Otolaryngology Department of Yueyang Hospital of Shanghai University for tinnitus between September 2015 and August 2017. The patients included 541 males (47.9%) and 588 females (52.1%). Of all patients, 285 patients (25.2%) received only primary education (at or below junior high school), 430 patients (38.1%) received secondary education (at or above junior high school), and 414 patients (36.7%) received higher education (at or above high school). Judging by the tinnitus frequency, 256 patients (22.7%) had low-frequency tinnitus, 82 patients (7.7%) had medium-frequency tinnitus, and 791 patients (70.7%) had high-frequency tinnitus. All subjects were native Chinese speakers with good oral communication and the ability to understand and provide informed consent.
Study Design and Statistical Analysis:
The patients were randomly divided into 2 groups without regard for gender, age, education level, or frequency of tinnitus. One group is the exploration group (group E, n = 565 cases), whose data were analyzed with the reliability analysis method using Statistical Package for the Social Sciences software 19.0 (IBM SPSS Corp.; Armonk, NY, USA). The other group is the validation group (group V, n = 564 cases), whose data were analyzed with the validity analysis method using AMOS21.0 (SPSS AMOS, which is a professional modeling software launched by IBM).
Reliability analysis
Statistical Package for the Social Sciences software 19.0 is used for the reliability analysis, and Cronbach’s alpha coefficient is used to investigate the internal consistency of the scale. The cross-item consistency of the scale is tested using split-half reliability. The data are reliable when the Cronbach’s alpha coefficient is >0.7.
Validity analysis
Exploratory factor analysis (EFA) is used to test the content validity of the THI. Tinnitus Handicap Inventory structure validity is tested using confirmatory factor analysis (CFA). Principal component analysis (PCA) method is used to analyze the content validity when the Kaiser–Meyer–Olkin (KMO) >0.9. The load matrix is rotated to be as close as possible to the simple structure, and the load amounts of the 25 questions on the 4 common factors (CFs) were obtained after the rotation of variance maximization using the Kaiser standardized orthogonal rotation method. The rotation converged after 7 iterations.
Results:
Tinnitus Handicap Inventory Data Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.
Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.
Reliability Analysis: Reliability Detection The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.
The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.
Exploratory Factor Analysis: Content Validity The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).
The coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.
The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).
The coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.
Confirmatory Factor Analysis: Structural Validity Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.
Researchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.
Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.
Researchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.
Statistical Analysis Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755.
Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755.
Reliability Analysis A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.
The split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.
After analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.
A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.
The split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.
After analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.
Validity Analysis Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Confirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Confirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Tinnitus Handicap Inventory Data:
Frequency distributions, means, standard deviations, and correlations of the Chinese THI data of 1129 patients (including the functional, emotional, and catastrophic subscales) are presented in Table 1.
Reliability Analysis: Reliability Detection:
The Cronbach’s coefficient of the group E is 0.94, which includes Cronbach’s coefficients of 0.87, 0.90, and 0.78 for the functional, emotional, and catastrophic subscales, respectively. The correlation coefficient between items in group E and the scale is 0.36-0.78, whereas the correlation coefficient between items in group V and the scale is 0.33-0.77. The Cronbach’s alpha coefficients of THI scale, group E, and group V are shown in Table 2.
Exploratory Factor Analysis: Content Validity:
The KMO value of the group E is 0.96, confirming that this scale is eligible for an EFA, which is then conducted using the PCA method. After the rotation of maximum variance, 4 CFs with feature roots larger than 1 were extracted; the cumulative variance contribution rate is 57.84%, the explanatory variance (EV) of the first factor is 23.94%, and the EVs of the other 3 factors were 14.52%, 11.06%, and 8.32%, respectively (Table 3).
The coefficients with load amounts less than 0.4 are deleted in Table 3. We see that the load of F24 on the four CFs is less than 0.4. The load capacities of questions F1, F4, E6, C11, F15, E21, E22, and C23 were all less than 0.5, whereas the load capacities of questions F1, E6, F9, C11, F15, E21, E22, and C23 were greater than 0.4 on 2 CFs at the same time in Table 4.
Confirmatory Factor Analysis: Structural Validity:
Using the structural equation model, the relationship between latent variables and the corresponding relationship between latent variables and explicit variables were represented using a path diagram. The rectangle represents the explicit variable, the ellipse or circle represents the implicit variable (data carrier), the 1-way arrow represents the 1-way influence or causality, and the 2-way arrow represents the correlation coefficient in the path diagram. The larger the data, the greater the correlation, but the smaller the data after the rectangle explicit variable, the greater the correlation. Analysis of Moment Structure (AMOS) is used to draw the structural equation model for the THI scale, as shown in Figure 1.
Researchers primarily use fitness indicators, such as chi square values, significance and root mean square error of approximation (RMSEA) values, expected cross-validation index values, Standardized Root Mean square Residual (SRMR) values, and goodness of fit index (GFI) and comparative fit index (CFI) values as the bases for deciding whether a model achieves a specific degree of overall fitness for practical applications because of their sufficient explanatory value. To determine the suitability of the various fitness indices of the model, the fitness indices of the AMOS structural equation model12 were compared to the parameters of the THI. These results are shown in Table 5.
Statistical Analysis:
Frequency distribution, mean value, standard deviation (significant dependence on functional, emotional, or disaster subscales) and question-total correlation (correlation between items and scales composed of other items) of each item of Tinnitus Handicap Inventory-China (THI-CN) are shown in Table 1. The range of question-total correlation coefficient for the THI-CN scale is 0.302-0.755.
Reliability Analysis:
A scale is considered to have good reliability when Cronbach’s alpha coefficient reaches 0.8-0.9. The THI scale showed good internal consistency from the reliability analyses of groups E and V. Cronbach’s alpha coefficient for both groups is 0.94, which is nearly the same as the original THI scale’s Cronbach’s alpha coefficient (0.93). In the 3 subscales, group E and V had good internal consistency reliability of functional factors (Cronbach’s alpha = 0.87) and emotional factors (Cronbach’s alpha = 0.90). The Cronbach’s alpha coefficient for the catastrophic factor is 0.78 and 0.76 in the group E and V, respectively, which were similar to those of the original THI scale as shown in Table 2. This low Cronbach’s alpha coefficient may be related to the small number of questions used in the questionnaire. We found that the internal consistency is still significant after the THI scale is introduced in China.
The split-half reliability reflects the degree of internal consistency of the test items in terms of similar content or characteristics. To analyze split-half reliability, the test items were divided into 2 equal groups after the test. An odd-even grouping method is adopted and then the correlation between the 2 items is calculated. A higher correlation is indicative of higher reliability. The coefficients of the 2 groups were both 0.87, which suggest a high correlation between the scores of the 2 items, that is, a significant degree of internal consistency is achieved after the THI scale is introduced in China.
After analyzing the reliability of groups T and Y, the data of the 2 groups were found to be almost identical, confirming that when previous researchers divided the total samples into 2 groups for analysis and comparison, any errors that may have been caused by artificial groupings were almost completely excluded, providing support for the authenticity of the validity analysis.
Validity Analysis:
Exploratory Factor Analysis Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Confirmatory Factor Analysis Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Exploratory Factor Analysis:
Exploratory factor analysis can be used to synthesize variables with complex relationships into key factors. In this study, PCA is used to analyze the THI scale. After rotation with maximized variance, 4 CFs with characteristic roots greater than 1 were extracted, which were inconsistent with the number of 3 CFs in the scale. The cumulative interpretation rate is 57.844%, as shown in Table 3.
There is a serious deviation from the classification of the original THI scale when the load amount is higher than 0.4 among the 4 CFs extracted, as shown in Table 4. None of the items in a CF were entirely functional, affective, or catastrophic. Therefore, it can be inferred that the use value of the THI scale is affected when it is introduced in China because of either translation or cultural issues.
The loads on the 4 CFs of question 24 (“Do you feel more ringing in your ears when you are stressed?”) were all less than 0.4. The loads on 2 CFs were greater than 0.4 for questions F1, E6, F9, C11, F15, E21, E22, and C23, suggesting that the choices of patients were ambiguous, and they did not know which CF to which they should be attributed. In other words, Chinese respondents have different understandings of the information they want to be asked in these 8 questions.
Confirmatory Factor Analysis:
Confirmatory factor analysis is used to determine whether the relationship between a factor and its corresponding measurement term conforms to the theoretical relationship designed by researchers. This study evaluated the validity of the THI scale using the path graph and model parameters.
According to the path map, there is a good correlation between explicit and implicit variables, with only 3 items showing a weak correlation. The correlation coefficient between question 2F (“Do you think tinnitus is too loud to affect your hearing of others?”) and latent variable F is only 0.30, showing a weak correlation. The correlation coefficient between question 24F (“Do you feel more ringing in your ears when you are stressed?”) and latent variable F is 0.49, showing a weak correlation. The correlation coefficient between question 19C of the catastrophic factors (“Do you feel unable to control tinnitus?”) and latent variable C is 0.49, showing a weak correlation.
The latent variables of the functional, affective, and catastrophic factors are also highly correlated with each other, as their correlation coefficients are all greater than 0.90, indicating that these 3 factors are interrelated and reflective of the internal consistency mentioned above.
The model of the THI is then evaluated according to the fitness degree index of the structural model, and the data showed that it is significant (P = .000). The sample size used in this confirmatory factor analysis is 545. The increase of sample size may lead to the increase of chi square value and degree of freedom, which indicates that the model is rejected. In this paper, the chi square degree of freedom (NC) ratio is used for analysis, which is one of the indicators for the fitness of the model. The adaptation of the THI scale model is good (NC = 3.363 > 3), as reflected by the PCA. The RMSEA is an absolute index that does not require a reference model: the smaller the value is, the better the fit of the model. The RMSEA value of group V is 0.065, which indicates that the model can be further optimized.13 A model is considered acceptable with an RMR value below 0.05, but the RMR value of the group V is 0.114, which indicates that the degree of fitness of the THI scale is questionable. The purpose of the non-centrality parameter (NCP) value is to minimize the parameter value: the larger its value, the worse the degree of fitness of the model. When the NCP value is 0, the model has perfect fitness. The NCP value of group V is 640.088, which is far from 0. The CFI, GFI, and adjusted GFI of group V are all near the critical value, which indicates that the degree of fitness of the THI scale model is good.
There are many indices of the degree of fitness of a model, and many combinations of indices exist from which researchers can choose. It is not possible to judge whether a model has a good degree of fitness according to any single index; researchers instead should combine other indices and statistical theories to show the degree of fitness of a model.
Discussion:
The linguistic validation of the Chinese THI from the original THI consisted of 3 phases: (1) The scale was translated and tested by Qiulan Shi14 in 2007. After the full text was translated into English, 3 professionals who were proficient in English and familiar with the scale independently translated it into Chinese and then 1 English professional with no knowledge of the scale and 1 doctor translated it back into Chinese and compared both scales to ensure the Chinese version of the scale was equivalent to the original scale. (2) A cultural adjustment of the scale was then performed. As the original scale was established under a specific cultural background,13 it was discussed among tinnitus patients, otologists, nursing staff, and professionals engaged in quality-of-life research, and the questions were determined to be readily understood by Chinese patients with different education levels. (3) The validation was then finalized, according to the reference method.4
Previous studies have reported that the THI performs comparably well in several countries and languages.7,9,15-21 The findings of our study suggest that the Chinese THI has good internal consistency reliability for both the total scale (Cronbach’s alpha = 0.94) and the 3 subscales-functional (Cronbach’s alpha = 0.87), emotional (Cronbach’s alpha = 0.90), and catastrophic (Cronbach’s alpha = 0.78-0.76). The internal consistency reliability of the Chinese THI is similar to the validation of the scale in other languages. For example, Cronbach’s alpha is 0.930 for the United States, Danish, German, Dutch, Lithuanian, and Hebrew THI scales; and it is 0.79-0.95 for the Korean THI, 0.72-0.94 for the Chinese THI (Cantonese), 0.91 for the Italian THI, and 0.94 for the Portuguese THI (Lithuanian). Cronbach’s alpha coefficients for the functional, emotional, and catastrophic subscales corresponded well with those of the original THI version. Our study found that the mean score of the question “Do you feel that your tinnitus problem has placed stress on your relationships with members of your family and friends?” (17E) is 0.6, which is the minimum of all items and consistent with previous Lithuanian research.8 Therefore, Tinnitus appears to have a little emotional impact on family and friends.
The THI is widely used in clinical practice and research as a 3-dimensional measure of tinnitus severity. Despite its extensive use, its factor structure remains unclear. Furthermore, the THI can be considered a reliable measure only if both Cronbach’s alpha coefficient and classical test theory are used. Recently, Gos22 analyzed 1115 patients with tinnitus using the more modern and robust item response theory, finding that this bifactor model had the best fit (RMSEA = 0.055; CFI = 0.976; and SRMR = 0.040) and identifying 1 strong general factor and several weak specific factors. Further, Mokken scaling generated a reliable unidimensional scale (Loevinger’s H = 0.463). To refine the THI, these authors proposed that 5 items have to be removed. Their findings support the use of the THI to evaluate tinnitus severity as a reliable, unidimensional scale. However, clinicians and researchers should rely only on its overall score, which reflects global tinnitus severity. Our study confirmed the utility of the 3-factor structure of the THI, which is consistent with an earlier German study of 373 patients with tinnitus.23 On the bases of several combined criteria, we propose that certain THI items be removed to refine the scale, which is consistent with Elżbieta’s study in which THI2, THI8, THI13, THI19, and THI24 were removed. Newman et al24 proposed a shorter version of the THI, consisting of only 10 items, which were selected on the bases of just 3 criteria: a high item/total correlation, the representativeness of the 3 content domains, and face validity. We find that such criteria are insufficient and propose refining the THI instrument by removing just those items with a low degree of fitness. We believe that short-form questionnaires are essential in busy clinical practices and extensive research protocols.
Additionally, Kennedy et al25 noted that the THI, compared to other tinnitus-related questionnaires, contains a disproportionately large number of items related to the psychological/emotional aspects of tinnitus. The results of our study also suggest that tinnitus severity, as measured by the Chinese THI, captures mainly the emotional aspects of tinnitus, which have a high Cronbach’s coefficient for the emotional subscale (0.90) in both the E and V groups as shown in Table 2.
It should be noted that the THI uses a 3-label nominal or category scale. Many psychophysicists would argue that this makes it inappropriate to average across scores and users. Stevens,26 often referred to as the father of human psychophysics, summarized the different scales (nominal, ordinal, interval, and ratio) and noted their appropriate use and limitations. Numerical scales are appropriate for equal differences and equal ratios. This does not apply to ordinal scales. The sensitivity of the THI has been challenged.27 Other considerations for tinnitus trials designs were proposed by professor Tyler.27,28 He created a questionnaire (developed on emotions, hearing, sleep, and concentration) focused on the primary activities impaired by tinnitus and therefore more sensitive to treatments and the questions and found that the tinnitus primary function questionnaire is valid, reliable, and sensitive and can be used to determine.29 Many scales attempt to quantify the assessment of “quality of life”. However, we believe that understanding the quality of life is complex, and many widely used questionnaires do not capture the broad range of factors that we believe are important. Many do not include questions about communicating. Professor Tyler also developed a preliminary questionnaire designed to measure “The Meaning of Life” from a broader perspective in 2020 year. Four factors were prominent in this initial sample, which called (1) friendship and positive outlook, (2) physical health, (3) hearing and mental health, and (4) satisfaction with life. Participants with tinnitus reported more trouble sleeping than participants with cochlear implants, whereas both groups had lower scores on hearing. Older patients reported more difficulty with remembering things but were more satisfied with their financial situation.30 Therefore, based on the complexity and particularity of tinnitus disease, the evaluation of tinnitus is also complex, and the scale is widely used rather than limited.
Conclusions:
The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study.
| Background:
The aim of this study is to observe the application of the Chinese version of Tinnitus Handicap Inventory-China in Tinnitus patients and verify its reliability and validity.
Methods:
About 1129 patients with tinnitus as the first complaint were selected as subjects. The patients were randomly divided into 2 groups: exploration group (n = 565), whose data were analyzed with reliability analysis method using Statistical Package for the Social Sciences software 19.0, validation group (n = 564), whose data were analyzed with validity analysis method using AMOS21.0.
Results:
(1) Reliability test: The Cronbach's α coefficients of the Tinnitus Handicap Inventory-China scale in both groups were 0.94, among which, the Cronbach's α coefficients of functional factor (F), emotion factor (E), and catastrophic factor (C) in group E were 0.87, 0.90, and 0.78, respectively. The half-reliability of the 2 components is 0.87. The correlation coefficient between items and the scale in group E and group V is 0.36-0.78 and 0.33-0.77, respectively. (2) Content validity: The Kaiser-Meyer-Olkin value of group E is 0.96, a total of 4 common factors were extracted, and the cumulative interpretation rate is 57.844%. The number of factors with load less than 0.4 on the 4 common factors is only 1 (F24), suggesting that this factor had little significance; the number of factors with load more than 0.4 on the 2 common factors is 8 (F1, E6, F9, C11, F15, E21, E22, and C23), suggesting that patients had different understandings of these 8 questions. (3) Structural validity: The root mean square error of approximation value of the AMOS structural model in group V is 0.065, and the root mean square residual value is 0.114, indicating low fitness; the NC value is 3.353, indicating good fitness of the scale, but it still needed to be simplified.
Conclusions:
The Chinese version of Tinnitus Handicap Inventory-China has a high reliability when applied in China, but the content validity and structure validity are not high, and the clinical practicability needs to be improved. | Introduction:
Tinnitus is defined as the sensation of hearing sound when no external sound is present; it can produce many symptoms of sleep disorders, difficulty hearing, an inability to concentrate, irritability anxiety, depression, and other adverse psychological reactions. If improperly treated, a patient with tinnitus can develop severe mental illness, sometimes with suicidal tendencies.1 Tinnitus can be categorized as either objective or subjective; objective tinnitus (often originating in the middle ear) appears to come from inside the body via the conduction of sound to the ear, for example, from the musculature or from pulsing blood vessels. People with subjective tinnitus (of sensorineural origon) may be with hearing loss or have no detectable signs of disease and almost no detectable physical ailments. Objective tinnitus can be detected using a stethoscope or radiological technology, whereas subjective tinnitus can only be heard by the patient themselves.
Data from the National Center for Health Statistics in the U.S. Department of Health, Education, and Social Welfare (1968) indicates that 30% of the general population is affected by tinnitus, with 6% of those affected (1.8% of the general population) suffering disabling symptoms.2 However, there are no current large-scale epidemiological investigations of tinnitus in China, although 10% of people in China have experienced tinnitus and 5% have sought medical treatment. Additionally, the lives, sleep, work, and social activities of 2% of these individuals are adversely affected, and 0.5% of patients with tinnitus become disabled due to severe tinnitus.3
Because of its characteristics, it is difficult for doctors to diagnose the severity of subjective tinnitus according to objective indicators. In terms of its epidemiology, one-half of tinnitus patients will seek medical treatment, 2% will experience serious disruptions to their social lives, and 0.5% will even become disabled. Newman4 developed the Tinnitus Handicap Inventory (THI) self-rated scale in 1996; its purpose is to comprehensively determine the severity of tinnitus affecting patients by assessments of their daily behaviors. At present, the THI scale has been widely used in Korea,5 Persia,6 Brazil,7 Lithuania,8 Denmark,9 and elsewhere. Further, there are studies reporting the use of THI in China, including both Cantonese10 and Mandarin versions.11 However, there are no current large-scale clinical studies of the applicability of the Chinese THI; thus, its reliability and validity require further verification. Therefore, this article aims to verify the application of the Chinese THI in China using a large clinical sample and observe whether there are situations in which the Chinese THI cannot be used as a result of its translation or due to cultural differences.12
Conclusions:
The THI is widely used in countries around the world to examine the severity of tinnitus in affected patients. Applied studies in most countries have shown that this scale has good reliability and validity, and many studies have shown that the THI scale has certain applicability in China. However, our study found some discrepancies between the original THI and the Chinese THI. The results obtained by extracting CFs using the PCA were completely inconsistent with the dimensions of the scale itself, findings supported by research in other countries. But an analysis of the Chinese literature reveals that the total score of the THI scale is valuable. The THI scale has not been fully utilized, and the patient’s assessments are time-consuming if the Chinese THI scale is widely used. Also, there is a deviation in the understanding of some items. How to make patients accurately understand the meaning of each item and make a correct evaluation during consultation is the key issue for the application of THI-CN in China in the future. At present, our team is continuing to collect THI scores of tinnitus patients, with 952 new cases included now. We hope that further research results can supplement the deficiencies of this study. | Background:
The aim of this study is to observe the application of the Chinese version of Tinnitus Handicap Inventory-China in Tinnitus patients and verify its reliability and validity.
Methods:
About 1129 patients with tinnitus as the first complaint were selected as subjects. The patients were randomly divided into 2 groups: exploration group (n = 565), whose data were analyzed with reliability analysis method using Statistical Package for the Social Sciences software 19.0, validation group (n = 564), whose data were analyzed with validity analysis method using AMOS21.0.
Results:
(1) Reliability test: The Cronbach's α coefficients of the Tinnitus Handicap Inventory-China scale in both groups were 0.94, among which, the Cronbach's α coefficients of functional factor (F), emotion factor (E), and catastrophic factor (C) in group E were 0.87, 0.90, and 0.78, respectively. The half-reliability of the 2 components is 0.87. The correlation coefficient between items and the scale in group E and group V is 0.36-0.78 and 0.33-0.77, respectively. (2) Content validity: The Kaiser-Meyer-Olkin value of group E is 0.96, a total of 4 common factors were extracted, and the cumulative interpretation rate is 57.844%. The number of factors with load less than 0.4 on the 4 common factors is only 1 (F24), suggesting that this factor had little significance; the number of factors with load more than 0.4 on the 2 common factors is 8 (F1, E6, F9, C11, F15, E21, E22, and C23), suggesting that patients had different understandings of these 8 questions. (3) Structural validity: The root mean square error of approximation value of the AMOS structural model in group V is 0.065, and the root mean square residual value is 0.114, indicating low fitness; the NC value is 3.353, indicating good fitness of the scale, but it still needed to be simplified.
Conclusions:
The Chinese version of Tinnitus Handicap Inventory-China has a high reliability when applied in China, but the content validity and structure validity are not high, and the clinical practicability needs to be improved. | 12,963 | 424 | [
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] | [CONTENT] Tinnitus | Tinnitus Handicap Inventory | reliability | questionnaire [SUMMARY] | [CONTENT] Tinnitus | Tinnitus Handicap Inventory | reliability | questionnaire [SUMMARY] | [CONTENT] Tinnitus | Tinnitus Handicap Inventory | reliability | questionnaire [SUMMARY] | [CONTENT] Tinnitus | Tinnitus Handicap Inventory | reliability | questionnaire [SUMMARY] | [CONTENT] Tinnitus | Tinnitus Handicap Inventory | reliability | questionnaire [SUMMARY] | [CONTENT] Tinnitus | Tinnitus Handicap Inventory | reliability | questionnaire [SUMMARY] | [CONTENT] Humans | Tinnitus | Reproducibility of Results | Surveys and Questionnaires | China | Disease Progression | Psychometrics [SUMMARY] | [CONTENT] Humans | Tinnitus | Reproducibility of Results | Surveys and Questionnaires | China | Disease Progression | Psychometrics [SUMMARY] | [CONTENT] Humans | Tinnitus | Reproducibility of Results | Surveys and Questionnaires | China | Disease Progression | Psychometrics [SUMMARY] | [CONTENT] Humans | Tinnitus | Reproducibility of Results | Surveys and Questionnaires | China | Disease Progression | Psychometrics [SUMMARY] | [CONTENT] Humans | Tinnitus | Reproducibility of Results | Surveys and Questionnaires | China | Disease Progression | Psychometrics [SUMMARY] | [CONTENT] Humans | Tinnitus | Reproducibility of Results | Surveys and Questionnaires | China | Disease Progression | Psychometrics [SUMMARY] | [CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY] | [CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY] | [CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY] | [CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY] | [CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY] | [CONTENT] 27 considerations tinnitus | impaired tinnitus | severity subjective tinnitus | validity thi tinnitus | china experienced tinnitus [SUMMARY] | [CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY] | [CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY] | [CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY] | [CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY] | [CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY] | [CONTENT] thi | model | scale | correlation | value | analysis | tinnitus | fitness | thi scale | degree [SUMMARY] | [CONTENT] tinnitus | subjective | objective | sound | subjective tinnitus | large | social | china | thi | population [SUMMARY] | [CONTENT] patients | analysis | tinnitus | group | hospital | frequency tinnitus | frequency | education | software | high school [SUMMARY] | [CONTENT] model | correlation | value | fitness | degree | scale | thi scale | thi | degree fitness | group [SUMMARY] | [CONTENT] thi | countries | scale | thi scale | research | studies | widely | results | chinese | patients [SUMMARY] | [CONTENT] thi | model | scale | tinnitus | correlation | group | patients | value | thi scale | analysis [SUMMARY] | [CONTENT] thi | model | scale | tinnitus | correlation | group | patients | value | thi scale | analysis [SUMMARY] | [CONTENT] Chinese | Tinnitus Handicap Inventory-China | Tinnitus [SUMMARY] | [CONTENT] About 1129 | first ||| 2 | 565 | Statistical Package | Social Sciences | 19.0 | 564 [SUMMARY] | [CONTENT] 1 | Cronbach | the Tinnitus Handicap Inventory-China | 0.94 | Cronbach | F | 0.87 | 0.90 | 0.78 ||| half | 2 | 0.87 ||| 0.36-0.78 | 0.33 ||| 2 | The Kaiser-Meyer-Olkin | 0.96 | 4 | 57.844% ||| less than 0.4 | 4 | only 1 | F24 | more than 0.4 | 2 | 8 | F1 | E6 | F9 | C11 | F15 | E21 | E22 | C23 | 8 ||| 3 | AMOS | 0.065 | 0.114 | NC | 3.353 [SUMMARY] | [CONTENT] Chinese | Tinnitus Handicap Inventory-China | China [SUMMARY] | [CONTENT] Chinese | Tinnitus Handicap Inventory-China | Tinnitus ||| About 1129 | first ||| 2 | 565 | Statistical Package | Social Sciences | 19.0 | 564 ||| 1 | Cronbach | the Tinnitus Handicap Inventory-China | 0.94 | Cronbach | F | 0.87 | 0.90 | 0.78 ||| half | 2 | 0.87 ||| 0.36-0.78 | 0.33 ||| 2 | The Kaiser-Meyer-Olkin | 0.96 | 4 | 57.844% ||| less than 0.4 | 4 | only 1 | F24 | more than 0.4 | 2 | 8 | F1 | E6 | F9 | C11 | F15 | E21 | E22 | C23 | 8 ||| 3 | AMOS | 0.065 | 0.114 | NC | 3.353 ||| Chinese | Tinnitus Handicap Inventory-China | China [SUMMARY] | [CONTENT] Chinese | Tinnitus Handicap Inventory-China | Tinnitus ||| About 1129 | first ||| 2 | 565 | Statistical Package | Social Sciences | 19.0 | 564 ||| 1 | Cronbach | the Tinnitus Handicap Inventory-China | 0.94 | Cronbach | F | 0.87 | 0.90 | 0.78 ||| half | 2 | 0.87 ||| 0.36-0.78 | 0.33 ||| 2 | The Kaiser-Meyer-Olkin | 0.96 | 4 | 57.844% ||| less than 0.4 | 4 | only 1 | F24 | more than 0.4 | 2 | 8 | F1 | E6 | F9 | C11 | F15 | E21 | E22 | C23 | 8 ||| 3 | AMOS | 0.065 | 0.114 | NC | 3.353 ||| Chinese | Tinnitus Handicap Inventory-China | China [SUMMARY] |
||||||
Does intravascular ultrasound provide clinical benefits for percutaneous coronary intervention with bare-metal stent implantation? A meta-analysis of randomized controlled trials. | 22999055 | The role of intravascular ultrasound (IVUS) in percutaneous coronary interventions (PCI) is still controversial despite several previously published meta-analyses. A meta-analysis to evaluate the controversial role of IVUS-guided PCI with bare-metal stenting was performed and a previous published meta-analysis was re-evaluated in order to clarify the discrepancy between results of these studies. | BACKGROUND | A systematic review was performed by an electronic search of the PubMed, Embase and Web of Knowledge databases and by a manual search of reference lists for randomized controlled trials published until April 2011, with clinical outcomes and, at least, six months of clinical follow-up. A meta-analysis based on the intention to treat was performed with the selected studies. | METHODS | Five studies and 1,754 patients were included. There were no differences in death (OR = 1.86; 95% CI = 0.88-3.95; p = 0.10), non-fatal myocardial infarction (OR = 0.65; 95% CI = 0.27-1.58; p = 0.35) and major adverse cardiac events (OR = 0.74; 95% CI = 0.49-1.13; p = 0.16). An analysis of the previous published meta-analysis strongly suggested the presence of publication bias. | RESULTS | There is no evidence to recommend routine IVUS-guided PCI with bare-metal stent implantation. This may be explained by the paucity and heterogeneity of the studies published so far. | CONCLUSIONS | [
"Humans",
"Percutaneous Coronary Intervention",
"Randomized Controlled Trials as Topic",
"Stents",
"Treatment Outcome",
"Ultrasonography, Interventional"
] | 3534608 | Background | Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.
The first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].
Since IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature. | Methods | The protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).
Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.
A manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.
We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.
A manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.
Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.
Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.
Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.
The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.
Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.
Publication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.
The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.
Publication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method. | Results | Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.
Article selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].
Patient characteristics
* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.
A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.
Article selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].
Patient characteristics
* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.
Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].
Study characteristics
*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.
The criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].
There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].
Study characteristics
*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.
The criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].
Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).
The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).
Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.
Duval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).
We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.
Duval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).
Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).
Clinical Outcomes
*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.
Meta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).
A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).
Clinical Outcomes
*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.
Meta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).
Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].
Original data from the re-evaluated meta-analysis - MACE[13].
A funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].
A cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.
Cumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.
One-study-removed method. Each line excludes the corresponding study from the combined analysis.
In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].
Original data from the re-evaluated meta-analysis - MACE[13].
A funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].
A cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.
Cumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.
One-study-removed method. Each line excludes the corresponding study from the combined analysis. | Conclusion | The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.
Therefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.
This research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1). | [
"Background",
"Strategy search",
"Eligibility criteria",
"Study selection",
"Statistical analysis",
"Literature search",
"Qualitative study analysis",
"Heterogeneity",
"Publication bias",
"Meta-analysis results",
"Reviewing published data",
"Differences between selected studies",
"Study biases",
"Limitations",
"Competing interest",
"Authors’ contributions"
] | [
"Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.",
"We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.",
"Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.",
"The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.",
"The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.",
"A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.",
"There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].",
"The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).",
"We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).",
"A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).",
"In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.",
"The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].",
"The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.",
"The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.",
"The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.",
"LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript."
] | [
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] | [
"Background",
"Methods",
"Strategy search",
"Eligibility criteria",
"Study selection",
"Statistical analysis",
"Results",
"Literature search",
"Qualitative study analysis",
"Heterogeneity",
"Publication bias",
"Meta-analysis results",
"Reviewing published data",
"Discussion",
"Differences between selected studies",
"Study biases",
"Limitations",
"Conclusion",
"Competing interest",
"Authors’ contributions"
] | [
"Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.\nThe first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].\nSince IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.",
"The protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).\n Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\nWe performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.\n Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\nOnly randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.\n Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\nThe titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.\n Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.\nThe intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.",
"We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.\nA manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.",
"Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.",
"The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.",
"The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.\nPublication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.",
" Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\nA total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.\n Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\nThere were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].\n Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\nThe heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).\n Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\nWe also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).\n Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\nA total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).\n Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.\nIn order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.",
"A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.\nArticle selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].\nPatient characteristics\n* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.",
"There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].\nStudy characteristics\n*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.\nThe criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].",
"The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).",
"We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.\nDuval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).",
"A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).\nClinical Outcomes\n*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.\nMeta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).",
"In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].\nOriginal data from the re-evaluated meta-analysis - MACE[13].\nA funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].\nA cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.\nCumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.\nOne-study-removed method. Each line excludes the corresponding study from the combined analysis.",
"In this rigorously conducted meta-analysis of randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI using bare metal stents, we did not find any advantage of the IVUS-guided strategy over the standard method in clinically relevant outcomes. Indeed, we found evidence of publication bias and of significant heterogeneity among the studies regarding the outcomes MI and MACE. These results diverge from the last two meta-analyses on this topic [12,13], which included studies with provisional stenting, considered surrogate outcomes, and did not evaluate the presence of publication bias. However, they are in concordance with recently published studies of IVUS-guided PCI with drug-eluting stent implantation, which were not associated with significant clinical benefits [30,31].\n Differences between selected studies The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\nThe five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].\n Study biases The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\nThe data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.\n Limitations The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.\nThe paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.",
"The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].",
"The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].\nSignificance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].\nSelective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].\nAnother major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].\nStudy funding and conflicts of interest of the authors of original articles included in this meta-analysis\nN/A, not applicable.\nMeta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.",
"The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].\nThe low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.",
"The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.\nTherefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.\nThis research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1).",
"The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.",
"LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript."
] | [
null,
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
null,
"discussion",
null,
null,
null,
"conclusions",
null,
null
] | [
"Intravascular ultrasound",
"Meta-analysis",
"Publication bias",
"Bare-metal stent",
"Percutaneous coronary intervention",
"Coronary artery disease"
] | Background:
Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.
The first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].
Since IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.
Methods:
The protocol for the present systematic review was based on the PRISMA Statement [14] and it was registered in the PROSPERO database (CRD42012002767).
Strategy search We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.
A manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.
We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.
A manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.
Eligibility criteria Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.
Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.
Study selection The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.
The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.
Statistical analysis The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.
Publication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.
The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.
Publication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.
Strategy search:
We performed an electronic search of PubMed, Embase and Web of Knowledge databases with the following terms: Myocardial Ischemia; Ischemic Heart Disease; Acute Coronary Syndrome; Angina; Coronary Disease; Coronary Artery Disease; Coronary Occlusion; Coronary Thrombosis and Myocardial Infarction, in association with the terms Interventional Ultraso*; Intravascular Ultraso*; Intracoronary Ultraso*; IVUS and ICUS.
A manual search was also performed to retrieve potential articles cited in previous meta-analyses, in review articles and those considered to be relevant by the reviewers. The electronic search, which evaluated the articles included in the databases through April 2011, was limited neither by publication date nor by language.
Eligibility criteria:
Only randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI, with clinical outcomes, and at least six months of clinical follow-up, were included in quantitative synthesis. The clinical outcomes considered were death, nonfatal MI and the combined endpoint of MACE (death, nonfatal MI, or repeat revascularization). For repeat revascularization, a report of any new coronary revascularization (surgical or percutaneous) was considered, regardless of the lesion and of the vessel treated. Surrogate outcomes, such as angiographic outcomes, were not taken into account because these can show a positive result with no effect (or harmful effect) on clinical outcomes [15]. These clinical outcomes (death, nonfatal MI and MACE) were considered primary endpoints in our meta-analysis.
Study selection:
The titles and abstracts from the articles retrieved by the search strategy had been independently evaluated by two reviewers (LCP, LLJ). All articles in which IVUS was mentioned were selected. These articles were fully read, and those that met the criteria were included. Disagreements were solved by consensus. If consensus was not achieved, a third reviewer (ALR) defined the question.
Statistical analysis:
The intention-to-treat meta-analysis that followed the systematic review was performed by the random-effects model of the Comprehensive Meta-Analysis software (Borenstein M, Hedges L, Higgins J, Rothstein H. Version 2.2.048, Biostat, Englewood NJ, USA 2005), with the odds ratio (OR), 95% confidence intervals and two-sided P-values calculated for each outcome. The analysis of heterogeneity between studies was estimated by the I2 statistic.
Publication bias evaluation was performed by Duval and Tweedie’s Trim and Fill method [16]. Egger's test was also performed to analyze the impact of several factors on the size of the treatment effect [17]. The small study effect was also evaluated by cumulative analysis (from largest to smallest sample size) and by the one-study-removed method.
Results:
Literature search A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.
Article selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].
Patient characteristics
* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.
A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.
Article selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].
Patient characteristics
* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.
Qualitative study analysis There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].
Study characteristics
*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.
The criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].
There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].
Study characteristics
*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.
The criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].
Heterogeneity The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).
The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).
Publication bias We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.
Duval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).
We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.
Duval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).
Meta-analysis results A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).
Clinical Outcomes
*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.
Meta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).
A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).
Clinical Outcomes
*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.
Meta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).
Reviewing published data In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].
Original data from the re-evaluated meta-analysis - MACE[13].
A funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].
A cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.
Cumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.
One-study-removed method. Each line excludes the corresponding study from the combined analysis.
In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].
Original data from the re-evaluated meta-analysis - MACE[13].
A funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].
A cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.
Cumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.
One-study-removed method. Each line excludes the corresponding study from the combined analysis.
Literature search:
A total of 4,247 articles in PubMed, 869 in Embase and 4,260 in Web of Knowledge databases were identified. Eight studies were selected according to the inclusion criteria (Figure 1) [18-26]. After a comprehensive analysis, three studies were excluded because they used a provisional stenting technique [19,25,26], which is no longer performed because of its higher restenosis rate [27]. Table 1 summarizes the clinical and angiographic characteristics of the patients included in the selected studies.
Article selection flowchart. Flowchart based on the PRISMA Flow Diagram [14].
Patient characteristics
* In the RESIST study, the reference diameter average was calculated. MI, myocardial infarction; CABG, coronary artery bypass graft; PCI, percutaneous coronary intervention; LV, left ventricular; ACA, American College of Cardiology; AHA, American Heart Association; IVUS, intravascular ultrasound; N/A, not applicable, QCA, Quantitative Coronary Angiography.
Qualitative study analysis:
There were significant differences between the five studies included in the final analysis (Table 2). One of the current indications of IVUS-guided PCI is for patients with long lesions (greater than 15 or 25 mm) [8,28,29], who have been excluded from most studies [18,20,24]. Unlike the others, the TULIP study excluded those patients who had focal lesions (less than 20 mm in length). Every study but the AVID trial excluded patients with a current or past history of acute coronary syndrome (ACS). In the RESIST study, randomization was performed only after the intervention, which may have caused a selection bias. In the AVID trial, the IVUS analysis was only performed after implantation of the stent, excluding the initial assessment of the target lesion [8].
Study characteristics
*Angiographic follow-up was left to the discretion of the operator: 87.9% (IVUS-guided group) and 83.7% (angio-guided group). †CABG or repeat PCI for any reason. IVUS, interventional ultrasound; PCI, percutaneous coronary intervention; MACE, major adverse cardiovascular events; CSA, cross sectional area; MI, myocardial infarction; TIMI, thrombolysis in myocardial infarction; CABG, coronary artery bypass graft; MLD, minimum lumen diameter; clinical TLR, ischemia-driven target lesion revascularization.
The criteria for optimal stent implantation were heterogeneous. Only the OPTICUS study used the criteria proposed by the MUSIC study [10]. The majority of patients underwent angiographic assessment after six months (angiographic follow-up) [20,22,24]. Another difference between the studies was in the criteria used for MACE. In the RESIST study, MI was not included. In the TULIP study, the MACE criteria included death, nonfatal MI and ischemia-driven target lesion revascularization (TLR). In the AVID trial, the composition of this outcome was not explained. In the other studies, the criteria for repeat revascularization were more comprehensive and included coronary artery bypass grafting (CABG) or a repeated PCI for any reason [18,20,21,24].
Heterogeneity:
The heterogeneity among the studies showed intermediate values in nonfatal MI (I2 = 48.82%) and MACE (I2 = 57.38%). For death, no heterogeneity was observed among the studies (I2 = 0%).
Publication bias:
We also evaluated the possibility of publication bias (B0) for MACE. Egger’s Test (B0 = −3.43; 95% CI − 6.40 to −0.47, one-tailed P-value 0.02) and the trim and fill test (observed OR 0.74, 95% CI 0.49 to 1.13; two studies imputed: adjusted OR 0.93, 95% CI 0.60 to 1.44) (Figure 2) were positive, suggesting the presence of small studies effects, which can be attributable to differences in design (not detected) or to publication bias.
Duval and Tweedie’s trim and fill test. The funnel plot shows the observed studies (white circles) and the imputed studies (black circles) in addition to the observed (white diamond) and adjusted combined effect (black diamond).
Meta-analysis results:
A total of 1,754 patients were randomized in five studies. There was no statistically significant difference between the IVUS-guided group and the angiography-guided group (Table 3) for death (OR 1.86, 95% CI 0.88 to 3.95, P = 0.10) (Figure 3-A), nonfatal MI (OR 0.65, 95% CI 0.27 to 1.58, P = 0.35) (Figure 3-B) or MACE (OR 0.74, 95% CI 0.49 to 1.13, P = 0.16) (Figure 3-C).
Clinical Outcomes
*The analysis of nonfatal MI excludes the RESIST Study, where this outcome was not calculated. MI, myocardial infarction; MACE, major adverse cardiovascular events; IVUS, interventional ultrasound.
Meta-analysis by outcomes (random effects). (A) Death. (B) Myocardial infarction (MI). (C) Major adverse cardiovascular events (MACE).
Reviewing published data:
In order to clarify the discrepancy in MACE results found in this analysis compared to the medical literature, the data of a previously published meta-analysis [13] were re-evaluated (Figure 4). Among the studies selected by that meta-analysis, only two were not included in the present selection because the provisional stenting technique was employed in both of them [19,25].
Original data from the re-evaluated meta-analysis - MACE[13].
A funnel plot analysis was performed along with Egger’s Test (B0 = −3.66, 95% CI − 5.54 to −1.78, one-tailed P-value = 0.002) and the trim and fill test (observed OR 0.70, 95% CI 0.50 to 0.98; three studies imputed: adjusted OR 0.89, 95% CI 0.62 to 1.27), which suggested the presence of publication bias [16,17].
A cumulative meta-analysis by reverse order of sample size was performed. The results only became positive when the last and smallest study was included in the analysis (Figure 5). Moreover, the one-study-removed analysis showed that the removal of any one of the smaller studies gave a neutral result from the meta-analysis (Figure 6). This makes it plausible to assume that one small unpublished study with negative results would be enough to nullify the effect of that meta-analysis.
Cumulative meta-analysis. Each line includes the combined analysis of the corresponding study and the other ones above. The studies were added from largest to smallest sample size.
One-study-removed method. Each line excludes the corresponding study from the combined analysis.
Discussion:
In this rigorously conducted meta-analysis of randomized controlled trials that compared IVUS-guided PCI with angiography-guided PCI using bare metal stents, we did not find any advantage of the IVUS-guided strategy over the standard method in clinically relevant outcomes. Indeed, we found evidence of publication bias and of significant heterogeneity among the studies regarding the outcomes MI and MACE. These results diverge from the last two meta-analyses on this topic [12,13], which included studies with provisional stenting, considered surrogate outcomes, and did not evaluate the presence of publication bias. However, they are in concordance with recently published studies of IVUS-guided PCI with drug-eluting stent implantation, which were not associated with significant clinical benefits [30,31].
Differences between selected studies The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].
The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].
Study biases The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].
Significance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].
Selective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].
Another major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].
Study funding and conflicts of interest of the authors of original articles included in this meta-analysis
N/A, not applicable.
Meta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.
The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].
Significance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].
Selective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].
Another major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].
Study funding and conflicts of interest of the authors of original articles included in this meta-analysis
N/A, not applicable.
Meta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.
Limitations The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].
The low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.
The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].
The low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.
Differences between selected studies:
The five selected studies have important differences that might lead to completely different outcomes in another context. For example, the exclusion of patients presenting with ACS may have led to a reduction in post-interventional adverse events [32-35]. In the AVID trial, pre-interventional IVUS was not performed, which excluded an important phase of the method because one of the roles of IVUS is to assess the target lesion to help in the choice of technique and devices for the PCI [8]. Only the OPTICUS study used the MUSIC study criteria for optimal stent implantation [10], which theoretically could be associated with a lower MACE rate [36]. Angiographic follow-up was performed in most studies, which may have led to an overestimated rate of repeat revascularization, due to the oculo-stenotic reflex [37], which is the predisposition to indicate a PCI for any significant luminal obstruction, despite the presence or absence of myocardial ischemia [38]. The fact that most studies have used more comprehensive criteria for repeat revascularization may have also increased MACE rates [18,20,24].
Study biases:
The data analysis suggested the presence of publication bias in both meta-analyses. This bias may have led to apparently positive results that could be easily modified by unpublished studies with small sample sizes. It may be harmful because it can maintain or amplify an apparent beneficial effect of the intervention [39].
Significance-chasing bias is an enticing term that refers to the clustering of the most common types of meta-analysis bias, including those in which the apparently negative results remain unpublished (study publication bias and selective outcome reporting bias), those in which negative results become positive (selective analysis reporting bias) and those in which no existing data are presented as positive (fabrication bias) [40,41].
Selective reporting bias is the most common problem in meta-analyses. In selective outcome reporting bias, specific data with a negative result are omitted from publication. In selective analysis reporting bias, which is even more frequent, a negative result calculated from a pre-determined analysis plan is replaced by a positive result achieved through post hoc data analysis [41].
Another major problem is the potential presence of interests other than scientific truth. This matter becomes critical when the object of the meta-analysis is an industry product, as in the present study, because most of the researches are conducted or funded by manufacturers (Table 4) [42,43]. There is a current trend towards opposing this practice [44].
Study funding and conflicts of interest of the authors of original articles included in this meta-analysis
N/A, not applicable.
Meta-analyses have gained prestige over time but they are still considered by some to be an ancillary method, accepted only when it corroborates the point of view of experts and of public policies [41]. They could play a fundamental role in changing (or in supporting) the evidence on relevant issues if conducted properly, with a pre-specified analysis plan and declared conflicts of interest for every study included, in addition to determining and reporting all possible biases.
Limitations:
The paucity of randomized controlled trials comparing IVUS-guided PCI and angio-guided PCI and the exclusion of groups with specific lesions (long lesions, small vessels, bifurcations or left main coronary artery) may have masked a possible benefit. The same point applies to the exclusion of patients presenting with ACS, whose rate of cardiovascular events is higher, and PCI, when indicated, may even reduce mortality [32-34].
The low statistical power of the present study and of the re-evaluated meta-analysis [13] is due to the presence of heterogeneity and to the possibility of study publication bias. The presence of other biases might be possible but that is even more difficult to prove.
Conclusion:
The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.
Therefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.
This research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1).
Competing interest:
The authors declare no conflicts of interest in the preparation and in the presentation of this meta-analysis.
Authors’ contributions:
LLJ participated in the design of the study, retrieved, reviewed and selected the articles from electronic and manual searches, helped to discuss the results and to draft the manuscript. MRS participated in the design of the study, performed the statistical analysis and helped to discuss the results and to draft the manuscript. LCP retrieved, reviewed and selected the articles from electronic and manual searches. SMBK participated in the design of the study and helped to retrieve articles from electronic searches, to discuss the results and to draft the manuscript. CFSA participated in the design of the study and helped to discuss the results and to draft the manuscript. ALR participated in the coordination of the study, helped to select the articles, to discuss the results and to draft the manuscript. All authors read and approved the final manuscript.
| Background:
The role of intravascular ultrasound (IVUS) in percutaneous coronary interventions (PCI) is still controversial despite several previously published meta-analyses. A meta-analysis to evaluate the controversial role of IVUS-guided PCI with bare-metal stenting was performed and a previous published meta-analysis was re-evaluated in order to clarify the discrepancy between results of these studies.
Methods:
A systematic review was performed by an electronic search of the PubMed, Embase and Web of Knowledge databases and by a manual search of reference lists for randomized controlled trials published until April 2011, with clinical outcomes and, at least, six months of clinical follow-up. A meta-analysis based on the intention to treat was performed with the selected studies.
Results:
Five studies and 1,754 patients were included. There were no differences in death (OR = 1.86; 95% CI = 0.88-3.95; p = 0.10), non-fatal myocardial infarction (OR = 0.65; 95% CI = 0.27-1.58; p = 0.35) and major adverse cardiac events (OR = 0.74; 95% CI = 0.49-1.13; p = 0.16). An analysis of the previous published meta-analysis strongly suggested the presence of publication bias.
Conclusions:
There is no evidence to recommend routine IVUS-guided PCI with bare-metal stent implantation. This may be explained by the paucity and heterogeneity of the studies published so far. | Background:
Since the first studies of intravascular ultrasound (IVUS) were published in 1989 [1-4], the technique has been widely used in clinical research and has contributed to technological improvements in interventional cardiology [5]. As a diagnostic tool, IVUS helps in the assessment of coronary lesions classified as moderate based on angiography, especially those located in the left main coronary artery [6,7], and in the assessment of long lesions, small artery lesions, bifurcations and in-stent restenosis [8,9]. As an ancillary technique in percutaneous coronary intervention (PCI), IVUS is useful in the evaluation of the target lesion and during stent implantation [10]. In theory, its use should reduce the risk of major adverse cardiovascular events (MACE) because of lower restenosis and stent thrombosis rates.
The first published systematic review evaluated the role of IVUS in PCI as well as its cost-effectiveness and did not show any difference between IVUS and angio-guided PCI [11]. A few years later, a meta-analysis did not show any reduction in death or myocardial infarction (MI) but revealed reductions in repeat revascularization and angiographic restenosis after a six-month follow-up [12]. This was corroborated by another meta-analysis that suggested an improvement in acute post-interventional results (larger minimal luminal diameter) and lower repeat revascularization, angiographic restenosis and MACE rates, but showed no effect on death or MI during the follow-up period of six to thirty months [13].
Since IVUS clinical benefit is still controversial and conclusions of meta-analyses may be misleading due to methodological issues, we performed a meta-analysis to assess the effect of IVUS in PCI with bare-metal stent implantation on clinically relevant outcomes, assessing the presence of publication bias. In addition, a critical review of the last published meta-analysis [13] was performed in order to clarify the discrepancy in the results found in this analysis comparing to medical literature.
Conclusion:
The clinical benefit of IVUS-guided PCI with bare-metal stent implantation could be determined neither by the meta-analysis presented in this study nor by the re-evaluated meta-analysis. This may be explained by the paucity and heterogeneity of the studies published so far. Furthermore, both meta-analyses showed possible publication biases.
Therefore, there is no evidence so far to recommend routine IVUS-guided PCI with bare-metal stent implantation. Studies on specific subgroups and performance of a simple large randomized trial could show different results.
This research was conducted by public funding from the National Council for Scientific and Technological Development (CNPq) (CNPq Project: 559584/2009-1). | Background:
The role of intravascular ultrasound (IVUS) in percutaneous coronary interventions (PCI) is still controversial despite several previously published meta-analyses. A meta-analysis to evaluate the controversial role of IVUS-guided PCI with bare-metal stenting was performed and a previous published meta-analysis was re-evaluated in order to clarify the discrepancy between results of these studies.
Methods:
A systematic review was performed by an electronic search of the PubMed, Embase and Web of Knowledge databases and by a manual search of reference lists for randomized controlled trials published until April 2011, with clinical outcomes and, at least, six months of clinical follow-up. A meta-analysis based on the intention to treat was performed with the selected studies.
Results:
Five studies and 1,754 patients were included. There were no differences in death (OR = 1.86; 95% CI = 0.88-3.95; p = 0.10), non-fatal myocardial infarction (OR = 0.65; 95% CI = 0.27-1.58; p = 0.35) and major adverse cardiac events (OR = 0.74; 95% CI = 0.49-1.13; p = 0.16). An analysis of the previous published meta-analysis strongly suggested the presence of publication bias.
Conclusions:
There is no evidence to recommend routine IVUS-guided PCI with bare-metal stent implantation. This may be explained by the paucity and heterogeneity of the studies published so far. | 8,615 | 301 | [
381,
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] | [
"ivus interventional ultrasound",
"coronary intervention pci",
"pci percutaneous coronary",
"ivus angio guided",
"intravascular ultrasound applicable"
] | [CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY] | [CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY] | [CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY] | [CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY] | [CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY] | [CONTENT] Intravascular ultrasound | Meta-analysis | Publication bias | Bare-metal stent | Percutaneous coronary intervention | Coronary artery disease [SUMMARY] | [CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY] | [CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY] | [CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY] | [CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY] | [CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY] | [CONTENT] Humans | Percutaneous Coronary Intervention | Randomized Controlled Trials as Topic | Stents | Treatment Outcome | Ultrasonography, Interventional [SUMMARY] | [CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY] | [CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY] | [CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY] | [CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY] | [CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY] | [CONTENT] ivus interventional ultrasound | coronary intervention pci | pci percutaneous coronary | ivus angio guided | intravascular ultrasound applicable [SUMMARY] | [CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY] | [CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY] | [CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY] | [CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY] | [CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY] | [CONTENT] analysis | study | studies | meta | bias | meta analysis | ivus | mace | coronary | pci [SUMMARY] | [CONTENT] restenosis | ivus | stent | meta | lesions | published | revascularization angiographic restenosis | revascularization angiographic | ivus pci | angiographic restenosis [SUMMARY] | [CONTENT] outcomes | articles | search | coronary | clinical outcomes | considered | disease | ultraso | clinical | performed [SUMMARY] | [CONTENT] analysis | studies | study | ci | 95 ci | 95 | figure | mi | mace | meta analysis [SUMMARY] | [CONTENT] cnpq | ivus guided pci bare | far | guided pci bare | guided pci bare metal | metal stent | metal stent implantation | pci bare metal stent | bare metal stent | bare metal stent implantation [SUMMARY] | [CONTENT] analysis | study | meta | studies | meta analysis | coronary | pci | ivus | bias | mi [SUMMARY] | [CONTENT] analysis | study | meta | studies | meta analysis | coronary | pci | ivus | bias | mi [SUMMARY] | [CONTENT] ||| IVUS [SUMMARY] | [CONTENT] PubMed | Embase | April 2011 | six months ||| [SUMMARY] | [CONTENT] Five | 1,754 ||| 1.86 | 95% | CI | 0.88 | 0.10 | 0.65 | 95% | CI | 0.27-1.58 | 0.35 | 0.74 | 95% | CI | 0.49 | 0.16 ||| [SUMMARY] | [CONTENT] IVUS ||| [SUMMARY] | [CONTENT] ||| IVUS ||| PubMed | Embase | April 2011 | six months ||| ||| Five | 1,754 ||| 1.86 | 95% | CI | 0.88 | 0.10 | 0.65 | 95% | CI | 0.27-1.58 | 0.35 | 0.74 | 95% | CI | 0.49 | 0.16 ||| ||| IVUS ||| [SUMMARY] | [CONTENT] ||| IVUS ||| PubMed | Embase | April 2011 | six months ||| ||| Five | 1,754 ||| 1.86 | 95% | CI | 0.88 | 0.10 | 0.65 | 95% | CI | 0.27-1.58 | 0.35 | 0.74 | 95% | CI | 0.49 | 0.16 ||| ||| IVUS ||| [SUMMARY] |
||||||
Application of Sigma metrics in the quality control strategies of immunology and protein analytes. | 34606652 | Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma. | BACKGROUND | Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values. | METHODS | While using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12). | RESULTS | The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool. | CONCLUSIONS | [
"Antibodies",
"Clinical Chemistry Tests",
"Humans",
"Proteins",
"Quality Control",
"Total Quality Management"
] | 8605144 | INTRODUCTION | Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.
1
,
2
As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐
3
, during‐
4
and post‐analytical
5
phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.
Quality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.
1
6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.
In this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI). | null | null | RESULTS | Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.
Mean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes
Abbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.
The TEa source did not cover the TEa of the analyte.
Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.
Mean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes
Abbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.
The TEa source did not cover the TEa of the analyte.
Distribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.
Sigma of 10 analytes based on four different TEa standards
σBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.
No calculated Sigma obtained.
Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.
Sigma of 10 analytes based on four different TEa standards
σBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.
No calculated Sigma obtained.
Composition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.
Sigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels
We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.
Sigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels
Resetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.
QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC. | null | null | [
"INTRODUCTION",
"Study design",
"Instruments and reagents",
"Evaluation of imprecision",
"Evaluation of bias",
"Allowable total error",
"Sigma metrics calculation",
"Composition of Sigma Method Decision Charts",
"Quality goal index ratio",
"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes",
"Distribution of Sigma metrics based on four TEa standards",
"Composition of Sigma Method Decision Charts",
"Resetting QC strategies and improvement measures",
"AUTHOR CONTRIBUTIONS"
] | [
"Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\n1\n, \n2\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\n3\n, during‐\n4\n and post‐analytical\n5\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\n1\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).",
"This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research",
"Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.",
"Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.",
"Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.",
"TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.",
"Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.",
"Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.",
"The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n",
"Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.",
"Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.",
"We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels",
"QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.",
"YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Study design",
"Instruments and reagents",
"Evaluation of imprecision",
"Evaluation of bias",
"Allowable total error",
"Sigma metrics calculation",
"Composition of Sigma Method Decision Charts",
"Quality goal index ratio",
"RESULTS",
"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes",
"Distribution of Sigma metrics based on four TEa standards",
"Composition of Sigma Method Decision Charts",
"Resetting QC strategies and improvement measures",
"DISCUSSIONS",
"CONFLICT OF INTEREST",
"AUTHOR CONTRIBUTIONS",
"Supporting information"
] | [
"Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.\n1\n, \n2\n As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐\n3\n, during‐\n4\n and post‐analytical\n5\n phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.\nQuality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.\n1\n 6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.\nIn this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).",
"Study design This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\nThis study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research\nInstruments and reagents Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\nRoche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.\nEvaluation of imprecision Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\nImprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.\nEvaluation of bias Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\nBias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.\nAllowable total error TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\nTEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.\nSigma metrics calculation Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\nSigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.\nComposition of Sigma Method Decision Charts Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\nLogging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.\nQuality goal index ratio The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n\nThe QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n",
"This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,\n6\n we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.\nFlow chart of this research",
"Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).\nTwo‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.\nFive‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.",
"Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.\n7\n, \n8\n, \n9\n The calculation equation is: RMS CV% = [(CV1\n2 + CV2\n2) /2]0.5.",
"Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows\n10\n, \n11\n:\nBias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.\nThe average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.",
"TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,\n12\n designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.\n(https://biologicalvariation.eu/)\n\n13\n was first adopted, secondly from other researches which EFLM does not cover (RF\n14\n and PA\n15\n). The TEaBV are calculated using the formula:\n\nTEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,\n\n\nTEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,\n\n\nTEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.\n\n\nHere CVI means CV within‐subject, CVG means CV between‐subject.",
"Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.\n6\n The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.\nWestgard Sigma multi‐rules\nN, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.",
"Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.\n10\n, \n16\n, \n17\n, \n18\n Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.",
"The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.\n10\n, \n16\n, \n17\n, \n18\n\n",
"Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nCumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.\nDistribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nTable 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.\nComposition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nWe constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels\nResetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.\nQC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.",
"Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.\nMean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes\nAbbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.\nThe TEa source did not cover the TEa of the analyte.",
"Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.\nSigma of 10 analytes based on four different TEa standards\nσBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.\nNo calculated Sigma obtained.",
"We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.\nSigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels",
"QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.",
"In this study, we evaluated the performance of 10 immunology and protein analytes (which named after their BIO‐RAD controls—“Immunology and Protein Control”) at the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine for a one year period based on Sigma metrics. The previous studies mainly focused on the evaluation of analytical performance of clinical biochemistry analytes,\n19\n, \n20\n, \n21\n endocrine analytes,\n10\n, \n22\n tumor marker analytes,\n11\n, \n22\n etc. using Sigma metrics. This study is the first time to focus on the application of Sigma metrics in immunology and protein analytes (IgG, IgA, IgM, C3, C4, CRP, RF, PA, ASO, and Cys C) based on four different sources of TEa. Further the Sigma Method Decision Charts were constructed in terms of TEaNCCL, providing us with a visual view of the analytes’ performance. For analytes with σ<6, the cause for poor performance was evaluated using QGI similar to previous studies.\n10\n, \n11\n\n\nAccording to the equation Sigma = (TEa% ‐ Bias%)/CV%, significantly different σ values are obtained using the same Bias% and CV% but different total allowable error targets similar to the previous studies,\n10\n, \n11\n the greater the TEa is, the greater the Sigma value is. In this study, initially five different TEa targets were included. Most TEa selected from the Clinical Laboratory Improvements Amendments (CLIA)‐88 requirements from the United States are exhibited as 3 times the standard deviation or as semi‐quantitative results\n23\n (data not shown), regardless of whether the standard deviation comes from IQC or the same group variation of the EQA, the variable standard deviations bring difficulties to calculation, and there may be an illusion that analytes with large TEa have better performance and analytes with small TEa have poor performance. Therefore, we would not include TEaCLIA in subsequent calculations. The Sigma metrics of 10 immunology and protein analytes were shown in Table 3 based on four different TEa standards.\nAs mentioned above, surprisingly, while using the TEaNCCL, 9 analytes for IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. On the other hand, while we chose the TEaBV, the Sigma values were quite different, even negative (C3). TEa sources play an important role in the calculation of Sigma value.\n24\n According to the 2015 Strategic Conference on Quality Specifications convened in Milan consensus, there are three more compact levels or models, that is clinical outcomes, biological variabilities, and state‐of‐the‐art, to determine performance specifications in clinical laboratories.\n25\n The actual clinical use of the test results is the gold standard for setting TEa. The optimal TEa should be established depending on the conditions and requirements of the individual laboratory. The laboratory should assess the suitability of the TEa for clinical use and determine if that would allow a larger TEa choice. TEaBV is too strict for our laboratory, after a comprehensive analysis of laboratory status, analytes’ performance, and clinical recognition, therefore, in the subsequent analysis, we chose TEaNCCL as performance specifications.\nAfter calculating the Sigma value, how can we have a more intuitive impression of these results? At this time, Sigma Method Decision Charts are on the stage, which converts all the Sigma metrics into a simple intuitive dashboard.\n25\n Nine Sigma in this research were in the “bull's eye” zone, suggesting so good performance as to world‐class. Cys C fallen in the 3σ‐4σ zone, suggesting there may be more defects. The Sigma Method Decision Charts allows us to get a succinct comprehensive view of an entire instrument's performance, provide us with a visual view of the analytes’ performance. We could intuitively judge the performance of the analytes through this chart. \nThe personalized QC strategies, QGI and Prioritize improvement of 10 analytes according to σNCCL\n\nAnalytes with σ>6 does not need to calculate QGI, and no need for improvement.\nSigma metrics are not just a tool to ascertain the performance of analytes but also a beacon for designing a more cost‐effective QC strategy. Through the assessment of Sigma metrics, we can specify the control rules scientifically, including the number of control materials and the necessary frequency of running those controls. The closer the operating dot of an analyte is to the chart's origin, the easier the QC program is. In our laboratory, we chose 13s/22s QC procedure with two QC levels once per day for the 10 immunology and protein analytes empirically to supervise the performance of analytes in the past. By using Sigma metrics, nine analytes for σ>6 can safely use the 13s procedure with one measurement of two QC levels to gain an appropriate level of analytical quality assurance, avoiding economic costs, and overwork. On the contrary, with the decline in performance, more quality control rules, more different levels of quality controls, and higher quality control frequency are required. As Cys C in this study, that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) were needed and the QC frequency could be increased to one control per 45 clinical samples. As Cys C had 38 average daily measurements in our laboratory,one time QC frequency per day is needed.\nThough the Westgard Sigma multi‐rules provide a scientific and reasonable method for setting QC procedure, it could not fully reflect the precision and accuracy of the method. QGI is a tool to provide easy insights into the reasons for quality errors such as those caused by imprecision, inaccuracy, or both.\n21\n, \n26\n Cys C (QGI = 0.12) had low precision and some action must be taken. We make continuous improvements from five potential causal factors: personnel, machine, material, method, and environment: (Ⅰ) Strengthen personnel training, including QC knowledge and analyzer operation training; (Ⅱ) Perform regular maintenance and calibration of analyzers to ensure the performance of the analyzer, and timely retire the analyzer with poor performance; (Ⅲ) Monitor the temperature of reagent transportation and storage, avoid reagent expiration and excessive on‐board time; (Ⅳ) Revise the operation protocols, perform the machine in strict accordance with the operation protocols; (Ⅴ) Ensure that the working environment of the instrument meets the requirements, such as temperature and humidity. In fact, Cys C used in this research is a third‐party reagent, not Roche original, the reagent batch is updated so quickly, and the difference between batches is large, resulting in a large CV. Aware of this, our laboratory try our best to use the same batch reagents. We would not replace the new batch reagents until the expiration date. In fact, combined with the increase calibration frequency, our laboratory has reduced the average CV of Cys C to below 4% from January to July 2021, leading improvement of precision. Using Westgard Advisor subfunction of BIO‐RAD Unity Real Time, Cys C has higher probability of error detection (Ped), from 0.5 using 13s/22s QC procedure increased to 0.995 using 13s/22s/R4s/41s/6X rules, but also higher probability of false rejection (Pfr), from 0.006 using 13s/22s QC procedure increased to 0.095 using 13s/22s/R4s/41s/6X rules. In future studies, the other aspects should be prioritized to generate more conclusive results.\nNevertheless, there were three aspects of limitations in this research as follows: (Ⅰ).\nBecause the target means in PT/EQA plans were derived from statistical results of peer groups without measurement traceability,\n21\n, \n27\n those using this approach to calculate bias should be aware of possible limitations, including statistical methods used to generate the data and the number of laboratories that participate. There may be increased imprecision due to the small number of laboratories. There also might be concerns about the commutability of PT samples,\n28\n because they are not the same as real patient specimens. (Ⅱ) Another weakness is that the bias evaluation in our research (analyze PT samples at five different concentrations daily for 3 days to obtain 15 results) was not conducted strictly to the method described in the CLSI EP15‐A2 document (analyze one run per day with 3 replicate samples at each of different concentrations daily for 5 days to obtain 15 results), which may have underestimated bias.\n29\n (Ⅲ) The performance specification used to benchmark the methods is the most common limitation in the Sigma researches. Even the Milan consensus details only ideal considerations, there is no one source of analytical goals for all tests, laboratories must choose appropriate practical goals for each individual analytes.\nThis research can provide support for laboratories to select detection systems for these 10 analytes and aid individual laboratories in their choice of proper TEa goals and in working out a detailed troubleshooting action plan as a part of their quality improvement tool. Laboratory staffs can use these tools to help them select high‐quality products, further contributing to the delivery of excellent quality healthcare for patients. The result is more efficient instrument operation, more optimized laboratory workflow, and more reliable test results, ultimately helping clinicians better diagnose and treat their patients.",
"None declared.",
"YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript.",
"Table S1\nClick here for additional data file.\nSupplementary Material\nClick here for additional data file."
] | [
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"materials-and-methods",
null,
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"COI-statement",
null,
"supplementary-material"
] | [
"allowable total error",
"immunology and protein analytes",
"quality goal index",
"Sigma Method Decision Charts",
"Sigma metrics"
] | INTRODUCTION:
Detection of immunology and protein analytes is widely conducted in medical laboratories in China. How to ensure test performance, provide patients with accurate and reliable results, and provide support for doctors’ diagnosis and treatment are the primary goals of medical laboratories. For this reason, Six Sigma is obviously a rare good tool for quality control (QC). Sigma, which has a statistical appellation—“Standard Deviation,” represents the data dispersion.
1
,
2
As we all know, the higher the Sigma value is, the better the quality is. In medical laboratories, Sigma metrics have been widely used for the quality control of the whole clinical test processes, including pre‐
3
, during‐
4
and post‐analytical
5
phases. A scientific and reasonable quality control strategy of medical laboratories can be achieved by combining Sigma quality management with Westgard multirules quality control charts.
Quality control is an important part of clinical laboratory management. As a commonly used quality management tool, Six Sigma management program can effectively evaluate the performance indicators of analytes, help the laboratories find problems in time. The “Six” in Six Sigma represents the ideal goal that anything beyond those tolerance specifications is considered a defect.
1
6σ means 3.4 defect per million with world class performance, while 3σ means 66800 defect per million with marginal performance, that is, for example, if the minimum standard of quality control is set at the 3σ level, 66800 out of 1 million human immunodeficiency virus (HIV) carriers may be misdiagnosed. Therefore, the minimum standard set at the 3σ level is not fully applicable to clinical laboratories. The laboratory needs to increase the value of Sigma, minimize those defects, and increase the probability of error detection. Six Sigma quality management provides a new perspective for the quality control strategy of clinical laboratories. Not only through Six Sigma can we identify whether our methods are appropriate for clinical but also it can help determine the QC rules, guide our risk management efforts.
In this study, we evaluated the performance of 10 immunology and protein analytes by calculating their Sigma values based on Bias%, CV%, and four different sources of TEa%. Based on the calculated Sigma value, the QC strategies were personalized and Sigma Method Decision Charts were established. Improvement measures of analytes with σ below 6 were recommended according to the quality goal index (QGI).
MATERIALS AND METHODS:
Study design This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,
6
we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.
Flow chart of this research
This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,
6
we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.
Flow chart of this research
Instruments and reagents Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).
Two‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.
Five‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.
Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).
Two‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.
Five‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.
Evaluation of imprecision Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.
7
,
8
,
9
The calculation equation is: RMS CV% = [(CV1
2 + CV2
2) /2]0.5.
Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.
7
,
8
,
9
The calculation equation is: RMS CV% = [(CV1
2 + CV2
2) /2]0.5.
Evaluation of bias Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows
10
,
11
:
Bias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.
The average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.
Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows
10
,
11
:
Bias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.
The average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.
Allowable total error TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,
12
designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.
(https://biologicalvariation.eu/)
13
was first adopted, secondly from other researches which EFLM does not cover (RF
14
and PA
15
). The TEaBV are calculated using the formula:
TEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,
TEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,
TEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.
Here CVI means CV within‐subject, CVG means CV between‐subject.
TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,
12
designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.
(https://biologicalvariation.eu/)
13
was first adopted, secondly from other researches which EFLM does not cover (RF
14
and PA
15
). The TEaBV are calculated using the formula:
TEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,
TEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,
TEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.
Here CVI means CV within‐subject, CVG means CV between‐subject.
Sigma metrics calculation Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.
6
The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.
Westgard Sigma multi‐rules
N, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.
Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.
6
The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.
Westgard Sigma multi‐rules
N, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.
Composition of Sigma Method Decision Charts Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.
10
,
16
,
17
,
18
Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.
Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.
10
,
16
,
17
,
18
Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.
Quality goal index ratio The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.
10
,
16
,
17
,
18
The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.
10
,
16
,
17
,
18
Study design:
This study included four steps (Figure 1). The study was conducted in the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. According to the formula Sigma = (TEa%‐Bias%)/CV%,
6
we first determined the source of TEa%, CV%, and the sample used for Bias% calculation, second calculated the Sigma, and then Sigma Method Decision Charts were constructed, and finally the QGI analysis and corrective actions were performed to find and eliminate the potential causes of poor clinical performance of the analytes.
Flow chart of this research
Instruments and reagents:
Roche c8000 (Roche, Switzerland) automatic biochemical analysis system was used to quantify the 10 analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C‐reactive protein (CRP), and Cystatin C (Cys C). All analytes adopted the principle of immunoturbidimetry. All the reagents and calibrators were Roche original except CRP from Sekisui (Osaka, Japan), PA from Kehua (Shanghai, China), and Cys C from Mindray (Shenzhen, China).
Two‐level internal quality controls (IQC) (Liquichek Immunology and protein Control, lot: 68931and 68932, BIO‐RAD, US) were analyzed, all controls were operated in strict accordance with the manufacturer's instructions.
Five‐level materials of external quality assessment (EQA) with different concentrations of the analytes were provided by the National Center for Clinical Laboratories (NCCL, China). Cys C (NCCL‐C‐14) had only one EQA plan in year 2020 with lots of 202011, 202012, 202013, 202014, and 202015. The remaining nine analytes, which was called “Special proteins plan (NCCL‐C‐06),” had two EQA plans: lots for the first EQA plan were 202011, 202012, 202013, 202014, and 202015; lots for the second EQA plan were 202021, 202022, 202023, 202024, and 202025. Each level material was dissolved in pure water according to the manufacturer's instructions.
Evaluation of imprecision:
Imprecision is estimated using the coefficient of variation (CV%) which is a measure of variability and indicator of random errors. The two IQC levels of cumulative coefficient of variation (CV%) were collected from the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine from January 1 to December 31, 2020. Each analyte had two concentration levels. The root mean square coefficient of variation (RMS CV%) was calculated.
7
,
8
,
9
The calculation equation is: RMS CV% = [(CV1
2 + CV2
2) /2]0.5.
Evaluation of bias:
Bias is an indicator of systematic errors. The laboratory was involved in the EQA program by analyzing five different concentration proficiency test (PT) samples provided by NCCL. PT samples were dissolved in pure water according to the NCCL’s instructions, each PT sample was tested for 3 days to obtain three results, and the mean of the three results was calculated and assigned as “Measured mean in our laboratory.” NCCL groups the submitted data according to the instruments or reagent manufacturers that participants use, and takes the ISO13528 robust average value in the group as the target mean. Seven analytes (C3, C4, IgG, IgM, IgA, ASO, and RF) were grouped according to the instrument, while the other three analytes (CRP, PA, and Cys C) were grouped according to reagent manufacturers due to non‐Roche reagents. The target mean assigned by NCCL of each analyte was considered as target value. Excluding unqualified PT data (Data exceeding two standard deviations of the mean), the calculation equation was as follows
10
,
11
:
Bias%=│Measured mean in our laboratory‐Target mean assigned by NCCL│/(Target mean assigned by NCCL)×100%.
The average bias of each analyte, which was calculated by 1‐year accumulative bias of each analyte sourced from the NCCL plans in 2020, was used in the calculation of Sigma.
Allowable total error:
TEa (or “total allowable variation”) represents the allowable difference between measured value and trueness. Four different TEa targets were used in this study: (Ⅰ) TEa derived from the quality goals issued by the China National Center for Clinical Laboratories (NCCL) in 2017,
12
designated as TEaNCCL. (Ⅱ, Ⅲ, Ⅳ) the biological variation database specifications (minimum, desirable, optimal), designated as TEaBVmin, TEaBVdes and TEaBVopt. BV provided by EFLM.
(https://biologicalvariation.eu/)
13
was first adopted, secondly from other researches which EFLM does not cover (RF
14
and PA
15
). The TEaBV are calculated using the formula:
TEaBVmin=1.65×0.75CVI+0.375CVI2+CVG20.5,
TEaBVdes=1.65×0.5CVI+0.25CVI2+CVG20.5,
TEaBVopt=1.65×0.25CVI+0.125CVI2+CVG20.5.
Here CVI means CV within‐subject, CVG means CV between‐subject.
Sigma metrics calculation:
Sigma value is calculated using the standard equation: Sigma = (TEa%‐Bias%)/CV%.
6
The performance evaluation standards are divided into 6 levels: world‐class: σ≥6; excellent: 5≤σ<6; good: 4≤σ<5; marginal: 3≤σ<4; poor: 2≤σ<3; Unacceptable: σ<2. 3σ was considered as the minimum acceptable limit. The quality control rules were selected as per Westgard Sigma multi‐rules (shown in Table 1) based on Sigma value.
Westgard Sigma multi‐rules
N, the number of quality control determinations per batch, N = 2, represented two measurements of a single QC level or one measurement of two QC levels, N = 2 similar definitions apply to N = 4 and N = 6. R, the number of batch. Batch length: The maximum number of samples in a round of quality control.
Composition of Sigma Method Decision Charts:
Logging in the NCCLnet (https://www.nccl.org.cn/loginCn), entering TEa%, Bias%, and CV% of each analyte which is obtained through the above steps in the interface of the Six Sigma management menu, the Sigma Method Decision Charts are composed with CV%/TEa% along the x‐axis and Bias%/TEa% along the y‐axis.
10
,
16
,
17
,
18
Five lines divide the chart into 6 levels: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). The specific Sigma value of the analyte is displayed as a point in the chart, providing us with a visual view of the analyte's performance.
Quality goal index ratio:
The QGI ratio was calculated from the analyte with a Sigma<6. The calculation equation is as follows: QGI =Bias%/(1.5×CV%). A QGI value less than 0.8 (QGI <0.8) indicates that the precision needs to be improved, whereas a value greater than 1.2 (QGI >1.2) indicates that the accuracy needs to be improved. A QGI value between 0.8 and 1.2 (0.8 ≤ QGI ≤1.2) indicates that both accuracy and precision need to be improved. Analytes with lower Sigma were analyzed according to QGI, that is, the problem was due to precision or accuracy or combination of both, helping the laboratories to take targeted measures.
10
,
16
,
17
,
18
RESULTS:
Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.
Mean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes
Abbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.
The TEa source did not cover the TEa of the analyte.
Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.
Mean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes
Abbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.
The TEa source did not cover the TEa of the analyte.
Distribution of Sigma metrics based on four TEa standards Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.
Sigma of 10 analytes based on four different TEa standards
σBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.
No calculated Sigma obtained.
Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.
Sigma of 10 analytes based on four different TEa standards
σBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.
No calculated Sigma obtained.
Composition of Sigma Method Decision Charts We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.
Sigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels
We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.
Sigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels
Resetting QC strategies and improvement measures QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.
QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.
Cumulative Mean, SD, CV%, Bias%, and TEa% derived from four standards for 10 immunology and protein analytes:
Cumulative Mean, SD, and CV% of two IQC levels were shown in Table 2. The RMS CV% ranged from 1.83% (IgG) to 4.96% (Cys C). The EQA data, which were all falling within ±2SD of the mean, were all satisfactory. The average bias values were displayed in Table 2. They ranged from 0.93% (Cys C) to 4.42% (C3). Cys C had the highest CV% and lowest Bias%. Four different source of TEa were also shown in Table 2. Mean assigned by NCCL and relative bias% were shown in Table S1.
Mean, SD, RMS CV%, Bias% and TEa% derived from four standards for 10 analytes
Abbreviations: Cumulative Mean, the concentration of two levels of IQC; SD, Standard Deviation; CV%, Cumulative coefficient of variation; RMS CV%, Root Mean Square coefficient of variation; TEaBVmin, TEaBVdes, TEaBVopt, represented the TEa sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; TEaNCCL represented the TEa sourced from the NCCL.
The TEa source did not cover the TEa of the analyte.
Distribution of Sigma metrics based on four TEa standards:
Table 3 showed the Sigma values of 10 immunology and protein analytes based on four different TEa standards. While using the TEaNCCL, 90% (9/10) analytes had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. However, while we chose the TEa based on biological variation database specifications, the Sigma values were not so satisfactory. Since the TEaBVmin of only two analytes IgM and CRP were looser than TEaNCCL, σBVmin of IgM, and CRP were greater than σNCCL. The other 8 analytes had lower σBVmin than σNCCL. Using minimum biological variation of TEa, seven of the nine analytes (excluded ASO) exhibited a performance of at least 3σ (marginal),and three of these analytes (IgG, IgM, and CRP) had a world‐class performance. While using desirable and optimal biological variation of TEa, only one analytes (CRP) reached 6σ level, with poor or unacceptable Sigma values of the remaining nine analytes. TEaBV is too strict for our laboratory, we chose the TEaNCCL specifications for the follow‐up analysis, such as QGI analysis, the QC strategies construction, and composition of Sigma Method Decision Charts.
Sigma of 10 analytes based on four different TEa standards
σBVmin, σBVdes, σBVopt, represented the calculated Sigma sourced from the minimum, desirable, and optimal biological variation database specifications, respectively; σNCCL represented the calculated Sigma sourced from the NCCL.
No calculated Sigma obtained.
Composition of Sigma Method Decision Charts:
We constructed Sigma Method Decision Charts of the ten analytes as per TEaNCCL (Figure 2). Nine out of ten analytes were displayed in the Six Sigma zone, while Cys C appeared in the 3σ‐ 4σ zone. This chart could provide us with a visual view of the analytes' performance. We could intuitively judge the performance of the analytes through this chart.
Sigma Method Decision Charts of 10 analytes based on TEaNCCL. The chart was drawn with CV/TEa along the x‐axis and Bias/TEa along the y‐axis, which divided into six zones by five performance lines. The zones from bottom left to top right were: world‐class (σ> 6), excellent (5≤σ< 6), good (4 ≤σ< 5), marginal (3≤σ< 4), poor (2≤σ< 3), and unacceptable (σ< 2). Different colored dots indicated different Sigma levels
Resetting QC strategies and improvement measures:
QC strategies were reset according to σNCCL and the QGI of analytes with σ<6 were calculated (Table 4). For analytes with σ>6 (world‐class performance), those were IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO, only one QC rule (13s), one measurement of two QC levels (N = 2) per QC event, and up to 1000 clinical samples in a round of quality control (Batch length: 1000) were adopted for QC management. For Cys C that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) with N = 6 and up to 45 clinical samples in a round of quality control (Batch length: 45) were adopted for QC management. The calculated QGI (0.12) showed that Cys C had low precision and required a close monitoring and troubleshooting from the aspects of personnel, equipment, material, method, and environment in the daily QC.
DISCUSSIONS:
In this study, we evaluated the performance of 10 immunology and protein analytes (which named after their BIO‐RAD controls—“Immunology and Protein Control”) at the Laboratory Department of Guangdong Provincial Hospital of Chinese Medicine for a one year period based on Sigma metrics. The previous studies mainly focused on the evaluation of analytical performance of clinical biochemistry analytes,
19
,
20
,
21
endocrine analytes,
10
,
22
tumor marker analytes,
11
,
22
etc. using Sigma metrics. This study is the first time to focus on the application of Sigma metrics in immunology and protein analytes (IgG, IgA, IgM, C3, C4, CRP, RF, PA, ASO, and Cys C) based on four different sources of TEa. Further the Sigma Method Decision Charts were constructed in terms of TEaNCCL, providing us with a visual view of the analytes’ performance. For analytes with σ<6, the cause for poor performance was evaluated using QGI similar to previous studies.
10
,
11
According to the equation Sigma = (TEa% ‐ Bias%)/CV%, significantly different σ values are obtained using the same Bias% and CV% but different total allowable error targets similar to the previous studies,
10
,
11
the greater the TEa is, the greater the Sigma value is. In this study, initially five different TEa targets were included. Most TEa selected from the Clinical Laboratory Improvements Amendments (CLIA)‐88 requirements from the United States are exhibited as 3 times the standard deviation or as semi‐quantitative results
23
(data not shown), regardless of whether the standard deviation comes from IQC or the same group variation of the EQA, the variable standard deviations bring difficulties to calculation, and there may be an illusion that analytes with large TEa have better performance and analytes with small TEa have poor performance. Therefore, we would not include TEaCLIA in subsequent calculations. The Sigma metrics of 10 immunology and protein analytes were shown in Table 3 based on four different TEa standards.
As mentioned above, surprisingly, while using the TEaNCCL, 9 analytes for IgG, IgA, IgM, C3, C4, CRP, RF, PA, and ASO had a world‐class performance with σ>6, Cys C showed marginal performance with σ<4. On the other hand, while we chose the TEaBV, the Sigma values were quite different, even negative (C3). TEa sources play an important role in the calculation of Sigma value.
24
According to the 2015 Strategic Conference on Quality Specifications convened in Milan consensus, there are three more compact levels or models, that is clinical outcomes, biological variabilities, and state‐of‐the‐art, to determine performance specifications in clinical laboratories.
25
The actual clinical use of the test results is the gold standard for setting TEa. The optimal TEa should be established depending on the conditions and requirements of the individual laboratory. The laboratory should assess the suitability of the TEa for clinical use and determine if that would allow a larger TEa choice. TEaBV is too strict for our laboratory, after a comprehensive analysis of laboratory status, analytes’ performance, and clinical recognition, therefore, in the subsequent analysis, we chose TEaNCCL as performance specifications.
After calculating the Sigma value, how can we have a more intuitive impression of these results? At this time, Sigma Method Decision Charts are on the stage, which converts all the Sigma metrics into a simple intuitive dashboard.
25
Nine Sigma in this research were in the “bull's eye” zone, suggesting so good performance as to world‐class. Cys C fallen in the 3σ‐4σ zone, suggesting there may be more defects. The Sigma Method Decision Charts allows us to get a succinct comprehensive view of an entire instrument's performance, provide us with a visual view of the analytes’ performance. We could intuitively judge the performance of the analytes through this chart.
The personalized QC strategies, QGI and Prioritize improvement of 10 analytes according to σNCCL
Analytes with σ>6 does not need to calculate QGI, and no need for improvement.
Sigma metrics are not just a tool to ascertain the performance of analytes but also a beacon for designing a more cost‐effective QC strategy. Through the assessment of Sigma metrics, we can specify the control rules scientifically, including the number of control materials and the necessary frequency of running those controls. The closer the operating dot of an analyte is to the chart's origin, the easier the QC program is. In our laboratory, we chose 13s/22s QC procedure with two QC levels once per day for the 10 immunology and protein analytes empirically to supervise the performance of analytes in the past. By using Sigma metrics, nine analytes for σ>6 can safely use the 13s procedure with one measurement of two QC levels to gain an appropriate level of analytical quality assurance, avoiding economic costs, and overwork. On the contrary, with the decline in performance, more quality control rules, more different levels of quality controls, and higher quality control frequency are required. As Cys C in this study, that had “marginal” performance, five multi‐rules (13s/22s/R4s/41s/6X) were needed and the QC frequency could be increased to one control per 45 clinical samples. As Cys C had 38 average daily measurements in our laboratory,one time QC frequency per day is needed.
Though the Westgard Sigma multi‐rules provide a scientific and reasonable method for setting QC procedure, it could not fully reflect the precision and accuracy of the method. QGI is a tool to provide easy insights into the reasons for quality errors such as those caused by imprecision, inaccuracy, or both.
21
,
26
Cys C (QGI = 0.12) had low precision and some action must be taken. We make continuous improvements from five potential causal factors: personnel, machine, material, method, and environment: (Ⅰ) Strengthen personnel training, including QC knowledge and analyzer operation training; (Ⅱ) Perform regular maintenance and calibration of analyzers to ensure the performance of the analyzer, and timely retire the analyzer with poor performance; (Ⅲ) Monitor the temperature of reagent transportation and storage, avoid reagent expiration and excessive on‐board time; (Ⅳ) Revise the operation protocols, perform the machine in strict accordance with the operation protocols; (Ⅴ) Ensure that the working environment of the instrument meets the requirements, such as temperature and humidity. In fact, Cys C used in this research is a third‐party reagent, not Roche original, the reagent batch is updated so quickly, and the difference between batches is large, resulting in a large CV. Aware of this, our laboratory try our best to use the same batch reagents. We would not replace the new batch reagents until the expiration date. In fact, combined with the increase calibration frequency, our laboratory has reduced the average CV of Cys C to below 4% from January to July 2021, leading improvement of precision. Using Westgard Advisor subfunction of BIO‐RAD Unity Real Time, Cys C has higher probability of error detection (Ped), from 0.5 using 13s/22s QC procedure increased to 0.995 using 13s/22s/R4s/41s/6X rules, but also higher probability of false rejection (Pfr), from 0.006 using 13s/22s QC procedure increased to 0.095 using 13s/22s/R4s/41s/6X rules. In future studies, the other aspects should be prioritized to generate more conclusive results.
Nevertheless, there were three aspects of limitations in this research as follows: (Ⅰ).
Because the target means in PT/EQA plans were derived from statistical results of peer groups without measurement traceability,
21
,
27
those using this approach to calculate bias should be aware of possible limitations, including statistical methods used to generate the data and the number of laboratories that participate. There may be increased imprecision due to the small number of laboratories. There also might be concerns about the commutability of PT samples,
28
because they are not the same as real patient specimens. (Ⅱ) Another weakness is that the bias evaluation in our research (analyze PT samples at five different concentrations daily for 3 days to obtain 15 results) was not conducted strictly to the method described in the CLSI EP15‐A2 document (analyze one run per day with 3 replicate samples at each of different concentrations daily for 5 days to obtain 15 results), which may have underestimated bias.
29
(Ⅲ) The performance specification used to benchmark the methods is the most common limitation in the Sigma researches. Even the Milan consensus details only ideal considerations, there is no one source of analytical goals for all tests, laboratories must choose appropriate practical goals for each individual analytes.
This research can provide support for laboratories to select detection systems for these 10 analytes and aid individual laboratories in their choice of proper TEa goals and in working out a detailed troubleshooting action plan as a part of their quality improvement tool. Laboratory staffs can use these tools to help them select high‐quality products, further contributing to the delivery of excellent quality healthcare for patients. The result is more efficient instrument operation, more optimized laboratory workflow, and more reliable test results, ultimately helping clinicians better diagnose and treat their patients.
CONFLICT OF INTEREST:
None declared.
AUTHOR CONTRIBUTIONS:
YL and SO designed the study, drafted the work, analyzed and interpreted the data, and wrote this article; XY, QX, YL, and JP searched the literature, performed the experimental procedure; QL, YC, YC, and HZ supervised this study and reviewed this article; CC reviewed this article. All authors have read and approved the final manuscript.
Supporting information:
Table S1
Click here for additional data file.
Supplementary Material
Click here for additional data file.
| Background:
Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma.
Methods:
Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values.
Results:
While using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12).
Conclusions:
The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool. | null | null | 9,241 | 375 | [
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|||
Comparing Results of Five Glomerular Filtration Rate-Estimating Equations in the Korean General Population: MDRD Study, Revised Lund-Malmö, and Three CKD-EPI Equations. | 27578504 | Estimated glomerular filtration rate (eGFR) is a widely used index of kidney function. Recently, new formulas such as the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations or the Lund-Malmö equation were introduced for assessing eGFR. We compared them with the Modification of Diet in Renal Disease (MDRD) Study equation in the Korean adult population. | BACKGROUND | The study population comprised 1,482 individuals (median age 51 [42-59] yr, 48.9% males) who received annual physical check-ups during the year 2014. Serum creatinine (Cr) and cystatin C (CysC) were measured. We conducted a retrospective analysis using five GFR estimating equations (MDRD Study, revised Lund-Malmö, and Cr and/or CysC-based CKD-EPI equations). Reduced GFR was defined as eGFR <60 mL/min/1.73 m². | METHODS | For the GFR category distribution, large discrepancies were observed depending on the equation used; category G1 (≥90 mL/min/1.73 m²) ranged from 7.4-81.8%. Compared with the MDRD Study equation, the other four equations overestimated GFR, and CysC-based equations showed a greater difference (-31.3 for CKD-EPI(CysC) and -20.5 for CKD-EPI(Cr-CysC)). CysC-based equations decreased the prevalence of reduced GFR by one third (9.4% in the MDRD Study and 2.4% in CKD-EPI(CysC)). | RESULTS | Our data shows that there are remarkable differences in eGFR assessment in the Korean population depending on the equation used, especially in normal or mildly decreased categories. Further prospective studies are necessary in various clinical settings. | CONCLUSIONS | [
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] | 5011104 | INTRODUCTION | Early detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.
There have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults. | METHODS | 1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.
The number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.
During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.
The number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.
2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:
a) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);
b) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;
c) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;
d) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;
e) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.
Serum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.
Serum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.
GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:
a) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);
b) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;
c) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;
d) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;
e) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.
Serum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.
Serum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.
3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].
GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].
4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].
Categorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.
Data were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.
The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].
Categorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.
Data were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant. | RESULTS | 1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.
The frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).
The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.
The frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).
2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.
For diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).
The mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).
Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.
For diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).
The mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).
3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.
The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation. | null | null | [
"1. Study population",
"2. Estimation of GFR",
"3. GFR categories",
"4. Statistical analyses",
"1. GFR category distribution in the study population",
"2. Concordance between MDRD Study equation and other equations",
"3. Prevalence of reduced eGFR"
] | [
"During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.",
"GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.",
"GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].",
"The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.",
"The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).",
"Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).",
"The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation."
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"1. Study population",
"2. Estimation of GFR",
"3. GFR categories",
"4. Statistical analyses",
"RESULTS",
"1. GFR category distribution in the study population",
"2. Concordance between MDRD Study equation and other equations",
"3. Prevalence of reduced eGFR",
"DISCUSSION"
] | [
"Early detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.\nThere have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults.",
" 1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\nDuring the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.\n 2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\nGFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.\n 3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\nGFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].\n 4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.\nThe eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.",
"During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.\nThe number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.",
"GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:\na) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);\nb) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;\nc) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;\nd) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;\ne) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.\nSerum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.\nSerum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.",
"GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].",
"The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].\nCategorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.\nData were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.",
" 1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\nThe baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).\n 2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\nCategorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).\n 3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.\nThe prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.",
"The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.\nThe frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).",
"Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.\nFor diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).\nThe mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).",
"The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.",
"The Cr-based CKD-EPI equation is recommended for the initial assessment of GFR, and CysC-based CKD-EPI equations can be used for confirmation of kidney disease according to the KDIGO guidelines [423]. In this study, we compared five eGFR equations, including CysC-based formulas, in the Korean population. The prevalence of reduced GFR by CysC-based CKD-EPI equations has not been reported in Korea yet.\nThe eGFR classification differed considerably according to the equation used for estimation, especially between CKD-EPICysC or CKD-EPICr-CysC equations compared with the MDRD Study equation. Most of the study population (>80%) were in the G2 category (60-89 mL/min/1.73 m2) according to the MDRD Study equation, but in G1 (≥90 mL/min/1.73 m2) according to the CKD-EPICysC equation. The reason for this discrepancy might be related to the eGFR distribution of the study population. Mean eGFRMDRD was 73.5 mL/min/1.73 m2 with a standard deviation of 12.2 mL/min/1.73 m2; thus, there were many results around the cutoff value of 90 mL/min/1.73 m2. In addition, the CKD-EPICysC and CKD-EPICr-CysC equations yielded systematically higher eGFR results (mean difference 31.4 mL/min/1.73 m2 and 20.6 mL/min/1.73 m2, respectively) in comparison with the MDRD Study equation. This finding was in line with previous studies. In the US National Health and Nutrition Examination Survey (NHANES) 1999-2002 data, the distribution of eGFRCKD-EPI CysC was broader and shifted to the right compared with that of eGFRMDRD [24]. Thus, upward reclassification might be common in CysC-based equations.\nAll equations, except for the CKD-EPICysC equation, showed good correlation with the MDRD Study equation. CKD-EPICysC showed only a moderate correlation (r=0.49). The three different CKD-EPI equations showed an overall low prevalence of reduced GFR compared with the MDRD Study equation, especially according to the two CysC-containing equations. The LMRevised equation was recently reported to outperform the MDRD Study and CKD-EPI equations in a Swedish population [15]; however, there has been no evaluation of this equation in the Asian population. It yielded similar mean eGFR results compared with the MDRD Study equation; these two equations showed a very high correlation and a similar prevalence of reduced GFR. However, eGFR was underestimated in patients ≥ 60 yr, when using the LMRevised equation. This observation needs to be subjected to further studies because of the increased possibility of co-morbidities in older patients. The LMRevised equation was generated only from the Swedish population; hence, ethnic differences might have influenced GFR estimation as well.\nAlthough the CKD-EPICr equation is recommended by KDIGO for initial GFR assessment, newer GFR estimating equations have been developed and validated, including a Korean version of the CKD-EPICr equation [25], a serum Cr-based full age spectrum equation [26], and a CysC-based equation based on a Caucasian, Asian, pediatric, and adult population (CAPA) [27]. Our study did not aim to compare all recent equations; however, we analyzed the CysC-based CAPA equation briefly. The mean eGFRCAPA (106.3 mL/min/1.73 m2), eGFR difference in comparison with MDRD Study equation (-32.9), and prevalence of eGFR less than 60 mL/min/1.73 m2 (2.7%) were similar when compared with CKD-EPICysC equation.\nThe prevalence of reduced GFR has been reported differently depending on the study population and the GFR-estimating equation used. In several previous studies, there were clinically significant differences in the prevalence of stage 3 or higher CKD depending on the equation used to estimate GFR. Delanaye et al [20] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRMDRD (13%), intermediate for eGFRCKD-EPI Cr (9.8%), and the lowest for eGFRCKD-EPI Cr-CysC (5%) and eGFRCKD-EPI CysC (4.7%) in 4,189 Belgian patients over 50 yr old. This prevalence trend was similar to ours. One Japanese study showed a 2-fold difference of prevalence between the MDRD Study and CKD-EPICr equations (12.8 vs 6.5%), by studying over 26,000 participants who underwent annual health check-ups [16]. Lujambio et al [28] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRCKD-EPI CysC (21.8%), intermediate for eGFRCKD-EPI Cr-CysC (11.8%), and the lowest for eGFRMDRD (5.9%) and eGFRCKD-EPI Cr (3.4%) in 119 Uruguayans.\nIn the US, the prevalence of reduced GFR (eGFR <60 mL/min/1.73 m2) by CKD-EPICr equation was reported to be 4.7% from 1988-1994 and 6.5% from 1999-2002 from NHANES data [29]. In Korea, the prevalence of reduced GFR by the CKD-EPICr equation has been reported as 7.7% in 2007 and 2.6% in 2010 from Korea NHANES data [1718]. In this study, it was 5.1% of the whole study population. This difference could be due to the following reasons. First, the proportion of younger individuals under 40 yr old was lower than that in other studies (23% vs 30-32%). Compared with 2010 Korea NHANES data, the mean eGFR was relatively lower in this study (81.4 mL/min/1.73 m2 vs 95.9-96.8 mL/min/1.73 m2), resulting in higher prevalence of reduced GFR compared with other studies. Second, the study period was different (2014 vs 2007-2010), although the impact of this on the prevalence is still uncertain.\nOur retrospective study has several limitations. First, we com-pared all the eGFR equations to the MDRD Study equation, because of the absence of GFR data measured by the gold standard method [18]. Therefore, it was impossible to evaluate the accuracy. The magnitude of bias, calculated by measured GFR-calculated GFR was previously reported to be 2.5-5.8 mL/min/1.73 m2, and this difference could influence the prevalence of CKD stages [1724]. Second, our population might not be representative of the entire population of Korea. Third, we could not analyze albuminuria data or other markers of kidney damage. The classification of CKD was not performed, which is based on both GFR category and albuminuria category. Thus, there could be the CKD patients among the subjects with eGFR ≥60 mL/min/1.73 m2.\nIn conclusion, this is the first study that compared five eGFR equations in the Korean population. Our data demonstrated remarkable differences in GFR assessment depending on the equation used. The proportion of each GFR category varied considerably, and CysC-containing equations yielded higher eGFRs and showed larger differences compared with the MDRD Study equation. The prevalence of reduced GFR was lowered by the CKD-EPI equations. Further studies using prospective design and in various ethnicities are necessary."
] | [
"intro",
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion"
] | [
"Estimated glomerular filtration rate",
"MDRD Study equation",
"CKD-EPI equation",
"Revised Lund-Malmö equation"
] | INTRODUCTION:
Early detection of at-risk populations with decreased kidney function is important for both acute kidney injury and chronic kidney disease (CKD) [123]. Glomerular filtration rate (GFR) is the most widely used index for assessing kidney function, which is implicated in the guidelines for CKD diagnosis and staging [3456]. Various equations for estimating GFR (eGFR) have been introduced and are currently used; among them, 83% of clinical laboratories used the serum creatinine (Cr)-based Modification of Diet in Renal Disease (MDRD) Study equation in the 2013 College of American Pathologists survey [7]. However, the equation was derived from subjects with CKD, and had the limitation of systematically underestimating GFR in healthy individuals with GFR ≥60 mL/min/1.73 m2 [89]. To overcome this limitation, the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPICr) was developed in 2009 on the basis of serum Cr, and more accurate calculations for GFRs ≥60 mL/min/1.73 m2 were made possible [10]. In addition, CKD-EPI 2012 equations based on cystatin C (CysC) (CKD-EPICysC) or a combination of Cr and CysC (CKD-EPICr-CysC) were proposed in a standardized assay to better estimate GFR [1112]. Another new equation, the revised Lund-Malmö (LMRevised) equation was recently developed, and its performance was reported to be more consistent than the MDRD Study and CKD-EPI equations [131415]. However, this equation was derived from only a Swedish Caucasian population.
There have been several reports that compared the MDRD Study and CKD-EPI equations in the general population [1617181920]; however, only a few studies have included the CysC-containing equations. In this study, we compared five eGFR equations (MDRD Study equation, LMRevised equation, and three CKD-EPI equations) to explore the differences in the equations across the GFR categories in Korean adults.
METHODS:
1. Study population During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.
The number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.
During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.
The number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.
2. Estimation of GFR GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:
a) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);
b) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;
c) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;
d) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;
e) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.
Serum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.
Serum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.
GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:
a) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);
b) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;
c) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;
d) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;
e) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.
Serum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.
Serum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.
3. GFR categories GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].
GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].
4. Statistical analyses The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].
Categorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.
Data were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.
The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].
Categorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.
Data were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.
1. Study population:
During the year 2014, the recipients of annual physical check-ups at the Gangnam branch of Korean Association of Health Promotion (KAHP, Seoul, Korea) were consecutively included according to the following criteria: over 30 yr old and presence of serum Cr and/or CysC results. There were no exclusion criteria for the subject selection process. KAHP is a specialized health-screening center, which provides routine medical check-ups to over 1,000,000 individuals annually in 16 branch clinics nationwide in Korea.
The number of subjects with serum Cr and CysC results was 1,482 (51 [42-59] yr, 48.9% males). Besides serum Cr and/or CysC results, lipid profiles and HbA1c levels were also collected. We conducted a retrospective analysis of laboratory data using the five eGFR equations, and this study was approved by the Institutional Review Board of KAHP.
2. Estimation of GFR:
GFR (mL/min/1.73 m2) was estimated by using five different equations (MDRD Study, LM revised, and three CKD-EPI equations) as follows:
a) Four-variable MDRD Study equation [9], GFR=175×sCr-1.154×Age-0.203×0.742 (if female);
b) LMRevised equation [13], GFR=eX-0.0158×Age+0.438×ln(Age), where ln is natural logarithm and X=2.50+0.0121×(150-sCr) for females with sCr level <150 µmol/L, 2.50-0.926×ln (sCr/150) for females with sCr level ≥150 µmol/L, 2.56+0.00968×(180-sCr) for males with sCr level <180 µmol/L, and 2.56-0.926×ln (sCr/180) for males with sCr level ≥180 µmol/L;
c) CKD-EPICr equation [10], GFR=141×min (sCr/κ, 1)α× max (sCr/κ, 1)-1.209×0.993Age×1.018 [if female], where sCr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min is the minimum of sCr/κ or 1, and max is the maximum of sCr/κ or 1;
d) CKD-EPICysC equation [11], GFR=133×min (sCysC/0.8, 1)-0.499×max (sCysC/0.8, 1)-1.328×0.996Age×0.932 [if female], where sCysC is serum cystatin C, min indicates the minimum of sCysC/0.8 or 1, and max indicates the maximum of sCysC/0.8 or 1;
e) CKD-EPICr-CysC equation [11], GFR=135×min (sCr/κ, 1)α× max (sCr/κ, 1)-0.601×min (sCysC/0.8, 1)-0.375×max (sCysC/0.8, 1)-0.711×0.995Age×0.969 [if female], where Scr is serum creatinine, sCysC is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is -0.248 for females and -0.207 for males.
Serum Cr levels were determined by the kinetic Jaffe method using SICDIA CRE reagent (Shinyang Chemical, Seoul, Korea) on an automated chemistry analyzer (HITACHI 7600-110; Hitachi High-Technologies Co., Tokyo, Japan). Isotope-dilution mass spectrometry (IDMS)-traceable calibration was conducted weekly with a C.f.a.s. calibrator (Roche Diagnostics, Indianapolis, IN, USA). For internal quality control in the Cr assay, two levels of Lyphochek assayed chemistry quality control materials (Bio-Rad, Hercules, CA, USA) were tested once a day. The mean within-laboratory precision of the serum Cr assay was 2.1% during the study period. The laboratory participated in the external proficiency testing program organized by the Korean Association of Quality Assurance for Clinical Laboratory, and the results were all acceptable (variance index scores <150) during 2014.
Serum CysC levels were measured by the latex immunoturbidimetric method using Sekisui reagent (Sekisui Chemical, Tokyo, Japan) on the same analyzer. The reagent was traceable to the European Reference Material DA471/IFCC. Two levels of Liquichek Immunology Control materials (Bio-Rad) were tested once a day. The mean within-laboratory precision of the serum CysC assay was 1.4% during the study period. The inter-laboratory comparison was performed for CysC, and the differences in results between the two locations were within 10%. All measurements were performed according to the manufacturer's in structions and standard laboratory procedures.
3. GFR categories:
GFRs were categorized into ≥90 (G1), 60-89 (G2), 45-59 (G3a), 30-44 (G3b), 15-29 (G4), and <15 (G5) mL/min/1.73 m2 according to the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guideline [4]. The prevalence of reduced GFR, defined as eGFR <60 mL/min/1.73 m2, was compared among the equations on the basis of the levels of serum Cr, CysC, or both [4].
4. Statistical analyses:
The eGFRMDRD was regarded as the comparative GFR for com-parisons. Bland-Altman plots were used to identify mean differences and 95% limits of agreement of eGFRs between each equation and the MDRD Study equation. Pearson's correlation coefficients (r) were calculated to compare the equations. r coefficients ≤0.35 were considered low or weak correlations; 0.36-0.67, modest or moderate correlations; and 0.68-1.0, strong or high correlations; with r coefficients ≥0.90 being very high correlations [21].
Categorical agreement rates were calculated when eGFRMDRD and eGFR based on other equations were within the same GFR categories. Weighted kappa value was determined to evaluate the degree of categorical agreement, and kappa value was determined for diagnostic agreement with GFR cutoffs of 60 mL/min/1.73 m2 and 45 mL/min/1.73 m2 [4]. The kappa values were interpreted as follows: <0.20, poor; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, good; and >0.81, very good [22]. In general, reduced GFR is defined as an eGFR <60 mL/min/1.73 m2. Additionally, the KDIGO guideline recommends measuring CysC in adults with an eGFRCr of 45-59 mL/min/1.73 m2, who do not have markers of kidney damage for confirmation of CKD [4]. Thus, we used two GFR cutoff points. The overall concordance rate (positive and negative) was also calculated for all equations.
Data were analyzed by using Analyse-it (Analyse-it Software Ltd., Leeds, UK) and MedCalc Statistical Software version 15.2.2 (MedCalc Software, Ostend, Belgium). P values ≤0.05 were considered statistically significant.
RESULTS:
1. GFR category distribution in the study population The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.
The frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).
The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.
The frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).
2. Concordance between MDRD Study equation and other equations Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.
For diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).
The mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).
Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.
For diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).
The mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).
3. Prevalence of reduced eGFR The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.
The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.
1. GFR category distribution in the study population:
The baseline subject characteristics and calculated eGFR stratified by age groups are shown in Table 1. The CKD-EPI equations yielded higher mean eGFRs than the MDRD Study equation, and the degree of difference varied with the equations and age groups. The eGFRCKD-EPI CysC (104.7 mL/min/1.73 m2) was the highest, followed by eGFRCKD-EPI Cr-CysC (94.0 mL/min/1.73 m2), eGFRCKD-EPI Cr (81.4 mL/min/1.73 m2), eGFRLM Revised (74.5 mL/min/1.73 m2), and eGFRMDRD (73.5 mL/min/1.73 m2). In older patients (≥ 60 yr), the LMRevised equation yielded a lower eGFR than the MDRD Study equation. For all equations, a decreasing eGFR trend was observed as age increased.
The frequencies of the subjects in each GFR category are shown in Table 2. The proportion of each GFR category varied considerably between equations, especially for categories G1 and G2. The proportions of G1 were 8.0% (MDRD), 28.0% (CKD-EPICr), 81.7% (CKD-EPICysC), 66.2% (CKD-EPICr-CysC), and 7.4% (LMRevised), and the proportions of G2 were 82.5% (MDRD), 66.3% (CKD-EPICr), 15.8% (CKD-EPICysC), 31.1% (CKD-EPICr-CysC), and 84.6% (LMRevised).
2. Concordance between MDRD Study equation and other equations:
Categorical agreement rates between the MDRD Study and other equations ranged from 20.3 to 93.8%. Upward reclassification from G2 (MDRD Study equation) to G1 (CKD-EPI equations) was common, especially for CKD-EPICysC and CKD-EPICr-CysC equations (Table 3). Weighted kappa values were fair to good in serum Cr-based equations, but poor in CysC-containing equations.
For diagnostic agreement, kappa values at two eGFR cutoffs were variable depending on the equation. Kappa values were better at cutoff 45 mL/min/1.73 m2 than 60 mL/min/1.73 m2. For all equations, overall concordance rates were greater than 90% when the GFR cutoff of 60 mL/min/1.73 m2 was used (96.2% for CKD-EPICr, 91.6% for CKD-EPICysC, 93.1% for CKD-EPICr-CysC, and 95.8% for LMRevised), or when 45 mL/min/1.73 m2 was used (99.9% for CKD-EPICr, 98.5% for CKD-EPICysC, 98.9% for CKD-EPICr-CysC, and 99.9% for LMRevised).
The mean eGFR difference (calculated by: GFR by MDRD Study equation-GFR by other equations) was largest in the CKD-EPICysC equation (-31.4, Fig. 1); it was significantly larger than the differences of other equations compared with the MDRD Study equation (P<0.001). The r coefficients were 0.936 for LMRevised, 0.972 for CKD-EPICr, 0.494 for CKD-EPICysC, and 0.806 for CKD-EPICr-CysC equations (P<0.001).
3. Prevalence of reduced eGFR:
The prevalence of reduced GFR (<60 mL/min/1.73 m2, which corresponds to GFR categories G3a-G5) according to the age group and gender are presented in Table 4. In total, the prevalence of reduced GFR was the highest according to the MDRD Study equation (9.4%) and the lowest (2.4%) according to the CKD-EPICysC equation. The prevalence increased as age increased in all equations. Of note, a very high percentage of low GFR (45.9%) was noted in patients in their 70's according to the LMRevised equation.
DISCUSSION:
The Cr-based CKD-EPI equation is recommended for the initial assessment of GFR, and CysC-based CKD-EPI equations can be used for confirmation of kidney disease according to the KDIGO guidelines [423]. In this study, we compared five eGFR equations, including CysC-based formulas, in the Korean population. The prevalence of reduced GFR by CysC-based CKD-EPI equations has not been reported in Korea yet.
The eGFR classification differed considerably according to the equation used for estimation, especially between CKD-EPICysC or CKD-EPICr-CysC equations compared with the MDRD Study equation. Most of the study population (>80%) were in the G2 category (60-89 mL/min/1.73 m2) according to the MDRD Study equation, but in G1 (≥90 mL/min/1.73 m2) according to the CKD-EPICysC equation. The reason for this discrepancy might be related to the eGFR distribution of the study population. Mean eGFRMDRD was 73.5 mL/min/1.73 m2 with a standard deviation of 12.2 mL/min/1.73 m2; thus, there were many results around the cutoff value of 90 mL/min/1.73 m2. In addition, the CKD-EPICysC and CKD-EPICr-CysC equations yielded systematically higher eGFR results (mean difference 31.4 mL/min/1.73 m2 and 20.6 mL/min/1.73 m2, respectively) in comparison with the MDRD Study equation. This finding was in line with previous studies. In the US National Health and Nutrition Examination Survey (NHANES) 1999-2002 data, the distribution of eGFRCKD-EPI CysC was broader and shifted to the right compared with that of eGFRMDRD [24]. Thus, upward reclassification might be common in CysC-based equations.
All equations, except for the CKD-EPICysC equation, showed good correlation with the MDRD Study equation. CKD-EPICysC showed only a moderate correlation (r=0.49). The three different CKD-EPI equations showed an overall low prevalence of reduced GFR compared with the MDRD Study equation, especially according to the two CysC-containing equations. The LMRevised equation was recently reported to outperform the MDRD Study and CKD-EPI equations in a Swedish population [15]; however, there has been no evaluation of this equation in the Asian population. It yielded similar mean eGFR results compared with the MDRD Study equation; these two equations showed a very high correlation and a similar prevalence of reduced GFR. However, eGFR was underestimated in patients ≥ 60 yr, when using the LMRevised equation. This observation needs to be subjected to further studies because of the increased possibility of co-morbidities in older patients. The LMRevised equation was generated only from the Swedish population; hence, ethnic differences might have influenced GFR estimation as well.
Although the CKD-EPICr equation is recommended by KDIGO for initial GFR assessment, newer GFR estimating equations have been developed and validated, including a Korean version of the CKD-EPICr equation [25], a serum Cr-based full age spectrum equation [26], and a CysC-based equation based on a Caucasian, Asian, pediatric, and adult population (CAPA) [27]. Our study did not aim to compare all recent equations; however, we analyzed the CysC-based CAPA equation briefly. The mean eGFRCAPA (106.3 mL/min/1.73 m2), eGFR difference in comparison with MDRD Study equation (-32.9), and prevalence of eGFR less than 60 mL/min/1.73 m2 (2.7%) were similar when compared with CKD-EPICysC equation.
The prevalence of reduced GFR has been reported differently depending on the study population and the GFR-estimating equation used. In several previous studies, there were clinically significant differences in the prevalence of stage 3 or higher CKD depending on the equation used to estimate GFR. Delanaye et al [20] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRMDRD (13%), intermediate for eGFRCKD-EPI Cr (9.8%), and the lowest for eGFRCKD-EPI Cr-CysC (5%) and eGFRCKD-EPI CysC (4.7%) in 4,189 Belgian patients over 50 yr old. This prevalence trend was similar to ours. One Japanese study showed a 2-fold difference of prevalence between the MDRD Study and CKD-EPICr equations (12.8 vs 6.5%), by studying over 26,000 participants who underwent annual health check-ups [16]. Lujambio et al [28] reported that the prevalence of eGFR less than 60 mL/min/1.73 m2 was the highest for eGFRCKD-EPI CysC (21.8%), intermediate for eGFRCKD-EPI Cr-CysC (11.8%), and the lowest for eGFRMDRD (5.9%) and eGFRCKD-EPI Cr (3.4%) in 119 Uruguayans.
In the US, the prevalence of reduced GFR (eGFR <60 mL/min/1.73 m2) by CKD-EPICr equation was reported to be 4.7% from 1988-1994 and 6.5% from 1999-2002 from NHANES data [29]. In Korea, the prevalence of reduced GFR by the CKD-EPICr equation has been reported as 7.7% in 2007 and 2.6% in 2010 from Korea NHANES data [1718]. In this study, it was 5.1% of the whole study population. This difference could be due to the following reasons. First, the proportion of younger individuals under 40 yr old was lower than that in other studies (23% vs 30-32%). Compared with 2010 Korea NHANES data, the mean eGFR was relatively lower in this study (81.4 mL/min/1.73 m2 vs 95.9-96.8 mL/min/1.73 m2), resulting in higher prevalence of reduced GFR compared with other studies. Second, the study period was different (2014 vs 2007-2010), although the impact of this on the prevalence is still uncertain.
Our retrospective study has several limitations. First, we com-pared all the eGFR equations to the MDRD Study equation, because of the absence of GFR data measured by the gold standard method [18]. Therefore, it was impossible to evaluate the accuracy. The magnitude of bias, calculated by measured GFR-calculated GFR was previously reported to be 2.5-5.8 mL/min/1.73 m2, and this difference could influence the prevalence of CKD stages [1724]. Second, our population might not be representative of the entire population of Korea. Third, we could not analyze albuminuria data or other markers of kidney damage. The classification of CKD was not performed, which is based on both GFR category and albuminuria category. Thus, there could be the CKD patients among the subjects with eGFR ≥60 mL/min/1.73 m2.
In conclusion, this is the first study that compared five eGFR equations in the Korean population. Our data demonstrated remarkable differences in GFR assessment depending on the equation used. The proportion of each GFR category varied considerably, and CysC-containing equations yielded higher eGFRs and showed larger differences compared with the MDRD Study equation. The prevalence of reduced GFR was lowered by the CKD-EPI equations. Further studies using prospective design and in various ethnicities are necessary.
| Background:
Estimated glomerular filtration rate (eGFR) is a widely used index of kidney function. Recently, new formulas such as the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations or the Lund-Malmö equation were introduced for assessing eGFR. We compared them with the Modification of Diet in Renal Disease (MDRD) Study equation in the Korean adult population.
Methods:
The study population comprised 1,482 individuals (median age 51 [42-59] yr, 48.9% males) who received annual physical check-ups during the year 2014. Serum creatinine (Cr) and cystatin C (CysC) were measured. We conducted a retrospective analysis using five GFR estimating equations (MDRD Study, revised Lund-Malmö, and Cr and/or CysC-based CKD-EPI equations). Reduced GFR was defined as eGFR <60 mL/min/1.73 m².
Results:
For the GFR category distribution, large discrepancies were observed depending on the equation used; category G1 (≥90 mL/min/1.73 m²) ranged from 7.4-81.8%. Compared with the MDRD Study equation, the other four equations overestimated GFR, and CysC-based equations showed a greater difference (-31.3 for CKD-EPI(CysC) and -20.5 for CKD-EPI(Cr-CysC)). CysC-based equations decreased the prevalence of reduced GFR by one third (9.4% in the MDRD Study and 2.4% in CKD-EPI(CysC)).
Conclusions:
Our data shows that there are remarkable differences in eGFR assessment in the Korean population depending on the equation used, especially in normal or mildly decreased categories. Further prospective studies are necessary in various clinical settings. | null | null | 7,502 | 316 | [
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] | null | null | [CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY] | [CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY] | [CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY] | null | [CONTENT] Estimated glomerular filtration rate | MDRD Study equation | CKD-EPI equation | Revised Lund-Malmö equation [SUMMARY] | null | [CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY] | [CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY] | [CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY] | null | [CONTENT] Adult | Aged | Algorithms | Creatinine | Cystatin C | Female | Glomerular Filtration Rate | Humans | Male | Middle Aged | Renal Insufficiency, Chronic | Retrospective Studies [SUMMARY] | null | [CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY] | [CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY] | [CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY] | null | [CONTENT] estimating gfr egfr | diet renal | index assessing kidney | gfr estimation ckd | kidney disease epidemiology [SUMMARY] | null | [CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY] | [CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY] | [CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY] | null | [CONTENT] ckd | gfr | equation | equations | min | cysc | 73 | study | min 73 | ml [SUMMARY] | null | [CONTENT] ckd | equation | kidney | equations | gfr | disease | epi | ckd epi | cysc ckd | kidney function [SUMMARY] | [CONTENT] scr | scysc | serum | males | laboratory | max | females | gfr | min | 150 [SUMMARY] | [CONTENT] ckd | epicr | ckd epicr | equations | equation | 73 | mdrd | ml min | min 73 m2 | min 73 [SUMMARY] | null | [CONTENT] ckd | equation | gfr | equations | min | 73 | scr | cysc | 73 m2 | ml [SUMMARY] | null | [CONTENT] ||| the Chronic Kidney Disease Epidemiology Collaboration ||| the Modification of Diet | Korean [SUMMARY] | [CONTENT] 1,482 | age 51 ||| 42 | 48.9% | annual | the year 2014 ||| Cr ||| five | GFR | MDRD Study | Lund-Malmö | Cr and/or CysC | CKD-EPI ||| 60 mL [SUMMARY] | [CONTENT] GFR | G1 | 7.4-81.8% ||| MDRD Study | four | GFR ||| GFR | one third | 9.4% | the MDRD Study | 2.4% | CKD-EPI(CysC [SUMMARY] | null | [CONTENT] ||| the Chronic Kidney Disease Epidemiology Collaboration ||| the Modification of Diet | Korean ||| 1,482 | age 51 ||| 42 | 48.9% | annual | the year 2014 ||| Cr ||| five | GFR | MDRD Study | Lund-Malmö | Cr and/or CysC | CKD-EPI ||| 60 mL ||| GFR | G1 | 7.4-81.8% ||| MDRD Study | four | GFR ||| GFR | one third | 9.4% | the MDRD Study | 2.4% | CKD-EPI(CysC ||| Korean ||| [SUMMARY] | null |
||||
Acceptability of provider-initiated HIV testing as an intervention for prevention of mother to child transmission of HIV and associated factors among pregnant women attending at Public Health Facilities in Assosa town, Northwest Ethiopia. | 26553035 | Despite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors. | BACKGROUND | Institutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20. | METHODS | A total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted. | RESULTS | Acceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests. | CONCLUSION | [
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] | 4638027 | Background | Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].
In year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].
The most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].
To address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].
Early detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].
Thus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].
Even with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia. | null | null | Results | Of the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)
Age
≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)
Residence
Urban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)
Religion
Orthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)
Ethnicity
Berta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa
48 (84.2)9 (15.8)57 (14.7)
Educational status
Not able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)
Occupation
Merchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb
105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)
Marital status
Unmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)
Household expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)
aOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)
bEmployed include government employed and private employed
Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
aOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)
bEmployed include government employed and private employed
Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2). | Conclusion | In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.
Housewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study. | [
"Background",
"Operational definitions",
"Data collection and quality control",
"Ethical consideration",
"Data management and analysis",
"Association between acceptance of PITC and each explanatory variable",
"Socio-demographic factors",
"Association between knowledge, attitudes and partner communication variables with PITC acceptance"
] | [
"Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.",
"Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.",
"The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.",
"Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.",
"All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.",
" Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).",
"There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used",
"Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2)."
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Operational definitions",
"Data collection and quality control",
"Ethical consideration",
"Data management and analysis",
"Results",
"Association between acceptance of PITC and each explanatory variable",
"Socio-demographic factors",
"Association between knowledge, attitudes and partner communication variables with PITC acceptance",
"Discussion",
"Conclusion"
] | [
"Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].\nIn year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].\nThe most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].\nTo address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].\nEarly detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].\nThus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].\nEven with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.",
"Health facility based cross sectional study was conducted from February to March, 2014 in Assosa town. Assosa town is the capital city of Benshangul Gumuz National, Regional State, located Northwest of Ethiopia at 680 km from Addis Ababa. According to the 2007 central statistics agency report of Ethiopia, a total population of the town was 24,214 (12,463 were males and 11,751 were females) [19].\nAdministratively, the town is structured into four urban kebeles. It has one general hospital and one health centre. Both health facilities currently provide ANC, PMTCT and PITC for pregnant mothers. PITC is given under informed consent to all pregnant mothers attending to the facilities based on national testing strategy guideline [13]. Based on the record review prior to data collection, the average flow of pregnant mothers in the hospital and the heal center were 450 and 250 for all ANC visits (ANC visit one up to four) per month, respectively.\nThe source population was all pregnant women attended ANC in the health facilities. The study population was 15–49 years’ old pregnant women who attended ANC services during the study period. The study units were 15–49 years’ old pregnant women who attended ANC services during the study period in Assosa town public health facilities and who selected by sampling procedures. Pregnant women, Women who were critically sick, unable to hear, or had another disability that impaired their ability to communicate were excluded.\nThe sample size was calculated using single population proportion formula considering the following assumptions; 95 % confidence interval with the corresponding value 1.96, P = 56 % (proportion of pregnant women had comprehensive knowledge on HIV/AIDS [15]), the margin of error 0.05 and with the addition of 5 % non-response rate, the total sample size became 398.\nThe sample size was proportionally allocated to the two health facilities based on the flow of ANC clients. After routine ANC visits, participants were approached for consent using a systematic random sampling method. During selection, the first pregnant woman was selected randomly by lottery method and then every other ANC follower was included in the study after obtaining their consent. Finally, data was collected using interviewer administered questionnaires until the required sample size was achieved.\n Operational definitions Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\nAcceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.\n Data collection and quality control The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\nThe questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.\n Ethical consideration Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\nEthical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.\n Data management and analysis All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.\nAll collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.",
"Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.\nKnowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.\nAttitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.\nAttitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.",
"The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.\nTwo diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.",
"Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.",
"All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.",
"Of the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)\nAge\n≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)\nResidence\nUrban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)\nReligion\nOrthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)\nEthnicity\nBerta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa\n48 (84.2)9 (15.8)57 (14.7)\nEducational status\nNot able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)\nOccupation\nMerchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb\n105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)\nMarital status\nUnmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)\nHousehold expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\nbEmployed include government employed and private employed\nSocio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\n\naOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)\n\nbEmployed include government employed and private employed\n Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\n Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).",
" Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\nThere were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used\n Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).\nParticipants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).",
"There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo\nResidence\nRural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00\nEthnicity\nBerta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00\nOccupation\nMerchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00\nAttitude towards PITC\nHigh favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00\nStigmatization\nNo99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00\nPlanned to disclose results to partner after testing\nYes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00\nPerceived the pre- test counselling service\nGood278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00\nPartner reaction to positive result\nPositive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used\nFactors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)\nN.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)\nFor explanatory variables having more than 2 categories, the overall significance of P value used",
"Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).\nFactors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).",
"Provider-initiated HIV testing and counselling process is crucial to achieve high coverage of HIV testing among pregnant women attending ANC for the PMTCT. The overall acceptance of PITC in this study was (80.8 %) which is consistent with the acceptance rates of PITC reported by a study done in Gondar, North West Ethiopia (82.5 %) [15]. This also much higher than the acceptance rates of VCT studies conducted Southwest parts of Ethiopia; in Illubabor (27.0 %) and in Arba-Minch (74.4 %) [16, 17]. The higher acceptance rate could be due to pregnant women consider that PITC as a standard care for the prevention of mother to child HIV transmission or it may be due to the government gives highest emphasis about the availability of comprehensive HIV/AIDS care (availability of rapid testing kits and increased access of ART drugs in ANC clinics).\nHowever, this finding was less than a study done in Nekemte, West part of Ethiopia (87.7 %) [17]. Acceptability of PITC also differs for studies conducted in different parts of Africa. This finding was relatively comparable to a study conducted in two rural districts of Zimbabwe (79 %), but lower than studies in Cameroon (88.3 %), in Botswana (95 %), in rural Ugandan hospital (97 %) and in Urban Zimbabwe 99.9 % [21–25].\nIn the present study rural women acceptability of PITC were more likely than urban residents. In contrary to this a study conducted in Gondar showed that urban residents were more likely to accept PITC than rural residents [15]. The difference in this study could be many reasons. In our context, majority of rural women were farmer, no or less educated and may not get information from different sources so, rural women might not refuse PITC because they expect that their health care provider will react negatively for their next visit, delivery or they fear that they will receive inappropriate care as a result of their refusal.\nThe other possible reason could be rural women may consider refusing of testing may not be the right of pregnant women or they may consider testing is a must because they simply accept what the health provider told them without question. These possible explanations are in line with a study conducted in Kenya where high coverage of HIV testing at ANC may be achieved at the cost of women not understanding that testing is optional and the current set-up makes most women believe that HIV testing is a prerequisite for obtaining other ANC services [26].\nIn this finding; merchants, employed women and students were more likely to accept PITC as compared to housewives. It is in line with a study done in Dre Dawa where employed women were more likely to accept testing than unemployed ones [27]. This finding also indicated that women who had higher expenditure were more likely to accept PITC than women who had lower expenditure. In line with this, a study conducted in Northwest part of Ethiopia indicated that acceptance rate of PITC were higher among those who had higher expenditure [15]. But when expenditure was adjusted with other variables in this study, it did not retain its significance. The possible reasons for association between occupation and acceptance of PITC might be for those women who had been employed, merchants and students had got access to information about PMTCT, PITC and MTCT from their respective occupation places and friends. This information might lead to independently influence their decision for accept PITC.\nIn this study 84 and 65 % pregnant women had sufficient knowledge about HIV/AIDS and PMTCT respectively. These findings are higher than a study conducted in Northwest of Ethiopia where 60 and 59 % had knowledge about HIV/AIDS and PMTCT respectively [14]. Unlike studies conducted other parts of Ethiopia [15, 27], knowledge on HIV/AIDS and PMTCT was not significantly associated for the acceptance of PITC. It indicated that knowledge about HIV/AIDS and PMTCT may not sufficient to guarantee for behavioural change. According to Fisher and colleagues, as cited by Kalich-man and Simbayi, factual based education about HIV transmission is found to be necessary but insufficient in promoting HIV testing [28].\nPositive association was reported between acceptability of PITC during ANC and more favourable attitude towards PITC. This finding was consistent with studies conducted other parts of Ethiopia [15, 27]. This could be due to the reason that pregnant women who understand the importance of testing HIV for the prevention of mother to child transmission of HIV.\nNo risk perception of acquiring HIV was observed among 60 % of the respondents in the present study. This finding was much higher than Dire Dawa (30.3 %) and much lower than Nekemte (87.5 %) findings [18, 27]. Perceived risk of getting HIV was not found to be associated with acceptance of HIV testing. A similar finding was reported from a study conducted in Ethiopia [15]. This could be due to no risk perception observed among the majority of the study participants.\nThe other independently associated factor for acceptance of testing was stigmatizing attitudes towards people having HIV/AIDS. Stigmatization attitude was a negative impact for acceptability of PITC. In line with this, a study in Humera (Ethiopia) showed that people having stigmatized attitude were decline of testing [29]. Other studies showed that AIDS related stigma may be categorized into two types of reactions to people with HIV: “instrumental AIDS stigma” (originating from a desire to protect oneself from this illness) and “symbolic AIDS stigma” (related to moral judgment) [30] and in this study stigma index included both these aspects. Unwillingness to care for relatives with AIDS, unwillingness to buy vegetables from shop sellers with AIDS, unwillingness to eat together and not allowing a teacher with AIDS to continue teaching can be categorized as instrumental AIDS stigma. Keeping HIV/AIDS infection secret can be categorized under symbolic AIDS stigma.\nStigma in this study area could have emerged due to different reasons. In the early period of the HIV epidemic in Ethiopia, many of the educational materials contained pictures of emaciated persons with AIDS. These types of educational messages might have created fears about the disease which resulted in deep rooted stigma towards people living with HIV. But similar study conducted in Gondar (Ethiopia) showed that stigmatized attitude was not significantly associated for the acceptability of PITC [15]. In Gondar study PITC may result in an atmosphere of open discussion about HIV in health care settings. This openness may spread to communities where pregnant women feel comfortable to ask and learn about HIV transmission and prevention.\nAccording to this study women who did not plan to disclose their test results of HIV from their partner were declined testing. Similarly a study conducted in Gambella (Ethiopia) showed that those who did not want to disclose test results to partner were 6.9 times more likely to refused testing [20]. The possible reasons for not disclosing to their partners might be due to fear of divorce, demand for money from her partner, cultural influence or perceived lack of acceptance. These possible reasons were consistent with a study done in Gondar where 78.5 % pregnant women expect negative reaction like insult her, psychological harassment, physical violence, disruption of marriage and stop financial support [31]. Similarly a study in Gambella showed that cultural factors like the role of polygamy marriage in creating rival wives who want to keep their test result as secret from their husband for fear of losing him and the demand for compensation payment from the one assumed to bring the disease may enforce not to disclose test results [20].\nAnother important finding in this study was those who perceived pre-test counselling service as well were more likely to accept testing. In line for this a study done in Kenya showed that good quality HIV pre-test counselling is central for making pregnant women understand and accept the reasons for testing and encourage consent to HIV testing [26]. Another study done in Gambella showed that the pre-test counselling service being fair were 6 times more likely to refuse HIV testing than those who stated their impression on the pre-test counselling service being very good [20]. Now a day’s increased number of pregnant women flow and the strategy of testing that a country follows “testing followed by counselling” may not result full course of counselling service. Due to this reason pregnant women might be loose their confidence to accept testing and they perceive pre-test counselling service quality as poor and may end up with decline of testing.\nThe first limitation was since information in the survey was self reported; some degree of underreporting of socially unacceptable behaviours and attitudes such as stigma was likely. The second limitation may be since interview was made at the end of ANC service, they might be in stressful to wait extra time and they might be respond questions without understanding. As much as possible social desirability biases were reduced by using other than health professionals for data collection and presenting study aims to the respondents clearly. This study assessed many aspects for PITC acceptability of pregnant women so, the study provides useful information that will inform the implementation and strengthening of PMTCT in Benshangul Gumuz Region and areas with similar set ups.",
"In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.\nHousewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study."
] | [
null,
"materials|methods",
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusion"
] | [
"Pregnant women",
"Provider-initiated HIV testing and counselling"
] | Background:
Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].
In year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].
The most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].
To address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].
Early detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].
Thus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].
Even with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.
Methods:
Health facility based cross sectional study was conducted from February to March, 2014 in Assosa town. Assosa town is the capital city of Benshangul Gumuz National, Regional State, located Northwest of Ethiopia at 680 km from Addis Ababa. According to the 2007 central statistics agency report of Ethiopia, a total population of the town was 24,214 (12,463 were males and 11,751 were females) [19].
Administratively, the town is structured into four urban kebeles. It has one general hospital and one health centre. Both health facilities currently provide ANC, PMTCT and PITC for pregnant mothers. PITC is given under informed consent to all pregnant mothers attending to the facilities based on national testing strategy guideline [13]. Based on the record review prior to data collection, the average flow of pregnant mothers in the hospital and the heal center were 450 and 250 for all ANC visits (ANC visit one up to four) per month, respectively.
The source population was all pregnant women attended ANC in the health facilities. The study population was 15–49 years’ old pregnant women who attended ANC services during the study period. The study units were 15–49 years’ old pregnant women who attended ANC services during the study period in Assosa town public health facilities and who selected by sampling procedures. Pregnant women, Women who were critically sick, unable to hear, or had another disability that impaired their ability to communicate were excluded.
The sample size was calculated using single population proportion formula considering the following assumptions; 95 % confidence interval with the corresponding value 1.96, P = 56 % (proportion of pregnant women had comprehensive knowledge on HIV/AIDS [15]), the margin of error 0.05 and with the addition of 5 % non-response rate, the total sample size became 398.
The sample size was proportionally allocated to the two health facilities based on the flow of ANC clients. After routine ANC visits, participants were approached for consent using a systematic random sampling method. During selection, the first pregnant woman was selected randomly by lottery method and then every other ANC follower was included in the study after obtaining their consent. Finally, data was collected using interviewer administered questionnaires until the required sample size was achieved.
Operational definitions Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.
Knowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.
Attitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.
Attitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.
Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.
Knowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.
Attitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.
Attitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.
Data collection and quality control The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.
Two diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.
The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.
Two diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.
Ethical consideration Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.
Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.
Data management and analysis All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.
All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.
Operational definitions:
Acceptors Antenatal care attendants who accepted/tested to provider-initiated HIV counselling as an intervention for PMTCT during their ANC follow up.
Knowledge HIV/AIDS knowledge index was built from the answers to 13 questions: four questions on knowledge of HIV prevention, four questions on knowledge of HIV transmission and five on misconceptions about modes of HIV transmission. It was categorized as insufficient knowledge (score ≤ 6) and sufficient knowledge (score 7–13) questions.
Attitude towards PITC was measured by eleven items and each item has fives scales from strongly disagree to strongly agree. The scale was computed and categorized into low and high favourable attitudes.
Attitude towards PITC was measured by five items questions (having yes or no responses). Pregnant women who had no stigmatized among these questions categorized as “no stigmatized” attitude towards PLWHA, those had one to two stigmatized attitude among the responses were categorized as “marginally stigmatized” attitude and those pregnant women who responded more than two stigmatized responses categorized as “strongly stigmatized” attitude towards PLWHA.
Data collection and quality control:
The questionnaire was adopted from a study conducted in Gondar [15] and some others were developed from by reviewed of literatures [17, 18, 20], prepared in simple words and in coherent way. It was pre-tested in the same setup among women attending at public health facilities who were not included in the study prior to the actual data collection. After pre-testing, all analysis procedures were done, then minor modifications and omissions was performed in some of the ambiguous questions based on the findings of pre-testing.
Two diploma and one BSC health professional who were fluent speakers of Amharic and local language “Rutangna” were recruited, trained and assigned for data collection and supervision respectively. During data collection, completeness of questionnaires was checked by supervisors on a daily basis.
Ethical consideration:
Ethical clearance and approval was obtained from ethical review committee of Bahir Dar University, College of Medicine and Health Sciences. Informed written consents were obtained from Benshangul Gumuz Regional Health Bureau and from the health facilities. Informed consent was also obtained from each participant and confidentiality was assured before conducting the data collection. Participants were given the right to withdraw from the study at any time without any form of preconception.
Data management and analysis:
All collected raw data were entered, edited and cleaned using a computer by Epi Info 3.5.3 version software. Then, data were exported to SPSS version 20 statistical package software for analysis. Descriptive statistics such as proportion and crosstabs were used to describe the study population in relation to relevant variables. Bivariate analysis was done for all explanatory variables in relation to acceptance status of PITC and those variables with P < 0.2 were entered into multivariate logistic regression analysis using backward stepwise method for adjustment of confounders. The model was checked by using Hosmer and Lemeshow test of fitness.
Results:
Of the total 398 pregnant women recruited, 386 participated in the study, yielding a response rate of 97 %. Three hundred twelve (80.8 %) of the participants accepted PITC and the non-acceptors accounted for the remains 74 (19.2 %). Two hundred ninety-eight (74.6 %) of the respondents were urban residents while the rest 88 (25.4 %) were rural inhabitants. More than half of the study participants (57.3 %) were within the age range of 20–29 years with a mean (±SD) age of 24.30 (±4.64) years. Pertaining to the religion category of the study participants, 167 (43.3 %) and 165 (42.7 %) were Muslim and Orthodox Christian followers respectively. Regarding ethnicity, 175 (45.3 %) and 89 (23.1 %) were Amhara and Berta Ethnic groups respectively. With regard to education, the majority of the study participants (77.5 %) had formal education from elementary to higher level while the rest 87 (22.5 %) was not attended formal education. In case of occupation, 145 (37.6 %) were housewives and 123 (31.9 %) government/private employed. About half of pregnant mothers (50.8 %) had monthly income of greater than or equal to 1000 Ethiopian Birr for household expenditure and 94 (24 %) of them had lower than 1000 Ethiopian (Table 1).Table 1Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptance statusYes (%)No (%)Total (%)
Age
≤1985 (81.7)19 (18.3)104 (26.9)20–29181 (81.9)40 (18.1)221 (57.3)30–4546 (75.4)15 (24.6)61 (15.8)
Residence
Urban235 (78.9)63 (21.1)298 (74.6)Rural77 (87.5)11 (12.5)88 (25.4)
Religion
Orthodox christians130 (78.8)35 (21.2)165 (42.7)Muslim137 (82.0)30 (18.0)167 (43.3)Protestant40 (81.6)9 (18.4)49 (12.7)Catholic5 (100)0 (0.0)5 (1.3)
Ethnicity
Berta78 (87.6)11 (12.4)89 (23.1)Amhara133 (76.0)42 (24.0)175 (45.3)Oromo53 (81.5)12 (18.5)65 (16.8)Othersa
48 (84.2)9 (15.8)57 (14.7)
Educational status
Not able to read and write37 (78.7)10 (21.3)47 (12. 2)Able to read and write31 (77.5)9 (22.5)40 (10.4)Grade 1–870 (79.5)18 (20.5)88 (22.8)Grade 9–1272 (76.6)22 (23.4)94 (24.4)Above 12102 (87.2)15 (12.8)117 (30.3)
Occupation
Merchant29 (90.6)3 (9.4)32 (8.3)Farmer49 (84.5)9 (15.5)58 (15.0)Employedb
105 (85.4)18 (14.6)123 (31.9)Student25 (89.3)3 (10.7)28 (7.3)Housewife104 (71.7)41 (28.3)145 (37.6)
Marital status
Unmarried8 (66.7)4 (33.3)12 (3.1)Married/living together303 (83.2)68 (16.8)364 (96.1)Divorced1 (33.3)2 (66.7)3 (0.8)
Household expenditur (Birr/month)No response78 (81.2)18 (18.8)96 (24.9)<100070 (83.7)24 (16.3)94 (24.4)≥1000164 (74.5)32 (24.4)196 (50.8)
aOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)
bEmployed include government employed and private employed
Socio-demographic characteristics of PITC acceptors and non-acceptors among pregnant women attending ANC in governmental health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
aOthers include Shinasha (24), Gurage (13), Tigray (13), Maokomo (2), Keffa (3), Agew (1) and Gumuz (1)
bEmployed include government employed and private employed
Association between acceptance of PITC and each explanatory variable Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Association between acceptance of PITC and each explanatory variable:
Socio-demographic factors There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
Association between knowledge, attitudes and partner communication variables with PITC acceptance Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Socio-demographic factors:
There were differences between residents, ethnic groups and occupations in the acceptance of PITC. After adjusting with other variables, women who lived in the rural areas were four times (AOR 4.04; 95 % CI 1.24–13.11) more likely to accept PITC than urban residents. Respondents belonging to Berta ethnic group were 3.5 times (AOR 3.51; 95 % CI 1.29–9.56) more likely to accept testing than pregnant women of Amhara ethnicity. Regarding to their occupation; merchants, students and employed were more likely to accept PITC compared to housewives (AOR 4.43; 95 % CI 1.18–16.68), (AOR 6.00; 95 % CI 1.45–24.75) and (AOR 2.15; 95 % CI 1.08–4.30) respectively (Table 2).Table 2Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)VariablesPITC acceptanceCOR (95 % CI)AOR (95 % CI)P valueYesNo
Residence
Rural77111.88 (0.94–3.74)4.04 (1.24–13.11)0.020Urban235631.001.00
Ethnicity
Berta78112.24 (1.09–4.60)3.51 (1.29–9.56)0.049Oromo53121.39 (0.68–2.86)2.10 (0.89–4.93)Others4891.68 (0.76–3.72)1.43 (0.59–3.43)Amhara133421.001.00
Occupation
Merchant2933.8 (1.10–13.20)4.43 (1.18-16.68)0.019Farmer4992.15 (0.97–4.76)1.80 (0.51–6.34)Employed105182.30 (1.24–4.26)2.15 (1.08–4.30)Student2533.29 (0.94–11.48)6.00 (1.45–24.75)Housewife104411.001.00
Attitude towards PITC
High favourable256531.81 (1.01–3.24)1.57 (1.08–6.25)0.048Less favourable56211.001.00
Stigmatization
No99252.34 (1.04–5.28)3.54 (1.23–10.16)0.019Low191363.14 (1.45–6.79)4.04 (1.52–10.72)High22131.001.00
Planned to disclose results to partner after testing
Yes297566.36 (3.03–13.37)14.85 (4.60–47.94)<0.001No15181.001.00
Perceived the pre- test counselling service
Good278523.46 (1.88–6.38)4.23 (2.01–8.89)<0.001Poor34221.001.00
Partner reaction to positive result
Positive211372.09 (1.25–3.49)1.7 (0.91–3.36)0.097Negative101371.001.00N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)For explanatory variables having more than 2 categories, the overall significance of P value used
Factors associated with acceptance of provider-initiated HIV testing among pregnant women attending ANC in public health facilities of Assosa town, Northwest Ethiopia, 2014 (N = 386)
N.B. The assumptions for the application of multivariate logistic regression analysis was fulfilled by using Hosmer and Lemeshow test and the model was adequately fitted (P = 0.747)
For explanatory variables having more than 2 categories, the overall significance of P value used
Association between knowledge, attitudes and partner communication variables with PITC acceptance:
Participants who had favourable attitudes towards PITC service were increased their acceptance of testing by 57 % than those had a low attitude towards PITC (AOR 1.57; 95 % CI 1.08–6.25). Compared to those who had a high stigmatized attitude towards PLWHA, respondents who had no stigmatized attitude were 3.5 times PITC (AOR 3.54; 95 % CI 1.23–10.16) more likely to accept PITC and those who had low stigmatized attitude were 4 times PITC (AOR 4.04; 95 % CI 1.50–10.72) more likely to accept PITC. Respondents who planned to disclose their HIV status after testing to their husbands were 14.8 times (AOR 14.85; 95 % CI 4.60–47.94) more likely to accept PITC than those who did not disclose their HIV test results (Table 2).
Factors associated with acceptance of provider-initiated HIV testing Participants who perceived the pre-test counselling service given by health professional as good were 3.5 times (COR 3.46; 95 % CI 1.88–6.38) more likely to accept PITC than those who perceived poor quality of service. While access to health facilities for HIV testing, access to transport services, preferred sex and age of counsellor were not statistically significant in the bivariate analysis (Table 2).
Discussion:
Provider-initiated HIV testing and counselling process is crucial to achieve high coverage of HIV testing among pregnant women attending ANC for the PMTCT. The overall acceptance of PITC in this study was (80.8 %) which is consistent with the acceptance rates of PITC reported by a study done in Gondar, North West Ethiopia (82.5 %) [15]. This also much higher than the acceptance rates of VCT studies conducted Southwest parts of Ethiopia; in Illubabor (27.0 %) and in Arba-Minch (74.4 %) [16, 17]. The higher acceptance rate could be due to pregnant women consider that PITC as a standard care for the prevention of mother to child HIV transmission or it may be due to the government gives highest emphasis about the availability of comprehensive HIV/AIDS care (availability of rapid testing kits and increased access of ART drugs in ANC clinics).
However, this finding was less than a study done in Nekemte, West part of Ethiopia (87.7 %) [17]. Acceptability of PITC also differs for studies conducted in different parts of Africa. This finding was relatively comparable to a study conducted in two rural districts of Zimbabwe (79 %), but lower than studies in Cameroon (88.3 %), in Botswana (95 %), in rural Ugandan hospital (97 %) and in Urban Zimbabwe 99.9 % [21–25].
In the present study rural women acceptability of PITC were more likely than urban residents. In contrary to this a study conducted in Gondar showed that urban residents were more likely to accept PITC than rural residents [15]. The difference in this study could be many reasons. In our context, majority of rural women were farmer, no or less educated and may not get information from different sources so, rural women might not refuse PITC because they expect that their health care provider will react negatively for their next visit, delivery or they fear that they will receive inappropriate care as a result of their refusal.
The other possible reason could be rural women may consider refusing of testing may not be the right of pregnant women or they may consider testing is a must because they simply accept what the health provider told them without question. These possible explanations are in line with a study conducted in Kenya where high coverage of HIV testing at ANC may be achieved at the cost of women not understanding that testing is optional and the current set-up makes most women believe that HIV testing is a prerequisite for obtaining other ANC services [26].
In this finding; merchants, employed women and students were more likely to accept PITC as compared to housewives. It is in line with a study done in Dre Dawa where employed women were more likely to accept testing than unemployed ones [27]. This finding also indicated that women who had higher expenditure were more likely to accept PITC than women who had lower expenditure. In line with this, a study conducted in Northwest part of Ethiopia indicated that acceptance rate of PITC were higher among those who had higher expenditure [15]. But when expenditure was adjusted with other variables in this study, it did not retain its significance. The possible reasons for association between occupation and acceptance of PITC might be for those women who had been employed, merchants and students had got access to information about PMTCT, PITC and MTCT from their respective occupation places and friends. This information might lead to independently influence their decision for accept PITC.
In this study 84 and 65 % pregnant women had sufficient knowledge about HIV/AIDS and PMTCT respectively. These findings are higher than a study conducted in Northwest of Ethiopia where 60 and 59 % had knowledge about HIV/AIDS and PMTCT respectively [14]. Unlike studies conducted other parts of Ethiopia [15, 27], knowledge on HIV/AIDS and PMTCT was not significantly associated for the acceptance of PITC. It indicated that knowledge about HIV/AIDS and PMTCT may not sufficient to guarantee for behavioural change. According to Fisher and colleagues, as cited by Kalich-man and Simbayi, factual based education about HIV transmission is found to be necessary but insufficient in promoting HIV testing [28].
Positive association was reported between acceptability of PITC during ANC and more favourable attitude towards PITC. This finding was consistent with studies conducted other parts of Ethiopia [15, 27]. This could be due to the reason that pregnant women who understand the importance of testing HIV for the prevention of mother to child transmission of HIV.
No risk perception of acquiring HIV was observed among 60 % of the respondents in the present study. This finding was much higher than Dire Dawa (30.3 %) and much lower than Nekemte (87.5 %) findings [18, 27]. Perceived risk of getting HIV was not found to be associated with acceptance of HIV testing. A similar finding was reported from a study conducted in Ethiopia [15]. This could be due to no risk perception observed among the majority of the study participants.
The other independently associated factor for acceptance of testing was stigmatizing attitudes towards people having HIV/AIDS. Stigmatization attitude was a negative impact for acceptability of PITC. In line with this, a study in Humera (Ethiopia) showed that people having stigmatized attitude were decline of testing [29]. Other studies showed that AIDS related stigma may be categorized into two types of reactions to people with HIV: “instrumental AIDS stigma” (originating from a desire to protect oneself from this illness) and “symbolic AIDS stigma” (related to moral judgment) [30] and in this study stigma index included both these aspects. Unwillingness to care for relatives with AIDS, unwillingness to buy vegetables from shop sellers with AIDS, unwillingness to eat together and not allowing a teacher with AIDS to continue teaching can be categorized as instrumental AIDS stigma. Keeping HIV/AIDS infection secret can be categorized under symbolic AIDS stigma.
Stigma in this study area could have emerged due to different reasons. In the early period of the HIV epidemic in Ethiopia, many of the educational materials contained pictures of emaciated persons with AIDS. These types of educational messages might have created fears about the disease which resulted in deep rooted stigma towards people living with HIV. But similar study conducted in Gondar (Ethiopia) showed that stigmatized attitude was not significantly associated for the acceptability of PITC [15]. In Gondar study PITC may result in an atmosphere of open discussion about HIV in health care settings. This openness may spread to communities where pregnant women feel comfortable to ask and learn about HIV transmission and prevention.
According to this study women who did not plan to disclose their test results of HIV from their partner were declined testing. Similarly a study conducted in Gambella (Ethiopia) showed that those who did not want to disclose test results to partner were 6.9 times more likely to refused testing [20]. The possible reasons for not disclosing to their partners might be due to fear of divorce, demand for money from her partner, cultural influence or perceived lack of acceptance. These possible reasons were consistent with a study done in Gondar where 78.5 % pregnant women expect negative reaction like insult her, psychological harassment, physical violence, disruption of marriage and stop financial support [31]. Similarly a study in Gambella showed that cultural factors like the role of polygamy marriage in creating rival wives who want to keep their test result as secret from their husband for fear of losing him and the demand for compensation payment from the one assumed to bring the disease may enforce not to disclose test results [20].
Another important finding in this study was those who perceived pre-test counselling service as well were more likely to accept testing. In line for this a study done in Kenya showed that good quality HIV pre-test counselling is central for making pregnant women understand and accept the reasons for testing and encourage consent to HIV testing [26]. Another study done in Gambella showed that the pre-test counselling service being fair were 6 times more likely to refuse HIV testing than those who stated their impression on the pre-test counselling service being very good [20]. Now a day’s increased number of pregnant women flow and the strategy of testing that a country follows “testing followed by counselling” may not result full course of counselling service. Due to this reason pregnant women might be loose their confidence to accept testing and they perceive pre-test counselling service quality as poor and may end up with decline of testing.
The first limitation was since information in the survey was self reported; some degree of underreporting of socially unacceptable behaviours and attitudes such as stigma was likely. The second limitation may be since interview was made at the end of ANC service, they might be in stressful to wait extra time and they might be respond questions without understanding. As much as possible social desirability biases were reduced by using other than health professionals for data collection and presenting study aims to the respondents clearly. This study assessed many aspects for PITC acceptability of pregnant women so, the study provides useful information that will inform the implementation and strengthening of PMTCT in Benshangul Gumuz Region and areas with similar set ups.
Conclusion:
In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.
Housewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study.
| Background:
Despite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors.
Methods:
Institutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20.
Results:
A total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted.
Conclusions:
Acceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests. | Background:
Worldwide rapid expansion of HIV/AIDS has a profound impact on the health sector and the socioeconomic development in general. Globally a total of 34 million people were living with HIV and of these about 69 % were in Sub Saharan Africa. In 2011, around 330,000 children acquired HIV infection mainly from mother to child transmission and of these more than 90 % were in sub-Saharan Africa [1].
In year 2011, the prevalence of HIV in Ethiopia was 1.5 % [2]. In year 2012 only, an estimated 37, 605 children in ager group of 0–4 years were HIV positive, more than 759, 268 people were living with HIV, around 20,158 newly infected in year 2012 and a total of 41,444 AIDS cases died. Of the total people living with HIV/AIDS (PLWHA), more than 22,057 were pregnant women [3]. The prevalence of HIV in Benshangul Gumuz Regional state, Ethiopia was 1.7 % [2] and ANC-based HIV prevalence among pregnant women and Assosa hospital was 17.8 and 7.6 % respectively [4].
The most significant source of HIV infection in children and infants are transmission of HIV from mother to child. Without interventions, the risk of transmission varies and ranging from 5–10 % during pregnancy, 10–20 % during labour/delivery and 10–20 % through mixed infant feeding [5, 6].
To address HIV/AIDS epidemics, prevention of mother to child transmission (PMTCT) of HIV becomes a priority for many developing country governments and agencies. PMTCT is a commonly used intervention designed to reduce the risk of mother to child transmission of HIV (MTCT). Among these, HIV testing and counselling (HTC) is a critical component and gate way for all pregnant mothers to learn whether they are infected, helped to understand the implications of their HIV status and make informed choices for the future to reduce morbidity, mortality and HIV transmission [7, 8].
Early detection of maternal HIV infection in pregnancy focused on voluntary counselling and testing (VCT) as the primary means of providing testing and encouraging people to become aware of their HIV status [9]. However Population surveys conducted in 2007–2009 in low- and middle-income countries showed that the median percentage of people living with HIV who know their status was estimated below 40 % [10]. Evidence showed that VCT coverage for target populations was low in Ethiopia [11].
Thus, World Health Organization (WHO) introduced provider-initiated HIV testing and counselling (PITC) approach and subsequently, the revised version of the Ethiopian PMTCT guideline adopted in 2007 recommends that provider-initiated HIV testing and counselling (PITC) as a routine care for pregnant women in antenatal care (ANC) clinics to decrease MTCT of HIV/AIDS [12, 13].
Even with great efforts have been done in Ethiopia after the introduction to PITC, only 11 % pregnant women tested for HIV in 2011 during their ANC visits [14]. Meanwhile limited studies conducted in other regions of Ethiopia, the acceptance rate and associated factors of PITC among pregnant women vary from one context to another [15–18] as well as some of the associated factors were contradicted. In addition the study area categorized as among the developing regions of Ethiopia and there was no study conducted about acceptance rate of PITC as an intervention of PMTCT among ANC attendants. Therefore, this study was done to assess acceptability of PITC as an intervention for prevention of mother to child transmission of HIV and to identify the associated factors that will help health planners and mangers for fair decision making process in resource limited setting in Ethiopia.
Conclusion:
In conclusion acceptability of PITC among pregnant women was relatively high. Ethnicity, residence, occupation, attitude towards PITC, stigmatized attitude towards PLWHA, perceived pre-test counselling and planned to disclosure of test results from their partner were independent factors for the acceptability of PITC among pregnant women in this study.
Housewife/farmer women engagement especially through education and skills to create their own work should be done by their family, community and the government so as to improve women’s access to economic resources to enable them to increase their own decision on accessing test of HIV. Provision of information and education during pre-test counselling service should be given for all pregnant women to increase their attitude towards PITC during their ANC visits. Couple counselling during ANC services should be taken as a strategy to minimize the difficulty that pregnant women face to disclose their HIV test result to their husband and encourage them to disclose their results to their partner. Behavioural approach to increase awareness of pregnant women to developed positive attitude towards PLWHA should be done by ANC providers, community health workers, Health offices, regional HIV/AIDS secretariat office and the community itself and Further studies including qualitative approach especially to assess ethnicity, residence and quality of pre-test counselling variables in relation to acceptance of PITC were recommended by this study. | Background:
Despite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors.
Methods:
Institutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20.
Results:
A total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted.
Conclusions:
Acceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests. | 10,248 | 391 | [
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Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Cross-Sectional Studies | Educational Status | Ethiopia | Female | HIV Infections | Health Knowledge, Attitudes, Practice | Humans | Infectious Disease Transmission, Vertical | Mass Screening | Middle Aged | Multivariate Analysis | Patient Acceptance of Health Care | Pregnancy | Pregnancy Complications, Infectious | Prenatal Care | Public Facilities | Regression Analysis | Social Class | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY] | null | [CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY] | [CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY] | [CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY] | [CONTENT] 2011 prevalence hiv | mother child hiv | hiv prevalence pregnant | hiv ethiopia year | hiv epidemic ethiopia [SUMMARY] | [CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY] | null | [CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY] | [CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY] | [CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY] | [CONTENT] pitc | hiv | testing | 95 | ci | 95 ci | women | aor | 00 | pregnant [SUMMARY] | [CONTENT] hiv | transmission | child | people | mother child | mother | ethiopia | living hiv | mother child transmission | people living hiv [SUMMARY] | null | [CONTENT] ci | 95 ci | 00 | aor | 95 | 24 | 001 | pitc | 001 00 | likely [SUMMARY] | [CONTENT] women | community | increase | test | pregnant women | pregnant | pitc | especially | acceptability pitc pregnant | acceptability pitc pregnant women [SUMMARY] | [CONTENT] hiv | pitc | ci | 95 ci | testing | women | 95 | aor | attitude | study [SUMMARY] | [CONTENT] hiv | pitc | ci | 95 ci | testing | women | 95 | aor | attitude | study [SUMMARY] | [CONTENT] ||| [SUMMARY] | null | [CONTENT] 386 | 97 % | 312 | 80.8 % ||| AOR 4.04 | 95 % | CI | 1.24-13.11 ||| AOR | 6.00 | 95 % | CI | 1.45-24.75 | AOR 4.43 | 95 % | CI | 1.18-16.68 | 2.15 | 95 % | CI | 1.08-4.30 ||| AOR | 95 % | CI | 1.23-10.16 ||| AOR | 14.85 | 95 % | 4.60-47.94 [SUMMARY] | [CONTENT] ||| ||| [SUMMARY] | [CONTENT] ||| ||| 398 | February to March 2014 | two | Assosa ||| ||| Bivariate | SPSS | 20 ||| 386 | 97 % | 312 | 80.8 % ||| AOR 4.04 | 95 % | CI | 1.24-13.11 ||| AOR | 6.00 | 95 % | CI | 1.45-24.75 | AOR 4.43 | 95 % | CI | 1.18-16.68 | 2.15 | 95 % | CI | 1.08-4.30 ||| AOR | 95 % | CI | 1.23-10.16 ||| AOR | 14.85 | 95 % | 4.60-47.94 ||| ||| ||| [SUMMARY] | [CONTENT] ||| ||| 398 | February to March 2014 | two | Assosa ||| ||| Bivariate | SPSS | 20 ||| 386 | 97 % | 312 | 80.8 % ||| AOR 4.04 | 95 % | CI | 1.24-13.11 ||| AOR | 6.00 | 95 % | CI | 1.45-24.75 | AOR 4.43 | 95 % | CI | 1.18-16.68 | 2.15 | 95 % | CI | 1.08-4.30 ||| AOR | 95 % | CI | 1.23-10.16 ||| AOR | 14.85 | 95 % | 4.60-47.94 ||| ||| ||| [SUMMARY] |
|||||
Molecular characterization of Blastocystis subtypes isolated in the city of Uberaba, Minas Gerais State, Brazil. | 34431950 | Blastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates. | INTRODUCTION | Blastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification. | METHODS | Polymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific. | RESULTS | The data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible. | CONCLUSIONS | [
"Animals",
"Blastocystis",
"Blastocystis Infections",
"Brazil",
"Cattle",
"Feces",
"Phylogeny",
"Swine"
] | 8405216 | INTRODUCTION | Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1
-
4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4.
Although underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1
,
5
,
6.
The molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7
-
11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124
,
10
,
12. Other subtypes have been found in pets, livestock, and zoo animals12.
Several studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13
,
14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94
,
12
,
15
-
19
,
20.
In distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21
-
25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6
,
26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.
This study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region. | METHODS |
Blastocystis samples
In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.
In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.
DNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7
,
29.
Bovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.
TABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA
Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7
,
29.
Bovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.
TABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA
DNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6.
SSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4.
The similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).
All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6.
SSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4.
The similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).
Ethical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804.
The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804. | RESULTS | PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates.
PCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.
TABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.
Blastocystis isolates
PCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†
Identity (%)PCR-STSSSU-rDNA amplificationSpeI digestion
H09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡
KX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.
*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).
†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.
‡Sample classified as ST1 in the study of Moura et al. (2018).
ND: Not determined.
Seven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined.
Amplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.
In this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.
Sequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1).
FIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.
In summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.
Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1). | null | null | [
"\nBlastocystis samples\n",
"DNA extraction and PCR",
"DNA sequencing and analysis",
"Ethical approval"
] | [
"In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.",
"Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n",
"All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n",
"The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804. "
] | [
null,
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"\nBlastocystis samples\n",
"DNA extraction and PCR",
"DNA sequencing and analysis",
"Ethical approval",
"RESULTS",
"DISCUSSION"
] | [
"Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1\n-\n4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4. \nAlthough underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1\n,\n5\n,\n6. \nThe molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7\n-\n11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124\n,\n10\n,\n12. Other subtypes have been found in pets, livestock, and zoo animals12. \nSeveral studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13\n,\n14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94\n,\n12\n,\n15\n-\n19\n,\n20.\nIn distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21\n-\n25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6\n,\n26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.\nThis study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region.",
"\nBlastocystis samples\n In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nIn this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.\nDNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nTotal DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n\nDNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nAll amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n\nEthical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804. \nThe project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804. ",
"In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.",
"Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7\n,\n29.\nBovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.\n\nTABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA\n\n\n",
"All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6. \nSSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4. \nThe similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).\n",
"The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804. ",
"PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates. \nPCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.\n\nTABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.\nBlastocystis isolates\nPCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†\nIdentity (%)PCR-STSSSU-rDNA amplificationSpeI digestion\n\n\n\nH09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡\nKX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.\n\n*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).\n†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.\n‡Sample classified as ST1 in the study of Moura et al. (2018). \nND: Not determined.\nSeven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined. \nAmplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.\nIn this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.\nSequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1). \n\nFIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.\n\nIn summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.\nPhylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1). ",
"In the present study, we confirmed that the SSU rDNA gene is polymorphic, as DNA products of different sizes from the described 1.1 kb fragment29 were amplified in human feces, indicating a similarity of approximately 99% with the Blastocystis sequences deposited in GenBank. Several indel events were observed in the generated sequences. \nThese results showed ST1-ST3, ST5, and ST10 in the city of Uberaba, with ST5 and ST10 present only in pigs and cattle, respectively. ST1 and ST3 were isolated from both humans and pigs, demonstrating the zoonotic potential of transmission of Blastocystis species, as reported by other authors31\n-\n34.\nFew studies have been carried out in Brazil regarding the geographic distribution of Blastocystis subtypes and their hosts35. ST1-ST4 have been reported to date, with a predominance of ST1 and ST321\n,\n26\n,\n35\n-\n36. In the world panorama, the most prevalent subtypes of Blastocystis spp. are ST1-ST4, but ST5-ST9 and ST12 have also been described and have different regional prevalence37\n-\n39. Mixed infections have also been reported11, showing that complex genotypes may occur in various regions of the world.\nPhylogenetic analysis showed that sequences originating from humans formed two distinct groups, one of which contained the sequences of the isolates characterized as ST1 and the other with the isolates characterized as ST3. In recent studies22\n,\n35, the grouping of subtypes into separate clades was shown by phylogenetic analysis of the different Blastocystis subtypes observed in Brazil in the South, Southeast, and Midwest regions. Pig isolates, although more similar to each other than human isolates, were divided into clades of the dendrogram, either isolated or belonging to one of the groups of human isolates. These data reinforce the potential for zoonotic transmission22\n,\n35\n,\n40. \nIn our study, 4 of the 18 water samples amplified with Blastocystis-specific primers followed by sequencing did not correspond to sequences of this parasite. Other studies in Brazil using PCR with primers other than those we used showed that amplified DNA fragments of water samples from the Tietê River, State of São Paulo, also did not correspond to the Blastocystis sequences deposited in GenBank35. These results show that the primers directed to rDNA targets, which are highly conserved regions among eukaryotes, are inadequate for investigating Blastocystis in environmental samples. Consequently, the use of primers directed to these regions is a limitation for investigating Blastocystis in environmental samples; their use in PCR tests may contribute to false-positive results if other techniques, such as DNA sequencing, are not employed. These observations point to the need for developing new specific primers and/or new techniques to investigate the presence of Blastocystis in environmental samples. \nBased on our study, we concluded that the SSU rDNA gene is polymorphic and may define intraspecific variations leading to the grouping of the isolates according to their genetic characteristics. In addition, it was verified that in the studied region, the subtypes of Blastocystis were ST1, ST3, and ST2, and zoonotic transmission is possible, as ST1 was found in both humans and pigs in the region. Finally, it was observed that the PCR of the SSU rDNA gene was not useful for the detection of Blastocystis in environmental samples, as it amplified the non-specific DNA of algae."
] | [
"intro",
"methods",
null,
null,
null,
null,
"results",
"discussion"
] | [
"Blastocystis species",
"Subtype",
"Genetic characterization",
"Brazil"
] | INTRODUCTION:
Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far1
-
4. Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known4.
Although underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition1
,
5
,
6.
The molecular characterization of Blastocystis isolates has been carried out using different techniques; however, the analysis of the small ribosomal subunit gene (SSU-rDNA) is the most commonly used method7
-
11. These studies showed that Blastocystis has broad genetic diversity and is classified into 17 subtypes (ST1-ST1712). Ten Blastocystis subtypes have been described in humans: ST1-ST9 and ST124
,
10
,
12. Other subtypes have been found in pets, livestock, and zoo animals12.
Several studies have attempted to establish a relationship between Blastocystis subtype and clinical symptoms in patients; however, this relationship remains inconclusive13
,
14. Humans are mainly infected by ST1-ST4 and rarely by ST5-ST94
,
12
,
15
-
19
,
20.
In distinct regions of Brazil, different authors have demonstrated the presence of ST1-ST4 and ST6-ST8 subtypes. The ST1-ST3 and the ST2 subtype are the most prevalent21
-
25. In the city of Uberaba, Minas Gerais (MG), an area of the Brazilian savannah, the parasite has been observed in humans (~17%), pigs (72.2%), sheep (33.3%), cattle (21.4%), and dogs (2,3%)6
,
26. However, the molecular epidemiological profile of Blastocystis isolated in this region remains unknown.
This study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region.
METHODS:
Blastocystis samples
In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.
In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.
DNA extraction and PCR Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7
,
29.
Bovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.
TABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA
Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7
,
29.
Bovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.
TABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA
DNA sequencing and analysis All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6.
SSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4.
The similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).
All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6.
SSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4.
The similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).
Ethical approval The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804.
The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804.
Blastocystis samples
:
In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin6, four pig feces, and one bovine26. In addition, the presence of Blastocystis DNA was tested in 18 water supply samples27 in the same area.
DNA extraction and PCR:
Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer’s instructions and protocol described by Moreira et al.27. Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described28. Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB (Table 1) was SpeI-digested to infer parasite classification28. PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined7
,
29.
Bovine and pig samples were obtained from another study by our team26. The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.
TABLE 1:Primers used to amplify the Small Subunit rDNA gene of Blastocystis isolates of humans, animals, and the water supply from the state of MG Brazil.Primer Sequence (5′→3′)Size (bp)Reference1FBGGAGGTAGTGACAATAAATC1100Yoshikawa et al. (2000)1RBACTAGGAATTCCTCGTTCATGSSU907 F-BHTGAAACTGCGAATGGCTCA907This studySSU907 R-BHCAAGAACGAAAGCTAGGGGASSU850 F-BHGCGAAAGCATTTACCAAGGA850This studySSU850 R-BHCCTACGGAAACCTTGTTACGA
DNA sequencing and analysis:
All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers (Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6.
SSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1)26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement4.
The similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).
Ethical approval:
The project was approved by the Universidade Federal do Triângulo Mineiro’s Research Ethics Committee, Avenida Getúlio Guaritá, 159, Casa da Comissões, Bairro Abadia, Uberaba/MG, CEP: 38.025-440, phone +55 (34) 3700-6803, e-mail: [email protected], under protocol number 1804.
RESULTS:
PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates.
PCR-RFLP showed the restriction profiles of 14 human isolates (Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 (Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.
TABLE 2:Blastocystis subtypes determined by molecular methods from human feces samples in the city of Uberaba, MG State, Brazil.
Blastocystis isolates
PCR-RFLP of SSU-rDNA* SSU-rDNA GenBank accession number Subtype inferred by sequence comparison†
Identity (%)PCR-STSSSU-rDNA amplificationSpeI digestion
H09YesNDKX257271ST199.1-99.9-H31YesST1, ST2 KX257272ST199.1-99.9ST1 H38YesST1, ST2KX257273ST199.1-100.0ST1 H40YesNDKX257274ST397.0-98.9-H42No----ST3 H46YesST1, ST2KX257275ST199.1-100.0ST1 H177YesNDKX257283ST397.0-99.2-H212YesNDKX257276ST397.0-99.2-H216YesNDKX257277ST397.0-99.6-H366YesNDKX257278ST199.6-99.8-H495YesST3, ST4, ST8/ST5, ST7KX257279ST397.1-99.9ST3 H496YesST1, ST2KX257280ST198.0-100.0ST1 H543YesST1, ST2KX257281ST199.1-100.0ST1 H595YesST1, ST2---ST2 H621YesNDKX257282ST199.6-99.8-B01YesNDKX257266ST1097.0-98.5-S01YesNDKX257267ST598.1-98.5-S02YesNDKX257268ST598.5-98.7-S03YesST1‡
KX257269 ST198.4-100.0ST1S06YesNDKX257270ST397.6-98.2-Ref.Bsp_ST1--MK801358ST198.4-100.0-Ref.Bsp_ST3--MK801403ST397.4-99.4-Ref.Bsp_ST5--MK801414ST599.1-99.9-Ref.Bsp_ST7--AF408427ST797.7-99.4-Ref.Bsp_ST10--MH507326ST1095.8-99.7-*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.‡Sample classified as ST1 in the study of Moura et al. (2018). ND: Not determined.
*PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length Polymorphism. SSU rDNA: Small Ribosomal Subunit; SpeI Restriction endonuclease (Yoshikawa et al., 2011).
†According to Stensvold and Clark (2016), Blastocystis subtypes can be inferred when the comparison of their sequence with the GenBank sequences has an alignment > 95% and error < 5%.
‡Sample classified as ST1 in the study of Moura et al. (2018).
ND: Not determined.
Seven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined.
Amplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.
In this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples (Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.
Sequences obtained from human fecal samples (KX257271 to KX257283) and animals (KX257266-KX257270) were deposited in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( >Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1).
FIGURE 1:Dendrogram obtained by multiple alignments of DNA sequences generated from Blastocystis species from humans and animals in the Uberaba city, MG, Brazil. A segment of the 18S rDNA gene of Thecamonas trahens (ATCC 50062, accession number XR_001290156) was used as an outgroup. Phylogenetic analysis was realized by Mega® version 6 software30, based on the SSU rRNA dataset of Blastocystis by the 95 ML method with 1000 replicates bootstrap values. Ref: reference strain; Bsp: Blastocystis spp.
In summary, in this study, the subtypes defined for human isolates were ST1 (8/15, 53.3%), ST3 (6/15, 40.0%), and ST2 (1/15, 6.7%); ST10 for the bovine isolate; ST5 (2/4, 50.0%), ST1 (1/4, 25%), and ST3 (1/4, 25%) for the pig isolates.
Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1).
DISCUSSION:
In the present study, we confirmed that the SSU rDNA gene is polymorphic, as DNA products of different sizes from the described 1.1 kb fragment29 were amplified in human feces, indicating a similarity of approximately 99% with the Blastocystis sequences deposited in GenBank. Several indel events were observed in the generated sequences.
These results showed ST1-ST3, ST5, and ST10 in the city of Uberaba, with ST5 and ST10 present only in pigs and cattle, respectively. ST1 and ST3 were isolated from both humans and pigs, demonstrating the zoonotic potential of transmission of Blastocystis species, as reported by other authors31
-
34.
Few studies have been carried out in Brazil regarding the geographic distribution of Blastocystis subtypes and their hosts35. ST1-ST4 have been reported to date, with a predominance of ST1 and ST321
,
26
,
35
-
36. In the world panorama, the most prevalent subtypes of Blastocystis spp. are ST1-ST4, but ST5-ST9 and ST12 have also been described and have different regional prevalence37
-
39. Mixed infections have also been reported11, showing that complex genotypes may occur in various regions of the world.
Phylogenetic analysis showed that sequences originating from humans formed two distinct groups, one of which contained the sequences of the isolates characterized as ST1 and the other with the isolates characterized as ST3. In recent studies22
,
35, the grouping of subtypes into separate clades was shown by phylogenetic analysis of the different Blastocystis subtypes observed in Brazil in the South, Southeast, and Midwest regions. Pig isolates, although more similar to each other than human isolates, were divided into clades of the dendrogram, either isolated or belonging to one of the groups of human isolates. These data reinforce the potential for zoonotic transmission22
,
35
,
40.
In our study, 4 of the 18 water samples amplified with Blastocystis-specific primers followed by sequencing did not correspond to sequences of this parasite. Other studies in Brazil using PCR with primers other than those we used showed that amplified DNA fragments of water samples from the Tietê River, State of São Paulo, also did not correspond to the Blastocystis sequences deposited in GenBank35. These results show that the primers directed to rDNA targets, which are highly conserved regions among eukaryotes, are inadequate for investigating Blastocystis in environmental samples. Consequently, the use of primers directed to these regions is a limitation for investigating Blastocystis in environmental samples; their use in PCR tests may contribute to false-positive results if other techniques, such as DNA sequencing, are not employed. These observations point to the need for developing new specific primers and/or new techniques to investigate the presence of Blastocystis in environmental samples.
Based on our study, we concluded that the SSU rDNA gene is polymorphic and may define intraspecific variations leading to the grouping of the isolates according to their genetic characteristics. In addition, it was verified that in the studied region, the subtypes of Blastocystis were ST1, ST3, and ST2, and zoonotic transmission is possible, as ST1 was found in both humans and pigs in the region. Finally, it was observed that the PCR of the SSU rDNA gene was not useful for the detection of Blastocystis in environmental samples, as it amplified the non-specific DNA of algae.
| Background:
Blastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates.
Methods:
Blastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification.
Results:
Polymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific.
Conclusions:
The data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible. | null | null | 4,248 | 329 | [
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] | null | null | [CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY] | [CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY] | [CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY] | null | [CONTENT] Blastocystis species | Subtype | Genetic characterization | Brazil [SUMMARY] | null | [CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY] | [CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY] | [CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY] | null | [CONTENT] Animals | Blastocystis | Blastocystis Infections | Brazil | Cattle | Feces | Phylogeny | Swine [SUMMARY] | null | [CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY] | [CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY] | [CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY] | null | [CONTENT] gene blastocystis isolates | seven blastocystis isolates | characterize isolates blastocystis | prevalence blastocystis spp | brazil blastocystis isolates [SUMMARY] | null | [CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY] | [CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY] | [CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY] | null | [CONTENT] blastocystis | pcr | samples | isolates | st1 | rdna | ssu | subtypes | sequences | dna [SUMMARY] | null | [CONTENT] blastocystis | relationship | 17 | st1 | epidemiological profile | epidemiological | relationship blastocystis | subtypes st1 | 12 | remains [SUMMARY] | [CONTENT] pcr | sequence | blastocystis | samples | sequenced | performed | rdna | ssu | analysis | dna [SUMMARY] | [CONTENT] 99 | pcr | isolates | blastocystis | ref | 100 | sequences | restriction | 98 | samples [SUMMARY] | null | [CONTENT] blastocystis | pcr | samples | isolates | st1 | sequences | rdna | sequence | subtypes | dna [SUMMARY] | null | [CONTENT] Blastocystis ||| Blastocystis | Uberaba | Minas Gerais | Brazil [SUMMARY] | [CONTENT] 26 | Uberaba ||| GenBank [SUMMARY] | [CONTENT] 21 | 18 ||| ST1 | 53.3% | 40.0% | 6.7% | 100% | 50.0% | 25% | 25% ||| 98%-99% | Blastocystis | GenBank ||| Blastocystis | two | one | ST1 ||| Blastocystis [SUMMARY] | null | [CONTENT] ||| Blastocystis | Uberaba | Minas Gerais | Brazil ||| 26 | Uberaba ||| GenBank ||| ||| 21 | 18 ||| ST1 | 53.3% | 40.0% | 6.7% | 100% | 50.0% | 25% | 25% ||| 98%-99% | Blastocystis | GenBank ||| Blastocystis | two | one | ST1 ||| Blastocystis ||| Blastocystis | Uberaba | Blastocystis [SUMMARY] | null |
||||
The relationship between cam morphology and hip and groin symptoms and signs in young male football players. | 32201993 | Conflicting and limited high-quality prospective data are available on the associations between cam morphology and hip and groin symptoms and range of motion (ROM). | BACKGROUND | Academy male football players (n = 49, 17-24 years) were included. Standardized antero-posterior pelvic and frog-leg lateral radiographs were obtained at baseline, 2.5- and 5-year follow-up. The femoral head-neck junction was quantified by: Visual score. Cam morphology (flattening or prominence), large cam (prominence). Alpha angle. Cam morphology (≥60°), large cam (≥78°). Cam morphology duration was defined as long (first present at baseline) or short (only from 2.5- to 5-year follow-up). Current symptoms at 5-year follow-up were assessed using a hip and groin pain question and by the "Hip and Groin Outcome Score" (HAGOS). HAGOS scores were categorized into: most symptoms (≥2 domains in lowest interquartile range [IQR]), least symptoms (≥2 domains in highest IQR). Hip ROM was measured by goniometry at 5-year follow-up. | METHODS | Large cam morphology based on visual score was associated with hip and groin pain (23.8% vs. 7.1%, OR: 3.17, CI: [1.15-8.70], P = .026), but not with HAGOS scores. Cam morphology presence, size, and duration were associated with limited flexion of around 6° and/or 3° to 6° for internal rotation. | RESULTS | Cam morphology presence, size, and duration were associated with limited hip flexion and/or internal rotation, but differences might not exceed the minimal clinical important difference. Whether cam morphology results in symptoms is uncertain. | CONCLUSION | [
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] | 7317829 | INTRODUCTION | Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,
1
while the incidence varies between 4% and 19%.
2
,
3
One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.
4
FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.
4
Imaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.
5
,
6
,
7
,
8
,
9
It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.
10
,
11
Cam morphology prevalence in football players is high.
5
,
6
,
7
,
9
,
12
Although there is an association between cam morphology and hip osteoarthritis (OA),
12
,
13
,
14
,
15
,
16
,
17
the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.
18
,
19
,
20
,
21
,
22
One available longitudinal case‐control study
23
showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study
24
found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA
13
and cartilage damage.
25
,
26
,
27
It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.
Therefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up. | METHODS | Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.
5
,
6
The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).
Participant characteristics at 5‐year follow‐up
Left
Right
Pain
Symptoms
Activities of daily living
Sports and recreational activity
Physical activity
Quality of life
Abbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.
At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.
5
,
6
The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).
Participant characteristics at 5‐year follow‐up
Left
Right
Pain
Symptoms
Activities of daily living
Sports and recreational activity
Physical activity
Quality of life
Abbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.
Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously
5
,
6
; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.
Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously
5
,
6
; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.
Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.
5
,
6
This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.
5
,
7
An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.
5
The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.
5
,
6
This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.
5
,
7
An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.
5
Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al
28
and was used previously.
5
,
6
In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).
13
The standard error of measurement (SEM) was 3.45.
The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al
28
and was used previously.
5
,
6
In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).
13
The standard error of measurement (SEM) was 3.45.
Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.
29
The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.
29
Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.
The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.
Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.
5
,
6
In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.
The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.
5
,
6
In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.
Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.
The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used. | RESULTS | Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.
9
Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.
Demographic baseline data of 5‐year follow‐up participants compared to dropouts
a
Baseline (n = 49)
5‐year follow‐up participants
Baseline (n = 40)
5‐year follow‐up dropouts
Visual score
Alpha angle
Flexion
Left
Right
Abduction
Left
Right
Adduction
Left
Right
Internal rotation
Left
Right
External rotation
Left
Right
Extension
Left
Right
Abbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.
Values are expressed as mean ± standard deviation.
Data of n = 38 are presented, due to missing data.
Bolded P‐values indicate a statistically significant difference.
Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.
9
Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.
Demographic baseline data of 5‐year follow‐up participants compared to dropouts
a
Baseline (n = 49)
5‐year follow‐up participants
Baseline (n = 40)
5‐year follow‐up dropouts
Visual score
Alpha angle
Flexion
Left
Right
Abduction
Left
Right
Adduction
Left
Right
Internal rotation
Left
Right
External rotation
Left
Right
Extension
Left
Right
Abbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.
Values are expressed as mean ± standard deviation.
Data of n = 38 are presented, due to missing data.
Bolded P‐values indicate a statistically significant difference.
Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.
Of 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).
Association between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up
Yes
Cam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.
Large cam morphology vs having no large cam morphology.
Bolded P‐values indicate a statistically significant difference.
Of 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).
Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.
Of 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).
Association between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up
Yes
Cam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.
Large cam morphology vs having no large cam morphology.
Bolded P‐values indicate a statistically significant difference.
Of 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).
Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1
. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).
Spreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up
a
Abbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.
Values are expressed as median (IQR, 25th‐75th centile).
Most symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.
In the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).
Long cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).
Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1
. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).
Spreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up
a
Abbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.
Values are expressed as median (IQR, 25th‐75th centile).
Most symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.
In the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).
Long cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).
Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).
Association between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
Association between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).
Association between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
Association between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference. | CONCLUSION | Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings. | [
"INTRODUCTION",
"Study participants",
"Radiographs",
"Visual scores",
"Alpha angle",
"Definition of cam morphology and large cam morphology",
"Cam morphology duration",
"Hip and groin pain/symptoms",
"Questionnaire on hip pain and participant characteristics",
"Hip and groin outcome score",
"Hip range of motion",
"Statistical analysis",
"Participant characteristics",
"Cam morphology and hip and groin pain",
"Cam morphology and hip and groin symptoms",
"Cam morphology and range of motion",
"Cam morphology and hip and groin pain/symptoms",
"Cam morphology and range of motion",
"Limitations",
"Range of motion",
"PERSPECTIVES",
""
] | [
"Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.",
"At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.",
"Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.",
"The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n",
"The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.",
"The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n",
"The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.",
" Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.",
"Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.",
"The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.",
"The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.",
"The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.",
"Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.",
"Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).",
"Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n",
"The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.",
"Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n",
"Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.",
"Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.",
"The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n",
"Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.",
"Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points"
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"INTRODUCTION",
"METHODS",
"Study participants",
"Radiographs",
"Visual scores",
"Alpha angle",
"Definition of cam morphology and large cam morphology",
"Cam morphology duration",
"Hip and groin pain/symptoms",
"Questionnaire on hip pain and participant characteristics",
"Hip and groin outcome score",
"Hip range of motion",
"Statistical analysis",
"RESULTS",
"Participant characteristics",
"Cam morphology and hip and groin pain",
"Cam morphology and hip and groin symptoms",
"Cam morphology and range of motion",
"DISCUSSION",
"Cam morphology and hip and groin pain/symptoms",
"Cam morphology and range of motion",
"Limitations",
"Range of motion",
"CONCLUSION",
"PERSPECTIVES",
"",
"Supporting information"
] | [
"Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,\n1\n while the incidence varies between 4% and 19%.\n2\n, \n3\n One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.\n4\n FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.\n4\n\n\nImaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.\n5\n, \n6\n, \n7\n, \n8\n, \n9\n It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.\n10\n, \n11\n\n\nCam morphology prevalence in football players is high.\n5\n, \n6\n, \n7\n, \n9\n, \n12\n Although there is an association between cam morphology and hip osteoarthritis (OA),\n12\n, \n13\n, \n14\n, \n15\n, \n16\n, \n17\n the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.\n18\n, \n19\n, \n20\n, \n21\n, \n22\n One available longitudinal case‐control study\n23\n showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study\n24\n found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA\n13\n and cartilage damage.\n25\n, \n26\n, \n27\n It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.\nTherefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.",
" Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\nAt baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.\n Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\nThree radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.\n Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\nThe femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n\n Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\nThe alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.\n Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\nThe independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n\n Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\nThe third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.\n Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\n Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\nThe researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.\n Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.\nThe association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.",
"At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.\n5\n, \n6\n The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).\nParticipant characteristics at 5‐year follow‐up\nLeft\nRight\nPain\nSymptoms\nActivities of daily living\nSports and recreational activity\nPhysical activity\nQuality of life\nAbbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.",
"Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously\n5\n, \n6\n; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.",
"The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.\n5\n, \n6\n This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.\n5\n, \n7\n An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.\n5\n\n",
"The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al\n28\n and was used previously.\n5\n, \n6\n In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).\n13\n The standard error of measurement (SEM) was 3.45.",
"The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.\n29\n\n",
"The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.",
" Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\nEvery participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.\n Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.\nThe “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.",
"Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.",
"The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.\n30\n The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).\n30\n, \n31\n, \n32\n This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.\n32\n The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.\n33\n The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.",
"The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.\n5\n, \n6\n In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.",
"The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.",
" Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\nDemographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.\n Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\nNine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).\n Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\nHip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n\n Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nThe average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.",
"Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.\n9\n Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.\nDemographic baseline data of 5‐year follow‐up participants compared to dropouts\na\n\n\nBaseline (n = 49)\n5‐year follow‐up participants\nBaseline (n = 40)\n5‐year follow‐up dropouts\nVisual score\nAlpha angle\nFlexion\nLeft\nRight\nAbduction\nLeft\nRight\nAdduction\nLeft\nRight\nInternal rotation\nLeft\nRight\nExternal rotation\nLeft\nRight\nExtension\nLeft\nRight\nAbbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.\nValues are expressed as mean ± standard deviation.\nData of n = 38 are presented, due to missing data.\nBolded P‐values indicate a statistically significant difference.",
"Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.\nOf 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).\nAssociation between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up\nYes\nCam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.\nLarge cam morphology vs having no large cam morphology.\nBolded P‐values indicate a statistically significant difference.\nOf 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).",
"Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1\n. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).\nSpreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up\na\n\n\nAbbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.\nValues are expressed as median (IQR, 25th‐75th centile).\nMost symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.\nIn the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).\nLong cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).\n",
"The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).\nAssociation between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.\nAssociation between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)\nValues are expressed as mean ± standard deviation.\nCam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.\nLarge cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.\nBolded P‐values indicate a statistically significant difference.",
"The relationship between cam morphology and hip and groin symptoms is inconsistent. A large cam morphology based on the visual score in young male academy football players showed an association with hip and groin pain, but not with more hip and groin symptoms as defined by the HAGOS score. A longer cam morphology duration was not significantly associated with more hip and groin symptoms. Cam morphology presence and size were associated with limited flexion and internal rotation, whereas a longer cam morphology duration was only associated with limited flexion.\n Cam morphology and hip and groin pain/symptoms Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\nLarge cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n\n Cam morphology and range of motion Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\nSignificant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.\n Limitations Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\nSome limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.\n Range of motion The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n\nThe ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n",
"Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al\n22\n did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,\n19\n who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al\n20\n who studied 3202 participants from the general population. A longitudinal study by Mosler et al\n24\n could not identify an association between cam morphology and groin injuries in professional athletes.\nHowever, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al\n23\n focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al\n21\n studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al\n18\n demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.\n34\n\n\nAn explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.\n12\n, \n29\n No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old\n5\n, \n6\n, \n7\n, \n8\n, \n9\n, \n35\n and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.\n11\n\n",
"Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al\n36\n observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al\n37\n found a significant association between alpha angle and limited internal rotation. Mosler et al\n38\n screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al\n39\n did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.\n33\n In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.\nNot all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.\n9\n This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.",
"Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence\n12\n, \n35\n and might have influenced symptoms.\n40\n A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.\nBy using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,\n4\n the risk of false‐negative measurements was minimalized.",
"The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.\n41\n Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.\n42\n\n",
"Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings.",
"Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.",
"Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points",
"Table S1\nClick here for additional data file."
] | [
null,
"methods",
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
null,
null,
null,
null,
"conclusions",
null,
null,
"supplementary-material"
] | [
"FAI syndrome",
"femoroacetabular impingement",
"pain",
"range of motion"
] | INTRODUCTION:
Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,
1
while the incidence varies between 4% and 19%.
2
,
3
One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.
4
FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.
4
Imaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.
5
,
6
,
7
,
8
,
9
It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.
10
,
11
Cam morphology prevalence in football players is high.
5
,
6
,
7
,
9
,
12
Although there is an association between cam morphology and hip osteoarthritis (OA),
12
,
13
,
14
,
15
,
16
,
17
the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.
18
,
19
,
20
,
21
,
22
One available longitudinal case‐control study
23
showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study
24
found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA
13
and cartilage damage.
25
,
26
,
27
It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.
Therefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.
METHODS:
Study participants At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.
5
,
6
The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).
Participant characteristics at 5‐year follow‐up
Left
Right
Pain
Symptoms
Activities of daily living
Sports and recreational activity
Physical activity
Quality of life
Abbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.
At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.
5
,
6
The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).
Participant characteristics at 5‐year follow‐up
Left
Right
Pain
Symptoms
Activities of daily living
Sports and recreational activity
Physical activity
Quality of life
Abbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.
Radiographs Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously
5
,
6
; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.
Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously
5
,
6
; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.
Visual scores The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.
5
,
6
This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.
5
,
7
An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.
5
The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.
5
,
6
This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.
5
,
7
An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.
5
Alpha angle The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al
28
and was used previously.
5
,
6
In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).
13
The standard error of measurement (SEM) was 3.45.
The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al
28
and was used previously.
5
,
6
In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).
13
The standard error of measurement (SEM) was 3.45.
Definition of cam morphology and large cam morphology The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.
29
The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.
29
Cam morphology duration The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.
The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.
Hip and groin pain/symptoms Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
Hip range of motion The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.
5
,
6
In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.
The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.
5
,
6
In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.
Statistical analysis The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.
The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.
Study participants:
At baseline, all academy male football players of the Feyenoord Academy aged between 12 and 19 years (n = 141) received an invitational letter of whom 89 finally participated. All 89 baseline participants were invited again to participate at 2.5‐year follow‐up (n = 63 participants) and the 5‐year follow‐up (n = 49 participants) (Figure 1). Inclusion and exclusion criteria were described previously.
5
,
6
The inclusion for the 5‐year follow‐up took place between June 2015 and October 2015. Ethical approval was obtained from the Medical Ethical Committee of the Erasmus Medical Center (Rotterdam, the Netherlands). Each participant gave written consent. For participants under 18 years, written consent was gathered from at least one parent. Participant characteristics, such as age, weight, height (and BMI), football experience, training intensity, and self‐reported hip and/or groin symptoms, were collected (Table 1).
Participant characteristics at 5‐year follow‐up
Left
Right
Pain
Symptoms
Activities of daily living
Sports and recreational activity
Physical activity
Quality of life
Abbreviations: HAGOS, Hip and Groin Outcome Score; IQR, interquartile range; SD, standard deviation.
Radiographs:
Three radiographs were obtained during this study by the same standardized radiographic protocol as described previously
5
,
6
; one supine antero‐posterior pelvic radiograph and a frog‐leg lateral radiograph of each hip.
Visual scores:
The femoral head‐neck junction of all hips was scored qualitatively as normal, flattening, or prominence.
5
,
6
This additional method was used because of alpha angle limitations, especially in hips with an open growth plate.
5
,
7
An experienced musculo‐skeletal radiologist and orthopedic surgeon determined all visual scores simultaneously, and any discrepancies were directly resolved based on consensus. Visual scores were obtained by scoring each hip of all three time points in one session. Visual scores showed a kappa of 0.68 for intra‐observer reliability in the baseline study.
5
Alpha angle:
The alpha angle was automatically calculated on all radiographs, as described by Nötzli et al
28
and was used previously.
5
,
6
In short, the shape of the proximal femur was outlined by a manually positioned anatomical set of points by one observer, by using Statistical Shape Modelling (ASM tool kit, Manchester University, Manchester, UK). The alpha angle was automatically calculated from this point set by using MATLAB v7.1.0 (MathWorks Inc). Intraclass correlation coefficient (ICC) for interobserver reliability was 0.73 (95% confidence interval [CI] 0.56‐0.86). Intra‐observer reliability ICC scores ranged from 0.85 (95% CI 0.49‐0.96) to 0.99 (95% CI 0.93‐1.00).
13
The standard error of measurement (SEM) was 3.45.
Definition of cam morphology and large cam morphology:
The independent variables cam morphology presence and size were analyzed on both the AP view and frog‐leg lateral view at 5‐year follow‐up. The highest score of one of both views was used for analysis. Cam morphology was defined twice, based on the visual score and alpha angle. Cam morphology based on the visual score was defined when either a flattening or prominence was present. Cam morphology based on the alpha angle was defined as alpha angle ≥60°. Large cam morphology based on the visual score was defined as having a prominence. Large cam morphology based on the alpha angle was defined as alpha angle ≥78°.
29
Cam morphology duration:
The third independent variable cam morphology duration was scored dichotomously as “long” or “short” for all radiographs from baseline, 2.5‐year follow‐up, and at 5‐year follow‐up. Long duration was defined as the first presence of cam morphology at baseline and short duration as having cam morphology for the first time at 2.5‐ and/or 5‐year follow‐up.
Hip and groin pain/symptoms:
Questionnaire on hip pain and participant characteristics Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Hip and groin outcome score The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
Questionnaire on hip pain and participant characteristics:
Every participant filled out a questionnaire on several participant characteristics at 5‐year follow‐up (Figure 1). This questionnaire contained a question about hip pain: “Do you sometimes have pain in your hips?” A dichotomous answer was possible, “yes” or “no”. When answered positive, the painful side was specified (left, right, and bilateral). They also filled in, if pain occurred during or after sporting activities, or in rest. As this question might include groin pain, we choose “hip and groin pain” as the overall term to define this outcome measure.
Hip and groin outcome score:
The “Copenhagen Hip and Groin Outcome Score” (HAGOS) is a valid patient‐reported outcome measure to quantify hip and groin symptomatology.
30
The validated Dutch HAGOS translation was filled out by all participants only at the 5‐year follow‐up (Figure 1).
30
,
31
,
32
This questionnaire obtained information from six domains, specified per person. Each domain is scaled between 0 and 100, with 100 as indicator for no problems, and a lower score for hip and groin symptoms.
32
The football players completed the questionnaires before or on the day at which the radiographs were obtained. All participants were divided into 3 groups based on the level of symptomatology, as described before by Tak et al.
33
The first group is the most symptomatic group in this cohort, defined by at least 2 domains in the lowest interquartile range (IQR) of the HAGOS scores. The group with the least symptomatic participants was defined as having at least 2 domains in the highest IQR of the HAGOS scores. The middle group was the remaining group.
Hip range of motion:
The researcher performing the physical examination was blinded to the outcome of the HAGOS scores and for the radiographs. The same physical examination protocol was used at all time points.
5
,
6
In short, while maintained in neutral rotation, the first resistance/end feel during passive flexion, abduction, adduction, internal rotation and external rotation were measured in supine position and extension in prone position on a flat examination table with a goniometer. Internal and external rotation were measured with 90° of flexion in the hip joint. Stabilization was provided by the free hand of the examiner to the adjacent joints and regions.
Statistical analysis:
The association between cam morphology presence, size and duration at 5‐year follow‐up and hip and groin pain (per hip) and most vs least hip and groin symptoms (based on HAGOS per person) were calculated by means of logistic regression and adjusted for age and body mass index (BMI). The association between cam morphology presence, size and duration at 5‐year follow‐up and ROM was calculated by a linear regression model, adjusted for age and BMI. All per hip regression analyses were performed in a Generalised Estimated Equations (GEE) model. These were all cross‐sectional associations at 5‐year follow‐up. The only longitudinal outcome of this study was the duration of cam morphology which was measured at baseline, 2.5‐year, and 5‐year follow‐up. Absolute rounded ROM averages are presented in Table 5 and 6, with differences observed in the statistical tests presented as estimated mean differences. Differences in baseline characteristics between included participants and dropouts were tested using an independent samples t test. A sensitivity analysis was performed to see if analyzing the HAGOS outcome defined as most symptoms vs middle and least symptoms, gave different results than defining the HAGOS outcome as most vs least symptoms (Table S1). SPSS25.0 (Windows) was used.
RESULTS:
Participant characteristics Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.
9
Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.
Demographic baseline data of 5‐year follow‐up participants compared to dropouts
a
Baseline (n = 49)
5‐year follow‐up participants
Baseline (n = 40)
5‐year follow‐up dropouts
Visual score
Alpha angle
Flexion
Left
Right
Abduction
Left
Right
Adduction
Left
Right
Internal rotation
Left
Right
External rotation
Left
Right
Extension
Left
Right
Abbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.
Values are expressed as mean ± standard deviation.
Data of n = 38 are presented, due to missing data.
Bolded P‐values indicate a statistically significant difference.
Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.
9
Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.
Demographic baseline data of 5‐year follow‐up participants compared to dropouts
a
Baseline (n = 49)
5‐year follow‐up participants
Baseline (n = 40)
5‐year follow‐up dropouts
Visual score
Alpha angle
Flexion
Left
Right
Abduction
Left
Right
Adduction
Left
Right
Internal rotation
Left
Right
External rotation
Left
Right
Extension
Left
Right
Abbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.
Values are expressed as mean ± standard deviation.
Data of n = 38 are presented, due to missing data.
Bolded P‐values indicate a statistically significant difference.
Cam morphology and hip and groin pain Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.
Of 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).
Association between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up
Yes
Cam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.
Large cam morphology vs having no large cam morphology.
Bolded P‐values indicate a statistically significant difference.
Of 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).
Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.
Of 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).
Association between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up
Yes
Cam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.
Large cam morphology vs having no large cam morphology.
Bolded P‐values indicate a statistically significant difference.
Of 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).
Cam morphology and hip and groin symptoms Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1
. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).
Spreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up
a
Abbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.
Values are expressed as median (IQR, 25th‐75th centile).
Most symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.
In the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).
Long cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).
Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1
. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).
Spreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up
a
Abbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.
Values are expressed as median (IQR, 25th‐75th centile).
Most symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.
In the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).
Long cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).
Cam morphology and range of motion The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).
Association between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
Association between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).
Association between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
Association between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
Participant characteristics:
Demographic data of the participants are summarized in Table 1. The mean follow‐up time was 5.3 ± 0.1 years (range 5.0‐5.6 years). Of the 89 baseline participants (12‐19 years old), 49 (55%) participated at 5‐year follow‐up. No differences in baseline demographic data between these 49 participants and 40 dropouts were observed, Table 2.
9
Participants dropped out for various reasons: 24 rejected the invitation, 11 were unreachable, 4 lived abroad, and 1 person accepted the invitation but did not appear during the allocated time‐slot. All 49 included participants still played football at the time of the 5‐year follow‐up study. Of those, 28 (57%) were still active in a first or second team of a professional football club. All other 21 football players (43%) played football at an amateur level. Cam morphology prevalence was 82% (80 of 98 hips) based on visual score and 80% (78 of 98 hips) based on the alpha angle.
Demographic baseline data of 5‐year follow‐up participants compared to dropouts
a
Baseline (n = 49)
5‐year follow‐up participants
Baseline (n = 40)
5‐year follow‐up dropouts
Visual score
Alpha angle
Flexion
Left
Right
Abduction
Left
Right
Adduction
Left
Right
Internal rotation
Left
Right
External rotation
Left
Right
Extension
Left
Right
Abbreviations: AA, alpha angle; ROM, range of motion; VS, visual score.
Values are expressed as mean ± standard deviation.
Data of n = 38 are presented, due to missing data.
Bolded P‐values indicate a statistically significant difference.
Cam morphology and hip and groin pain:
Nine players (18.4%) reported hip and groin pain (5 bilateral and 4 unilateral). Of these 14 hips, 10 hips were painful at one occasion and 4 at two occasions; 1 hip both during sports and at rest and 3 hips directly after sports and at rest. In total, 4 hips were painful during sports, 6 hips directly after sports, and 8 hips at rest.
Of 80 hips with cam morphology based on visual score, 11 hips (13.8%) had hip and groin pain compared to 3 of 18 hips (16.7%) without cam (OR: 0.51, CI: [0.15‐1.69]). Of 78 hips with cam morphology based on alpha angle, 9 hips (11.5%) had hip and groin pain, compared to 5 of 20 hips (25.0%) without cam (OR: 0.42, CI: [0.13‐1.32]). Of the 42 hips with large cam morphology based on visual score, 10 hips (23.8%) had hip and groin pain compared to 4 of 56 hips (7.1%) without large cam (OR: 3.17, CI: [1.15‐8.70], P = .026). Of 25 hips with large cam morphology based on alpha angle, 4 hips (16.0%) had hip and groin pain compared to 10 of 73 hips (13.7%) without large cam (OR: 1.21, CI: [0.60‐2.43]) (Table 3).
Association between cam morphology based on both visual score (VS) and alpha angle (AA) and symptoms at 5‐year follow‐up
Yes
Cam morphology vs having no cam morphology. For associations between cam morphology presence and size and HAGOS, “most vs least symptoms” is used.
Large cam morphology vs having no large cam morphology.
Bolded P‐values indicate a statistically significant difference.
Of 47 hips with long cam morphology duration based on visual score, 7 hips (14.9%) had hip and groin pain compared to 4 of the 33 hips (12.1%) with short cam duration (OR: 1.99, CI: [0.19‐21.19]). Long cam morphology duration defined by the alpha angle, resulted in 6 of 47 hips (12.8%) with hip and groin pain, compared to 3 of 31 hips (9.7%) with short cam duration (OR: 1.63, CI: [0.24‐10.93]).
Cam morphology and hip and groin symptoms:
Hip and Groin Outcome scores were not normally distributed. The median and IQRs of all 6 HAGOS domains of this cohort are presented in Table 1
. An overview of the distribution of the HAGOS domains per HAGOS group (most, middle, and least symptoms) in this cohort is presented in Table 4. The group with most symptoms consisted of 12 of 49 football players (25%), the group with the least symptoms consisted of 33 football players (67%) and the middle group consisted of 4 football players (8%).
Spreading of all 6 HAGOS domain medians for the 3 different HAGOS groups at 5‐year follow‐up
a
Abbreviations: DL, daily living; PA, physical activities; QoL, quality of life; S&R, sports & recreation.
Values are expressed as median (IQR, 25th‐75th centile).
Most symptoms are defined by at least 2 domains in the lowest interquartile range (IQR), least symptoms by at least 2 domains in the highest IQR and the middle group is the remaining group.
In the group with cam morphology based on visual score, 10 of 41 persons (24.4%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 2 of 4 persons (50.0%) (OR: 0.24, CI: [0.03‐2.20]). In the group with cam morphology based on alpha angle, 9 of 40 persons (22.5%) were classified into the group with most symptoms. In the group without cam, most symptoms were observed in 3 of 5 persons (60.0%) (OR: 0.22, CI: [0.03‐1.67]). Large cam morphology based on visual score was observed in 25 persons (51.0%), and 7 of them (28.0%) were classified in the group with most symptoms. In the group without large cam, most symptoms were observed in 5 of 20 persons (25.0%) (OR: 1.12, CI: [0.28‐4.46]). Large cam morphology based on alpha angle was observed in 18 persons (36.7%), and 4 of them (22.2%) were classified into the group with most symptoms. In the group without large cam, most symptoms were observed in 8 of 27 persons (29.6%) (OR: 0.95, CI: [0.21‐4.30]) (Table 3).
Long cam morphology duration defined by the visual score, resulted in 9 of 27 persons (33.3%) in the group with most symptoms. Short cam duration was observed in 1 of 14 persons (7.1%) in the group with most symptoms (OR: 12.92, CI: [0.88‐188.93], P = .062). Long cam morphology duration defined by the alpha angle resulted in 8 of 29 persons (27.6%) in the group with most symptoms. Short cam duration was observed in 1 of 11 persons (9.1%) in the group with most symptoms (OR: 4.27, CI: [0.40‐45.52]).
Cam morphology and range of motion:
The average flexion was lower in hips with cam morphology than in hips without cam based on visual score (116° ± 6° vs 121° ± 8°, P = .001) and alpha angle (116° ± 6° vs 122° ± 9°, P = .032) (Tables 5 and 6). Lower average internal rotation was observed in hips with cam morphology based on alpha angle, compared to hips without cam (24° ± 7° vs 30° ± 9°, P = .005) (Table 6). The average internal rotation in hips with large cam morphology based on visual score was lower than in hips without large cam (24 ± 8° vs 27° ± 7°, P = .033) (Table 5). Limited flexion was observed in hips with large cam morphology based on alpha angle, compared to hips without large cam (113° ± 7° vs 118° ± 7°, P = .049) (Table 6). Lower flexion was observed in hips with cam morphology based on alpha angle for at least 5 years (long duration), than hips with cam for 2.5 years or less (short duration) (115° ± 6° vs 116° ± 7°, P = .016).
Association between cam morphology based on visual score and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presented in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
Association between cam morphology based on alpha angle (AA) and range of motion at 5‐year follow‐up (n = hips)
Values are expressed as mean ± standard deviation.
Cam morphology vs having no cam morphology. Difference between groups is also presentsed in degrees range of motion.
Large cam morphology vs having no large cam morphology. Difference between groups is also presented as the estimated mean difference in degrees range of motion.
Bolded P‐values indicate a statistically significant difference.
DISCUSSION:
The relationship between cam morphology and hip and groin symptoms is inconsistent. A large cam morphology based on the visual score in young male academy football players showed an association with hip and groin pain, but not with more hip and groin symptoms as defined by the HAGOS score. A longer cam morphology duration was not significantly associated with more hip and groin symptoms. Cam morphology presence and size were associated with limited flexion and internal rotation, whereas a longer cam morphology duration was only associated with limited flexion.
Cam morphology and hip and groin pain/symptoms Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al
22
did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,
19
who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al
20
who studied 3202 participants from the general population. A longitudinal study by Mosler et al
24
could not identify an association between cam morphology and groin injuries in professional athletes.
However, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al
23
focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al
21
studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al
18
demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.
34
An explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.
12
,
29
No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old
5
,
6
,
7
,
8
,
9
,
35
and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.
11
Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al
22
did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,
19
who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al
20
who studied 3202 participants from the general population. A longitudinal study by Mosler et al
24
could not identify an association between cam morphology and groin injuries in professional athletes.
However, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al
23
focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al
21
studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al
18
demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.
34
An explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.
12
,
29
No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old
5
,
6
,
7
,
8
,
9
,
35
and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.
11
Cam morphology and range of motion Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al
36
observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al
37
found a significant association between alpha angle and limited internal rotation. Mosler et al
38
screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al
39
did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.
33
In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.
Not all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.
9
This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.
Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al
36
observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al
37
found a significant association between alpha angle and limited internal rotation. Mosler et al
38
screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al
39
did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.
33
In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.
Not all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.
9
This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.
Limitations Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence
12
,
35
and might have influenced symptoms.
40
A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.
By using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,
4
the risk of false‐negative measurements was minimalized.
Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence
12
,
35
and might have influenced symptoms.
40
A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.
By using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,
4
the risk of false‐negative measurements was minimalized.
Range of motion The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.
41
Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.
42
The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.
41
Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.
42
Cam morphology and hip and groin pain/symptoms:
Large cam morphology was significantly associated with hip and groin pain, but not with the HAGOS scores. Other cross‐sectional studies on this association showed conflicting results. Mayes et al
22
did not find an association between cam morphology and with HAGOS scores < 100 in ballet dancers. Anderson et al,
19
who investigated 547 individuals (1081 hips, mean age 67 years), did not find a significant association between cam morphology and the “modified Harris Hip Scores” or “Hip Outcome Scores.” Also, no association between cam morphology and self‐reported hip pain was found by Gosvig et al
20
who studied 3202 participants from the general population. A longitudinal study by Mosler et al
24
could not identify an association between cam morphology and groin injuries in professional athletes.
However, other studies did show an association between cam morphology and hip and groin symptoms. A longitudinal study by Khanna et al
23
focused on the development of hip pain at 4.4‐year follow‐up in 170 asymptomatic volunteers (mean age 29.5 years) at baseline. Seven of 14 (50.0%) painful hips had cam morphology compared with 37 of 318 (11.6%) painless hips at follow‐up (RR: 4.3, P = .0002). Other cross‐sectional studies also found an association. Larson et al
21
studied 125 National Football League prospects and observed a significantly higher cam morphology prevalence in the symptomatic group (P = .009). Allen et al
18
demonstrated a significant association between higher alpha angles in painful hips (mean 69.9°) than in asymptomatic hips (mean 63.1°). In a retrospective study of 334 patients, a significant association between hip symptoms and increased alpha angles (P < .001) was observed as well.
34
An explanation for the absence of association between cam morphology (flattening or prominence and/or alpha angle ≥60°) and symptoms within 5‐year follow‐up could be that only larger cam morphology can cause rapid intra‐articular damage. This is in line with the higher risk of developing hip OA when cam morphology is bigger.
12
,
29
No association between cam morphology and HAGOS scores was observed in this study. HAGOS scores in the group classified as most symptoms in this cohort were ranging between 66.07 and 90.00, indicating that symptoms were mild. Also, the HAGOS score is a score per person rather than per hip, which might dilute the association in the presence of unilateral cam morphology. Also, the HAGOS questionnaire captures hip and groin symptoms and not only hip‐related pain, as other entities of groin pain might be more prevalent in football players than pain arising from the hip joint. Another explanation could be that participants were young. Cam morphology arises from 12 to 14 years old
5
,
6
,
7
,
8
,
9
,
35
and continues to grow thereafter until growth plate closure. During 5‐year follow‐up, participants were aged 20.5 years (17‐24 years) on average. Therefore, the cam morphology duration might have been too short to create hip damage and symptoms. This is supported by our findings that 33.3% of the group with long cam morphology duration based on visual score was classified in the most symptoms group, compared to 7.1% in the short duration group. Although not statistically significant (P = .06), future studies on the relationship between cam morphology and symptoms should also take into account the duration of cam morphology. It could also be that football players are not keen to report about their (hip and groin) complaints as they may be afraid of losing their place on the pitch. Finally, the pathway from having cam morphology into developing the clinical entity of FAI syndrome and thus pain is complex and also involves the amount of femoral and acetabular version, soft‐tissue structures, activities which a person undertakes and many other person‐specific factors. Obviously, it can also be that the presence of cam morphology itself is not associated with symptoms or reduced range of motion, as cam morphology is also highly prevalent in asymptomatic populations.
11
Cam morphology and range of motion:
Significant associations between cam morphology presence and limited flexion and internal rotation were observed and influenced by cam morphology size. A longer cam morphology duration only negatively influenced the amount of flexion. Our findings partly correspond with current available literature. Audenaert et al
36
observed a significantly lower range of internal rotation in the cam morphology group (based on CT) vs a group without cam morphology. In collegiate football players, Kapron et al
37
found a significant association between alpha angle and limited internal rotation. Mosler et al
38
screened 426 male professional football players in Qatar for 2 consecutive seasons and observed that asymptomatic hips with cam morphology and large cam morphology were associated with lower internal rotation. Interestingly, a systematic review of Freke et al
39
did only find limited and conflicting evidence on the association of cam morphology and limited ROM in symptomatic patients. However, ROM in symptomatic hips might also be influenced by pain rather than cam morphology only.
33
In the current study, the average differences between hips with and without cam morphology were around 6° for flexion and 3°‐6° for internal rotation. This raises questions on whether these differences are clinically relevant.
Not all growth plates were closed (93.9%) at 5‐year follow‐up. This means that hips with open growth plates might still have the potential to develop cam morphology or increase to a large cam morphology.
9
This can possibly cause more severe impingement, and therefore result in more symptoms and limited ROM in the future.
Limitations:
Some limitations in this study have to be acknowledged. During 5‐year follow‐up, 40 participants (44.9%) were lost to follow‐up. Although there were no differences in baseline characteristics between participants and dropouts, it has bias, as it resulted in a relatively small sample size. Due to the small sample size and low proportion of hips without cam morphology, the resulting findings have wide confidence intervals. Of the included 49 participants, 42.9% played football at an amateur level, with lower intensity and training hours per week. This could have resulted in lower cam morphology prevalence
12
,
35
and might have influenced symptoms.
40
A possible limitation of the patient characteristics questionnaire is that it cannot be excluded that the question “Do you sometimes have pain in your hips?” also included patients with groin symptoms and made no distinction between long standing and acute hip and groin symptoms. However, large cam morphology based on the alpha angle was associated with hip and groin pain based on this question, which indicates that type II errors are not likely. As the HAGOS‐domain scores were not normally distributed and as the median scores in 3 out of 6 domains are having the maximum score of 100, a ceiling effect cannot be ruled out.
By using radiographs, the prevalence of cam morphology might have been underestimated as compared to cross‐sectional imaging such as magnetic resonance imaging (MRI) or computed tomography (CT). However, by using two radiographic views (AP and frog‐leg lateral), as recommended by the Warwick Agreement,
4
the risk of false‐negative measurements was minimalized.
Range of motion:
The ROM was obtained before or after training, which could have resulted in different outcomes. Range of motion measurement by goniometry could result in measurement errors and can give an overestimation of the ROM.
41
Beside this limitation, range of motion is an acceptable and reliable measurement method for longitudinal studies in FAI syndrome patients. The reliability of range of motion testing of the hip is described in literature as good to excellent by Prather et al.
42
CONCLUSION:
Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings.
PERSPECTIVES:
Our study showed that large cam morphology is only associated with hip and groin symptoms but not with HAGOS scores. The presence, size, and duration of cam morphology are associated with limited flexion and/or internal rotation, although the clinical relevance of these differences is questionable. This suggests that a bony cam morphology has a direct but small effect on the range of motion and symptoms which might develop in some players several years after cam morphology has developed. More factors are involved in the complex pathway between cam morphology and developing the clinical entity of FAI syndrome with symptoms and limited function, such as femoral and acetabular orientation, soft‐tissue condition (eg, labrum, cartilage, ligamentum teres), activity level, and many other person‐specific factors. This needs further investigation in a larger cohort.
:
Flowchart of all analysed HAGOS questionnaires, hip pain questionnaires and ROM at all time‐points
Supporting information:
Table S1
Click here for additional data file.
| Background:
Conflicting and limited high-quality prospective data are available on the associations between cam morphology and hip and groin symptoms and range of motion (ROM).
Methods:
Academy male football players (n = 49, 17-24 years) were included. Standardized antero-posterior pelvic and frog-leg lateral radiographs were obtained at baseline, 2.5- and 5-year follow-up. The femoral head-neck junction was quantified by: Visual score. Cam morphology (flattening or prominence), large cam (prominence). Alpha angle. Cam morphology (≥60°), large cam (≥78°). Cam morphology duration was defined as long (first present at baseline) or short (only from 2.5- to 5-year follow-up). Current symptoms at 5-year follow-up were assessed using a hip and groin pain question and by the "Hip and Groin Outcome Score" (HAGOS). HAGOS scores were categorized into: most symptoms (≥2 domains in lowest interquartile range [IQR]), least symptoms (≥2 domains in highest IQR). Hip ROM was measured by goniometry at 5-year follow-up.
Results:
Large cam morphology based on visual score was associated with hip and groin pain (23.8% vs. 7.1%, OR: 3.17, CI: [1.15-8.70], P = .026), but not with HAGOS scores. Cam morphology presence, size, and duration were associated with limited flexion of around 6° and/or 3° to 6° for internal rotation.
Conclusions:
Cam morphology presence, size, and duration were associated with limited hip flexion and/or internal rotation, but differences might not exceed the minimal clinical important difference. Whether cam morphology results in symptoms is uncertain. | INTRODUCTION:
Hip and groin symptoms are frequently observed in professional sports and football in particular. The prevalence of hip and groin symptoms in (elite) football is reported as 49% per season,
1
while the incidence varies between 4% and 19%.
2
,
3
One of the causes of hip and groin symptoms in athletes is femoroacetabular impingement (FAI) syndrome.
4
FAI syndrome is defined by a triad of symptoms, clinical signs, and imaging findings.
4
Imaging findings consistent with FAI syndrome include cam and/or pincer morphology. Cam morphology is an extra bone formation on the anterolateral side of the head‐neck junction of the femur which arises during growth.
5
,
6
,
7
,
8
,
9
It can potentially damage intra‐articular structures such as the cartilage and acetabular labrum and might cause symptoms.
10
,
11
Cam morphology prevalence in football players is high.
5
,
6
,
7
,
9
,
12
Although there is an association between cam morphology and hip osteoarthritis (OA),
12
,
13
,
14
,
15
,
16
,
17
the association between cam morphology and symptoms in athletes remains contradictory. Several cross‐sectional studies showed conflicting results.
18
,
19
,
20
,
21
,
22
One available longitudinal case‐control study
23
showed an association between cam morphology and development of hip pain in the general population with a relative risk of 4.3 (95% confidence interval [CI]: 2.3‐7.8). Another prospective cohort study
24
found no association between cam morphology and groin injuries in professional football players. A large cam morphology is associated with a higher risk for developing hip OA
13
and cartilage damage.
25
,
26
,
27
It might therefore hypothetically result in more symptoms and more limited range of motion (ROM). The influence of cam morphology duration on both symptoms and ROM has never been investigated and can only be assessed when information on when cam morphology arises is available.
Therefore, the study aims of this cohort with young academy male football players were to assess the association between the cam morphology presence, size and duration and hip and groin symptoms and ROM within 5‐year follow‐up.
CONCLUSION:
Data of this cohort study suggest that the presence, size, and duration of a bony cam morphology have a direct but small effect on the range of motion. Symptoms might develop in some football players with large cam morphology or several years after cam morphology development. A larger prospective cohort is needed to further elucidate these findings. | Background:
Conflicting and limited high-quality prospective data are available on the associations between cam morphology and hip and groin symptoms and range of motion (ROM).
Methods:
Academy male football players (n = 49, 17-24 years) were included. Standardized antero-posterior pelvic and frog-leg lateral radiographs were obtained at baseline, 2.5- and 5-year follow-up. The femoral head-neck junction was quantified by: Visual score. Cam morphology (flattening or prominence), large cam (prominence). Alpha angle. Cam morphology (≥60°), large cam (≥78°). Cam morphology duration was defined as long (first present at baseline) or short (only from 2.5- to 5-year follow-up). Current symptoms at 5-year follow-up were assessed using a hip and groin pain question and by the "Hip and Groin Outcome Score" (HAGOS). HAGOS scores were categorized into: most symptoms (≥2 domains in lowest interquartile range [IQR]), least symptoms (≥2 domains in highest IQR). Hip ROM was measured by goniometry at 5-year follow-up.
Results:
Large cam morphology based on visual score was associated with hip and groin pain (23.8% vs. 7.1%, OR: 3.17, CI: [1.15-8.70], P = .026), but not with HAGOS scores. Cam morphology presence, size, and duration were associated with limited flexion of around 6° and/or 3° to 6° for internal rotation.
Conclusions:
Cam morphology presence, size, and duration were associated with limited hip flexion and/or internal rotation, but differences might not exceed the minimal clinical important difference. Whether cam morphology results in symptoms is uncertain. | 16,550 | 348 | [
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"athletes femoroacetabular impingement"
] | [CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY] | [CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY] | [CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY] | [CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY] | [CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY] | [CONTENT] FAI syndrome | femoroacetabular impingement | pain | range of motion [SUMMARY] | [CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Athletes | Groin | Hip Joint | Humans | Male | Musculoskeletal Pain | Pain Measurement | Range of Motion, Articular | Soccer | Surveys and Questionnaires | Young Adult [SUMMARY] | [CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY] | [CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY] | [CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY] | [CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY] | [CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY] | [CONTENT] hip groin symptoms | causes hip groin | groin symptoms athletes | athletes femoroacetabular | athletes femoroacetabular impingement [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | groin | group | pain | hip groin [SUMMARY] | [CONTENT] cam | morphology | cam morphology | symptoms | association cam | association | association cam morphology | hip | football | groin [SUMMARY] | [CONTENT] hip | groin | year | pain | participants | year follow | follow | outcome | hip groin | group [SUMMARY] | [CONTENT] cam | hips | cam morphology | morphology | group | persons | group symptoms | vs | cam morphology based | morphology based [SUMMARY] | [CONTENT] morphology | cam morphology | cam | cohort | develop football | size duration bony cam | suggest presence size | suggest presence size duration | years cam morphology development | cohort study suggest presence [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | group | groin | pain | follow [SUMMARY] | [CONTENT] cam | morphology | cam morphology | hip | hips | symptoms | group | groin | pain | follow [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] 49 | 17-24 years ||| 2.5- | 5-year ||| ||| ||| ||| ||| first | 2.5- | 5-year ||| 5-year ||| IQR ||| ROM | 5-year [SUMMARY] | [CONTENT] 23.8% | 7.1% | 3.17 | CI ||| 1.15 | .026 | HAGOS ||| 6° and/or 3° to 6 [SUMMARY] | [CONTENT] ||| [SUMMARY] | [CONTENT] ||| 49 | 17-24 years ||| 2.5- | 5-year ||| ||| ||| ||| ||| first | 2.5- | 5-year ||| 5-year ||| IQR ||| ROM | 5-year ||| 23.8% | 7.1% | 3.17 | CI ||| 1.15 | .026 | HAGOS ||| 6° and/or 3° to 6 ||| ||| [SUMMARY] | [CONTENT] ||| 49 | 17-24 years ||| 2.5- | 5-year ||| ||| ||| ||| ||| first | 2.5- | 5-year ||| 5-year ||| IQR ||| ROM | 5-year ||| 23.8% | 7.1% | 3.17 | CI ||| 1.15 | .026 | HAGOS ||| 6° and/or 3° to 6 ||| ||| [SUMMARY] |
||||||
Complications Associated With Nasopharyngeal COVID-19 Testing: An Analysis of the MAUDE Database and Literature Review. | 34547903 | Nasopharyngeal swab testing, which has greatly increased in utilization due to the COVID-19 pandemic, is generally safe and well-tolerated, although it may be rarely associated with adverse events. | BACKGROUND | Publicly reported adverse events associated with nasopharyngeal COVID-19 testing within the Manufacturer and User Facility Device Experience (MAUDE) database and the published literature were queried. | METHODS | A total of 129 adverse events were reported, including 66 from the MAUDE database and 63 from literature review. The most common complications were swab fracture resulting in retained foreign body (47%), followed by epistaxis (17%), and headache (11%). Seven (12%) of the reported retained foreign body cases required removal under general anesthesia, while 1 (5%) of the epistaxis cases required surgical intervention. The most serious adverse event was meningitis following cerebrospinal fluid leak. | RESULTS | Patients and healthcare providers should be aware of the potential risks associated with testing, with attention to ensuring proper technique, and be prepared to recognize and manage adverse events. | CONCLUSIONS | [
"COVID-19",
"COVID-19 Testing",
"Databases, Factual",
"Humans",
"Nasopharynx",
"Pandemics",
"SARS-CoV-2"
] | 8808139 | Introduction | Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged. | Methods | This study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test. | Results | The MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8
Summary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.
Incidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.
MAUDE, Manufacturer and User Facility Device Experience. | Conclusion | Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery. | [
"Conclusion"
] | [
"Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery."
] | [
null
] | [
"Introduction",
"Methods",
"Results",
"Discussion",
"Conclusion"
] | [
"Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.",
"This study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test.",
"The MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8\nSummary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.\nIncidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.\nMAUDE, Manufacturer and User Facility Device Experience.",
"Nasopharyngeal testing for COVID-19 is generally safe and well-tolerated, with the risk of any adverse event ranging from 0.02% to 0.16% in previous single-center retrospective reviews.9,10 Nevertheless, healthcare providers should be cognizant of and well-equipped to manage complications associated with testing. In general, most of these adverse events may likely be mitigated by proper sampling technique, where the swab is inserted parallel to the nasal floor as opposed to a more superiorly oriented axis.\nSwab fracture resulting in a retained foreign body was the most common adverse event reported both in the MAUDE database and the literature. Swabs have an inherent breakpoint mechanism to aid in easy transfer to the transport vial. However, this breakpoint is vulnerable to accidental fragmentation during sample collection, especially in uncooperative patients or sedated patients upon whom undue force is applied.11 Furthermore, when not inserted along the nasal floor axis, the swab may contact structures that can increase the risk of fracture, such as septal spurs and the inferior and middle turbinates. Retrieval was generally performed with or without local anesthesia under direct visualization with direct rhinoscopy or nasal endoscopy. However, if the fragmented swab is not visualized, patients must be carefully monitored for foreign body ingestion or aspiration. One report discussed the need for an upper GI endoscopy to avoid possible perforation given the ingested swab's body length and sharp pointed form.12\nAlthough epistaxis was a rare and largely self-limited adverse event, its incidence warrants caution when testing individuals at increased bleeding risk. Mucosal trauma is the most likely etiology, which may be avoided by staying low in the nose. COVID-19 disproportionally affects the elderly, many of whom are treated with oral anticoagulants. Studies examining epistaxis rates following nasopharyngeal testing among such at-risk patients are needed, and a risk assessment should be considered prior to testing. Alternatively, less invasive testing methods including throat swabbing or saliva sampling can be considered.\nIatrogenic CSF leak is an unusual yet potentially fatal complication following testing. Whereas one case involved a patient with a pre-existing encephalocele7, 4 patients did not have radiographic or visual evidence of a skull base defect before testing.8–10,20 These reports illustrate the importance of proper nasopharyngeal swab technique which has been described through simulation models,13 videos,14 and graphics.15 Furthermore, providers should exhibit increased caution in patients with history of sinus/skull base surgery and consider alternative testing methods including throat swabbing or saliva sampling. As many nasopharyngeal swabs are performed in a drive-through format, patients should be queried about prior endonasal endoscopic sinus or skull base operations prior to testing.\nWe recognize that the MAUDE database is limited, especially in describing patient characteristics and comorbidities which may help identify patients who are more susceptible to experiencing a nasopharyngeal swab-related adverse event. Reports of device-related complications are mandatory for industry and voluntary for providers and patients, thus data may be under-reported. Additionally, MAUDE reports may contain erroneous narrative information. There is also a possibility that some of the complications published in the literature may have also been submitted via the MAUDE database and therefore duplicated; however, the rate of complications found through the MAUDE database and the literature review do not suggest that this happened at a significant degree. While future peer-reviewed studies will continue to provide the strongest evidence on this topic, the MAUDE database helps fill the current gap in knowledge, making this study the most comprehensive overview of adverse events following nasopharyngeal COVID-19 testing.",
"Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery."
] | [
"intro",
"methods",
"results",
"discussion",
null
] | [
"COVID-19",
"nasopharyngeal swab",
"MAUDE",
"adverse events",
"complications"
] | Introduction:
Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.
Methods:
This study did not require approval from the University of California Irvine's Institutional Review Board since data were collected from a publicly accessible database and previously published studies. The MAUDE database was queried for cases involving nasopharyngeal COVID-19 testing from December 1, 2019 to February 28, 2021 using the product class “Applicator, Absorbent Tipped, Sterile.” Manufacturers reported in each event were recorded and individually searched in the database. Furthermore, we performed a literature search of published articles in PubMed, Ovid MEDLINE, and Cochrane databases using keywords [“covid19” OR “covid-19”] AND “swab” AND [“complication” OR “complications” OR “cerebrospinal fluid leak” OR “CSF leak” OR “foreign body” OR “adverse events”]. The full abstract of each article was independently evaluated by 2 authors (AAH and KG). Additionally, the reference list of included studies and all articles that referenced these studies were screened. Inclusion criteria mandated the article was written in English and that the authors reported an adverse event relating to a COVID-19 nasopharyngeal swab test.
Results:
The MAUDE search returned 416 results, of which 66 unique adverse events (from 8 swab manufacturers) were included. Fifteen of the 130 published studies generated from the literature search were included (Table 1), describing 63 adverse events. All reported adverse events are categorized in Table 2, demonstrating the most commonly reported complications following COVID-19 nasopharyngeal swab testing are swab fracture resulting in retained foreign body (47%), epistaxis (17%), and headache (11%). Of the 60 reported retained foreign bodies, 7 (12%) required removal under general anesthesia. The fragmented swab was not visualized on nasal endoscopy or imaging in 6 (10%) cases, increasing suspicion for possible foreign body ingestion. Only 1 patient (4.5%) experiencing epistaxis following testing required surgical intervention after failing clinical management with external pressure, ice packs, and cauterization. The most serious complication was cerebrospinal fluid (CSF) leak,4–7 of which 1 case led to meningitis.8
Summary of Studies Reporting Adverse Events Associated With COVID-19 Nasopharyngeal Swab Testing in the Literature.
Incidence of Adverse Events Related to COVID-19 Nasopharyngeal Swab Testing Reported in the MAUDE Database and Literature.
MAUDE, Manufacturer and User Facility Device Experience.
Discussion:
Nasopharyngeal testing for COVID-19 is generally safe and well-tolerated, with the risk of any adverse event ranging from 0.02% to 0.16% in previous single-center retrospective reviews.9,10 Nevertheless, healthcare providers should be cognizant of and well-equipped to manage complications associated with testing. In general, most of these adverse events may likely be mitigated by proper sampling technique, where the swab is inserted parallel to the nasal floor as opposed to a more superiorly oriented axis.
Swab fracture resulting in a retained foreign body was the most common adverse event reported both in the MAUDE database and the literature. Swabs have an inherent breakpoint mechanism to aid in easy transfer to the transport vial. However, this breakpoint is vulnerable to accidental fragmentation during sample collection, especially in uncooperative patients or sedated patients upon whom undue force is applied.11 Furthermore, when not inserted along the nasal floor axis, the swab may contact structures that can increase the risk of fracture, such as septal spurs and the inferior and middle turbinates. Retrieval was generally performed with or without local anesthesia under direct visualization with direct rhinoscopy or nasal endoscopy. However, if the fragmented swab is not visualized, patients must be carefully monitored for foreign body ingestion or aspiration. One report discussed the need for an upper GI endoscopy to avoid possible perforation given the ingested swab's body length and sharp pointed form.12
Although epistaxis was a rare and largely self-limited adverse event, its incidence warrants caution when testing individuals at increased bleeding risk. Mucosal trauma is the most likely etiology, which may be avoided by staying low in the nose. COVID-19 disproportionally affects the elderly, many of whom are treated with oral anticoagulants. Studies examining epistaxis rates following nasopharyngeal testing among such at-risk patients are needed, and a risk assessment should be considered prior to testing. Alternatively, less invasive testing methods including throat swabbing or saliva sampling can be considered.
Iatrogenic CSF leak is an unusual yet potentially fatal complication following testing. Whereas one case involved a patient with a pre-existing encephalocele7, 4 patients did not have radiographic or visual evidence of a skull base defect before testing.8–10,20 These reports illustrate the importance of proper nasopharyngeal swab technique which has been described through simulation models,13 videos,14 and graphics.15 Furthermore, providers should exhibit increased caution in patients with history of sinus/skull base surgery and consider alternative testing methods including throat swabbing or saliva sampling. As many nasopharyngeal swabs are performed in a drive-through format, patients should be queried about prior endonasal endoscopic sinus or skull base operations prior to testing.
We recognize that the MAUDE database is limited, especially in describing patient characteristics and comorbidities which may help identify patients who are more susceptible to experiencing a nasopharyngeal swab-related adverse event. Reports of device-related complications are mandatory for industry and voluntary for providers and patients, thus data may be under-reported. Additionally, MAUDE reports may contain erroneous narrative information. There is also a possibility that some of the complications published in the literature may have also been submitted via the MAUDE database and therefore duplicated; however, the rate of complications found through the MAUDE database and the literature review do not suggest that this happened at a significant degree. While future peer-reviewed studies will continue to provide the strongest evidence on this topic, the MAUDE database helps fill the current gap in knowledge, making this study the most comprehensive overview of adverse events following nasopharyngeal COVID-19 testing.
Conclusion:
Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery.
| Background:
Nasopharyngeal swab testing, which has greatly increased in utilization due to the COVID-19 pandemic, is generally safe and well-tolerated, although it may be rarely associated with adverse events.
Methods:
Publicly reported adverse events associated with nasopharyngeal COVID-19 testing within the Manufacturer and User Facility Device Experience (MAUDE) database and the published literature were queried.
Results:
A total of 129 adverse events were reported, including 66 from the MAUDE database and 63 from literature review. The most common complications were swab fracture resulting in retained foreign body (47%), followed by epistaxis (17%), and headache (11%). Seven (12%) of the reported retained foreign body cases required removal under general anesthesia, while 1 (5%) of the epistaxis cases required surgical intervention. The most serious adverse event was meningitis following cerebrospinal fluid leak.
Conclusions:
Patients and healthcare providers should be aware of the potential risks associated with testing, with attention to ensuring proper technique, and be prepared to recognize and manage adverse events. | Introduction:
Diagnostic testing to identify individuals infected with SARS-CoV-2 is critical to COVID-19 containment and providing adequate and timely patient care. Over 363 million tests were performed in the United States as of March 7, 2021, with over 1 million new tests performed daily.1 The nasopharyngeal swab is the recommended and most commonly deployed testing method.2 We hypothesize that complications associated with nasopharyngeal testing are rare but do occur. Accordingly, their characterization is warranted to ensure that practitioners are aware of and equipped to manage such adverse events. We sought to identify publicly reported adverse events associated with nasopharyngeal COVID-19 testing. We queried the Manufacturer and User Facility Device Experience (MAUDE) database utilized by the Food and Drug Administration to report medical device-related complications.3 Additionally, we performed a comprehensive literature review to identify complications reported in the published literature since the pandemic emerged.
Conclusion:
Nasopharyngeal COVID-19 testing is rarely associated with complications, which nevertheless may result in morbidity and medical emergencies. Patients and providers should be aware of the potential risks associated with testing as well as the methods to manage complications. A risk assessment should be considered in patients who are at increased risk of bleeding, or those with a history of rhinologic or skull base disorders and/or surgery. | Background:
Nasopharyngeal swab testing, which has greatly increased in utilization due to the COVID-19 pandemic, is generally safe and well-tolerated, although it may be rarely associated with adverse events.
Methods:
Publicly reported adverse events associated with nasopharyngeal COVID-19 testing within the Manufacturer and User Facility Device Experience (MAUDE) database and the published literature were queried.
Results:
A total of 129 adverse events were reported, including 66 from the MAUDE database and 63 from literature review. The most common complications were swab fracture resulting in retained foreign body (47%), followed by epistaxis (17%), and headache (11%). Seven (12%) of the reported retained foreign body cases required removal under general anesthesia, while 1 (5%) of the epistaxis cases required surgical intervention. The most serious adverse event was meningitis following cerebrospinal fluid leak.
Conclusions:
Patients and healthcare providers should be aware of the potential risks associated with testing, with attention to ensuring proper technique, and be prepared to recognize and manage adverse events. | 1,333 | 206 | [
71
] | 5 | [
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"19",
"covid 19",
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"maude",
"patients"
] | [
"nasopharyngeal testing risk",
"covid 19 swab",
"conclusion nasopharyngeal covid",
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"nasopharyngeal covid 19"
] | [CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY] | [CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY] | [CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY] | [CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY] | [CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY] | [CONTENT] COVID-19 | nasopharyngeal swab | MAUDE | adverse events | complications [SUMMARY] | [CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 [SUMMARY] | [CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 [SUMMARY] | [CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 [SUMMARY] | [CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 [SUMMARY] | [CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 [SUMMARY] | [CONTENT] COVID-19 | COVID-19 Testing | Databases, Factual | Humans | Nasopharynx | Pandemics | SARS-CoV-2 [SUMMARY] | [CONTENT] nasopharyngeal testing risk | covid 19 swab | conclusion nasopharyngeal covid | associated nasopharyngeal covid | nasopharyngeal covid 19 [SUMMARY] | [CONTENT] nasopharyngeal testing risk | covid 19 swab | conclusion nasopharyngeal covid | associated nasopharyngeal covid | nasopharyngeal covid 19 [SUMMARY] | [CONTENT] nasopharyngeal testing risk | covid 19 swab | conclusion nasopharyngeal covid | associated nasopharyngeal covid | nasopharyngeal covid 19 [SUMMARY] | [CONTENT] nasopharyngeal testing risk | covid 19 swab | conclusion nasopharyngeal covid | associated nasopharyngeal covid | nasopharyngeal covid 19 [SUMMARY] | [CONTENT] nasopharyngeal testing risk | covid 19 swab | conclusion nasopharyngeal covid | associated nasopharyngeal covid | nasopharyngeal covid 19 [SUMMARY] | [CONTENT] nasopharyngeal testing risk | covid 19 swab | conclusion nasopharyngeal covid | associated nasopharyngeal covid | nasopharyngeal covid 19 [SUMMARY] | [CONTENT] testing | swab | nasopharyngeal | adverse | 19 | covid 19 | covid | complications | maude | patients [SUMMARY] | [CONTENT] testing | swab | nasopharyngeal | adverse | 19 | covid 19 | covid | complications | maude | patients [SUMMARY] | [CONTENT] testing | swab | nasopharyngeal | adverse | 19 | covid 19 | covid | complications | maude | patients [SUMMARY] | [CONTENT] testing | swab | nasopharyngeal | adverse | 19 | covid 19 | covid | complications | maude | patients [SUMMARY] | [CONTENT] testing | swab | nasopharyngeal | adverse | 19 | covid 19 | covid | complications | maude | patients [SUMMARY] | [CONTENT] testing | swab | nasopharyngeal | adverse | 19 | covid 19 | covid | complications | maude | patients [SUMMARY] | [CONTENT] identify | performed | associated nasopharyngeal | million | tests performed | tests | testing | nasopharyngeal | complications | device [SUMMARY] | [CONTENT] studies | authors | article | articles | database | event | 19 | covid 19 | covid | leak [SUMMARY] | [CONTENT] swab | swab testing | 19 nasopharyngeal swab testing | nasopharyngeal swab testing | events | adverse events | adverse | covid 19 nasopharyngeal swab | covid 19 nasopharyngeal | 19 nasopharyngeal swab [SUMMARY] | [CONTENT] patients | risk | associated | providers aware | risk assessment considered patients | testing methods manage | manage complications risk | manage complications risk assessment | rhinologic | rhinologic skull [SUMMARY] | [CONTENT] testing | swab | patients | adverse | nasopharyngeal | 19 | covid | covid 19 | complications | risk [SUMMARY] | [CONTENT] testing | swab | patients | adverse | nasopharyngeal | 19 | covid | covid 19 | complications | risk [SUMMARY] | [CONTENT] Nasopharyngeal | COVID-19 [SUMMARY] | [CONTENT] COVID-19 | Manufacturer | User Facility Device Experience [SUMMARY] | [CONTENT] 129 | 66 | 63 ||| 47% | 17% | 11% ||| Seven | 12% | anesthesia | 1 | 5% ||| [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] Nasopharyngeal | COVID-19 ||| COVID-19 | Manufacturer | User Facility Device Experience ||| 129 | 66 | 63 ||| 47% | 17% | 11% ||| Seven | 12% | anesthesia | 1 | 5% ||| ||| [SUMMARY] | [CONTENT] Nasopharyngeal | COVID-19 ||| COVID-19 | Manufacturer | User Facility Device Experience ||| 129 | 66 | 63 ||| 47% | 17% | 11% ||| Seven | 12% | anesthesia | 1 | 5% ||| ||| [SUMMARY] |
||||||
Novel biomarkers identifying hypertrophic cardiomyopathy and its obstructive variant based on targeted amino acid metabolomics. | 36156511 | Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM. | BACKGROUND | Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM. | METHODS | The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively. | RESULTS | The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive. | CONCLUSIONS | [
"Humans",
"Amino Acids",
"Cysteine",
"Cardiomyopathy, Hypertrophic",
"Biomarkers",
"Proline",
"Arginine",
"Ornithine",
"Glycine",
"Choline"
] | 9746752 | Introduction | Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]
To date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.
Circulatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.
In the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM. | Methods | Ethical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.
This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.
Study population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.
Study workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.
HCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]
A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.
Study workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.
HCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]
Inclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.
The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.
Targeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.
To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.
Statistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.
Variables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).
Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.
Variables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/). | Results | Baseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.
Baseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.
Data are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.
There was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.
A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.
Baseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.
Data are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.
There was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.
Plasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.
Heatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.
Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.
Heatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.
Screening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.
First, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:
Screening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.
Statistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.
Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.
First, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:
Screening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.
Statistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.
Screening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:
Screening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.
Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:
Screening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM. | Conclusions | The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care. | [
"Ethical approval",
"Study population",
"Inclusion and exclusion criteria",
"Targeted AA metabolomics in plasma",
"Statistical analysis",
"Baseline characteristics",
"Plasma AA and derivatives levels and their correlations with baseline characteristics",
"Screening model to discriminate HCM from NC",
"Screening model to discriminate HOCM from HNCM",
"Limitations",
"Funding"
] | [
"This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.",
"A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]",
"The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.",
"To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.",
"Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).",
"A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.",
"Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.",
"Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.",
"Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.",
"The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.",
"This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08)."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Ethical approval",
"Study population",
"Inclusion and exclusion criteria",
"Targeted AA metabolomics in plasma",
"Statistical analysis",
"Results",
"Baseline characteristics",
"Plasma AA and derivatives levels and their correlations with baseline characteristics",
"Screening model to discriminate HCM from NC",
"Screening model to discriminate HOCM from HNCM",
"Discussion",
"Limitations",
"Conclusions",
"Funding",
"Conflicts of interest",
"Supplementary Material",
"Supplementary Material"
] | [
"Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]\nTo date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.\nCirculatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.\nIn the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.",
"Ethical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nThis prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.\nStudy population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nA total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]\nInclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nThe inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.\nTargeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nTo exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.\nStatistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).\nContinuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).",
"This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.",
"A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.\nStudy workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.\nHCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]",
"The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.",
"To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.",
"Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.\nVariables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).",
"Baseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nA total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.\nPlasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nTargeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.\nScreening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nIntegrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.\nScreening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.\nAnother major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.",
"A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.\nBaseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.\nData are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.\nThere was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.",
"Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.\nHeatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.",
"Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.\nFirst, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:\nScreening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.\nStatistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.",
"Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:\nScreening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.\nThe AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.",
"HCM is an underdiagnosed monogenetic cardiovascular disease accompanied by metabolic disorders. Growing evidence indicates that aberrations in myocardial energy metabolism contribute to the development of hypertrophy and heart failure.[27,28] In the present study, we profiled plasma AAs and derivatives, and identified AA metabolite panels with high discriminative accuracy to distinguish HCM individuals. Furthermore, multiple AAs and derivatives were significantly correlated with clinical parameters, such as the MWT and LVOTGrest. These findings provide an opportunity for developing panels of plasma biomarkers to help physicians initially identify HCM.\nHCM may be defined clinically, genetically, and histologically with high clinical heterogeneity. Some individuals remain asymptomatic throughout their lifetime with little need for treatment, while some may exhibit a progressive course, such as severe heart failure, stroke, chest pain, or even SCD.[29] It remains challenging to diagnose HCM promptly in clinical practice. Thus, a tremendous clinical challenge exists to differentiate or predict which individuals experience HCM. Even though HCM is diagnosed, it is still difficult to discern HOCM and HNCM, owing to the vulnerable LVOTG. Developing novel non-invasive, objective, and available screening biomarkers may help identify HCM in the population and improve care quality, especially in medically underserved patients. Metabolites are promising tools to understand the pathophysiological changes involved in disease onset and progression, and they can be used as biomarkers for disease prediction, diagnosis, and prognosis.[30,31] Metabolomic technologies, such as ultra-performance liquid chromatography–mass spectrometry, (semi)quantitatively measure hundreds of unique metabolites, identifying a broad range of metabolic pathways, and only small volumes of biofluids are needed.[32] Moreover, sophisticated machine learning algorithms are utilized to analyze heterogeneous datasets to discover useful patterns that would be difficult for even well-trained professionals to identify, and they have been generally recognized as an effective approach in clinical diagnostics, precision treatments, and health monitoring.[33]\nSpecifically, aberrations in cardiac AA metabolism have been shown to be associated with cardiac hypertrophy and heart failure progression.[27,28] Emerging evidence has demonstrated that metabolic aberrations may participate in the initiation and development of HCM, even in preclinical HCM.[34–36] By utilizing targeted AA metabolomics, the present study investigated the circulating AA and derivatives profile in HCM patients. Interestingly, plasma arginine, phenylalanine, tyrosine, ornithine, proline, alanine, asparagine, creatine, tryptophan, and choline levels correlated with LVOTGrest, and ornithine levels correlated with MWT, indicating that the above metabolites are associated with the severity of HCM. These results suggested that systemic AA and derivatives dysmetabolism may be involved in the pathogenesis of HCM and that changes in the plasma AA and derivatives profile may have the potential to detect or diagnose HCM.\nUnivariate analysis showed that 10 AAs and derivatives (serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid) in the HCM groups were significantly different from those in the NC group. After systematically profiling AA metabolic alterations in HCM, four metabolites (proline, glycine, cysteine, and choline) were selected as a potential AA and derivatives panel for discerning HCM from the NC, considering discriminative efficacy and clinical utility. In the training and validation sets, the AUC values of the ROC analysis were 0.83 and 0.79, respectively, demonstrating the good value of detecing HCM.\nTo minimize the confounding factors that may affect the metabolomic differences, we included a “pure” NC group accompanied by no common comorbidities. The mean age of the “pure” NC group was 34 years, which may limit generalizability to the general population. Among the different factors that may influence systemic metabolism, age and sex were not matched between HCM patients and their NC counterparts. In the past 10 years, the average age of patients diagnosed with HCM has increased by 10 years to 51 years, and male patients account for 60% of the cases.[37] In the present study, the average age of HCM patients was nearly 47 years, and males accounted for 72% of the cases. Although the baseline characteristics were mismatched in the NC and HCM groups, the sensitivity analysis stratified by age, sex, and BMI showed a consistent AUC value of approximately 0.80 across all the subgroups. Similarly, the previous history of HP had a limited effect on the discriminative performance. These results indicated that age, sex, BMI, and previous history of HP exerted a limited influence on the establishment and discriminating efficacy of the model.\nSeveral AAs and derivatives have been reported to be involved in the regulation of cardiovascular pathology. Plasma homocysteine levels are associated with the incidence of cardiovascular diseases. Homocysteine reduction improves cardiac function and ameliorates pathological structural remodeling.[38] Choline ameliorates cardiac dysfunction by regulating the expression of genes involved in the ketone body and fatty acid metabolism. This process may be involved in the activation of the sirtuin 3/adenosine monophosphate (AMP)-activated protein kinase pathway.[39] Accumulating evidence demonstrates that choline supplementation prevents myocardial hypertrophy, fibrosis, and the inflammatory response induced by pressure overload.[40,41] The elevation in plasma choline levels is associated with the incidence of heart failure.[42] Glycine promotes the synthesis of the antioxidant metabolite, glutathione, and it reduces the production of mitochondrial oxidized protein and increases adenosine triphosphate, thereby improving cardiac function and ventricular remodeling.[43] The decrease in plasma glycine levels observed in HCM patients indicates that HCM patients are in a state of oxidative stress. Proline improves myocardial remodeling and reduces infarct size and cardiomyocyte death after myocardial infarction. Proline plays a protective role in postischemic heart failure by promoting oxidative phosphorylation and improving the redox state.[44] The above studies suggest that cysteine, choline, glycine, and proline may play an important role in the pathogenesis of HCM, and more research is needed to confirm these findings. The panel comprised of the four AAs and derivatives has the potential to be used for broad population-based screening of HCM.\nLeft ventricular outflow tract obstruction (LVOTO) has malignant effects on patients. A previous study has reported that HNCM patients have higher morbidity and arrhythmic risk than HOCM patients, while HOCM patients, who are generally much older, have a higher BMI, New York Heart Association class, and overall risk.[45] Additionally, therapy choice and prognosis are quite different between HOCM and HNCM patients, and septal incision therapy may be recommended for medical refractory patients with LVOTG ≥50 mmHg. Therefore, provocative maneuvers, despite their relatively high cost and limited availability, should be considered in patients with low peak resting gradients (ie, <30 mmHg) to induce the presence of LVOTO, particularly for those with symptoms, even if provocative tests have some lethal risks.\nIn the present study, among the 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between the HOCM and HNCM groups, three metabolites (arginine, proline, and ornithine) were validated as potential biomarkers to further differentiate HOCM from HNCM with similar AUC values of 0.83 and 0.82 in the training and validation sets, respectively. Arginine levels have been linked with heart failure in a rat model.[46] HCM patients show specific features of endothelial dysfunction detectable in peripheral blood that are more severe in HOCM than HNCM.[47] In the present study, the level of plasma arginine increased in HCM, particularly in HOCM, and the levels of proline and ornithine, as the main products of arginine metabolism, decreased, indicating that arginine metabolism disorders occur in HCM, particularly for those with HOCM. Therefore, this panel can be used as a supplement to distinguish whether there is an obstruction for HCM patients, especially those with inconclusive Echo results or contraindications to stress Echo. Recognizing the existence of obstruction is helpful to guide clinical treatment strategies.\nThe present study developed two AA and derivatives panels to discriminate HCM patients from NCs as well as HOCM from HNCM patients with high sensitivity and specificity. It should be noted that the AAs and derivatives selected in these panels are different, which may indicate that distinct pathological mechanisms exist in AA metabolism during HNCM and HOCM progression.",
"The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.",
"The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care.",
"This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08).",
"None.",
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"intro",
"methods",
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"results",
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] | [
"Hypertrophic cardiomyopathy",
"Amino acids",
"Targeted metabolomics",
"Biomarkers",
"Algorithm"
] | Introduction:
Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]
To date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.
Circulatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.
In the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.
Methods:
Ethical approval This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.
This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.
Study population A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.
Study workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.
HCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]
A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.
Study workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.
HCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]
Inclusion and exclusion criteria The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.
The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.
Targeted AA metabolomics in plasma To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.
To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.
Statistical analysis Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.
Variables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).
Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.
Variables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).
Ethical approval:
This prospective, single-center observational study was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No. KY20150120-1), and it complied with the Declaration of Helsinki. Written informed consent was obtained from all participants.
Study population:
A total of 166 fasting participants who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020 were consecutively recruited for the present study. The study flowchart is shown in Figure 1. For sample size calculation, at least 10 events per variable are needed.
Study workflow and design. HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LVOTG: Left ventricular outflow tract gradient.
HCM was diagnosed according to the 2014 European Society of Cardiology guidelines for the diagnosis and management of HCM.[6] LVOTG was measured with color-guided continuous-wave Doppler Echo (Philips Medical Systems, Bothell, Washington, USA) at rest and provocation, such as Valsalva maneuver or exercise stress with a supine bicycle exercise (semirecumbent and tilting bicycle ergometer; Lode BV, Groningen, Netherlands).[23,24] The presence of a peak LVOTG ≥30 mmHg at rest and with exercise stress is considered indicative of HOCM, while HNCM is defined by LVOTG <30 mmHg at rest and with exercise stress.[6,25] NCs were individuals who were suspected of having HCM and visited the International Cooperation Center for HCM in Xijing Hospital, but were eventually precluded from having HCM due to normal ventricular wall thickness. All participants underwent transthoracic echocardiography independently conducted by two experienced ultrasound technicians. All procedures were in accordance with the guidelines of the American Society of Echocardiography.[26]
Inclusion and exclusion criteria:
The inclusion criteria were as follows: (1) age between 18 and 80 years; (2) clinical diagnosis of HCM[6] as indicated by a maximal end-diastolic wall thickness ≥15 mm anywhere in the left ventricle that is not explained solely by loading conditions. More limited hypertrophy (13–14 mm) can be diagnostic when present in family members of a patient with HCM; and (3) informed consent for the present study. The exclusion criteria were as follows: (1) severe or acute inflammation within 1 month; (2) autoimmune diseases; (3) uncontrolled hypertension (HP); (4) moderate-severe aortic stenosis; (5) end-stage liver or renal failure; (6) other health behaviors that may affect the study, such as intemperance; (7) needle phobia and recent history (<1 month) of blood transfusion or hemodialysis; (8) severe non-cardiac disease with an expected survival of <1 year; and (9) unwillingness to participate.
Targeted AA metabolomics in plasma:
To exclude the dietary impact on plasma AA and derivatives levels, venous blood samples were collected from participants enrolled in the study after overnight fasting. Blood samples were collected in ethylenediaminetetraacetic acid vacutainer tubes and centrifuged at 644 ×g for 10 min, and aliquoted samples were stored at −80°C until processing. Plasma samples (100 μL) were added to 400 μL of precooled methanol acetonitrile solution (1:1, v/v), vortexed for 60 s, and placed at −20°C for 1 h to precipitate protein. The samples were then centrifuged at 14,000 ×g at 4°C for 20 min, and the supernatant was separated and tested by MS using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography sytem and 5500 QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA). A quality control (QC) sample was set up, among each interval of a certain number of experimental samples, to detect and evaluate the stability and repeatability of the detection system, and peaks with a relative standard deviation (RSD) >30% in QC samples were removed from the peak table. The standard mixture of AA metabolites was used for the correction of chromatographic retention time and the identification of metabolites. Multireaction monitoring mode was used to detect the ion pair. MultiQuant 3.0.3 (AB SCIEX, Framingham, MA, USA) was used to extract the chromatographic peak area and retention time.
Statistical analysis:
Continuous variables were presented as the mean ± standard deviation or median (Q1, Q3) when appropriate, and categorical variables were presented as counts (percentages). The independent t-test was used for the comparison of two continuous variables with normal distribution, and non-normally distributed data were compared using a two-sided nonparametric Mann–Whitney U-test. χ2 or Fisher's exact test was used for the comparison of unordered categorical variables. Spearman's rank correlation analysis was performed to analyze the correlations between AAs and other parameters in HCM.
Variables with P < 0.05 were considered significant AA metabolites and were used as alternatives for further selection analysis. To identify potential biomarkers for HCM, we used three integrated methods, including RF, SVM, and LR, based on multiple feature selection algorithms. We applied a voting procedure to select potential biomarkers, which rewarded features appearing in the optimal subsets by the ensemble methods. The selected variables were ranked in decreasing order by their contribution to the classification model. Several highly ranked metabolites that led to the highest and most stable area under the receiver operating characteristic (ROC) curve (AUC) value of the model were considered potential biomarkers for developing screening HCM panels. Thereafter, the screening models were developed and assessed according to LR, SVM, and RF. We performed a 5-fold cross-validation for each of the three classifiers. Finally, the screening model was presented as LR. Youden's index was used to define the optimal discriminative cutoff value. The ROC curve was applied to evaluate the performance of the statistical model. In addition, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. All statistical analyses were performed using SPSS statistics 26.0 (IBM, Armonk, NY, USA) and R software (version 4.0.2) (https://www.r-project.org/).
Results:
Baseline characteristics A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.
Baseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.
Data are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.
There was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.
A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.
Baseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.
Data are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.
There was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.
Plasma AA and derivatives levels and their correlations with baseline characteristics Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.
Heatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.
Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.
Heatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.
Screening model to discriminate HCM from NC Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.
First, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:
Screening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.
Statistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.
Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.
First, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:
Screening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.
Statistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.
Screening model to discriminate HOCM from HNCM Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:
Screening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.
Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:
Screening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.
Baseline characteristics:
A total of 166 participants were enrolled in this study, including 57 NCs and 109 cases of HCM (52 cases of HNCM and 57 cases of HOCM). The demographic and clinical characteristics were shown in Table 1. The mean age was 47.4 ± 14.3 years in the HCM group and 34.0 ± 11.8 years in the NC group (t = −6.078, P < 0.001). Males accounted for 72.5% (n = 79) and 43.9% (n = 25) in the HCM and NC groups (χ2 = 14.046, P < 0.001), respectively. Body mass index (BMI, 25.14 ± 3.93 kg/m2vs. 23.38 ± 3.87 kg/m2, t = −2.536, P = 0.012) and systolic blood pressure (125.61 ± 18.73 mmHg vs. 120.00 ± 13.23 mmHg, t = −2.222, P = 0.028) were higher in HCM than NC. 36.7% of HCM patients had a previous history of HP, which was significantly higher than that of NC (36.7% vs. 0, χ2 = 27.558, P < 0.001), and all received regular antihypertensive drugs and the blood pressure was under control.
Baseline characteristics of patients with HCM and normal controls as well as those with HNCM and HOCM.
Data are presented as n (%) or mean ± SD; otherwise, the data are presented as median (Q1, Q3). ∗t value. †χ2 value. ‡Z value. A: Atrial contraction mitral value velocity; ACEI/ARB: Angiotensin-converting enzyme inhibitor/angiotensin receptor antagonist; BMI: Body mass index; BSA: Body surface area; CCB: Calcium channel blocker; DBP: Diastolic blood pressure; DM: Diabetes mellitus; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; HP: Hypertension; LAD: Anteroposterior linear diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTGmax: Left ventricular outflow tract gradient at provocation; LVOTGrest: Left ventricular outflow tract gradient at rest; MWT: Maximum wall thickness; NC: Normal control; SAM: Systolic anterior motion of the mitral valve; SBP: Systolic blood pressure; SD: Standard deviation; –: Not available.
There was no significant difference in end-diastolic volume, end-systolic volume, or left ventricular ejection fraction between the HCM and NC groups. The ratio of early diastolic mitral valve velocity to atrial contraction mitral valve velocity (E/A) was significantly lower (0.85 [0.68, 1.35] vs. 1.48 [1.21, 1.66], Z = −4.893, P < 0.001), while the ratio of early diastolic mitral valve velocity to early diastolic mitral annular velocity (E/e′) was significantly higher in HCM as compared with NC (13.86 [10.45, 17.27] vs. 8.14 [6.91, 9.34], Z = −7.054, P < 0.001), which reflected left ventricular diastolic dysfunction. The diameters of the left atrium (39.06 ± 6.07 mm vs. 30.29 ± 3.90 mm, t = −3.770, P < 0.001), the maximum wall thickness (MWT; 22.57 ± 5.51 mm vs. 8.12 ± 1.43 mm, t = −25.783, P < 0.001), LVOTGrest (21.00 [4.00, 77.00] mmHg vs. 3.20 [2.75, 4.00] mmHg, Z = −6.928, P < 0.001), and LVOTGmax (53.00 [16.75, 124.00] mmHg vs. 9.00 [8.00, 14.00] mmHg, Z = −7.465, P < 0.001) in the HCM group were significantly higher than those in the NC group. The MWT (23.84 ± 5.22 mm vs. 21.17 ± 5.52 mm, t = −2.594, P = 0.011), LVOTGrest (76.00 [43.00, 103.00] mmHg vs. 4.00 [3.00, 6.00] mmHg, Z = −8.974, P < 0.001), and LVOTGmax (122.00 [92.00, 149.50] mmHg vs. 16.25 [8.95, 22.00] mmHg, Z = −8.992, P < 0.001) were also significantly different between the HOCM and HNCM groups.
Plasma AA and derivatives levels and their correlations with baseline characteristics:
Targeted AA metabolomics was performed, and 31 AA metabolites were measured in the plasma samples. The total ion chromatogram indicated the reliability of the metabolomics analysis [Supplementary Figure 1]. The results revealed that, except for spermidine and putrescine, 29 metabolites were absolutely quantified in the samples. There were significant differences in plasma serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels between the HCM and NC groups. In addition, there were also significant differences in plasma arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline levels between the HOCM and HNCM groups [Supplementary Table 1]. Correlations between 29 metabolites levels and baseline parameters (clinical and echocardiographic characteristics) in the HCM group are shown as a heatmap [Figure 2]. Among these metabolites, the plasma ornithine level was correlated with the MWT (R = −0.243, P = 0.011), while arginine (R = 0.253, P = 0.008), phenylalanine (R = −0.267, P = 0.005), tyrosine (R = −0.227, P = 0.017), ornithine (R = −0.442, P < 0.001), proline (R = −0.194, P = 0.043), alanine (R = −0.208, P = 0.030), asparagine (R = −0.241, P = 0.012), creatine (R = 0.252, P = 0.008), tryptophan (R = −0.321, P = 0.001), and choline (R = −0.242, P = 0.011) levels were correlated with the LVOTGrest.
Heatmap of correlations between plasma AA and derivatives levels and baseline parameters in HCM. The red denoted positive correlation, while the blue indicated negative correlation, and the size of the ball represented the value of correlation coefficient. AA: Amino acid; BMI: Body mass index; BSA: Body surface area; e′: Early diastolic mitral annular velocity; E: Early diastolic mitral valve velocity; EDV: End-diastolic volume; ESV: End-systolic volume; HCM: Hypertrophic cardiomyopathy; LAD: Anteroposterior diameter of left atrium; LVEF: Left ventricular ejection fraction; LVOTG: Left ventricular outflow tract gradient; MWT: Maximum wall thickness.
Screening model to discriminate HCM from NC:
Integrated approaches based on multiple feature selection algorithms were used to identify the perfect candidate AA metabolites, which were evaluated by accumulative AUC. Based on 5-fold cross-validation, RF, LR, and SVM were used to construct the optimal combination of classifier models. The screening model was presented as an LR. Moreover, 70% of the samples were randomly selected as the training set, and the remaining 30% of the samples were selected as the validation set in each group. The ROC curve was applied to evaluate the performance of the classifiers. Independent panels with distinct combinations of AA metabolites were developed for the discrimination of HCM from NC and HOCM from HNCM.
First, to differentiate the HCM and NC groups, four metabolites (Panel A: proline, glycine, cysteine, and choline) were filtered out because their combination showed the optimum discrimination efficacy and practical performance [Figure 3A, B and Supplementary Figure 2A]. The correlation of selected metabolites was poor in Panel A (all R < 0.70; Supplementary Figure 3A). The ROC analyses, utilizing RF, SVM, and LR, showed similar efficacy with an approximate AUC of 0.80 in distinguishing the HCM and NC groups [Figure 3C and Supplementary Figure 2B]. The following screening formula was deduced using LR to differentiate the HCM and NC groups:
Screening model to discriminate HCM patients from NCs. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HCM patients and NCs. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acid; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The expression level of the AAs and derivatives after log10 conversion was brought into the probability P value calculated by the above panel formula. The best cutoff value for classification was derived from Youden's index. Figures 3D and 3E show the relationships among the accuracy, sensitivity, specificity, and cutoff value. If the Y-value was >0.54, it indicated HCM; otherwise, it indicated NC. The corresponding accuracy, sensitivity, and specificity were 0.78, 0.80, and 0.75. Finally, 70% of the samples were randomly selected as the training set, and the remainder was selected as the validation set. The AUC values of the training and validation sets were 0.83 (0.75–0.91) and 0.79 (0.65–0.94), respectively [Figure 3F]. These results demonstrated that this screening model has good efficacy in the discrimination of HCM patients from NCs.
Statistical differences in age, sex, BMI, and previous history between the NC and HCM groups were observed in the baseline characteristics [Table 1]. To test whether the mismatch of clinical characteristics influenced the model establishment and discriminating efficacy, we performed a sensitivity analysis stratified by age, sex, and BMI. The analysis exhibited a consistent AUC value of approximately 0.80 across all the subgroups [Supplementary Figure 4]. These results indicated that the establishment and discriminating efficacy of the model was not influenced by age, sex, or BMI. Furthermore, to test the effect of a previous history of HP on discriminative ability, we performed an additional exploratory analysis by removing 40 cases of HCM combined with HP history. There was still a significant difference between the HCM without HP and NC groups in terms of ten differential AAs and derivatives [Supplementary Table 2]. In addition, there was no evident difference between HCM patients with HP and without HP in terms of the ten AAs and derivatives [Supplementary Table 3]. The subgroup analysis showed that in HCM patients with or without HP, the AUC values were 0.79 (0.69–0.89) and 0.84 (0.76–0.91), respectively [Supplementary Figure 5]. The DeLong test showed that there was no statistical significance between the overall AUC and the AUC of HCM patients with or without HP (P = 0.636 and 0.739, respectively), or between the AUC of HCM patients with HP and without HP (P = 0.463) [Supplementary Table 4]. These results suggested that the history of HP had limited influence on the discriminative ability of the screening model.
Screening model to discriminate HOCM from HNCM:
Another major clinical challenge is to distinguish HOCM from HNCM. Therefore, we aimed to construct a predition panel to further differentiate HOCM from HNCM. Only arginine, proline, and ornithine were left in the model (Panel B) [Figure 4A, B and Supplementary Figure 2C]. The poor correlation of the selected metabolites is presented in Supplementary Figure 3B, and the following screening formula was deduced:
Screening model to discriminate HOCM from HNCM. (A) Weight ranking of candidate AAs. (B) Cumulative AUC of AAs in the discrimination of HOCM and HNCM. (C) ROC curves analyzed by LR, SVM, and RF. (D) Curve of the accuracy and the corresponding cutoff values. (E) Curve of specificity, sensitivity, and the corresponding cutoff values. (F) ROC curves of the training and validation sets. AAs: Amino acids; AUC: Area under the ROC curve; CI: Confidence interval; HCM: Hypertrophic cardiomyopathy; HNCM: Non-obstructive HCM; HOCM: Obstructive HCM; LR: Logistic regression; NCs: Normal controls; RF: Random forest; ROC: Receiver operating characteristic; SVM: Support vector machine.
The AUC values and discriminative efficacy analyzed by LR and SVM were better than those analyzed by RF [Figure 4C and Supplementary Figure 2D]. If the Y-value was >0.53, it indicated HOCM; otherwise, it indicated HNCM. The corresponding accuracy, sensitivity, and specificity were 0.80, 0.81, and 0.79, respectively [Figures 4D and E]. The AUC values of the training and validation sets were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively [Figure 4F]. These results highlighted that this screening model has promising potential to differentiate HOCM from HNCM.
Discussion:
HCM is an underdiagnosed monogenetic cardiovascular disease accompanied by metabolic disorders. Growing evidence indicates that aberrations in myocardial energy metabolism contribute to the development of hypertrophy and heart failure.[27,28] In the present study, we profiled plasma AAs and derivatives, and identified AA metabolite panels with high discriminative accuracy to distinguish HCM individuals. Furthermore, multiple AAs and derivatives were significantly correlated with clinical parameters, such as the MWT and LVOTGrest. These findings provide an opportunity for developing panels of plasma biomarkers to help physicians initially identify HCM.
HCM may be defined clinically, genetically, and histologically with high clinical heterogeneity. Some individuals remain asymptomatic throughout their lifetime with little need for treatment, while some may exhibit a progressive course, such as severe heart failure, stroke, chest pain, or even SCD.[29] It remains challenging to diagnose HCM promptly in clinical practice. Thus, a tremendous clinical challenge exists to differentiate or predict which individuals experience HCM. Even though HCM is diagnosed, it is still difficult to discern HOCM and HNCM, owing to the vulnerable LVOTG. Developing novel non-invasive, objective, and available screening biomarkers may help identify HCM in the population and improve care quality, especially in medically underserved patients. Metabolites are promising tools to understand the pathophysiological changes involved in disease onset and progression, and they can be used as biomarkers for disease prediction, diagnosis, and prognosis.[30,31] Metabolomic technologies, such as ultra-performance liquid chromatography–mass spectrometry, (semi)quantitatively measure hundreds of unique metabolites, identifying a broad range of metabolic pathways, and only small volumes of biofluids are needed.[32] Moreover, sophisticated machine learning algorithms are utilized to analyze heterogeneous datasets to discover useful patterns that would be difficult for even well-trained professionals to identify, and they have been generally recognized as an effective approach in clinical diagnostics, precision treatments, and health monitoring.[33]
Specifically, aberrations in cardiac AA metabolism have been shown to be associated with cardiac hypertrophy and heart failure progression.[27,28] Emerging evidence has demonstrated that metabolic aberrations may participate in the initiation and development of HCM, even in preclinical HCM.[34–36] By utilizing targeted AA metabolomics, the present study investigated the circulating AA and derivatives profile in HCM patients. Interestingly, plasma arginine, phenylalanine, tyrosine, ornithine, proline, alanine, asparagine, creatine, tryptophan, and choline levels correlated with LVOTGrest, and ornithine levels correlated with MWT, indicating that the above metabolites are associated with the severity of HCM. These results suggested that systemic AA and derivatives dysmetabolism may be involved in the pathogenesis of HCM and that changes in the plasma AA and derivatives profile may have the potential to detect or diagnose HCM.
Univariate analysis showed that 10 AAs and derivatives (serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid) in the HCM groups were significantly different from those in the NC group. After systematically profiling AA metabolic alterations in HCM, four metabolites (proline, glycine, cysteine, and choline) were selected as a potential AA and derivatives panel for discerning HCM from the NC, considering discriminative efficacy and clinical utility. In the training and validation sets, the AUC values of the ROC analysis were 0.83 and 0.79, respectively, demonstrating the good value of detecing HCM.
To minimize the confounding factors that may affect the metabolomic differences, we included a “pure” NC group accompanied by no common comorbidities. The mean age of the “pure” NC group was 34 years, which may limit generalizability to the general population. Among the different factors that may influence systemic metabolism, age and sex were not matched between HCM patients and their NC counterparts. In the past 10 years, the average age of patients diagnosed with HCM has increased by 10 years to 51 years, and male patients account for 60% of the cases.[37] In the present study, the average age of HCM patients was nearly 47 years, and males accounted for 72% of the cases. Although the baseline characteristics were mismatched in the NC and HCM groups, the sensitivity analysis stratified by age, sex, and BMI showed a consistent AUC value of approximately 0.80 across all the subgroups. Similarly, the previous history of HP had a limited effect on the discriminative performance. These results indicated that age, sex, BMI, and previous history of HP exerted a limited influence on the establishment and discriminating efficacy of the model.
Several AAs and derivatives have been reported to be involved in the regulation of cardiovascular pathology. Plasma homocysteine levels are associated with the incidence of cardiovascular diseases. Homocysteine reduction improves cardiac function and ameliorates pathological structural remodeling.[38] Choline ameliorates cardiac dysfunction by regulating the expression of genes involved in the ketone body and fatty acid metabolism. This process may be involved in the activation of the sirtuin 3/adenosine monophosphate (AMP)-activated protein kinase pathway.[39] Accumulating evidence demonstrates that choline supplementation prevents myocardial hypertrophy, fibrosis, and the inflammatory response induced by pressure overload.[40,41] The elevation in plasma choline levels is associated with the incidence of heart failure.[42] Glycine promotes the synthesis of the antioxidant metabolite, glutathione, and it reduces the production of mitochondrial oxidized protein and increases adenosine triphosphate, thereby improving cardiac function and ventricular remodeling.[43] The decrease in plasma glycine levels observed in HCM patients indicates that HCM patients are in a state of oxidative stress. Proline improves myocardial remodeling and reduces infarct size and cardiomyocyte death after myocardial infarction. Proline plays a protective role in postischemic heart failure by promoting oxidative phosphorylation and improving the redox state.[44] The above studies suggest that cysteine, choline, glycine, and proline may play an important role in the pathogenesis of HCM, and more research is needed to confirm these findings. The panel comprised of the four AAs and derivatives has the potential to be used for broad population-based screening of HCM.
Left ventricular outflow tract obstruction (LVOTO) has malignant effects on patients. A previous study has reported that HNCM patients have higher morbidity and arrhythmic risk than HOCM patients, while HOCM patients, who are generally much older, have a higher BMI, New York Heart Association class, and overall risk.[45] Additionally, therapy choice and prognosis are quite different between HOCM and HNCM patients, and septal incision therapy may be recommended for medical refractory patients with LVOTG ≥50 mmHg. Therefore, provocative maneuvers, despite their relatively high cost and limited availability, should be considered in patients with low peak resting gradients (ie, <30 mmHg) to induce the presence of LVOTO, particularly for those with symptoms, even if provocative tests have some lethal risks.
In the present study, among the 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between the HOCM and HNCM groups, three metabolites (arginine, proline, and ornithine) were validated as potential biomarkers to further differentiate HOCM from HNCM with similar AUC values of 0.83 and 0.82 in the training and validation sets, respectively. Arginine levels have been linked with heart failure in a rat model.[46] HCM patients show specific features of endothelial dysfunction detectable in peripheral blood that are more severe in HOCM than HNCM.[47] In the present study, the level of plasma arginine increased in HCM, particularly in HOCM, and the levels of proline and ornithine, as the main products of arginine metabolism, decreased, indicating that arginine metabolism disorders occur in HCM, particularly for those with HOCM. Therefore, this panel can be used as a supplement to distinguish whether there is an obstruction for HCM patients, especially those with inconclusive Echo results or contraindications to stress Echo. Recognizing the existence of obstruction is helpful to guide clinical treatment strategies.
The present study developed two AA and derivatives panels to discriminate HCM patients from NCs as well as HOCM from HNCM patients with high sensitivity and specificity. It should be noted that the AAs and derivatives selected in these panels are different, which may indicate that distinct pathological mechanisms exist in AA metabolism during HNCM and HOCM progression.
Limitations:
The present study had several limitations. First, the study was performed in a single center, even though the center is the largest HCM center in northwestern China. Second, a total of 166 participants were enrolled in the study, resulting in a relatively small sample size. Third, because plasma metabolites reflect systemic inflammation and metabolic changes, the mechanistic understanding of such differential AA metabolites involved in HCM pathophysiology and whether the changes in plasma AA and derivatives profiles in HCM contribute to disease progression is still undetermined. Fourth, the present study used targeted AA metabolomics to discriminate HCM patients from NCs. The adaptation of non-targeted metabolomics may adequately map the metabolic changes in these patients. Fifth, genotypes were not tested for all the patients, even though the clinical HCM phenotypes may be independent of the genotypes. Finally, validation of these results in a large prospective cohort is required to address the value for future clinical application in HCM initial screening. Therefore, large-scale, multiple-center, and non-targeted metabolomics studies combined with genomics are needed to validate the application of metabolic changes detection in HCM screening and detection.
Conclusions:
The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care.
Funding:
This research was funded by the National Key Research & Development Program of China (No. 2018YFA0107400) and Program for Chang-Jiang Scholars and Innovative Research Team in University (No. PCSIRT-14R08).
Conflicts of interest:
None.
Supplementary Material:
Supplementary Material:
| Background:
Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM.
Methods:
Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.
Results:
The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively.
Conclusions:
The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive. | Introduction:
Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease worldwide with an unexpectedly high prevalence of 1:500 to 1:200.[1,2] It is estimated that HCM affects nearly 20 million people worldwide. However, only 10% of patients with HCM are clinically identified by a non-invasive imaging approach.[1,3] According to the left ventricular outflow tract gradient (LVOTG), obstructive HCM (HOCM) is defined as a maximum LVOTG (LVOTGmax) ≥30 mmHg; otherwise, it is classified as non-obstructive HCM (HNCM). HOCM is a common but catastrophic cause of sudden cardiac death (SCD) in young athletes and results in tremendous emotional, social, financial, and medical burdens for families across the country.[4] However, LVOTG is dynamic and is greatly influenced by myocardial contractility and preload, and enhanced myocardial contractility or decreased preload increases LVOTG.[5]
To date, the screening and diagnosis of HCM and even HOCM is still largely dependent on imaging modalities, including echocardiography (Echo), cardiac magnetic resonance and computed tomography, and genetic testing.[6–11] These imaging modalities have a series of limitations,[12–14] such as high interobserver variability, high intraobserver variability, high false-negative rate, high medical cost, high dependence on professional guidance, low popularity, increased radiation exposure, and intolerance among certain patients. These weaknesses profoundly limit the universal screening, especially among those living in undeveloped regions or countries. Therefore, it is necessary to explore an objective, convenient, and easily available approach to apply in clinical practice for HCM initial screening.
Circulatory biomarkers have been widely used in the early diagnosis or large-scale screening of diseases. However, biomarkers for HCM screening remain clinically lacking. Non-targeted and targeted metabolomics have emerged as powerful tools for mapping circulating metabolite changes and screening candidate biomarkers across a spectrum of cardiovascular diseases.[15] To date, the potential of metabolomics in the discovery and establishment of HCM biomarkers has not been studied. We have provided evidence that the dysregulation of amino acid (AA) metabolism is tightly associated with pathological cardiac remodeling in response to ischemic insult.[16,17] Recent clinical observations have also indicated that alterations in circulating AAs correlate with adverse cardiovascular events.[18–21] A multi-omics analysis has demonstrated that myocardial AA metabolism is robustly changed in HCM.[22] Thus, we explored whether circulating AA and derivatives concentrations are changed in patients with HCM and if they could serve as promising biomarkers for HCM screening.
In the present study, we recruited 166 participants, including 57 normal controls (NCs), 57 cases of HOCM, and 52 cases of HNCM, to depict the AA and derivatives profiles, and identify possible screening biomarkers utilizing high-performance liquid chromatography–mass spectrometry (HPLC–MS)-based targeted AA metabolomics. Three separate classification algorithms, including random forest (RF), support vector machine (SVM), and logistic regression (LR), were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.
Conclusions:
The present study delineated the distinct AA and derivatives profiles in HCM patients compared to NCs. Utilizing multiple algorithms, two separate AA and derivatives panels were established to discriminate HCM patients from NCs as well as HOCM from HNCM. These easily available screening models may have potential value as alternative non-invasive and objective biomarkers for HCM screening and diagnosis. Further proper identification of HOCM from HNCM by health professionals may assist in prompt diagnosis and precise clinical care. | Background:
Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM.
Methods:
Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.
Results:
The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively.
Conclusions:
The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive. | 13,656 | 454 | [
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] | [CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY] | [CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY] | [CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY] | [CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY] | [CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY] | [CONTENT] Hypertrophic cardiomyopathy | Amino acids | Targeted metabolomics | Biomarkers | Algorithm [SUMMARY] | [CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY] | [CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY] | [CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY] | [CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY] | [CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY] | [CONTENT] Humans | Amino Acids | Cysteine | Cardiomyopathy, Hypertrophic | Biomarkers | Proline | Arginine | Ornithine | Glycine | Choline [SUMMARY] | [CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY] | [CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY] | [CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY] | [CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY] | [CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY] | [CONTENT] hypertrophic cardiomyopathy hncm | cardiomyopathy hncm non | hcm left ventricular | hcm normal ventricular | echocardiographic characteristics hcm [SUMMARY] | [CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY] | [CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY] | [CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY] | [CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY] | [CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY] | [CONTENT] hcm | hocm | hncm | 00 | nc | patients | screening | aa | figure | auc [SUMMARY] | [CONTENT] high | hcm | screening | biomarkers | imaging | aa | myocardial | circulating | lvotg | hocm [SUMMARY] | [CONTENT] hcm | samples | variables | exercise | study | usa | exercise stress | model | lvotg | potential biomarkers [SUMMARY] | [CONTENT] 00 | hcm | 001 | nc | figure | vs | supplementary | auc | diastolic | mitral [SUMMARY] | [CONTENT] diagnosis | hcm patients | hcm | patients | aa derivatives | derivatives | screening | algorithms separate aa derivatives | prompt | easily available screening [SUMMARY] | [CONTENT] hcm | hocm | patients | screening | 00 | aa | hncm | samples | figure | study [SUMMARY] | [CONTENT] hcm | hocm | patients | screening | 00 | aa | hncm | samples | figure | study [SUMMARY] | [CONTENT] HCM ||| HCM | HCM | two ||| HCM ||| HCM [SUMMARY] | [CONTENT] Plasma | 166 | 57 | HOCM | 52 | 57 | first | the International Cooperation Center for HCM | Xijing Hospital | between December 2019 and September 2020 | AA metabolomics ||| Three | HCM | HCM | NC | HNCM [SUMMARY] | [CONTENT] HCM | NC ||| Four | Panel A | HCM ||| ROC | Panel A | ROC | 0.83 | 0.75-0.91 | 0.79 | 0.65 ||| 10 | HOCM | HNCM | 3 | two ||| AUC | 0.83 | 0.74-0.93 | 0.82 | 0.66 [SUMMARY] | [CONTENT] HCM | NC ||| two | HCM [SUMMARY] | [CONTENT] HCM ||| HCM | HCM | two ||| HCM ||| HCM | 166 | 57 | HOCM | 52 | 57 | first | the International Cooperation Center for HCM | Xijing Hospital | between December 2019 and September 2020 | AA metabolomics ||| Three | HCM | HCM | NC | HNCM ||| ||| HCM | NC ||| Four | Panel A | HCM ||| ROC | Panel A | ROC | 0.83 | 0.75-0.91 | 0.79 | 0.65 ||| 10 | HOCM | HNCM | 3 | two ||| AUC | 0.83 | 0.74-0.93 | 0.82 | 0.66 ||| HCM | NC ||| two | HCM [SUMMARY] | [CONTENT] HCM ||| HCM | HCM | two ||| HCM ||| HCM | 166 | 57 | HOCM | 52 | 57 | first | the International Cooperation Center for HCM | Xijing Hospital | between December 2019 and September 2020 | AA metabolomics ||| Three | HCM | HCM | NC | HNCM ||| ||| HCM | NC ||| Four | Panel A | HCM ||| ROC | Panel A | ROC | 0.83 | 0.75-0.91 | 0.79 | 0.65 ||| 10 | HOCM | HNCM | 3 | two ||| AUC | 0.83 | 0.74-0.93 | 0.82 | 0.66 ||| HCM | NC ||| two | HCM [SUMMARY] |
||||||
Prevalence survey of healthcare-associated infections and antimicrobial use at the University Hospital "Paolo Giaccone", Palermo, Italy. | 24779280 | Healthcare-associated infections (HAIs) and antimicrobial resistance are well known major public health threats. The first goal of our study was to describe the prevalence of HAI, while the second goal was to describe the antibiotic consumption at our University Hospital, "P. Giaccone" in Palermo, Italy. | INTRODUCTION | A standardized methodology for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use in European acute care hospital developed by the European Centre for Disease Prevention and Control (ECDC) was piloted across Europe. The teaching Hospital "P. Giaccone" in Palermo, Italy, participated in the study. | METHODS | Out of 328 surveyed patients, 12 (3.6%) had an HAI and 159 (48.5%) were receiving at least one antimicrobial agent. Prevalence results were highest in intensive care units, with 17.6% patients with HAI. Bloodstream infections represented the most common type (50%) of HAI. Surgical prophylaxis was the indication for antimicrobial prescribing in 59 (37.1%) out of 159 patients and exceeded 24 hours in 54 (91.5%) cases. | RESULTS | The results suggest that in our hospital there was a frequent and inappropriate use of antimicrobials, especially in the setting of surgical prophylaxis. | DISCUSSION | [
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"Humans",
"Intensive Care Units",
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] | 4718321 | Introduction | Healthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.
Excessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.
On September 2011, the teaching Hospital "Paolo Giaccone" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.
The specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level. | Methods | SETTING The Teaching Hospital "P. Giaccone" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.
The Teaching Hospital "P. Giaccone" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.
POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].
A series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: "Hospital data form" including general information on the type of surveyed hospital and "Patient data form" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.
In the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.
The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].
A series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: "Hospital data form" including general information on the type of surveyed hospital and "Patient data form" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.
In the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.
CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:
signs and symptoms fulfilled the survey definitions of HAI and were present on the survey date
or
signs and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.
European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:
signs and symptoms fulfilled the survey definitions of HAI and were present on the survey date
or
signs and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.
DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:
community-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;
healthcare infection acquired in long term care facility or chronic care hospital (LI);
acute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;
Surgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;
Medical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;
other indications (e.g. use of erythromycin as a prokinetic agent);
unknown indication/reason (assessed during PPS);
unknown/missing information on indication no verified during PPS.
The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:
community-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;
healthcare infection acquired in long term care facility or chronic care hospital (LI);
acute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;
Surgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;
Medical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;
other indications (e.g. use of erythromycin as a prokinetic agent);
unknown indication/reason (assessed during PPS);
unknown/missing information on indication no verified during PPS. | Results | A total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).
Twelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).
Overall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).
Pattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.
ECDC: European Centre for Disease Prevention and Control.
Total of patients treated for relative indication.
Percent of the total.
Patients receiving the indicated number of antimicrobial agents for each indication of treatment.
Percent each within category.
In 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.
Distribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).
Anatomical Therapeutic Chemical
In MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).
Data on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae. | null | null | [
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"The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.",
"The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.",
"European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.",
"The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS."
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null,
null,
null,
null
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"CASE DEFINITION",
"DEFINITIONS OF ANTIMICROBIAL USE DATA",
"Results",
"Discussion"
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"Healthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.\nExcessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.\nOn September 2011, the teaching Hospital \"Paolo Giaccone\" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.\nThe specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level.",
" SETTING The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\nThe Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.\n POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\nThe standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.\n CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\nEuropean case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.\n DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.\nThe antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.",
"The Teaching Hospital \"P. Giaccone\" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.",
"The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].\nA series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: \"Hospital data form\" including general information on the type of surveyed hospital and \"Patient data form\" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.\nIn the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.",
"European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:\nsigns and symptoms fulfilled the survey definitions of HAI and were present on the survey date\nor\nsigns and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.",
"The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:\ncommunity-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;\nhealthcare infection acquired in long term care facility or chronic care hospital (LI);\nacute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;\nSurgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;\nMedical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;\nother indications (e.g. use of erythromycin as a prokinetic agent);\nunknown indication/reason (assessed during PPS);\nunknown/missing information on indication no verified during PPS.",
"A total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).\nTwelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).\nOverall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).\nPattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.\nECDC: European Centre for Disease Prevention and Control.\nTotal of patients treated for relative indication.\nPercent of the total.\nPatients receiving the indicated number of antimicrobial agents for each indication of treatment.\nPercent each within category.\nIn 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.\nDistribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).\nAnatomical Therapeutic Chemical\nIn MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).\nData on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae.",
"HAIs are a serious problem, contributing heavily to the burden of the hospital costs [7]. These infections have a negative impact on patient because of the worsening of underlying medical condition and the increased morbidity and mortality [7].\nThe rapidly escalating prevalence of antimicrobial resistance is a global concern. The reduced susceptibility to most current of available antimicrobial agents coupled with the progressive shortage of newly approved compounds is a worrisome situation [7]. Major problems are encountered for a growing number of Gram-positive organisms (i.e., Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus spp.) and Gram-negative pathogens (i.e., Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae) [7]. Serious infections caused by resistant bacteria do not respond to therapy and are often associated with worse outcomes, including increased rates of complications, additional costs, higher associated mortality rates and prolonged hospital stays [8].\nHAIs' prevalence of 3.6% observed in our survey is lower than that reported in other European studies and that observed in 2008 (6.7%) in the same hospital. The limits of the HAI's estimate by PPS are well known. However, because case-mix and admission rules are not changed in the last years, it can be presumed that some prevention and control interventions have been to some extent effective. In particular\nsome guidelines were issued to implement infection control procedures, such as Antibiotic perisurgical prophylaxis in adults, Ambulances hygiene, handwashing (Clean care is safer care), Isolation measures in the AOUP P. Giaccone, Palermo, isolation of patients with colonization/infection by multi-resistant pathogens. So healthcare workers training programs were carried out as well.\nThe European prevalence has been estimated to be 7.1% by ECDC, based on a review of 30 national or multicentre PPS in 19 countries in its annual epidemiological report for 2008 [4, 6]. However an important issue that should be considered for the interpretation of the epidemiological results of this and future surveys is the standardization of data collection in participating hospitals to compare results between hospitals, regions and countries. Indeed, HAI prevalence may depend on differences in methodology and patients case-mix and not only on performance variations.\nHigh rates of antibiotic usage were observed in our hospital. In particular our survey revealed use of broad spectrum antibiotics, sometimes combined in multidrug protocols, because of emergence of antibiotic-resistant microorganisms [9]. On the other hand, due to the widespread diffusion of MDR organisms in our healthcare settings, it is often necessary to start an initial antibiotic therapy with multidrug protocols or last resource molecules, such as colistin. New diagnostic methods that allow to obtain microbiological confirmation in short time could be very useful. It is known that the excessive and unnecessary use of antibiotics is the main driving force of the high rates of drug resistant infections we are seeing in recent years [10, 11].\nThe results of this survey confirm those of a parallel analysis with another classical approach in which the antibiotic use in the hospital was measured by evaluating the numbers of defined daily doses (DDD) (Malta R et al., 2010) for 100 bed days or for number of admissions. The DDD consumed in 2011 were in fact 82.06 as per 100 bed-days and 5.41 per admission. For the surgical wards, overall the DDD were 94.00 per100 bed-days and 5.98 per admission; quinolones accounted for 25.6% of the DDD consumed, cephalosporins for 23.7%, penicillins for 16.2% and metronidazole for 14.4%. Moreover, DDD consumed for 100 bed-days in our hospital proved to be higher than those observed in a sample of five hospitals in Emilia Romagna despite increasing by 18% in the three years period, 64.9 DDD/100 bed days in 2002 to 76.7 in 2004 [9, 12, 14].\nResults showed also that a significant area of antibacterial overuse was SP. According to the international consensus, antibiotic prophylaxis in surgery involves the administration of an antibacterial agent for a very short time, temporally located just before the beginning of intervention [12]. However, our study revealed that the antibiotics were used for longer intervals of times. A reason of inappropriate antibacterial drug use was the frequent administration as SP of drugs, such as 3rd generation cephalosporins, which are not recommended for surgical prophylaxis and should be reserved to the treatment of severe active infections.\nOur study has some limitation. Indeed the PP study design has some inherent limits, including reduced periods of observation and the possibility of obtaining biased results. It has the advantage to be very economical in terms of both time and human and financial resources. Repeated PPS represent a more feasible alternative for hospital-wide surveillance of all HAIs, while still allowing the estimation of HAIs burden in acute hospitals, and helping to prioritize areas requiring interventions [1]. Therefore, although it was carried out in full compliance with the European protocol, the study has likely some limitations inherent to the study design. Continuous surveillance, especially prospective active surveillance, is the gold standard to improve patient safety.\nThe HAI prevalence rate detected in our PPS was lower than expected. However, our investigation highlighted a large misuse of antibacterial drugs, in particular in the area of SP. Prophylactic use of antibiotics in surgery is an example of how a potentially useful practice can be misused. More in general, inappropriate antibiotic use can lead to increased morbidity, prolonged hospital stay and need for more expensive drugs [13, 14]. The main aim of antibiotic stewardship is to bring a change in prescribing which could lead to control of drug resistance, decreased costs and improved quality of antibiotic use. Approaches that have to be, also locally, adopted include educational programs, development of prescribing guidelines (e.g. Clean Care is Safer Care, WHO) [15], monitoring of drug resistance patterns, turning to restrictive hospital formularies [16, 17]."
] | [
"introduction",
"methods",
null,
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] | [
"Healthcare-associated infections",
"Antimicrobials drug resistance",
"Point Prevalence Survey"
] | Introduction:
Healthcare-associated infections (HAIs) are an important cause of morbidity and mortality worldwide [1]. The annual prevalence of HAIs in Italy is estimated to range between 5% and 8% by year. The most frequent HAIs are the lower respiratory tract infections (LRTIs), followed by urinary tract infections (UTIs), bloodstream infections (BSIs) and surgical site infections (SSIs) [1]. In our country about 450,000-700,000 patients acquire at least one HAI during hospital stay [1-3]. Thus, HAI's surveillance is recognized as a crucial activity for the prevention and control programs.
Excessive and inappropriate use of antibacterial drugs is a major public health problem worldwide, since it is associated to an alarming increase in drug resistance and adverse drug reactions and causes also huge economical costs [4]. Available international and national data about amount and pattern of antibiotic use in both community and hospital setting are not always sufficiently informative because national databases may use different and no standardized methods to measure antibiotic use. To this aim, PPS (Point Prevalence Survey) can provide baseline information about occurrence and distribution of HAI and antibiotic use in healthcare institutions.
On September 2011, the teaching Hospital "Paolo Giaccone" of Palermo, Italy, participated to the Europeanscale Prevalence Survey for the Surveillance of Healthcare- Associated Infections (HAI) and Antimicrobial Use in Acute Care Hospitals [2]. This project was implemented by the European Center for Disease Prevention and Control (ECDC) through a protocol providing a standardized methodology to the Member States and the participating hospitals.
The specific objectives of Prevalence Survey were to estimate the total burden of HAI and antimicrobial use, by applying a standardized European methodology, and to disseminate the results at a local, regional, national and EU level.
Methods:
SETTING The Teaching Hospital "P. Giaccone" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.
The Teaching Hospital "P. Giaccone" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.
POINT PREVALENCE SURVEY (PPS) The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].
A series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: "Hospital data form" including general information on the type of surveyed hospital and "Patient data form" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.
In the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.
The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].
A series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: "Hospital data form" including general information on the type of surveyed hospital and "Patient data form" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.
In the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.
CASE DEFINITION European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:
signs and symptoms fulfilled the survey definitions of HAI and were present on the survey date
or
signs and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.
European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:
signs and symptoms fulfilled the survey definitions of HAI and were present on the survey date
or
signs and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.
DEFINITIONS OF ANTIMICROBIAL USE DATA The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:
community-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;
healthcare infection acquired in long term care facility or chronic care hospital (LI);
acute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;
Surgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;
Medical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;
other indications (e.g. use of erythromycin as a prokinetic agent);
unknown indication/reason (assessed during PPS);
unknown/missing information on indication no verified during PPS.
The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:
community-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;
healthcare infection acquired in long term care facility or chronic care hospital (LI);
acute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;
Surgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;
Medical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;
other indications (e.g. use of erythromycin as a prokinetic agent);
unknown indication/reason (assessed during PPS);
unknown/missing information on indication no verified during PPS.
SETTING:
The Teaching Hospital "P. Giaccone" of Palermo, Italy, is a tertiary care hospital with 575 beds, of which 497 reserved to acute and 16 to intensive care, with nearly 20,400 admissions annually. The number of patient-days per year is about 151,700. This hospital in the Europeanscale Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use accounted for western Sicily.
POINT PREVALENCE SURVEY (PPS):
The standardized protocol for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use that we used, represents the final protocol (version 4) defined in 2011-2012 by ECDC, which carried out a review of 17 national or regional point prevalence surveys of HAI (and antimicrobial use) in European countries (ECDC Annual Epidemiological report 2008) [2, 5, 6].
A series of inclusion requirements had to be met. All acute care units were included, but long-term care units, accident and emergency (A&E) Departments, patients admitted to same-day treatment or surgery, the outpatient departments, emergency room and dialysis were ruled out. Data recovered for each patient aimed to identify an active HAI and/or the use of antimicrobial drugs at the time of the survey. Data had to be collected in a single day for each ward/unit. The total time frame of data collection for all units of a single hospital had not to exceed 2-3 weeks. Data were collected using two forms: "Hospital data form" including general information on the type of surveyed hospital and "Patient data form" subdivided in three parts: the first one dedicated to demographic and clinical data, the second one describing antimicrobials use and the last one regarding HAI.
In the pilot study, ECDC recommended that data collection was to be carried out by experienced staff in reading patient charts/notes and in HAIs' identifying (e.g. infection control professionals, clinical microbiologists, infectious disease specialists). In particular, in our survey, data collectors (HAIs' prevention and surveillance staff, residents) were previously trained by the national PPS coordinators to become familiar with protocol and case definition.
CASE DEFINITION:
European case definition for HAI was used according to previous surveillance projects (HELICS or other European projects), whereas case definitions from the National Healthcare safety, Centers for Disease Control and Prevention (CDC), were otherwise used. In the HAIs section, data on microorganisms and their resistance phenotype were collected. Only results that were already available at the time of the survey were included. Data were also collected for patients showing an active HAI on the day of the survey. A HAI was defined as active when:
signs and symptoms fulfilled the survey definitions of HAI and were present on the survey date
or
signs and symptoms fulfilling the survey definitions of HAI had been present in the past and the patient was still receiving treatment on the survey date.
DEFINITIONS OF ANTIMICROBIAL USE DATA:
The antimicrobial-related informations were only collected if patient was receiving antimicrobials at the time of the survey or alternatively had an active HAI. Both generic and brand names were allowed. The drugs included the ATC classes J01 (antibacterials), J02 (antifungals) and J04 (antimycobacterials). The route of administration was also recorded. The patients could receive systemic antimicrobials for:
community-acquired infection (CI): all infections already present at admission except for those correlated to a previous hospitalization;
healthcare infection acquired in long term care facility or chronic care hospital (LI);
acute hospital acquired infection (HI): this also applied to hospital-acquired infections occurring after discharge and for occupational infections among staff of the structure;
Surgical Prophylaxis (SP): any single dose of an antimicrobial agent given within the 24-hour period before 8:00 am on the day of the survey. This time window for surgical prophylaxis allowed for distinguish single dose prophylaxis, one day prophylaxis or prophylactic doses given over more than one day;
Medical Prophylaxis (MP): it included antibiotic therapy administered to prevent a disease or its recurrence;
other indications (e.g. use of erythromycin as a prokinetic agent);
unknown indication/reason (assessed during PPS);
unknown/missing information on indication no verified during PPS.
Results:
A total of 328 patients, of whom 161 females (49.1%), were included in the PPS. Twenty-one patients were infants aged 0-10 months. The mean age of the remaining 307 patients was 61.8 [standard deviation (SD) 19.4] years for females and 60.4 (SD 18.8 years) for males. One hundred ninety patients out of 328 were hospitalized in clinical medicine departments (57.9%), 112 in surgical departments (34.1%) and 26% in intensive care units (ICUs).
Twelve (3.6%) out of the 328 patients had an HAI. One patient had a triple healthcare infection, so the total burden of HAIs was 14. BSIs represented the commonest type of HAI (7 out of 14), followed by UTIs (four out of 14), pneumonia, decubitus ulcer and SSIs. One (7.1%) central vascular catheter (CVC)-related infection was also identified. All UTIs were occurring in patients with urinary catheter. All of the six BSIs arose in patients with at least an invasive device in situ (CVC and/or peripheral vascular catheter, PVC) within for at least 48-hour. Prevalence was highest in ICUs (17.6%). In the department of gastroenterology two out of 17 patients had an active infection at the moment of survey similarly to the Department of Clinical Medicine Respiratory, where one out of nine patients had an HAI. The remaining infections were detected in the General Medicine ward (three out of 33 patients), Orthopedics and Traumatology ward (one out of 11 patients), Hematology (one out of 14 patients) and Emergency Surgery ward (one out of 20 patients had an HAI).
Overall, 159 (48.5%) out of 328 of the surveyed patients were prescribed one or more antimicrobials. Among the patients receiving at least one antimicrobial, 98 (61.6%) out of 159 were administrated with a single agent, 47 (29.5%) two and 14 (8.8%) received three agents. Overall 59 (37.1%) patients were prescribed antibiotics for surgical prophylaxis, 58 (36.5%) for a community infections, 27 (17.0%) for medical prophylaxis, 12 (7.5%) for HAI, three (1.9%) for LI (Tab. I).
Pattern of administration of antibiotics and indications of treatment in the surveyed patients during the PPS, 2011.
ECDC: European Centre for Disease Prevention and Control.
Total of patients treated for relative indication.
Percent of the total.
Patients receiving the indicated number of antimicrobial agents for each indication of treatment.
Percent each within category.
In 36 out of 73 (49.3%) of the cases of SP, cephalosporins were used, alone or in combination with other agents, followed by quinolones 16.4% (12 cases) and metronidazole 13.7% (10 cases) (Tab. II). Among 3rd generation cephalosporins, ceftriaxone, was the most frequently used.
Distribution of antimicrobial agents (ATC* 4th levels) by main indication for use, ECDC pilot point prevalence survey, 2011 (n = 223 antimicrobial agents).
Anatomical Therapeutic Chemical
In MP the quinolones were administered at a rate of 27.0% (10 out of 37), cephalosporins of 21.6% (8 out of 37) and metronidazole of 16.2% (6 out of 37). Of the 59 patients treated for surgical prophylaxis, only in one case the antibiotics were administered in single dose (1.7%), while in four cases for one day (6.8%) and in all the other cases (54 cases, 91.5%) for more than one day. In these last cases, the antibiotic administration was also prolonged after surgery. One antibiotic was administered to 37 (68.5%) out of 54 patients, while more than one antibiotic was given to the remaining 17 (31.5%).
Data on causal agents and their respective resistant phenotype were included only if they were already available on the date of the survey. Therefore, data on the species of microorganisms causing HAI were unavailable, except for six cases. Enterobacteriaceae (E. cloacae) and enterococci (E. faecalis and E. faecium) were associated with urinary tract, blood, decubitus ulcers, including both superficial and deep infections and SSI. One BSI was associated with Cytomegalovirus. Candida albicans and Acinetobacter baumannii were also identified. This last one affected three infection sites in a single patient (decubitus ulcer, bloodstream and urinary tract). Resistance to third generation cephalosporin and carbapenem was reported for E. cloacae.
Discussion:
HAIs are a serious problem, contributing heavily to the burden of the hospital costs [7]. These infections have a negative impact on patient because of the worsening of underlying medical condition and the increased morbidity and mortality [7].
The rapidly escalating prevalence of antimicrobial resistance is a global concern. The reduced susceptibility to most current of available antimicrobial agents coupled with the progressive shortage of newly approved compounds is a worrisome situation [7]. Major problems are encountered for a growing number of Gram-positive organisms (i.e., Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus spp.) and Gram-negative pathogens (i.e., Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae) [7]. Serious infections caused by resistant bacteria do not respond to therapy and are often associated with worse outcomes, including increased rates of complications, additional costs, higher associated mortality rates and prolonged hospital stays [8].
HAIs' prevalence of 3.6% observed in our survey is lower than that reported in other European studies and that observed in 2008 (6.7%) in the same hospital. The limits of the HAI's estimate by PPS are well known. However, because case-mix and admission rules are not changed in the last years, it can be presumed that some prevention and control interventions have been to some extent effective. In particular
some guidelines were issued to implement infection control procedures, such as Antibiotic perisurgical prophylaxis in adults, Ambulances hygiene, handwashing (Clean care is safer care), Isolation measures in the AOUP P. Giaccone, Palermo, isolation of patients with colonization/infection by multi-resistant pathogens. So healthcare workers training programs were carried out as well.
The European prevalence has been estimated to be 7.1% by ECDC, based on a review of 30 national or multicentre PPS in 19 countries in its annual epidemiological report for 2008 [4, 6]. However an important issue that should be considered for the interpretation of the epidemiological results of this and future surveys is the standardization of data collection in participating hospitals to compare results between hospitals, regions and countries. Indeed, HAI prevalence may depend on differences in methodology and patients case-mix and not only on performance variations.
High rates of antibiotic usage were observed in our hospital. In particular our survey revealed use of broad spectrum antibiotics, sometimes combined in multidrug protocols, because of emergence of antibiotic-resistant microorganisms [9]. On the other hand, due to the widespread diffusion of MDR organisms in our healthcare settings, it is often necessary to start an initial antibiotic therapy with multidrug protocols or last resource molecules, such as colistin. New diagnostic methods that allow to obtain microbiological confirmation in short time could be very useful. It is known that the excessive and unnecessary use of antibiotics is the main driving force of the high rates of drug resistant infections we are seeing in recent years [10, 11].
The results of this survey confirm those of a parallel analysis with another classical approach in which the antibiotic use in the hospital was measured by evaluating the numbers of defined daily doses (DDD) (Malta R et al., 2010) for 100 bed days or for number of admissions. The DDD consumed in 2011 were in fact 82.06 as per 100 bed-days and 5.41 per admission. For the surgical wards, overall the DDD were 94.00 per100 bed-days and 5.98 per admission; quinolones accounted for 25.6% of the DDD consumed, cephalosporins for 23.7%, penicillins for 16.2% and metronidazole for 14.4%. Moreover, DDD consumed for 100 bed-days in our hospital proved to be higher than those observed in a sample of five hospitals in Emilia Romagna despite increasing by 18% in the three years period, 64.9 DDD/100 bed days in 2002 to 76.7 in 2004 [9, 12, 14].
Results showed also that a significant area of antibacterial overuse was SP. According to the international consensus, antibiotic prophylaxis in surgery involves the administration of an antibacterial agent for a very short time, temporally located just before the beginning of intervention [12]. However, our study revealed that the antibiotics were used for longer intervals of times. A reason of inappropriate antibacterial drug use was the frequent administration as SP of drugs, such as 3rd generation cephalosporins, which are not recommended for surgical prophylaxis and should be reserved to the treatment of severe active infections.
Our study has some limitation. Indeed the PP study design has some inherent limits, including reduced periods of observation and the possibility of obtaining biased results. It has the advantage to be very economical in terms of both time and human and financial resources. Repeated PPS represent a more feasible alternative for hospital-wide surveillance of all HAIs, while still allowing the estimation of HAIs burden in acute hospitals, and helping to prioritize areas requiring interventions [1]. Therefore, although it was carried out in full compliance with the European protocol, the study has likely some limitations inherent to the study design. Continuous surveillance, especially prospective active surveillance, is the gold standard to improve patient safety.
The HAI prevalence rate detected in our PPS was lower than expected. However, our investigation highlighted a large misuse of antibacterial drugs, in particular in the area of SP. Prophylactic use of antibiotics in surgery is an example of how a potentially useful practice can be misused. More in general, inappropriate antibiotic use can lead to increased morbidity, prolonged hospital stay and need for more expensive drugs [13, 14]. The main aim of antibiotic stewardship is to bring a change in prescribing which could lead to control of drug resistance, decreased costs and improved quality of antibiotic use. Approaches that have to be, also locally, adopted include educational programs, development of prescribing guidelines (e.g. Clean Care is Safer Care, WHO) [15], monitoring of drug resistance patterns, turning to restrictive hospital formularies [16, 17].
| Background:
Healthcare-associated infections (HAIs) and antimicrobial resistance are well known major public health threats. The first goal of our study was to describe the prevalence of HAI, while the second goal was to describe the antibiotic consumption at our University Hospital, "P. Giaccone" in Palermo, Italy.
Methods:
A standardized methodology for a combined Point Prevalence Survey (PPS) on healthcare-associated infections (HAIs) and antimicrobial use in European acute care hospital developed by the European Centre for Disease Prevention and Control (ECDC) was piloted across Europe. The teaching Hospital "P. Giaccone" in Palermo, Italy, participated in the study.
Results:
Out of 328 surveyed patients, 12 (3.6%) had an HAI and 159 (48.5%) were receiving at least one antimicrobial agent. Prevalence results were highest in intensive care units, with 17.6% patients with HAI. Bloodstream infections represented the most common type (50%) of HAI. Surgical prophylaxis was the indication for antimicrobial prescribing in 59 (37.1%) out of 159 patients and exceeded 24 hours in 54 (91.5%) cases.
Conclusions:
The results suggest that in our hospital there was a frequent and inappropriate use of antimicrobials, especially in the setting of surgical prophylaxis. | null | null | 4,818 | 247 | [
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||||
Clinical assessment of liver metabolism during hypothermic oxygenated machine perfusion using microdialysis. | 34516020 | While growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood. | BACKGROUND | To assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome. | METHODS | Median (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2nd-hour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN. These correlations were not apparent when perfusate levels were considered. In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts. | RESULTS | MD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment. | CONCLUSION | [
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] | 9292750 | INTRODUCTION | Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),
1
,
2
,
3
extended‐criteria donors after brain death (DBD)
4
,
5
, and steatotic grafts.
6
The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,
7
better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,
8
,
9
and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,
10
HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,
11
,
12
thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.
Little is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.
Microdialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.
13
,
14
,
15
,
16
,
17
In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,
18
,
19
,
20
,
21
,
22
and explored as a tool for early detection of ischemic complications and acute rejection.
23
,
24
,
25
,
26
,
27
,
28
,
29
As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.
30
MD has been occasionally used in the setting of kidney machine perfusion
31
,
32
,
33
but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.
The aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT. | null | null | RESULTS | D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.
Baseline characteristics of recipients and donors
Abbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.
Developed early allograft dysfunction.
Developed graft failure.
Operational characteristics of liver grafts
Abbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.
Cold ischemia time from cold perfusion in the donor to D‐HOPE start.
Time from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.
Median duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.
Study variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).
Study variables in the whole series and according to the onset of early allograft dysfunction
Data are presented as median [IQR] or number (%), as appropriate.
Abbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.
Concerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.
With regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.
Four (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.
Median follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.
Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.
Baseline characteristics of recipients and donors
Abbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.
Developed early allograft dysfunction.
Developed graft failure.
Operational characteristics of liver grafts
Abbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.
Cold ischemia time from cold perfusion in the donor to D‐HOPE start.
Time from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.
Median duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.
Study variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).
Study variables in the whole series and according to the onset of early allograft dysfunction
Data are presented as median [IQR] or number (%), as appropriate.
Abbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.
Concerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.
With regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.
Four (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.
Median follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.
Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.
Glucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η
2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]
Kinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.
Line plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]
2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).
Correlation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]
Levels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.
Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.
Glucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η
2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]
Kinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.
Line plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]
2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).
Correlation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]
Levels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.
Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).
Non‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]
Correlation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]
Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).
Non‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]
Correlation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]
Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).
Panel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]
Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).
Panel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com] | null | null | [
"INTRODUCTION",
"PATIENTS AND METHODS",
"Study design",
"Outcome measures",
"Statistical analysis",
"D‐HOPE procedure and clinical outcome",
"Microdialysate and perfusate metabolites",
"Microdialysate and perfusate FMN",
"Histology and expression of inflammatory cytokines",
"AUTHOR CONTRIBUTIONS"
] | [
"Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\n1\n, \n2\n, \n3\n extended‐criteria donors after brain death (DBD)\n4\n, \n5\n, and steatotic grafts.\n6\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\n7\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\n8\n, \n9\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\n10\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\n11\n, \n12\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\n13\n, \n14\n, \n15\n, \n16\n, \n17\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\n18\n, \n19\n, \n20\n, \n21\n, \n22\n and explored as a tool for early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\n30\n MD has been occasionally used in the setting of kidney machine perfusion\n31\n, \n32\n, \n33\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT.",
" Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\nPrimary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).",
"This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.",
"Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.",
"Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).",
"Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.",
"Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.",
"Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]",
"Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]",
"Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision."
] | [
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null,
null,
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null,
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] | [
"INTRODUCTION",
"PATIENTS AND METHODS",
"Study design",
"Outcome measures",
"Statistical analysis",
"RESULTS",
"D‐HOPE procedure and clinical outcome",
"Microdialysate and perfusate metabolites",
"Microdialysate and perfusate FMN",
"Histology and expression of inflammatory cytokines",
"DISCUSSION",
"CONFLICT OF INTEREST",
"AUTHOR CONTRIBUTIONS",
"Supporting information"
] | [
"Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),\n1\n, \n2\n, \n3\n extended‐criteria donors after brain death (DBD)\n4\n, \n5\n, and steatotic grafts.\n6\n The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,\n7\n better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,\n8\n, \n9\n and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,\n10\n HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,\n11\n, \n12\n thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.\nLittle is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.\nMicrodialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.\n13\n, \n14\n, \n15\n, \n16\n, \n17\n In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,\n18\n, \n19\n, \n20\n, \n21\n, \n22\n and explored as a tool for early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.\n30\n MD has been occasionally used in the setting of kidney machine perfusion\n31\n, \n32\n, \n33\n but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.\nThe aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT.",
" Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\nThis was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.\n Outcome measures Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\nPrimary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.\n Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).\nVariables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).",
"This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.\n34\n\n\nOur machine perfusion and LT protocols have been previously described.\n4\n, \n34\n, \n35\n, \n36\n At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.\nMD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.\n37\n The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.\n13\n, \n38\n, \n39\n The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.\nSynopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]\nDuring D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.\n34\n Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.\n40\n, \n41\n FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.\nD‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.\nCollected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.",
"Primary outcome measures were early allograft dysfunction (EAD)\n42\n and L‐GrAFT score.\n43\n Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),\n44\n and comprehensive complication index\n45\n (CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.",
"Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).",
" D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\nRecipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.\n Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\nFigure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.\n Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\nFigure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]\n Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]\nHistological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]",
"Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.\nBaseline characteristics of recipients and donors\nAbbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.\nDeveloped early allograft dysfunction.\nDeveloped graft failure.\nOperational characteristics of liver grafts\nAbbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.\nCold ischemia time from cold perfusion in the donor to D‐HOPE start.\nTime from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.\nMedian duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.\nStudy variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).\nStudy variables in the whole series and according to the onset of early allograft dysfunction\nData are presented as median [IQR] or number (%), as appropriate.\nAbbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.\nConcerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.\nWith regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.\nFour (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.\nMedian follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.",
"Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.\nGlucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η\n2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]\nKinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.\nLine plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]\n2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).\nCorrelation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]\nLevels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.",
"Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).\nNon‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]\nCorrelation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]",
"Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).\nPanel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]",
"In the setting of clinical LT, the use of MD has been explored mainly as a tool for monitoring and early detection of ischemic complications and acute rejection.\n23\n, \n24\n, \n25\n, \n26\n, \n27\n, \n28\n, \n29\n However, one fascinating feature of this technology is the possibility of evaluating liver metabolism during different phases of organ retrieval, static cold storage, implantation, and early postoperative course.\n18\n, \n19\n, \n21\n, \n22\n In particular, the studies by Nowak et al.\n19\n and Silva et al.\n21\n have clearly shown that both glucose and lactate slowly build up during SCS, whereas their levels increase steeply during implantation and organ reperfusion, to return progressively to baseline levels within 3 h following reperfusion. Perera et al.\n46\n have shown that end‐ischemic interstitial lactate level and lactate/pyruvate ratio are higher in grafts from DCD donors, this finding being associated with more severe glycogen depletion. Similarly, glutamate also appears to accumulate during SCS, reaching end‐ischemic levels of approximately 300 μmol/L, which progressively normalize in the hours following graft reperfusion.\n20\n In contrast, pyruvate quickly becomes undetectable during SCS (reflecting lack of metabolic activity during this phase), rises above pre‐SCS values upon reperfusion (suggesting a phase of hypermetabolism) to return to baseline in the following hours.\n19\n\n\nThe release of glucose in the extracellular space represents a hepatic‐specific response to ischemia and it has been interpreted as a result of glycogen breakdown.\n19\n In other tissues, ischemic events are associated with very low interstitial glucose. Interestingly, the rate of glucose and lactate accumulation increases during implantation\n19\n and back table preparation,\n21\n suggesting a potential role for temperature during these phases. Previous studies have suggested a prognostic value of MD metabolites, as patients developing initial poor function had higher MD lactate level during back table preparation and delayed clearance after graft reperfusion.\n21\n, \n46\n\n\nIn this study, we sought to use MD to deepen our understanding of liver metabolism during SCS and D‐HOPE. Our main finding is that glucose and lactate are released in the extracellular space upon initiation of D‐HOPE and their levels correlate with other known predictors of graft function (cold ischemia time and macrosteatosis), FMN perfusate level, graft dysfunction, and worse clinical outcome.\nIn our study, D‐HOPE did not alter pyruvate and glutamate levels, as compared to the expected kinetics during SCS. In particular, pyruvate levels were almost undetectable and glutamate levels were constantly high, in keeping with findings from previous studies.\n20\n The lower end‐ischemic lactate (~1900 μmol/L) and glutamate (~200 μmol/L) concentrations observed in our study (~200 μmol/L), as compared to those observed in previous studies by Silva et al.\n20\n, \n21\n can be explained by the relatively short ischemia time before D‐HOPE (5 h 44 min) in our series. Interestingly, the only graft that failed in our series had constantly very low levels of MD glutamate during back table and D‐HOPE.\nWhy D‐HOPE was associated with a significant increase of glucose and lactate into microdialysate is open to several interpretations. Temperature may have played a role, as the device employed for machine perfusion operates at ~10°C. During rewarming, it is possible that increased metabolism enhanced glycogenolysis, which in turn resulted in increased glucose release. The composition of the perfusion fluid used for D‐HOPE, containing 180 mg/ml of dextrose, may also have influenced MD glucose level. However, it should be noted that glucose levels in microdialysate were lower than those in perfusate and their kinetics was different (Figure 3). In addition, perfusion fluid composition was unlikely to affect lactate levels and progressive accumulation of lactate in the renal cortex has also been observed in a study using MD in a model of hypothermic perfusion of porcine kidneys.\n33\n\n\nWhatever the mechanism, glucose and lactate released in the extracellular space during D‐HOPE can be interpreted as markers of graft injury. The rise of MD glucose and lactate during the first 2 h of D‐HOPE was more important in grafts that developed early dysfunction, whereas it was mild or absent in those exhibiting primary function (Figure 3). However, in the few cases that had a 3‐h MD sample available, glucose and lactate levels started to decrease also in EAD group (Table S2). Consequently, it seems that restoration of aerobic metabolism during D‐HOPE could take longer in more severely damaged grafts.\nImportantly, there was a significant discrepancy between perfusate and MD kinetics of glucose and lactate (Figure 3). Perfusate analysis, as a measure of larger and global compartment, did not seem to entirely capture metabolic changes happening at a cellular level, a finding that was more evident for glucose and which is in keeping with our previous study on ex‐vivo lung perfusion.\n30\n Perfusate glucose level increased during D‐HOPE independently of graft function, whereas MD levels of glucose were higher in EAD patients, with a significant difference during the 2nd hour of machine perfusion. As lactate is produced during anaerobic glycolysis and glucose is released during ischemia due to hepatocytes glycogenolysis, we might hypothesize that the grafts that developed EAD did not fully recover after SCS and continued to experience a certain degree of hypoxia during machine perfusion, as demonstrated by their metabolic profile. In line with this observation, glycogen content was reduced in EAD livers, likely due to glycogen depletion during cold preservation, an observation which is in keeping with findings from Perera et al.\n46\n\n\nThe next logical step was comparing these findings with perfusate and MD levels of FMN, a marker of mitochondrial injury that recently emerged as a promising tool for graft viability assessment.\n40\n, \n41\n As opposed to glucose and lactate, D‐HOPE start determined a steep decrease of MD FMN levels, suggesting its rapid washout from the extracellular space and confirming protection of mitochondria during D‐HOPE. In contrast, FMN appeared to accumulate in perfusate, with an incremental trend in grafts who developed EAD and with the highest levels observed in the only failing graft of this series (Figure 5). This finding, which is in keeping with those from Mueller et al.,\n40\n also suggests a mechanistic interpretation to the association between microdialysate metabolites and graft dysfunction. Taken as a whole, our data seem to point to the same direction: mitochondrial injury sustained during SCS, which is proportional to cold ischemia time and macrosteatosis, is reflected by the accumulation of FMN in perfusate and release of glucose and lactate in the extracellular space during D‐HOPE, highlighting their potential as viability markers. Noteworthy, these metabolites can be measured point‐of‐care using MD equipment during D‐HOPE.\nThis study has numerous implications. First, it confirms that precious information on graft function and injury can be gathered also during hypothermic perfusion, challenging the concept that normothermic perfusion is a prerequisite for graft viability assessment.\n47\n, \n48\n Second, while perfusate analysis appears handier, this study and previous experiences\n23\n, \n24\n, \n27\n, \n28\n suggest that MD is feasible without major modifications of routine practice. In our opinion, microdialysate and perfusate analysis should be seen as complementary rather than alternative techniques, as metabolites kinetics appears to be different in these two compartments. Real‐time assessment of graft function could drive not only graft acceptance but also influence the duration of machine perfusion, as more severely damaged grafts may require longer perfusion time to recover from ischemic injury.\nThird, this pilot study represents a baseline based on which other potential applications of MD during machine perfusion could be explored. In a recent study on perfusate analysis during D‐HOPE,\n34\n we identified alanine aminotransferase as the most reliable predictor of EAD. Unfortunately, MD ALT could not be evaluated due to its molecular weight (100 kDa). Further studies on the subject should ideally employ available 100 kDa MD catheters to allow measurement of other molecules, including inflammatory cytokines, as also suggested by a recent study.\n49\n Finally, MD could be used to assess liver metabolism and explore new viability criteria during normothermic machine perfusion, a setting mimicking normal physiology and characterized by longer perfusion times.\n50\n\n\nLimitations of our study include the small sample size of 10 grafts with rather heterogeneous characteristics, the lack of a power analysis, and the choice of EAD as an endpoint, which has been discouraged in machine perfusion trials as it strongly relies on transaminase level after transplant and could be influenced by the washout phenomenon during machine perfusion.\n51\n\n\nIn this study, microdialysis time was limited to less than 4 h by design, allowing complete measurement of MD metabolites at only 3 time points. As previous studies have shown a quick recovery of aerobic metabolism after liver reperfusion\n21\n, \n46\n and due to concerns about sterility breaches, we did not consider leaving the MD in place during this phase, possibly missing relevant metabolic changes.\n31\n Rapid sampling MD\n33\n could improve the yield of MD as applied to procedures of limited duration, whereas the use of an MD catheter with higher molecular weight cutoff membrane would allow measuring MD concentration of other potentially important molecules.\nIn conclusion, this study expands previous knowledge on liver metabolism during D‐HOPE and confirms that liver graft injury and function can be assessed during hypothermic machine perfusion. These preliminary findings require validation in larger studies allowing correlation of MD parameters with clinically relevant endpoints and possibly exploring further applications of MD in machine perfusion.",
"The authors of this manuscript have no conflicts of interest to disclose.",
"Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision.",
"Supplementary Material\nClick here for additional data file."
] | [
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"COI-statement",
null,
"supplementary-material"
] | [
"extracellular fluid",
"flavin mononucleotide",
"liver metabolism",
"liver viability assessment",
"machine perfusion",
"microdialysis"
] | INTRODUCTION:
Hypothermic oxygenated machine perfusion (HOPE) is gaining increasing interest as a tool to reduce ischemia‐reperfusion injury and to improve outcomes of liver transplantation (LT). In clinical studies, HOPE use has been associated with improved outcomes of grafts from donors after circulatory death (DCD),
1
,
2
,
3
extended‐criteria donors after brain death (DBD)
4
,
5
, and steatotic grafts.
6
The protective mechanism of HOPE appears to be manifold, being related to adenosine‐triphosphate replenishment, immunomodulation,
7
better preservation of endothelial cells glycocalyx, peri‐biliary vascular plexus, and peribiliary glands,
8
,
9
and, more importantly, modulation of mitochondrial respiration. As shown in 2013 by Schlegel et al.,
10
HOPE progressively decreases the rate of mitochondrial respiration and determines an oxidized state in mitochondria, limiting the production of reactive oxygen species by reverse electron transfer upon organ reperfusion,
11
,
12
thus resulting in diminished mitochondrial, nuclear, hepatocyte, and sinusoidal injury.
Little is known, however, about other aspects of liver metabolism during HOPE, especially concerning the transition between the phase of static cold storage (SCS) and HOPE.
Microdialysis (MD) is a technique by which interstitial fluid (microdialysate) can be sampled from a variety of tissues to measure the concentration of metabolites such as glucose, lactate, pyruvate, and glutamate, which diffuse into the extracellular space. MD has been extensively used in a variety of settings, especially for bedside sampling of cerebral interstitial fluid in critically ill patients.
13
,
14
,
15
,
16
,
17
In LT, MD has been used to assess liver metabolism during the phases of liver retrieval, cold preservation and after graft implantation,
18
,
19
,
20
,
21
,
22
and explored as a tool for early detection of ischemic complications and acute rejection.
23
,
24
,
25
,
26
,
27
,
28
,
29
As applied to machine perfusion, MD has the potential of allowing monitoring of liver metabolism throughout SCS and HOPE, overcoming one limitation of perfusate analysis, which cannot assess metabolism during SCS. In a study from our group, MD was used to assess the metabolism of lungs perfused ex‐vivo and emerged as a potential tool to discriminate lung function after transplantation.
30
MD has been occasionally used in the setting of kidney machine perfusion
31
,
32
,
33
but, to the best of our knowledge, its use in HOPE‐treated livers has not been reported.
The aim of this study was to assess the time course of liver metabolism biomarkers during SCS and HOPE using MD and to explore the potential role of MD for liver graft viability assessment in LT.
PATIENTS AND METHODS:
Study design This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.
34
Our machine perfusion and LT protocols have been previously described.
4
,
34
,
35
,
36
At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.
MD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.
37
The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.
13
,
38
,
39
The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.
Synopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]
During D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.
34
Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.
40
,
41
FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.
D‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.
Collected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.
This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.
34
Our machine perfusion and LT protocols have been previously described.
4
,
34
,
35
,
36
At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.
MD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.
37
The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.
13
,
38
,
39
The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.
Synopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]
During D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.
34
Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.
40
,
41
FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.
D‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.
Collected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.
Outcome measures Primary outcome measures were early allograft dysfunction (EAD)
42
and L‐GrAFT score.
43
Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),
44
and comprehensive complication index
45
(CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.
Primary outcome measures were early allograft dysfunction (EAD)
42
and L‐GrAFT score.
43
Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),
44
and comprehensive complication index
45
(CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.
Statistical analysis Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).
Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).
Study design:
This was a prospective, open‐label observational pilot study on 10 consecutive grafts treated with dual‐HOPE (i.e., double cannulation of both portal vein and hepatic artery—D‐HOPE) in the period October 2019–January 2020 at our Institution. The study was approved by the local ethics committee (resolution nr. 739 of June 10, 2019). Patients signed a consent form for receiving an organ treated with machine perfusion and for participating in the study. All study procedures complied with the Declaration of Helsinki and the Declaration of Istanbul (https://www.wma.net). Recipients of DBD grafts included in this study were also included in a recently published study on the value of perfusate analysis during D‐HOPE in predicting outcome after LT.
34
Our machine perfusion and LT protocols have been previously described.
4
,
34
,
35
,
36
At our Institution, the use of D‐HOPE is systematic for grafts from DCD donors, whereas it is evaluated on a case‐by‐case basis for grafts from DBD donors, mainly based on donor age and steatosis. In this study, end‐ischemic D‐HOPE using LiverAssist® (XVIVO, Groningen, The Netherlands) primed with 3 L of Belzer MP® fluid (Bridge to Life Europe Ltd. London, UK) was applied for a minimum of 90 min during recipient hepatectomy. The liver graft was weighed before and after machine perfusion to detect swelling during machine perfusion.
MD was used to sample extracellular fluid during back table preparation and subsequent machine perfusion. Study design and timing of sample collection are summarized in Figure 1. Briefly, the liver was perfused with Celsior® (IGL, Lisseu, France) at retrieval and transported in SCS at our center. The liver was unpacked upon arrival and, before the start of back table preparation, a 61 hepatic microdialysis catheter® with membrane length = 30 mm and membrane cutoff ~20 kDa (M Dialysis AB, Stockholm, Sweden) was inserted at a ~4 cm depth by the mean of a splitable introducer into liver segment 6 and secured by a 5/0 Prolene suture. The MD catheter was connected to a 107 Microdialysis pump® charged with normal saline with a flow set at 2 μl/min (after an initial 5‐min flush at 15 μl/min). By this setting, the concentration of metabolites in microdialysate represents roughly 40% of extracellular fluid concentration.
37
The first MD vial was connected to the MD catheter after the first drop of microdialysate appeared at the tip of the connection needle and discarded after 30 min to allow fluid equilibration, as recommended.
13
,
38
,
39
The second vial, which was the first to be analyzed, was collected at the end of backtable preparation, at least one hour after having been positioned, and was therefore representative of liver metabolism during the last part of SCS. Subsequently, the liver was connected to the perfusion device with the MD catheter in place and MD vials were changed hourly during D‐HOPE. Thus, at least 3 MD samples were available for every single procedure: the first representing the terminal phase of SCS, and the remaining two samples the D‐HOPE phase. When D‐HOPE time was <120 min, the last MD sample was collected at the end of machine perfusion, thus representing extracellular fluid concentration during the 2nd hour of D‐HOPE.
Synopsis of study design [Color figure can be viewed at wileyonlinelibrary.com]
During D‐HOPE, perfusate samples were collected every 30 min from the sampling port on the hepatic artery circuit and stored in cryotubes.
34
Both MD and perfusate samples were snap frozen and subsequently analyzed using CMA 600 Microdialysis Analyzer. The concentration of glucose, lactate, pyruvate, and glutamate was measured, and lactate/pyruvate ratio was calculated. Perfusate parameters were normalized to liver weight. Perfusate and microdialysate flavin mononucleotide (FMN) level was measured with Synergy HTX microplate reader (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 460/40 nm and recording the fluorescence with wavelength of 528/20 nm with 100% gain, as previously described.
40
,
41
FMN levels were expressed as relative fluorescing units (RFU) and, given the methodology used for their measurement, are presented both as non‐adjusted and adjusted values. The MD catheter was removed at the end of machine perfusion. Liver biopsies were collected at the beginning of backtable preparation, before and after D‐HOPE, and at the end of the transplant. Biopsies were immediately immersed in RNA Later solution (Invitrogen, Thermofisher) and freezed at −20°C for subsequent determination of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα), toll‐like receptor‐4 (TLR‐4), intercellular adhesion molecule 1 (ICAM1) and C‐X‐C Motif Chemokine Ligand 12 (CXCL12) expression (Table S1). Succinate level was measured on hepatic biopsies using the Succinate Colorimetric Assay Kit (K649, Biovision Incorporated, Milpitas, CA, USA) (Supporting Information). Histological ischemia‐reperfusion injury and steatosis were determined on liver biopsies obtained at the end of transplant. Tissue samples were fixed and processed to obtain 5‐µm‐thick sections. Hematoxylin and eosin, periodic acid‐Schiff, and periodic acid‐Schiff‐diastase staining were performed to evaluate steatosis, necrosis, as well as glycogen content and distribution.
D‐HOPE and MD were not used for graft evaluation and all livers in this series were transplanted. A t‐tube was routinely positioned during transplant operation and removed at 3 months after a control cholangiogram.
Collected variables included indication for LT, recipient, and donor features, as well as times and technical details about retrieval, initial SCS, D‐HOPE, and transplant operation. Levels and trend of microdialysate and perfusate metabolites were described in the whole cohort and analyzed with regards to their correlation with outcome measures, as outlined below.
Outcome measures:
Primary outcome measures were early allograft dysfunction (EAD)
42
and L‐GrAFT score.
43
Secondary outcome measures were duration of hospital stay, onset and grade of acute kidney injury (AKI),
44
and comprehensive complication index
45
(CCI) calculated at hospital discharge and at 6‐month follow‐up. Graft survival and occurrence of biliary complications were also evaluated. Minimum follow‐up was 6 months.
Statistical analysis:
Variables were expressed as number (%) or median (interquartile range) and compared using standard parametric and non‐parametric tests. All perfusate values were normalized to liver weight. For repeated measures ANOVA, normal distribution of variables was verified by Shapiro–Wilk test and by visual inspection using quantile‐quantile plots. p‐values of pairwise t‐tests between different timepoints were adjusted using the Bonferroni multiple testing correction method. Correlation between variables was evaluated using Pearson correlation coefficient. Optimal cutoff points in ROC analyses were calculated using Youden method. Considering the exploratory nature of this study, a convenience sample size = 10 was chose based on the number of D‐HOPE procedures per year at our Institution. All analyses were performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/).
RESULTS:
D‐HOPE procedure and clinical outcome Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.
Baseline characteristics of recipients and donors
Abbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.
Developed early allograft dysfunction.
Developed graft failure.
Operational characteristics of liver grafts
Abbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.
Cold ischemia time from cold perfusion in the donor to D‐HOPE start.
Time from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.
Median duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.
Study variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).
Study variables in the whole series and according to the onset of early allograft dysfunction
Data are presented as median [IQR] or number (%), as appropriate.
Abbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.
Concerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.
With regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.
Four (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.
Median follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.
Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.
Baseline characteristics of recipients and donors
Abbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.
Developed early allograft dysfunction.
Developed graft failure.
Operational characteristics of liver grafts
Abbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.
Cold ischemia time from cold perfusion in the donor to D‐HOPE start.
Time from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.
Median duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.
Study variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).
Study variables in the whole series and according to the onset of early allograft dysfunction
Data are presented as median [IQR] or number (%), as appropriate.
Abbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.
Concerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.
With regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.
Four (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.
Median follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.
Microdialysate and perfusate metabolites Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.
Glucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η
2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]
Kinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.
Line plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]
2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).
Correlation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]
Levels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.
Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.
Glucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η
2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]
Kinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.
Line plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]
2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).
Correlation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]
Levels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.
Microdialysate and perfusate FMN Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).
Non‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]
Correlation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]
Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).
Non‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]
Correlation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]
Histology and expression of inflammatory cytokines Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).
Panel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]
Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).
Panel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]
D‐HOPE procedure and clinical outcome:
Recipient and donor features are summarized in Table 1. Machine perfused grafts were preferentially allocated to low MELD (12.1 [9.1, 14.5]) patients, of whom 7 (70%) had hepatocellular carcinoma. The indication for D‐HOPE was based on advanced donor age (cases 6, 7, 8, and 10), high BMI and steatotic graft appearance (cases 3, 4, 5, and 9), advanced donor age in association with graft steatosis (case 1) and donation after circulatory death (case 2). Table 2 summarizes times and graft weight before and after machine perfusion. Cold ischemia and D‐HOPE time were 344 (295, 367) and 116 (103, 143) min. In 3 grafts weight increased ≥5% during D‐HOPE. Total preservation time never exceeded 10 h 15 min.
Baseline characteristics of recipients and donors
Abbreviations: BMI, body mass index; Crea, creatinine; DBD, donation after brain death; DCD, Maastricht category 3 donation after circulatory death; ETOH, alcoholic cirrhosis; GGT, gamma glutamyl transferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; M, macrovesicular steatosis; Na, sodium; NASH, non‐alcoholic steatohepatitis; PBC, primary biliary cholangitis; S, sex; μ, microvesicular steatosis.
Developed early allograft dysfunction.
Developed graft failure.
Operational characteristics of liver grafts
Abbreviations: BT, back table preparation; CIT, cold ischemia time; D‐HOPE, dual hypothermic oxygenated machine perfusion; Retr, donor hepatectomy time; rWIT, recipient warm ischemia time; Tot, total preservation time.
Cold ischemia time from cold perfusion in the donor to D‐HOPE start.
Time from start of vascular anastomoses to graft reperfusion into recipient. The liver was weighed before and after machine perfusion and the weight variation was expressed as a percentage of the initial weight.
Median duration of MD monitoring, from insertion of the first microvial to removal of the last one, was 228 (210, 245) min. No major adverse events occurred and occasional mild bleeding from MD catheter entry site was easily controlled by diathermy. No patient developed catheter‐related bleeding or intra‐or extra‐hepatic hematoma, as assessed by ultrasound scan performed after LT.
Study variables and outcome measures are summarized in Table 3. Three patients developed EAD after LT and one later developed delayed non‐function and required retransplantation. All three patients had a transaminase peak ≥2000 IU, whereas only the patient who subsequently suffered from graft failure had bilirubin level ≥10 mg/dl on day 7th after LT. Patients who developed EAD had higher transaminase peak, as well as higher day 7th bilirubin, INR, and alkaline phosphatases levels (Table 3). L‐GrAFT score was higher in the EAD group, with a difference approaching statistical significance (estimated risk of graft loss 22.1% vs. 7.9%, p = 0.09).
Study variables in the whole series and according to the onset of early allograft dysfunction
Data are presented as median [IQR] or number (%), as appropriate.
Abbreviations: AKI, acute kidney injury; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CCI, comprehensive complication index; D‐HOPE, dual hypothermic oxygenated machine perfusion; EAD, early allograft dysfunction; GGT, gamma glutamyl transferase; ICU, intensive care unit; INR, international normalized ratio; IS, immunosuppression; L‐GrAFT. Liver graft assessment following transplantation; PVT, portal vein thrombosis; TIPS, transjugular intrahepatic portosystemic shunt.
Concerning baseline variables, the only significant difference between EAD and non‐EAD cases was a higher percentage of macrovesicular steatosis (25% vs. 3%, p = 0.04) in EAD group. Degree of macrovesicular steatosis was 25% and 10% in patients who recovered from EAD, whereas it was 40% in the patient who developed graft failure.
With regards to clinical outcome, patients in the EAD group had in the general poorer outcome and suffered from a higher rate of surgical complication postoperatively and at 6‐month follow‐up, as demonstrated by a higher CCI (47.4 vs. 22.6, p = 0.05). Only patient 4 developed Clavien‐Dindo ≥3b complications, represented by coagulopathy‐related bleeding requiring relaparotomy and temporary packing on postoperative day 1, followed by renal failure requiring renal replacement therapy and graft failure leading to retransplantation on postoperative day 31st.
Four (40%) patients developed biliary complications (anastomotic, n = 3; ischemic‐type, n = 1). Patients 3, 8, and 10 presented at 3‐month cholangiogram with sludge and anastomotic stricture, which were successfully managed endoscopically. Patient 6 developed an ischemic‐type stricture of the biliary confluence, which was successfully managed by percutaneous balloon bilioplasty and no evidence of recurrence thereafter.
Median follow‐up was 10.9 (9.8, 11.6) months. Patient and graft survival were 90%. Patient 4, after making a good recovery after re‐LT, died 6 months after LT due to complications of HHV8 infection.
Microdialysate and perfusate metabolites:
Figure 2 depicts values of MD metabolites during SCS and D‐HOPE. Only 3 patients had D‐HOPE lasting >2 h and had samples representative of this time frame. We observed a significant rise of MD glucose level upon initiation of D‐HOPE, which increased from 49 (42, 68) mg/dl to 133 (118, 146) and 152 (119, 216) mg/dl at 1st and 2nd hour of D‐HOPE, respectively (p = 0.01). A similar trend was observed for lactate, which increased from 1. 9 (1.4, 2.3) mmol/L to 2.8 (2.2, 3.8) mmol/L at 1st hour and 2.9 (2.2, 4.4) mmol/L at 2nd hour (p = 0.08). Levels of glutamate in MD fluid persisted high throughout the procedure (200 [189, 206], 192 [187, 194], and 196 [190, 199] μmol/L during SCS, D‐HOPE 1st and 2nd hour, respectively) and were unaffected by D‐HOPE, whereas pyruvate levels were persistently low (4 [2, 4], 4 [2, 6], and 6 [4, 8] μmol/L at all timepoints, respectively). As an effect of low pyruvate levels, lactate/pyruvate ratio trend closely mimicked the lactate trend.
Glucose, lactate, glutamate, pyruvate levels and lactate/pyruvate ratio in microdialysate during backtable preparation and subsequent machine perfusion. In the left column, levels across different timepoints are compared using ANOVA for repeated measures. Degrees of freedom, F‐statistic (F), p‐value, and generalized effect size squared (η
2) are provided for each biomarker. Levels of significance of pairwise t‐test across different time points is indicated as non‐significant (ns), <0.01 (**) or <0.001 (***). Y axis scale changes across different plots to improve data visualization. As only 3 grafts had a D‐HOPE time exceeding 2 h, the 3‐h timepoint is not visualized. In the right column, line plots depicting the trend of study metabolites in each patient are provided. Line colors identify patients who had primary graft function (light blue), early allograft dysfunction (orange), or required retransplantation (red). 2nd hour samples were collected 2 h after the beginning of D‐HOPE or at the end of machine perfusion when D‐HOPE time was <120 min. Asterisks indicate that glucose and lactate levels during the 2nd hour of D‐HOPE were significantly higher in patients developing early allograft dysfunction. pwc, pairwise comparison [Color figure can be viewed at wileyonlinelibrary.com]
Kinetics of MD lactate and glucose was different in grafts that developed EAD (Figure 2). In particular, levels of glucose and lactate were significantly higher during 2nd hour of D‐HOPE (244 vs. 121 mg/dl, p = 0.03 and 4.9 vs. 2.7 mmol/L, p = 0.03) (Table S2). In contrast with glucose and lactate levels on perfusate, levels of MD metabolites clearly diverged, being significantly higher in dysfunctioning grafts at two hours of machine perfusion (Figure 3) (Tables S2 and S3). For 2nd‐hour MD glucose, the area under the receiver operating characteristic curve evaluating its association with EAD was 0.952. With a cutoff value of 215 mg/dl, 2nd hour MD glucose had 100% sensitivity, 86% specificity, 75% positive predictive value, and 100% negative predictive value for the development of EAD.
Line plots depicting trend of glucose and lactate in perfusate (green and light green) and microdialysate (blue and light blue), according to subsequent development of early allograft dysfunction. In cases in which D‐HOPE time was <120 min, 2nd hour samples were collected at the end of machine perfusion. Values are represented as mean ± standard error (vertical error bars) [Color figure can be viewed at wileyonlinelibrary.com]
2nd‐hour MD glucose and lactate level were positively correlated with L‐GrAFT score, 6‐month CCI, graft weight variation during D‐HOPE, cold ischemia time, and macrosteatosis (Figure 4).
Correlation of 2nd hour MD metabolites with L‐GrAFT score, 6‐month CCI, and graft characteristics [Color figure can be viewed at wileyonlinelibrary.com]
Levels of MD metabolites were not associated with the development of biliary complications. However, both surviving grafts that initially developed EAD subsequently developed biliary complications, including one case of ischemic cholangiopathy.
Microdialysate and perfusate FMN:
Figure 5 depicts FMN levels in perfusate and microdialysate. As opposed to glucose and lactate, FMN levels in microdialysate significantly dropped after initiation of D‐HOPE, independently of graft function. In contrast, adjusted and non‐adjusted perfusate FMN levels were higher in grafts who developed early dysfunction, although this difference did not achieve statistical significance (Table S4). The only graft that developed delayed non‐function and required re‐LT was characterized by the highest perfusate FMN levels, which progressively increased throughout D‐HOPE. Perfusate FMN levels were positively correlated with L‐GrAFT score, 2nd‐hour MD glucose and lactate, and with clinical outcome measures (Figure 6).
Non‐adjusted (left column) and adjusted (middle column) flavin mononucleotide level in perfusate and in microdialysate (right column). Individual trends for each patient are presented in the first row, with colors differentiating cases according to early graft function. Levels across different timepoints are compared with ANOVA for repeated measures in the second row. In the third row, levels (mean ± standard error) are presented according to the development of early allograft dysfunction [Color figure can be viewed at wileyonlinelibrary.com]
Correlation of 2nd hour perfusate FMN with L‐GrAFT score, 6‐month CCI, and 2nd hour MD metabolites [Color figure can be viewed at wileyonlinelibrary.com]
Histology and expression of inflammatory cytokines:
Histological injury (Figure 7—Panel A) correlated with EAD onset and was more severe in the graft that required retransplantation. Interestingly, reduced glycogen content was observed in grafts that subsequently developed EAD. Upon reperfusion, a significant increase of inflammatory cytokines (IL‐6, IL‐8, and TNFα) and adhesion molecules (ICAM1) expression was observed, with no significant differences according to EAD development (Figure 7—Panel B). There was no correlation between MD and perfusate parameters and the expression of inflammatory cytokines or adhesion molecules. Succinate tissue content exhibited a downward trend from cold preservation to reperfusion into recipient, with no significant differences between study groups (Figure S1).
Panel A. Representative histological images of liver grafts at the end of transplant (100× original magnification). Non‐EAD case (case 10; images A–C) showed mild signs of steatosis and reperfusion injury (A). PAS staining (B) enhanced cytoplasmatic hepatocytes’ glycogen deposits, confirmed with PAS‐D staining (C). Glycogen was diffusely distributed with a zone‐1‐to‐zone‐3 gradient pattern. EAD case (case 3; images D–F) was characterized by mild steatosis and focal parcellar necrosis (D). Glycogen was present in few periportal hepatocytes (E,F). Graft failure case (case 4; images G–I) showed severe steatosis (G) and minimal signs of glycogen deposits (H,I). Microscope liver histologic slides were scanned with the NanoZoomer S210 Digital slide scanner (Hamamatsu Photonics K.K.) using an objective lens with a numerical aperture of 0.75. Slides were focused at 400× original magnification (scanning resolution: 0.23 μm/pixel), and images were acquired with the NDP.scan image acquisition software (Hamamatsu Photonics K.K.). Then, contrast and brightness corrections were performed to the whole image and data exported with the NDP.view2 viewing software (Hamamatsu Photonics K.K.). Panel B. Cytokines levels at the start of backtable preparation (T1), before (T2) and after (T3) machine perfusion, and at the end of transplant operation (T4). No significant differences were observed between EAD and non‐EAD patients at any timepoint [Color figure can be viewed at wileyonlinelibrary.com]
DISCUSSION:
In the setting of clinical LT, the use of MD has been explored mainly as a tool for monitoring and early detection of ischemic complications and acute rejection.
23
,
24
,
25
,
26
,
27
,
28
,
29
However, one fascinating feature of this technology is the possibility of evaluating liver metabolism during different phases of organ retrieval, static cold storage, implantation, and early postoperative course.
18
,
19
,
21
,
22
In particular, the studies by Nowak et al.
19
and Silva et al.
21
have clearly shown that both glucose and lactate slowly build up during SCS, whereas their levels increase steeply during implantation and organ reperfusion, to return progressively to baseline levels within 3 h following reperfusion. Perera et al.
46
have shown that end‐ischemic interstitial lactate level and lactate/pyruvate ratio are higher in grafts from DCD donors, this finding being associated with more severe glycogen depletion. Similarly, glutamate also appears to accumulate during SCS, reaching end‐ischemic levels of approximately 300 μmol/L, which progressively normalize in the hours following graft reperfusion.
20
In contrast, pyruvate quickly becomes undetectable during SCS (reflecting lack of metabolic activity during this phase), rises above pre‐SCS values upon reperfusion (suggesting a phase of hypermetabolism) to return to baseline in the following hours.
19
The release of glucose in the extracellular space represents a hepatic‐specific response to ischemia and it has been interpreted as a result of glycogen breakdown.
19
In other tissues, ischemic events are associated with very low interstitial glucose. Interestingly, the rate of glucose and lactate accumulation increases during implantation
19
and back table preparation,
21
suggesting a potential role for temperature during these phases. Previous studies have suggested a prognostic value of MD metabolites, as patients developing initial poor function had higher MD lactate level during back table preparation and delayed clearance after graft reperfusion.
21
,
46
In this study, we sought to use MD to deepen our understanding of liver metabolism during SCS and D‐HOPE. Our main finding is that glucose and lactate are released in the extracellular space upon initiation of D‐HOPE and their levels correlate with other known predictors of graft function (cold ischemia time and macrosteatosis), FMN perfusate level, graft dysfunction, and worse clinical outcome.
In our study, D‐HOPE did not alter pyruvate and glutamate levels, as compared to the expected kinetics during SCS. In particular, pyruvate levels were almost undetectable and glutamate levels were constantly high, in keeping with findings from previous studies.
20
The lower end‐ischemic lactate (~1900 μmol/L) and glutamate (~200 μmol/L) concentrations observed in our study (~200 μmol/L), as compared to those observed in previous studies by Silva et al.
20
,
21
can be explained by the relatively short ischemia time before D‐HOPE (5 h 44 min) in our series. Interestingly, the only graft that failed in our series had constantly very low levels of MD glutamate during back table and D‐HOPE.
Why D‐HOPE was associated with a significant increase of glucose and lactate into microdialysate is open to several interpretations. Temperature may have played a role, as the device employed for machine perfusion operates at ~10°C. During rewarming, it is possible that increased metabolism enhanced glycogenolysis, which in turn resulted in increased glucose release. The composition of the perfusion fluid used for D‐HOPE, containing 180 mg/ml of dextrose, may also have influenced MD glucose level. However, it should be noted that glucose levels in microdialysate were lower than those in perfusate and their kinetics was different (Figure 3). In addition, perfusion fluid composition was unlikely to affect lactate levels and progressive accumulation of lactate in the renal cortex has also been observed in a study using MD in a model of hypothermic perfusion of porcine kidneys.
33
Whatever the mechanism, glucose and lactate released in the extracellular space during D‐HOPE can be interpreted as markers of graft injury. The rise of MD glucose and lactate during the first 2 h of D‐HOPE was more important in grafts that developed early dysfunction, whereas it was mild or absent in those exhibiting primary function (Figure 3). However, in the few cases that had a 3‐h MD sample available, glucose and lactate levels started to decrease also in EAD group (Table S2). Consequently, it seems that restoration of aerobic metabolism during D‐HOPE could take longer in more severely damaged grafts.
Importantly, there was a significant discrepancy between perfusate and MD kinetics of glucose and lactate (Figure 3). Perfusate analysis, as a measure of larger and global compartment, did not seem to entirely capture metabolic changes happening at a cellular level, a finding that was more evident for glucose and which is in keeping with our previous study on ex‐vivo lung perfusion.
30
Perfusate glucose level increased during D‐HOPE independently of graft function, whereas MD levels of glucose were higher in EAD patients, with a significant difference during the 2nd hour of machine perfusion. As lactate is produced during anaerobic glycolysis and glucose is released during ischemia due to hepatocytes glycogenolysis, we might hypothesize that the grafts that developed EAD did not fully recover after SCS and continued to experience a certain degree of hypoxia during machine perfusion, as demonstrated by their metabolic profile. In line with this observation, glycogen content was reduced in EAD livers, likely due to glycogen depletion during cold preservation, an observation which is in keeping with findings from Perera et al.
46
The next logical step was comparing these findings with perfusate and MD levels of FMN, a marker of mitochondrial injury that recently emerged as a promising tool for graft viability assessment.
40
,
41
As opposed to glucose and lactate, D‐HOPE start determined a steep decrease of MD FMN levels, suggesting its rapid washout from the extracellular space and confirming protection of mitochondria during D‐HOPE. In contrast, FMN appeared to accumulate in perfusate, with an incremental trend in grafts who developed EAD and with the highest levels observed in the only failing graft of this series (Figure 5). This finding, which is in keeping with those from Mueller et al.,
40
also suggests a mechanistic interpretation to the association between microdialysate metabolites and graft dysfunction. Taken as a whole, our data seem to point to the same direction: mitochondrial injury sustained during SCS, which is proportional to cold ischemia time and macrosteatosis, is reflected by the accumulation of FMN in perfusate and release of glucose and lactate in the extracellular space during D‐HOPE, highlighting their potential as viability markers. Noteworthy, these metabolites can be measured point‐of‐care using MD equipment during D‐HOPE.
This study has numerous implications. First, it confirms that precious information on graft function and injury can be gathered also during hypothermic perfusion, challenging the concept that normothermic perfusion is a prerequisite for graft viability assessment.
47
,
48
Second, while perfusate analysis appears handier, this study and previous experiences
23
,
24
,
27
,
28
suggest that MD is feasible without major modifications of routine practice. In our opinion, microdialysate and perfusate analysis should be seen as complementary rather than alternative techniques, as metabolites kinetics appears to be different in these two compartments. Real‐time assessment of graft function could drive not only graft acceptance but also influence the duration of machine perfusion, as more severely damaged grafts may require longer perfusion time to recover from ischemic injury.
Third, this pilot study represents a baseline based on which other potential applications of MD during machine perfusion could be explored. In a recent study on perfusate analysis during D‐HOPE,
34
we identified alanine aminotransferase as the most reliable predictor of EAD. Unfortunately, MD ALT could not be evaluated due to its molecular weight (100 kDa). Further studies on the subject should ideally employ available 100 kDa MD catheters to allow measurement of other molecules, including inflammatory cytokines, as also suggested by a recent study.
49
Finally, MD could be used to assess liver metabolism and explore new viability criteria during normothermic machine perfusion, a setting mimicking normal physiology and characterized by longer perfusion times.
50
Limitations of our study include the small sample size of 10 grafts with rather heterogeneous characteristics, the lack of a power analysis, and the choice of EAD as an endpoint, which has been discouraged in machine perfusion trials as it strongly relies on transaminase level after transplant and could be influenced by the washout phenomenon during machine perfusion.
51
In this study, microdialysis time was limited to less than 4 h by design, allowing complete measurement of MD metabolites at only 3 time points. As previous studies have shown a quick recovery of aerobic metabolism after liver reperfusion
21
,
46
and due to concerns about sterility breaches, we did not consider leaving the MD in place during this phase, possibly missing relevant metabolic changes.
31
Rapid sampling MD
33
could improve the yield of MD as applied to procedures of limited duration, whereas the use of an MD catheter with higher molecular weight cutoff membrane would allow measuring MD concentration of other potentially important molecules.
In conclusion, this study expands previous knowledge on liver metabolism during D‐HOPE and confirms that liver graft injury and function can be assessed during hypothermic machine perfusion. These preliminary findings require validation in larger studies allowing correlation of MD parameters with clinically relevant endpoints and possibly exploring further applications of MD in machine perfusion.
CONFLICT OF INTEREST:
The authors of this manuscript have no conflicts of interest to disclose.
AUTHOR CONTRIBUTIONS:
Damiano Patrono: concept and design, data collection, analysis, and interpretation, statistics, drafting article; Dorotea Roggio: data analysis and interpretation, drafting article; Anna Teresa Mazzeo: concept and design, data interpretation, funding, approval of article; Giorgia Catalano, Elena Mazza, Giorgia Rizza, Alessandro Gambella, Federica Rigo, Nicola Leone, Vincenzo Elia, Daniele Dondossola, Caterina Lonati: data collection, analysis, and interpretation; article revision; Vito Fanelli: concept and design, article revision; Renato Romagnoli: concept and design, funding, article revision, study supervision.
Supporting information:
Supplementary Material
Click here for additional data file.
| Background:
While growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood.
Methods:
To assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome.
Results:
Median (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2nd-hour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN. These correlations were not apparent when perfusate levels were considered. In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts.
Conclusions:
MD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment. | null | null | 14,165 | 301 | [
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] | null | null | null | [CONTENT] extracellular fluid | flavin mononucleotide | liver metabolism | liver viability assessment | machine perfusion | microdialysis [SUMMARY] | null | [CONTENT] extracellular fluid | flavin mononucleotide | liver metabolism | liver viability assessment | machine perfusion | microdialysis [SUMMARY] | null | [CONTENT] extracellular fluid | flavin mononucleotide | liver metabolism | liver viability assessment | machine perfusion | microdialysis [SUMMARY] | null | [CONTENT] Aged | Cold Ischemia | Female | Glucose | Graft Survival | Humans | Lactic Acid | Liver | Liver Transplantation | Male | Microdialysis | Middle Aged | Organ Preservation | Perfusion | Pilot Projects | Prospective Studies [SUMMARY] | null | [CONTENT] Aged | Cold Ischemia | Female | Glucose | Graft Survival | Humans | Lactic Acid | Liver | Liver Transplantation | Male | Microdialysis | Middle Aged | Organ Preservation | Perfusion | Pilot Projects | Prospective Studies [SUMMARY] | null | [CONTENT] Aged | Cold Ischemia | Female | Glucose | Graft Survival | Humans | Lactic Acid | Liver | Liver Transplantation | Male | Microdialysis | Middle Aged | Organ Preservation | Perfusion | Pilot Projects | Prospective Studies [SUMMARY] | null | [CONTENT] protection mitochondria hope | ischemia reperfusion | metabolism liver reperfusion | transfer organ reperfusion | perfusion donor hope [SUMMARY] | null | [CONTENT] protection mitochondria hope | ischemia reperfusion | metabolism liver reperfusion | transfer organ reperfusion | perfusion donor hope [SUMMARY] | null | [CONTENT] protection mitochondria hope | ischemia reperfusion | metabolism liver reperfusion | transfer organ reperfusion | perfusion donor hope [SUMMARY] | null | [CONTENT] hope | md | graft | levels | perfusion | ead | perfusate | machine | lactate | machine perfusion [SUMMARY] | null | [CONTENT] hope | md | graft | levels | perfusion | ead | perfusate | machine | lactate | machine perfusion [SUMMARY] | null | [CONTENT] hope | md | graft | levels | perfusion | ead | perfusate | machine | lactate | machine perfusion [SUMMARY] | null | [CONTENT] hope | metabolism | md | liver | assess | liver metabolism | mitochondrial | tool | potential | scs [SUMMARY] | null | [CONTENT] graft | ead | hope | levels | developed | hour | 2nd | 2nd hour | figure | time [SUMMARY] | null | [CONTENT] hope | md | graft | levels | ead | perfusion | liver | lactate | perfusate | machine [SUMMARY] | null | [CONTENT] [SUMMARY] | null | [CONTENT] IQR | MD | 228 | 210 | 245 | 116 | 103 | 143 | Three | EAD ||| MD | 9.88 ||| 0.01 | 3.71 | 0.08 ||| 2nd-hour | EAD ||| 2nd-hour | MD | FMN ||| ||| MD FMN | FMN [SUMMARY] | null | [CONTENT] ||| MD | 10 ||| ||| IQR | MD | 228 | 210 | 245 | 116 | 103 | 143 | Three | EAD ||| MD | 9.88 ||| 0.01 | 3.71 | 0.08 ||| 2nd-hour | EAD ||| 2nd-hour | MD | FMN ||| ||| MD FMN | FMN ||| [SUMMARY] | null |
|||
Awareness of HIV functional cure and willingness in participating in related clinical trials: comparison between antiretroviral naïve and experienced men who have sex with men living with HIV. | 35428275 | Human immunodeficiency virus (HIV) functional cure is a novel biomedical strategy characterized by sustained viral suppression without the need for life-long medications. The attitude of people living with HIV (PLHIV) towards functional cure and clinical trials are understudied. We aimed to examine the awareness and levels of anticipation for HIV functional cure among men who have sex with men (MSM) living with HIV, and their willingness to join trials as differentiated by their antiretroviral treatment status. | BACKGROUND | MSM living with HIV with and those without treatment history were recruited from Hong Kong's HIV specialist clinics. Self-administered questionnaires covering behavioral profile, perceived impact of HIV cure, attitude towards HIV functional cure and related clinical trials were collected. Clinical data were separately transcribed. Determinants of perceptions and attitudes were identified by logistic regression models. | METHODS | Of 356 MSM living with HIV recruited, less than half (42%) were aware of HIV functional cure, but they had a high level of anticipation for it. Treatment-experienced participants were more likely to be aware of HIV functional cure. Awareness was associated with continued engagement in sexual activities after HIV diagnosis and sexually transmitted infection (STI) diagnosis. Higher anticipation was observed among older MSM living with HIV but it was negatively associated with one's awareness. Over 90% were willing to join functional cure trials, especially those who had previously been diagnosed with STI and had engaged in chemsex in the past year. Advice from healthcare professional was an important factor considered by those willing to join clinical trials. Younger, better educated MSM, and those with lower CD4 counts were more concerned about potential risk of AIDS and potential complications upon trial participation. | RESULTS | MSM living with HIV, especially those sexually active, showed positive attitude towards functional cure and willingness to join related clinical trials despite low awareness. To enhance preparedness for HIV functional cure trials, community education, updated information and appropriate medical advice would be needed. Safety is a major concern for potential enrollees in HIV functional cure trials. | CONCLUSIONS | [
"Cross-Sectional Studies",
"Female",
"HIV Infections",
"Homosexuality, Male",
"Humans",
"Male",
"Sexual Behavior",
"Sexual and Gender Minorities",
"Sexually Transmitted Diseases"
] | 9013029 | Background | Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].
In the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART. | null | null | Results | Demographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.
Table 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data
Participants’ socio-demographics, sexual and clinical characteristics by treatment status
STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome
aTotal number for each variable may not add up to N = 356 due to missing data
Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.
Table 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data
Participants’ socio-demographics, sexual and clinical characteristics by treatment status
STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome
aTotal number for each variable may not add up to N = 356 due to missing data
Perceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).
Table 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)
n
%
n
%
OR (95% CI)
(a) Perceived impacts of HIV cure
No longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)
(b) Perception and attitude towards HIV functional cure
Awareness of HIV functional cure
Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*
Level of anticipation for HIV functional cure
< 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00
Willingness in joining a functional cure trial
Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00
(c) Considerations about HIV functional cure trial
Important factors to be considered about the trial
Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)
Concerns about consequences of joining a trial
CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001
Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status
HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome
*p < 0.05 ** p < 0.01 *** p < 0.001
Table 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure
IMP1a
IMP2aIMP3aIMP4aIMP5a
IMP6aIMP7a
Age group
16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)
Education level
Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)*
STI history in the past year
1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)
Sexual activity after HIV diagnosis
1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58)
Engagement in chemsex
1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)
CD4 cell count (cells/uL)
≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)
About Functional cure
Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001
Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome
aIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV
* p < 0.05 ** p < 0.01 *** p < 0.001
Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).
Table 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)
n
%
n
%
OR (95% CI)
(a) Perceived impacts of HIV cure
No longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)
(b) Perception and attitude towards HIV functional cure
Awareness of HIV functional cure
Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*
Level of anticipation for HIV functional cure
< 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00
Willingness in joining a functional cure trial
Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00
(c) Considerations about HIV functional cure trial
Important factors to be considered about the trial
Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)
Concerns about consequences of joining a trial
CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001
Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status
HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome
*p < 0.05 ** p < 0.01 *** p < 0.001
Table 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure
IMP1a
IMP2aIMP3aIMP4aIMP5a
IMP6aIMP7a
Age group
16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)
Education level
Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)*
STI history in the past year
1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)
Sexual activity after HIV diagnosis
1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58)
Engagement in chemsex
1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)
CD4 cell count (cells/uL)
≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)
About Functional cure
Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001
Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome
aIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV
* p < 0.05 ** p < 0.01 *** p < 0.001
Awareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).
Table 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Age group
16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–
Education level
Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)–
STI history in the past year
1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*
Sexual activity after HIV diagnosis
1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)
Engagement in chemsex
0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)
CD4 cell count (cells/uL)
≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–
About functional cure
Awareness
–
–
0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***
–
–
1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001
Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio
* p < 0.05 ** p < 0.01 *** p < 0.001
Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).
Table 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Age group
16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–
Education level
Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)–
STI history in the past year
1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*
Sexual activity after HIV diagnosis
1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)
Engagement in chemsex
0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)
CD4 cell count (cells/uL)
≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–
About functional cure
Awareness
–
–
0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***
–
–
1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001
Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio
* p < 0.05 ** p < 0.01 *** p < 0.001
Anticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).
Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).
Acceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.
Concerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).
Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.
Concerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29). | Conclusions | To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment. | [
"Background",
"Methods",
"Demographics",
"Perceived benefits of HIV cure",
"Awareness of HIV functional cure",
"Anticipation of HIV functional cure",
"Acceptance of an HIV functional cure trial",
""
] | [
"Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.",
"Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.",
"Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data",
"Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001",
"Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001",
"Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).",
"Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).",
"\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356)."
] | [
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] | [
"Background",
"Methods",
"Results",
"Demographics",
"Perceived benefits of HIV cure",
"Awareness of HIV functional cure",
"Anticipation of HIV functional cure",
"Acceptance of an HIV functional cure trial",
"Discussion",
"Conclusions",
"Supplementary Information",
""
] | [
"Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].\nIn the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.",
"Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.\nA self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.\nOn the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.\nDifferences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.",
"Demographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nBetween March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data\nPerceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nPerceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAwareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nOverall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001\nAnticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAmong all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).\nAcceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).\nShould there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).",
"Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.\n\nTable 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data\nParticipants’ socio-demographics, sexual and clinical characteristics by treatment status\nSTI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome\naTotal number for each variable may not add up to N = 356 due to missing data",
"Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).\n\nTable 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)\nn\n\n%\n\nn\n\n%\n\nOR (95% CI)\n\n(a) Perceived impacts of HIV cure\nNo longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)\n(b) Perception and attitude towards HIV functional cure\n\nAwareness of HIV functional cure\n Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*\nLevel of anticipation for HIV functional cure\n < 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00\nWillingness in joining a functional cure trial\n Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00\n(c) Considerations about HIV functional cure trial\n\nImportant factors to be considered about the trial\n Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)\nConcerns about consequences of joining a trial\n CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001\nParticipants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status\nHIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome\n*p < 0.05 ** p < 0.01 *** p < 0.001\n\nTable 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure\nIMP1a\nIMP2aIMP3aIMP4aIMP5a\nIMP6aIMP7a\nAge group\n 16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)\nEducation level\n Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)* \nSTI history in the past year\n1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)\nSexual activity after HIV diagnosis\n1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58) \nEngagement in chemsex\n1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)\nAbout Functional cure\n Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome\naIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV\n* p < 0.05 ** p < 0.01 *** p < 0.001",
"Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).\n\nTable 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nCrude OR\n\nAdjusted OR\n\nAge group\n 16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–\nEducation level\n Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)– \nSTI history in the past year\n1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*\n Sexual activity after HIV diagnosis\n1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)\n Engagement in chemsex\n0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)\nCD4 cell count (cells/uL)\n ≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–\nAbout functional cure\n Awareness\n–\n\n–\n0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***\n–\n\n–\n1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001\nCrude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)\nMSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio\n* p < 0.05 ** p < 0.01 *** p < 0.001",
"Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).",
"Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.\nConcerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).",
"HIV functional cure is a relatively new concept for PLHIV. Despite their generally low awareness, MSM living with HIV had, as shown in this study, a high level of anticipation for HIV functional cure, especially those who had been diagnosed and put on ART for some years. The effective achievement of functional cure carries the potential prospect of getting rid of repeated clinic visits and taking long-term medications [11]. The benefit of being untransmittable may, however, not be regarded as an important impact for HIV cure because in the current era of “undetectable equals untransmittable”, adherence to ART is already effective in minimizing the risk of transmission. In our cohorts of MSM living with HIV, the attitudes differed by one’s ART treatment experience. Treatment-naïve participants were more concerned about their immune function and AIDS progression than those ART-experienced, as deteriorated health was perceived by newly diagnosed patients an imminent consequence brought on by HIV [12]. Anticipation for HIV functional cure was apparently lower among those who were aware of it. Although some 42% had heard about functional cure, most had limited knowledge of it and cared little about the potential benefit of restoring their immune function and potential adverse effects arising from the new therapy. With the increasing likelihood that functional cure may soon become the next major advance in HIV treatment [7], there is the need for promoting community education on the rationale and strategy for HIV functional cure. As in many countries, MSM accounted for a high proportion of PLHIV in Hong Kong [13], and who continued to be significantly impacted by the epidemic [14]. The MSM community, especially those living with HIV would need to be targeted in anticipation of the introduction of functional cure therapy. In our study, MSM engaging in higher risk sexual behaviors were more likely to be aware of the recent advancement on HIV science, such as pre-exposure prophylaxis [15]. This echoed our results that sexually active PLHIV, particularly those engaged in condomless sex as indicated by their STI history, were more likely to be aware of HIV functional cure.\nThere are diverse reasons for PLHIV to look forward to receiving functional cure therapy. It is intuitive that PLHIV with a weaker immune system were less likely to be sexually active and they wished to restore and stabilize their immune function and were at the same time more concerned about adverse HIV outcomes after joining functional cure trials. The fact that younger PLHIV cared more about disease development and transmittability was likely because they were newly diagnosed and would remain sexually active for a longer time in the future. PLHIV with higher education level regarded de-labelling of HIV an important impact of HIV cure. In Hong Kong, public stigma against PLHIV [16] and perceived discrimination [17] have been reported. It is therefore an ideal vision for them to remove the label of HIV after achieving some sort of cure. Discrimination in workplace and difficulty in career development could be experienced by PLHIV [18]. Although highly educated PLHIV were found to deal better with workplace discrimination and improve employment quality than those with lower education level [19], they were more concerned with their career development that removing stigma means removing a barrier in their career paths. From the perspective of the general public, increased awareness and knowledge in HIV science advancement might be able to reduce stigma. A previous study showed that heterosexually active adults who were aware of the untransmittable nature of PLHIV achieving undetectable viral load had a lower level of anticipated HIV stigma [20]. As this study focuses on PLHIV, a follow-up study in the general population and the MSM community would be needed to assess the impact of functional cure on general, and dating- and sex-related enacted stigmas.\nOverall, the idea of an HIV functional cure trial was well accepted by our cohorts of MSM living with HIV with over 90% indicating their willingness to join, similar to the high acceptance rate of 95% in a previous study [11]. Potential trial participants were more likely to have engaged in higher risk sexual activities as reflected by their report of STI in the preceding year. This might be precipitated by a prospect of continuation or re-engagement in adventurous condomless behaviors upon cure [21]. Safety is a major concern for clinical trials involving the use of novel agents like neutralizing antibodies or therapeutic vaccines. Careful monitoring of PLHIV in the trial would be warranted not just for protecting participants’ health but also easing their concerns of side effects and adverse HIV outcomes. Hesitation would arise if the trial requires interruption of ART or if it takes a long time, in relation to the risk of rebound and potential adverse effects [11]. Trusting relationship between PLHIV, researchers and healthcare workers could be beneficial for ensuring the delivery of optimal health outcomes [22]. Based on potential participants’ trust on healthcare professional advice, collaboration with HIV clinics on explaining the details and safety issues of the trial would be paramount.\nThis study carries some limitations. Like other epidemiological studies, there were inherent recall and social desirability biases in the administration of behavioral questionnaires. We had minimized these biases by adopting a shorter recall period and omitting fine details about sexual activities. The adoption of a self-administering approach should have reduced embarrassments in response to interviewers’ questions. Incorporation of clinical data by transcription benefited the study by providing objective measurements of participants’ HIV outcomes. In the absence of any functional cure trials, the exploration of one’s willingness to participate in a hypothetical trial without having one in place could be postulational. Nevertheless, the systematic inquiries into the awareness and anticipation of functional HIV cure among MSM living with HIV and their inclination in joining a hypothetical study have generated useful results which could be of reference for the planning of a functional cure trial.",
"To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment.",
" \nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\n\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).",
"\nAdditional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).\nAdditional file 1. Questionnaire.\nAdditional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356)."
] | [
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
"conclusion",
"supplementary-material",
null
] | [
"HIV",
"Functional cure",
"Men who have sex with men",
"Awareness"
] | Background:
Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].
In the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.
Methods:
Data for analyses came from a questionnaire survey administered on ART-naïve and ART-experienced men who have sex with men (MSM) living with HIV participating in two separate cohort studies in Hong Kong. The former study comprised the collection of data from newly diagnosed MSM attending any one of the three HIV specialist clinics over a two-year period; while the latter study involved treatment-experienced MSM receiving ART at the largest HIV clinic during the same period of time as a follow-up round of a study on the sex networking behaviors following HIV diagnosis [10]. Informed consents were obtained prior to the conduct of the surveys. Approval of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and Ethics Committee of Department of Health were obtained.
A self-administered questionnaire was designed with mutual topics in both surveys covering (a) demographics and HIV diagnosis year, (b) perceptions about HIV cure and their impacts, and (c) awareness and anticipation of HIV functional cure, considerations of and concerns about participating in an HIV functional cure clinical trial (Additional file 1). Functional cure was the specific theme for this survey, the definition of which was clearly described in order to avoid the misinterpretation of the results as there could be confusion between different forms of HIV curative treatment. Hypothesizing that sexually active PLHIV would have a higher awareness, anticipation and interest in HIV functional cure, sexual behaviors in the preceding year including sexually transmitted infection (STI) diagnosis, sexual activity, and use of psychotropic drugs for sex (chemsex), were inquired. Clinical data were separately transcribed, which included the age of HIV diagnosis, acquired immunodeficiency syndrome (AIDS) status, and longitudinal CD4 cell count and viral load. The most recent readings prior to the time of questionnaire for the latter two measures were recorded for analyses.
On the perceived impacts of HIV cure, participants were asked to choose from seven options: no longer needing to take HIV medications, restoration and stabilization of effective immune function, not getting HIV for a second time, no longer needing to visit a doctor for HIV, no longer at risk of AIDS or HIV-related morbidity, no longer transmitting HIV to the others, and being considered as a person not living with HIV. Awareness of HIV functional cure was categorized into: knew of it and understood what it is, heard of the idea but not the details, and had no idea of it. After giving a brief introduction on HIV functional cure, participants were asked to rate their anticipation on a scale of 0 to 10. Willingness to join an HIV functional cure study was assessed by a 6-point Likert scale (definitely no, probably no, maybe no, maybe yes, probably yes, and definitely yes). Participants were inquired about the importance of each of the following factors which they would consider in joining an HIV functional cure trial: safety, duration of the clinical trial, incentives for participation, view and support from family and peers, advice from healthcare professionals, credibility of the research institution, and need for interruption of ART medications. Potential adverse consequences of participating in the trial included CD4 count going down, HIV viral load going up, becoming infectious to the others, presence of AIDS or other related complications, and side effects arising from the therapy. The levels of importance and concern were ranked in descending order as: very important/concerned, moderately important/concerned, somewhat important/concerned, and a minimally important/concerned.
Differences in perceptions on HIV cure and HIV functional cure trial between ART-naïve and experienced groups were assessed by participants’ sociodemographic and behavioral factors in logistic regression. Determinants of anticipation of HIV cure, awareness of HIV functional cure, and willingness to join an HIV functional cure trial were analyzed by univariable and multivariable logistic regression models. Participants were defined as having a high degree of anticipation if they gave a score of 10. Awareness of HIV functional cure was reflected by at least knowing the idea without details. The importance of factors associated with joining functional cure trials and the concerns were dichotomized by defining the lower two levels (“a little important” and “somewhat important”, and “a little concerned” and “somewhat concerned”) as zero and the rest (“moderately important” and “very important”, and “moderately concerned” and “very concerned”) as one. Only variables with p < 0.10 were included in the initial multivariable models for stepwise backward elimination based on the AIC value. Inter-correlations between cure- and trial-related consideration factors and concerns were explored using logistic regression models. All statistical analyses were conducted in R.
Results:
Demographics Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.
Table 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data
Participants’ socio-demographics, sexual and clinical characteristics by treatment status
STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome
aTotal number for each variable may not add up to N = 356 due to missing data
Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.
Table 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data
Participants’ socio-demographics, sexual and clinical characteristics by treatment status
STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome
aTotal number for each variable may not add up to N = 356 due to missing data
Perceived benefits of HIV cure Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).
Table 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)
n
%
n
%
OR (95% CI)
(a) Perceived impacts of HIV cure
No longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)
(b) Perception and attitude towards HIV functional cure
Awareness of HIV functional cure
Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*
Level of anticipation for HIV functional cure
< 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00
Willingness in joining a functional cure trial
Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00
(c) Considerations about HIV functional cure trial
Important factors to be considered about the trial
Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)
Concerns about consequences of joining a trial
CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001
Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status
HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome
*p < 0.05 ** p < 0.01 *** p < 0.001
Table 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure
IMP1a
IMP2aIMP3aIMP4aIMP5a
IMP6aIMP7a
Age group
16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)
Education level
Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)*
STI history in the past year
1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)
Sexual activity after HIV diagnosis
1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58)
Engagement in chemsex
1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)
CD4 cell count (cells/uL)
≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)
About Functional cure
Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001
Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome
aIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV
* p < 0.05 ** p < 0.01 *** p < 0.001
Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).
Table 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)
n
%
n
%
OR (95% CI)
(a) Perceived impacts of HIV cure
No longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)
(b) Perception and attitude towards HIV functional cure
Awareness of HIV functional cure
Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*
Level of anticipation for HIV functional cure
< 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00
Willingness in joining a functional cure trial
Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00
(c) Considerations about HIV functional cure trial
Important factors to be considered about the trial
Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)
Concerns about consequences of joining a trial
CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001
Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status
HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome
*p < 0.05 ** p < 0.01 *** p < 0.001
Table 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure
IMP1a
IMP2aIMP3aIMP4aIMP5a
IMP6aIMP7a
Age group
16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)
Education level
Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)*
STI history in the past year
1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)
Sexual activity after HIV diagnosis
1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58)
Engagement in chemsex
1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)
CD4 cell count (cells/uL)
≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)
About Functional cure
Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001
Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome
aIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV
* p < 0.05 ** p < 0.01 *** p < 0.001
Awareness of HIV functional cure Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).
Table 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Age group
16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–
Education level
Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)–
STI history in the past year
1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*
Sexual activity after HIV diagnosis
1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)
Engagement in chemsex
0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)
CD4 cell count (cells/uL)
≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–
About functional cure
Awareness
–
–
0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***
–
–
1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001
Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio
* p < 0.05 ** p < 0.01 *** p < 0.001
Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).
Table 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Age group
16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–
Education level
Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)–
STI history in the past year
1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*
Sexual activity after HIV diagnosis
1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)
Engagement in chemsex
0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)
CD4 cell count (cells/uL)
≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–
About functional cure
Awareness
–
–
0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***
–
–
1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001
Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio
* p < 0.05 ** p < 0.01 *** p < 0.001
Anticipation of HIV functional cure Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).
Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).
Acceptance of an HIV functional cure trial Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.
Concerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).
Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.
Concerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).
Demographics:
Between March 2019 and January 2020, 153 treatment-naïve MSM were recruited, which accounted for about one-third of all newly diagnosed male PLHIV in Hong Kong; while data from 203 treatment-experienced MSM were collected between April 2019 and November 2020. The median age of MSM included in the analyses was 37 years (interquartile range [IQR] 29–47 years), with treatment-naïve ones at 30 years (IQR 26–38 years) and treatment-experienced ones at 42 years (IQR 35–50 years) of age (Table 1). Among those receiving treatment, the median duration of HIV diagnosis and receiving ART was 9 years (IQR: 7–13 years) and 8 years (IQR: 6–12 years), respectively. The median age of HIV diagnosis of all enrolled MSM was 31 years (IQR 26–38 years). As regards sexual behaviors, about one-third (38%) did not have any sexual activities in the preceding year, while 39% had engaged in chemsex, among whom 12% injected drugs. About 90% of treatment-experienced participants have achieved a viral load below 20 copies/mL, while almost all treatment-naïve counterparts gave a value of at least 1000 copies/mL. Regarding CD4 cell count, a smaller proportion (16%) of treatment-naïve participants had at least 500 cells/uL, whereas three-quarters in the treatment-experienced group had attained the same level.
Table 1Participants’ socio-demographics, sexual and clinical characteristics by treatment statusTreatment-naïve(n = 153)Treatment-experienced(n = 203)Totala(N = 356)n%n%n%Age group (median = 37, IQR = 29–47) 16–297448.42311.59727.5 30–394730.76030.010730.3 40–491711.15929.57621.5 50 or over159.85829.07320.7Education level Secondary or below5033.66532.011532.3 Post-secondary or above10367.313868.024167.7Employment status Full-time/self-employed10971.214877.925774.9 Part-time/freelancer149.212010.5349.9 Student127.800123.5 Others1811.72211.64011.7STI history in the past year No11675.813668.325271.6 Yes3724.26331.710028.4Sexual activity after HIV diagnosis No10367.33115.613438.1 Yes5032.716884.421861.9Engagement in chemsex in the past year No7649.711660.719255.8 Yes7750.37539.315244.2Age at HIV diagnosis (median = 31, IQR = 26–38) 16–297851.07437.015243.1 30–394328.17939.512234.6 40–491711.13919.55615.9 50 or over159.884.0236.5History of AIDS No14292.817686.731889.3 Yes117.22713.33810.7CD4 cell count (cells/uL) ≥ 5002516.315877.818351.4 < 50012883.74522.217348.6HIV viral load (copies/mL) < 2021.318089.618251.4 20–99953.3189.0236.5 1000–99,9997146.421.07320.6 ≥ 100,0007549.010.57621.5STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndromeaTotal number for each variable may not add up to N = 356 due to missing data
Participants’ socio-demographics, sexual and clinical characteristics by treatment status
STI sexually transmitted infections, HIV human immunodeficiency virus, MSM men who have sex with men, AIDS acquired immunodeficiency syndrome
aTotal number for each variable may not add up to N = 356 due to missing data
Perceived benefits of HIV cure:
Perceptions on HIV cure differed between the treatment-experienced and naïve group (Table 2). The most commonly perceived impact of HIV cure was “restoration and stabilization of effective immune function” (63%), with lower odds among those on ART compared to ART-naïve participants (odds ratio [OR] 0.36, 95% confidence interval [CI] 0.23–0.58). About half (56%) considered untransmittability as an important impact of curing HIV infection. Lowered risk of AIDS or related morbidity was considered more important in the treatment-naïve group (46% vs. 35%, OR 0.62, 95% CI 0.40–0.95), whereas reduced need for clinic visit was accorded higher importance among treatment-experienced participants (18% vs. 34%, OR 2.28, 95% CI 1.38–3.78). Those who considered immune function stabilization as an important impact of HIV cure had a lower CD4 cell count (OR 3.49, 95% CI 2.20–5.52) and were less likely to have recently been engaging in sex (OR 0.30, 95% CI 0.19–0.50) (Table 3). Perceived importance of reduction of clinic visits was associated with being sexually active (OR 1.85, 95% CI 1.11–3.08) and a higher CD4 level (OR 1.86, 95% CI 1.15–3.00). Participants having attained post-secondary education level (OR 1.75 95% CI 1.09–2.81) and younger than 40 years of age regarded being free from the risk of AIDS or HIV-related morbidity as an important impact for HIV cure. Older adults (those aged 50 years or above) did not consider reduced transmittability important (OR 0.23, 95% CI 0.12–0.43). De-labelling was an important impact for those with a higher education level (OR 1.73, 95% CI 1.00-2.97).
Table 2Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment statusTreatment-naïve (n = 153) (reference)Treatment-experienced (n = 203)
n
%
n
%
OR (95% CI)
(a) Perceived impacts of HIV cure
No longer needing to take HIV medications6240.59748.31.37 (0.89–2.09)Restoration and stabilization of effective immune function11675.810753.20.36 (0.23–0.58)***Not getting HIV for a second time2013.13316.41.31 (0.72–2.38)No longer needing to visit a doctor for HIV2818.36833.82.28 (1.38–3.78)**No longer at risk of AIDS or HIV-related morbidity7146.47034.80.62 (0.40–0.95)*No longer be transmitting HIV to others9562.110552.20.67 (0.44–1.02)Being considered as a person not living with HIV3422.25929.41.45 (0.89–2.37)
(b) Perception and attitude towards HIV functional cure
Awareness of HIV functional cure
Never heard about it9462.310954.01.00 Heard but didn’t know the details2113.97135.12.92 (1.67–5.10)*** Heard and understood what it is3623.82210.90.53 (0.29–0.96)*
Level of anticipation for HIV functional cure
< 105938.67840.00.94 (0.61–1.45) 109461.411760.01.00
Willingness in joining a functional cure trial
Maybe/probably/definitely no106.5167.90.82 (0.36–1.85) Maybe/probably/definitely yes14393.518792.11.00
(c) Considerations about HIV functional cure trial
Important factors to be considered about the trial
Safety of the therapy14896.719597.01.10 (0.33–3.67) Duration of the clinical trial12380.415979.10.92 (0.55–1.56) Incentives for participation4026.14020.30.72 (0.44–1.19) Views and support from family and peers5032.75126.00.72 (0.46–1.15) Advice from healthcare professionals13917490.887.00.67 (0.34–1.34) Credibility of the research institution13286.318089.61.36 (0.72–2.60) Interruption of HIV antiretroviral medications13588.216683.40.67 (0.36–1.24)
Concerns about consequences of joining a trial
CD4 count going down11877.114371.90.76 (0.47–1.23) HIV viral load going up13286.316381.50.70 (0.39–1.26) Becoming infectious to the others12380.414773.90.69 (0.41–1.15) Presence of AIDS or other related complications14091.516683.00.45 (0.23–0.89)* Adverse effects of therapy12783.016180.50.85 (0.49–1.46)HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome*p < 0.05 ** p < 0.01 *** p < 0.001
Participants’ (a) perceptions of the impacts of HIV cure, (b) perception and attitudes towards HIV functional cure, and (c) consideration about HIV functional cure trial, by treatment status
HIV human immunodeficiency virus; AIDS acquired immunodeficiency syndrome
*p < 0.05 ** p < 0.01 *** p < 0.001
Table 3Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)Impacts of perceived importance for HIV cure
IMP1a
IMP2aIMP3aIMP4aIMP5a
IMP6aIMP7a
Age group
16–291.001.001.001.001.001.001.00 30–390.85 (0.48–1.48)0.65 (0.36–1.15)1.18 (0.56–2.48)1.21 (0.64–2.30)0.58 (0.33–1.02)0.60 (0.34–1.06)1.80 (0.97–3.35) 40–491.46 (0.80–2.66)0.68 (0.36–1.27)0.83 (0.35–1.96)1.39 (0.70–2.76)0.54 (0.29-1.00)*0.90 (0.48–1.71)0.98 (0.48–2.02) 50 or over1.27 0.69–2.35)0.98 (0.51–1.89)0.69 (0.28–1.74)1.74 (0.88–3.45)0.50 (0.27–0.94)*0.23 (0.12–0.43)***1.07 (0.52–2.21)
Education level
Secondary or below1.001.001.001.001.001.001.00 Post-secondary or above1.51 (0.96–2.38)1.01 (0.64–1.60)0.61 (0.34–1.11)1.70 (1.00-2.9)1.75 (1.09–2.81)*0.65 (0.41–1.02)1.73 (1.00-2.97)*
STI history in the past year
1.16 (0.73–1.85)0.76 (0.47–1.23)1.24 (0.66–2.33)1.33 (0.80–2.22)1.22 (0.76–1.95)1.44 (0.89–2.32)0.90 (0.52–1.54)
Sexual activity after HIV diagnosis
1.07 (0.69–1.64)0.30 (0.19–0.50)***0.95 (0.52–1.75)1.85 (1.11–3.08)*0.94 (0.61–1.46)0.86 (0.56–1.33)1.55 (0.93–2.58)
Engagement in chemsex
1.03 (0.67–1.58)0.71 (0.46–1.11)0.88 (0.48–1.64)0.95 (0.58–1.54)1.24 (0.80–1.91)1.05 (0.68–1.62)0.90 (0.55–1.46)
CD4 cell count (cells/uL)
≥ 5001.001.001.001.001.001.001.00 < 5000.79 (0.52–1.20)3.49 (2.20–5.52)***0.78 (0.43–1.41)0.54 (0.33–0.87)*1.24 (0.81–1.89)1.04 (0.68–1.58)1.05 (0.65–1.68)
About Functional cure
Anticipation2.15 (1.38–3.36)***1.56 (1.00-2.43)0.87 (0.48–1.58)1.08 (0.66–1.76)1.34 (0.86–2.08)0.61 (0.39–0.94)*0.96 (0.59–1.58) Awareness1.04 (0.68–1.60)0.60 (0.39–0.93)*1.19 (0.66–2.16)1.01 (0.63–1.63)0.85 (0.55–1.31)0.73 (0.48–1.12)1.31 (0.81–2.11) Willingness0.68 (0.30–1.51)2.10 (0.94–4.70)0.97 (0.32–2.92)1.01 (0.41–2.49)1.87 (0.77–4.58)2.20 (0.97-5.00)0.79 (0.33–1.88)MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndromeaIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV* p < 0.05 ** p < 0.01 *** p < 0.001
Crude odds ratio for impacts of perceived importance for HIV cure among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, AIDS acquired immunodeficiency syndrome
aIMP1 No longer need to take HIV medications; IMP2 Restoration and stabilization of effective immune function; IMP3 Not getting HIV for a second time; IMP4 No longer need to visit a doctor for HIV; IMP5 No longer at risk of AIDS or HIV-related morbidity; IMP6 No longer transmitting HIV to the others; IMP7 Being considered as a person not infected with HIV
* p < 0.05 ** p < 0.01 *** p < 0.001
Awareness of HIV functional cure:
Overall, less than half (42%) of the participants were aware of HIV functional cure. Compared with those having no knowledge of it, treatment-experienced MSM were more likely to have heard of HIV functional cure but without detailed knowledge (adjusted odds ratio [aOR] 2.92, 95% CI 1.67–5.10). Awareness was associated with STI history in the past year (aOR 1.64, 95% CI 1.00-2.68), and being sexually active (aOR 1.90, 95% CI 1.14–3.18) (Table 4). Those who were aware of HIV functional cure did not consider restoration and stabilization of effective immune function an important impact (OR 0.60, 95% CI 0.39–0.93), nor were they concerned about potential adverse effects of therapies for functional cure (OR 0.54, 95% CI 0.31–0.93).
Table 4Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)AwarenessLevel of anticipationWillingness to participate in functional cure trial
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Crude OR
Adjusted OR
Age group
16–291.00–1.001.001.00– 30–391.01 (0.58–1.76)–0.87 (0.50–1.52)0.88 (0.50–1.56)0.72 (0.25–2.10)– 40–490.74 (0.40–1.36)–1.28 (0.69–2.37)1.18 (0.62–2.22)1.19 (0.32–4.37)– 50 or over0.98 (0.53–1.82)–2.34 (1.18–4.61)*2.55 (1.28–5.11)**0.62 (0.20–1.94)–
Education level
Secondary or below1.00–1.00–1.00– Post-secondary or above0.97 (0.62–1.52)–1.10 (0.69–1.74)–1.12 (0.48–2.59)–
STI history in the past year
1.54 (0.97–2.46)1.64 (1.00-2.68)*1.23 (0.75–1.99)1.46 (0.88–2.42)9.94 (1.32–74.65)*8.16 (1.06–62.77)*
Sexual activity after HIV diagnosis
1.60 (1.03–2.50)*1.90 (1.14–3.18)*1.08 (0.70–1.69)–1.84 (0.82–4.17)2.11 (0.87–5.13)
Engagement in chemsex
0.82 (0.53–1.27)–1.25 (0.80–1.94)–3.42 (1.25–9.33)*2.35 (0.83–6.61)
CD4 cell count (cells/uL)
≥ 5001.001.001.00–1.00– < 5001.22 (0.80–1.87)1.59 (0.97–2.61)0.89 (0.58–1.37)–0.80 (0.36–1.78)–
About functional cure
Awareness
–
–
0.48 (0.31–0.75)**0.47 (0.30–0.74)**0.85 (0.38–1.90)0.52 (0.21–1.25) Anticipation0.48 (0.31–0.75)**0.46 (0.29–0.72)***
–
–
1.74 (0.77–3.93)– Willingness to participate in trial0.85 (0.38–1.90)–1.74 (0.77–3.93)–––MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio* p < 0.05 ** p < 0.01 *** p < 0.001
Crude and adjusted odds ratio for awareness and anticipation of HIV functional cure and willingness in participating in a trial among MSM (N = 356)
MSM men who have sex with men, HIV human immunodeficiency virus, STI sexually transmitted infections, OR odds ratio
* p < 0.05 ** p < 0.01 *** p < 0.001
Anticipation of HIV functional cure:
Among all participants, the median anticipation score for HIV functional cure was 10 (IQR 8–10) with more than half (59%) giving a score of 10, and it did not differ between ART treatment-naïve and experienced participants. Among treatment-experienced MSM, those having been diagnosed with HIV for more than 8 years had higher odds of scoring 10 for their level of anticipation for HIV cure (OR 1.93, 95% CI 1.08–3.46). Those anticipating HIV functional cure were more likely to be of age 50 years or above (aOR 2.55, 95% CI 1.28–5.11), and to have a lower awareness of the idea (aOR 0.47, 95% CI 0.30–0.74) (Table 4). Higher level of anticipation was associated with the consideration of not needing to take long-term HIV medications following functional cure (OR 2.15, 95% CI 1.38–3.36) but negatively with untransmittability of HIV (OR 0.61, 95% CI 0.39–0.94).
Acceptance of an HIV functional cure trial:
Should there be an HIV functional cure trial, the vast majority (93%) would consider joining. Therapy safety (96%), advice from healthcare professionals (88%), and credibility of the research institution (88%) were the top three factors participants took into account when considering whether to join such a trial. Participants interested in joining the trial were more likely to have been diagnosed with an STI in the past year (aOR 8.16, 95% CI 1.06–62.77), and to take advice from healthcare professionals when making such a decision (OR 4.64, 95% CI 1.84–11.69). When deciding whether to participate in an HIV functional cure trial, view and support from family and peers were important for those who ceased to have sex (OR 1.99, 95% CI 1.18–3.34) and who had attained lower education level (OR 2.31, 95% CI 1.42–3.74) (Additional file 2: Table S1). Credibility of the research institution was important to those who had received higher education (OR 2.11, 95% CI 1.10–4.04). Participating in such a trial is not without concerns. Major concerns included the potential risk of developing AIDS and complications (86%), HIV viral load going up (83%) and adverse effects of the therapy (81%). They were concerned about adverse effects on their CD4 level (OR 1.84, 95% CI 1.09–3.10) and complications (OR 2.16, 95% CI 1.06–4.42) after joining the trial. Treatment-experienced participants were less likely to be concerned about AIDS or complications (OR 0.45, 95% CI 0.23–0.89), whereas participants aged below 30 years (OR 4.76, 95% CI 1.66–13.64), those who had attained post-secondary level education (OR 2.08, 95% CI 1.11–3.88), and who had a CD4 cell count lower than 500 cells/uL (OR 2.02, 95% CI 1.06–3.84), expressed concerns about the risk of developing AIDS or related complications after or during trial participation.
Concerns arising from participating in an HIV functional cure study were intertwined with factors that were considered when deciding on whether one would join such a study. Those concerned about interruption of ART regimen were worried about CD4 count going down (OR 2.30, 95% CI 1.24–4.27), HIV viral load going up (OR 2.81, 95% CI 1.43–5.52) and becoming infectious (OR 2.01, 95% CI 1.06–3.80) (Additional file 2: Table S2) following functional cure therapy. Considering trial duration important was associated with concerns about adverse effects (OR 2.67, 95% CI 1.47–4.84). Participants who considered trial safety important were concerned about all five potential adverse consequences of participating in the trial. Those looking forward to the prospect of restoring effective immunity following HIV cure had concerns about CD4 count going down after joining the trial (OR 2.03, 95% CI 1.25–3.29).
Discussion:
HIV functional cure is a relatively new concept for PLHIV. Despite their generally low awareness, MSM living with HIV had, as shown in this study, a high level of anticipation for HIV functional cure, especially those who had been diagnosed and put on ART for some years. The effective achievement of functional cure carries the potential prospect of getting rid of repeated clinic visits and taking long-term medications [11]. The benefit of being untransmittable may, however, not be regarded as an important impact for HIV cure because in the current era of “undetectable equals untransmittable”, adherence to ART is already effective in minimizing the risk of transmission. In our cohorts of MSM living with HIV, the attitudes differed by one’s ART treatment experience. Treatment-naïve participants were more concerned about their immune function and AIDS progression than those ART-experienced, as deteriorated health was perceived by newly diagnosed patients an imminent consequence brought on by HIV [12]. Anticipation for HIV functional cure was apparently lower among those who were aware of it. Although some 42% had heard about functional cure, most had limited knowledge of it and cared little about the potential benefit of restoring their immune function and potential adverse effects arising from the new therapy. With the increasing likelihood that functional cure may soon become the next major advance in HIV treatment [7], there is the need for promoting community education on the rationale and strategy for HIV functional cure. As in many countries, MSM accounted for a high proportion of PLHIV in Hong Kong [13], and who continued to be significantly impacted by the epidemic [14]. The MSM community, especially those living with HIV would need to be targeted in anticipation of the introduction of functional cure therapy. In our study, MSM engaging in higher risk sexual behaviors were more likely to be aware of the recent advancement on HIV science, such as pre-exposure prophylaxis [15]. This echoed our results that sexually active PLHIV, particularly those engaged in condomless sex as indicated by their STI history, were more likely to be aware of HIV functional cure.
There are diverse reasons for PLHIV to look forward to receiving functional cure therapy. It is intuitive that PLHIV with a weaker immune system were less likely to be sexually active and they wished to restore and stabilize their immune function and were at the same time more concerned about adverse HIV outcomes after joining functional cure trials. The fact that younger PLHIV cared more about disease development and transmittability was likely because they were newly diagnosed and would remain sexually active for a longer time in the future. PLHIV with higher education level regarded de-labelling of HIV an important impact of HIV cure. In Hong Kong, public stigma against PLHIV [16] and perceived discrimination [17] have been reported. It is therefore an ideal vision for them to remove the label of HIV after achieving some sort of cure. Discrimination in workplace and difficulty in career development could be experienced by PLHIV [18]. Although highly educated PLHIV were found to deal better with workplace discrimination and improve employment quality than those with lower education level [19], they were more concerned with their career development that removing stigma means removing a barrier in their career paths. From the perspective of the general public, increased awareness and knowledge in HIV science advancement might be able to reduce stigma. A previous study showed that heterosexually active adults who were aware of the untransmittable nature of PLHIV achieving undetectable viral load had a lower level of anticipated HIV stigma [20]. As this study focuses on PLHIV, a follow-up study in the general population and the MSM community would be needed to assess the impact of functional cure on general, and dating- and sex-related enacted stigmas.
Overall, the idea of an HIV functional cure trial was well accepted by our cohorts of MSM living with HIV with over 90% indicating their willingness to join, similar to the high acceptance rate of 95% in a previous study [11]. Potential trial participants were more likely to have engaged in higher risk sexual activities as reflected by their report of STI in the preceding year. This might be precipitated by a prospect of continuation or re-engagement in adventurous condomless behaviors upon cure [21]. Safety is a major concern for clinical trials involving the use of novel agents like neutralizing antibodies or therapeutic vaccines. Careful monitoring of PLHIV in the trial would be warranted not just for protecting participants’ health but also easing their concerns of side effects and adverse HIV outcomes. Hesitation would arise if the trial requires interruption of ART or if it takes a long time, in relation to the risk of rebound and potential adverse effects [11]. Trusting relationship between PLHIV, researchers and healthcare workers could be beneficial for ensuring the delivery of optimal health outcomes [22]. Based on potential participants’ trust on healthcare professional advice, collaboration with HIV clinics on explaining the details and safety issues of the trial would be paramount.
This study carries some limitations. Like other epidemiological studies, there were inherent recall and social desirability biases in the administration of behavioral questionnaires. We had minimized these biases by adopting a shorter recall period and omitting fine details about sexual activities. The adoption of a self-administering approach should have reduced embarrassments in response to interviewers’ questions. Incorporation of clinical data by transcription benefited the study by providing objective measurements of participants’ HIV outcomes. In the absence of any functional cure trials, the exploration of one’s willingness to participate in a hypothetical trial without having one in place could be postulational. Nevertheless, the systematic inquiries into the awareness and anticipation of functional HIV cure among MSM living with HIV and their inclination in joining a hypothetical study have generated useful results which could be of reference for the planning of a functional cure trial.
Conclusions:
To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment.
Supplementary Information:
Additional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).
Additional file 1. Questionnaire.
Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).
Additional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).
Additional file 1. Questionnaire.
Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).
:
Additional file 1. Questionnaire.Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).
Additional file 1. Questionnaire.
Additional file 2: Table S1. Crude odds ratio for factors of importance to be considered and concerns related to participation in functional cure trial among MSM (N=356). Table S2. Crude odds ratio for important factors to be considered about and concerns related to the trial among MSM (N=356).
| Background:
Human immunodeficiency virus (HIV) functional cure is a novel biomedical strategy characterized by sustained viral suppression without the need for life-long medications. The attitude of people living with HIV (PLHIV) towards functional cure and clinical trials are understudied. We aimed to examine the awareness and levels of anticipation for HIV functional cure among men who have sex with men (MSM) living with HIV, and their willingness to join trials as differentiated by their antiretroviral treatment status.
Methods:
MSM living with HIV with and those without treatment history were recruited from Hong Kong's HIV specialist clinics. Self-administered questionnaires covering behavioral profile, perceived impact of HIV cure, attitude towards HIV functional cure and related clinical trials were collected. Clinical data were separately transcribed. Determinants of perceptions and attitudes were identified by logistic regression models.
Results:
Of 356 MSM living with HIV recruited, less than half (42%) were aware of HIV functional cure, but they had a high level of anticipation for it. Treatment-experienced participants were more likely to be aware of HIV functional cure. Awareness was associated with continued engagement in sexual activities after HIV diagnosis and sexually transmitted infection (STI) diagnosis. Higher anticipation was observed among older MSM living with HIV but it was negatively associated with one's awareness. Over 90% were willing to join functional cure trials, especially those who had previously been diagnosed with STI and had engaged in chemsex in the past year. Advice from healthcare professional was an important factor considered by those willing to join clinical trials. Younger, better educated MSM, and those with lower CD4 counts were more concerned about potential risk of AIDS and potential complications upon trial participation.
Conclusions:
MSM living with HIV, especially those sexually active, showed positive attitude towards functional cure and willingness to join related clinical trials despite low awareness. To enhance preparedness for HIV functional cure trials, community education, updated information and appropriate medical advice would be needed. Safety is a major concern for potential enrollees in HIV functional cure trials. | Background:
Life-long treatment with antiretroviral compounds is currently the gold standard in the clinical management of human immunodeficiency virus (HIV) infection. While antiretrovirtal therapy is effective in restoring health and minimizing secondary HIV transmission, it falls short of achieving virus eradication. Effective curative treatment could provide a sustainable solution to prevention and control [1]. Despite increasing research with the accumulation of scientific evidence for HIV cure, such therapy is not yet in sight [2]. Despite the uncertainties, a survey of over 400 people living with HIV (PLHIV) in the United States showed that more than half were willing to participate in different HIV cure studies [3]. Elimination of the HIV virus in the “Berlin patient” offered new hope in the PLHIV communities about the ultimate goal of cure [4]. De-stigmatization was particularly valued by PLHIV who favored curative treatment when it becomes available [5, 6].
In the interim, animal studies and human trials suggested that “functional cure” of HIV infection may become a reality in the coming years [7, 8]. Unlike “sterilizing cure”, functional cure strategy aims to achieve effective suppression of HIV viral load so that antiretroviral therapy (ART) becomes unnecessary. While “functional cure” could represent different strategies, the term is now consistently used in referring to the attainment of virus control without ART [7], while one’s HIV infection status remains unchanged. Willingness of PLHIV in receiving non-eradication cure treatment or joining functional cure research would be an important consideration, especially that current generations of antiretrovirals are safe and extremely effective. PLHIV’s decision may hinge on one’s understanding of the concept of cure and how this is explained [9]. In the United States, many participants in a study did not consider functional cure as an improvement to conventional ART [5]. Experiences with ART may also affect PLHIV’s decision about participation in functional cure research. In Hong Kong, ART coverage in PLHIV receiving care at the public service is high. With the increasing reports of the promising outcome of functional cure, it is timely that their attitude towards participation in functional cure research be explored. We hypothesized that the degree of knowledge and attitudes towards HIV cure and functional cure differed between ART-experienced and ART-naïve PLHIV. In addition, education needs in the specific area of functional cure among newly diagnosed patients could be identified, which may in turn ease subject recruitment for a clinical trial, and improve expectation management of participants. As such, we undertook to examine the attitude of MSM living with HIV towards functional cure in Hong Kong, and contrast the awareness and perceptions between newly diagnosed and veteran PLHIV who have been on long duration of ART.
Conclusions:
To PLHIV, HIV cure means restoring and stabilizing their immune systems that they no longer need to take long-term medications, nor do they need to visit HIV clinics repeatedly. While virus eradication cannot be achieved, HIV functional cure is a promising strategy in which MSM living with HIV had a high level of anticipation despite relatively low awareness. While a high acceptance rate of a functional cure clinical trial was elicited, MSM living with HIV were concerned about adverse HIV outcomes. Their appreciation of the objectives of functional cure, understanding of the study procedures, and recognition of potential adverse events are crucial, and all of which need to be accessible well before a functional cure trial begins enrolment. | Background:
Human immunodeficiency virus (HIV) functional cure is a novel biomedical strategy characterized by sustained viral suppression without the need for life-long medications. The attitude of people living with HIV (PLHIV) towards functional cure and clinical trials are understudied. We aimed to examine the awareness and levels of anticipation for HIV functional cure among men who have sex with men (MSM) living with HIV, and their willingness to join trials as differentiated by their antiretroviral treatment status.
Methods:
MSM living with HIV with and those without treatment history were recruited from Hong Kong's HIV specialist clinics. Self-administered questionnaires covering behavioral profile, perceived impact of HIV cure, attitude towards HIV functional cure and related clinical trials were collected. Clinical data were separately transcribed. Determinants of perceptions and attitudes were identified by logistic regression models.
Results:
Of 356 MSM living with HIV recruited, less than half (42%) were aware of HIV functional cure, but they had a high level of anticipation for it. Treatment-experienced participants were more likely to be aware of HIV functional cure. Awareness was associated with continued engagement in sexual activities after HIV diagnosis and sexually transmitted infection (STI) diagnosis. Higher anticipation was observed among older MSM living with HIV but it was negatively associated with one's awareness. Over 90% were willing to join functional cure trials, especially those who had previously been diagnosed with STI and had engaged in chemsex in the past year. Advice from healthcare professional was an important factor considered by those willing to join clinical trials. Younger, better educated MSM, and those with lower CD4 counts were more concerned about potential risk of AIDS and potential complications upon trial participation.
Conclusions:
MSM living with HIV, especially those sexually active, showed positive attitude towards functional cure and willingness to join related clinical trials despite low awareness. To enhance preparedness for HIV functional cure trials, community education, updated information and appropriate medical advice would be needed. Safety is a major concern for potential enrollees in HIV functional cure trials. | 12,661 | 393 | [
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|||||
The Impact of the COVID-19 Pandemic on an Israeli Acute Care Surgery Unit: Fewer Patients, More Disease. | 33856956 | The COVID-19 pandemic has transformed and affected every aspect of health care. Like any catastrophic event, the stress on hospitals to maintain a certain level of function is immense. Acute surgical pathologies cannot be prevented or curtailed; therefore, it is important to understand patterns and outcomes during catastrophes in order to optimize care and organize the health care system. | BACKGROUND | In a single urban tertiary care center, a retrospective study examined the first complete lockdown period of Israel during the COVID-19 pandemic. This was compared to the same time period the previous year. | METHODS | During the pandemic, time to hospitalization was significantly decreased. There was also an overall reduction in surgical admissions yet with a higher percentage being hospitalized for further treatment (69.2% vs 23.5%). The patients admitted during this time had a higher APACHE-II score and Charlson comorbidity index score. During the pandemic, time to surgery was decreased, there were less laparoscopic procedures, and more RBC units were used per patient. There were no differences in overall complications, except when sub-analyzed for major complications (9.7% vs 6.3%). There was no significant difference in overall in-house mortality or morbidity. Length of hospitalization was significantly decreased in the elderly population during the pandemic. | RESULTS | During the COVID-19 pandemic, despite a significantly less number of patients presenting to the hospital, there was a higher percentage of those admitted needing surgical intervention, and they were overall sicker than the previous year. | CONCLUSION | [
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] | 9629022 | Introduction | The COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6
A perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11
Understanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15
There is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic. | Methods | All patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.
Outline of study process.
During the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.
Statistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant. | Results | Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Demographics, Intervention, and Diagnosis.
Abbreviations: ED, emergency department.
Patients Who Had Surgical Intervention.
Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Comparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.
Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Demographics, Intervention, and Diagnosis.
Abbreviations: ED, emergency department.
Patients Who Had Surgical Intervention.
Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Comparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.
Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.
For the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.
When analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Elderly Patients Sub-Analysis.
Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Only ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10
10 Patients Originally Triaged as Potential COVID-19 Patients.
For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.
For the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.
When analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Elderly Patients Sub-Analysis.
Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Only ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10
10 Patients Originally Triaged as Potential COVID-19 Patients. | null | null | [
"Patient Demographics and Clinical Characteristics",
"Treatment and Outcome Characteristics"
] | [
"Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.",
"For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients."
] | [
null,
null
] | [
"Introduction",
"Methods",
"Results",
"Patient Demographics and Clinical Characteristics",
"Treatment and Outcome Characteristics",
"Discussion"
] | [
"The COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6\nA perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11\nUnderstanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15\nThere is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic.",
"All patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.\nOutline of study process.\nDuring the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.\nStatistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant.",
" Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\nOverall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.\n Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.\nFor the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.",
"Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nDemographics, Intervention, and Diagnosis.\nAbbreviations: ED, emergency department.\nPatients Who Had Surgical Intervention.\nAbbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.\nComparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.",
"For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.\nFor the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.\nWhen analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.\nElderly Patients Sub-Analysis.\nAbbreviation: LOS, length of stay; PRBC, packed red blood cells.\nOnly ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10\n10 Patients Originally Triaged as Potential COVID-19 Patients.",
"During this period, we have shown that despite a significant decrease in the number of patients presenting to our ER, a majority of them presented with more severe disease when compared to the previous year. In general, the patients undergoing an operation were in worse condition than the patients without operation. Clearly, during this pandemic, or any other natural disaster or military campaign, there is limited ability to curtail the inevitable presentation of acute surgical pathologies or traumatic events. During challenging circumstances, hospitals must be prepared for and must be able to maintain emergent surgical services in order to avoid preventable deaths and mortality. The results of this study have shown the effect of the COVID-19 pandemic on our patient population suffering from acute general surgery illnesses.\nThere was no difference in the pathologies presenting between the two periods, which is an important statistical finding, yet must be taken in proper context. Depending on the type of catastrophe, the required mobilization of specific medical specialties and strain on the systems will be drastically different. During times of war or natural disasters, hospitals will require a larger ratio of orthopedic surgeons, general surgeons, surgical specialties, OR access, and ICU beds.14 In contrast, pathogen-derived epidemics/pandemics will require more specialists in the fields of internal medicine (ie, infectious disease, pulmonology, intensivist, and hematologist), ICU beds, and potential access to extracorporeal membrane oxygenation.15 As seen with our results, this primarily respiratory pandemic did not significantly change the acute surgical pathologies.\nIn regard to the younger patient population, there were several observations that were of interest yet not statistically significant. These patients appeared to present later in their course of disease, with a higher rate of complicated pathologies (ie, perforated appendix and gangrenous cholecystitis), postoperative complications, and blood transfusions. These younger patients were therefore more likely able to tolerate their symptoms at home for a longer period and therefore had a delay in diagnosis and treatment. In contrast, the older population, with their lower physiological reserve, appeared less able to delay medical treatment and came to the hospital regardless of fears and inconveniences caused by the pandemic restrictions.\nThe “fear” of presenting to the ER with severe illness has been documented from this current COVID-19 pandemic21 and from previously published literature during catastrophes. For example, during the SARS epidemic in 2002, Taiwan and Hong Kong showed a decrease in routine care not related to the severe acute respiratory syndrome.22 During this COVID-19 pandemic, one recent study reported the patterns of patients presenting to the ER of a busy level-1 trauma center in America. This group showed a global decrease in arrivals to the ER, along with a decreased proportion of patients presenting with abdominal pain when compared to other systemic complaints before the pandemic.23 This cannot be accurately compared to our data because we were only looking at acute surgical pathologies. Nevertheless, what was reported in this reference might also be a result of avoiding going to the hospital with abdominal pain during a pandemic that involves primarily respiratory symptoms. In Italy, toward the beginning of the pandemic, a series of children, without COVID-19-related illness, were shown to have presented late and in more critical condition, due to the fear of presenting to a hospital or lack of provisional care because of closure of health consults/centers.24 This corresponds with our overall results in general of sicker patients being admitted to the surgical ward.\nThe country where this study took place, Israel, also presents a unique confounding factor to these data. Our hospital is a public hospital within a socialized system where every citizen is born with universal health care coverage25; therefore, lacking health care is rarely a reason for not seeking medical attention.26 Compared to the rest of the world, our mortality has been comparatively low.27,28 Our “lockdown” measures and monitoring methods were considered aggressive, but the country also progressed in easing the regulations and opening its economy relatively fast. Nevertheless, the cooperation and coordination of our Ministry of Health, security sectors, and private industry played a pivotal role in preventing our hospital infrastructure from being completely overwhelmed. Our results showed a different demographic in terms of ethnicity presenting to the hospital during the COVID-19 pandemic. There was a significant increase in the ratio of Israeli-Jewish to Israeli-non-Jewish patients presenting to the ED and a smaller proportion of minorities, tourist, and foreign workers when compared to 2019. As mentioned above, the TASMC is located in the center of Tel Aviv, where 91% of the population is Jewish and 9% are non-Jewish. This difference might be explained by the lack of transport, hesitancy to travel distances, and fear of larger medical centers because of their treatment of COVID-19 patients.29 Hence, smaller local medical centers were potentially receiving more patients, regardless of appropriateness of level of care.\nWe found a low incidence of mis-triage of surgical patients into the COVID-19 isolation wing by our ER triage protocol. Out of the 209 patients admitted during this time, only 10 were initially placed in isolation. None of these 10 isolated patients had a positive COVID-19 test result. Despite being placed in triage, the average time to hospitalization, despite being statistically different, was only 30 minutes longer than the non-isolated group. When intervention (surgical and nonsurgical) was needed in the isolated group, there was no increase in time when compared to the non-isolated patients and, on average, even occurred more rapidly. With many surgical pathologies presenting with fever, it was difficult, especially at the beginning of the pandemic, not to over triage these patients and potentially delay intervention for surgical emergencies. This became especially relevant as evidence began to show that a possible presenting symptom of COVID-19 is fever with diarrhea.30 The significant reduction in time to hospitalization during the pandemic may be a confounding effect of both a decrease in volume to the ED and also sicker patients presenting to the ED, which would require quicker diagnosis and appropriate therapy. This decrease in hospitalization time happened despite a reduction in working staff.\nMultiple studies have established that this elderly population is at an increased risk to clinically significant COVID-19.31 Our elderly population (>70 years old) during the pandemic had slightly more comorbidities and had a greater 30-day mortality than the previous year. Despite their increased comorbidities, they did not present to the hospital sicker than their counterparts the previous year. This trend might demonstrate the opposite of the “fear” phenomenon mentioned above for younger patients that sicker, elder patients present more easily to the hospital driven by the distress that something is wrong. With the elderly patients who did not undergo surgery, we were able to significantly reduce their hospitalization time. We have emphasized in our department not to neglect surgical pathologies during this time in this vulnerable population.\nAn interesting finding from the patients undergoing surgery during the pandemic is that they had a higher APACHE score, more complications, more blood given, yet more cases were open (verses laparoscopic), and there was an attending present more often. This seemingly contradicting findings might be a true indicator of how severely ill/complicated the patients were when compared to the previous year. Yet, this finding would need to be compared to other large tertiary centers in order to gather a significant conclusion. There are additional limitations to this study, including the fact that it is coming from a single urban referral center. These data may not be representative of other smaller and/or rural centers. Other limitations include the short study time and not having a cohort of surgical patients who were COVID-19 positive. The short study time was chosen in order to describe the initial effect of the pandemic and the most constricting limitations imposed by the government during the initial lockdown. Therefore, the initial month of true, full lockdown was taken and compared to the previous year. Clearly, during different stages and evolution of the pandemic, societies and medical systems have changed/adapted; therefore, the time when this study was conducted needs to be taken into consideration. There was no (knowingly) COVID-19-positive patient in our cohort and therefore no comparison between the differences in surgical pathology progression between positive and negative surgical patients. As there is more understanding of the COVID-19 pathophysiology, this would be an important patient population to analyze.\nIn conclusion, we have shown that during this COVID-19 pandemic, the patients presenting were sicker than the patients during the same time frame in the previous year despite there being fewer patients needing surgical admission. We were able to decrease the time until hospitalization and the length of hospitalization on the highly vulnerable elderly population. These data support the need for advocating and educating the public to present to hospitals without delay if there are symptoms of a possible acute surgical illness. Clearly, there are difficult times that may force the population to stay home (natural disasters and war), but fear alone should not delay the presentation of a patient with abdominal pain to the hospital. With well-organized systems and cooperation between governing and health sectors, hospitalization can be streamlined and physician/hospital workforce exposure can be minimized, while not compromising the level of care and treatment needed for acute surgical pathologies. Through understanding the surgical needs of a population during a catastrophic event, the public health sector may more rapidly and precisely distribute needed personnel, essential resources, and allocate certain medical centers as referral points for surgical emergencies."
] | [
"intro",
"methods",
"results",
null,
null,
"discussion"
] | [
"COVID-19",
"acute care surgery",
"pandemic"
] | Introduction:
The COVID-19 pandemic has affected every aspect of health care. Hospitals have quickly reorganized in order to accommodate the sudden needs of symptomatic COVID-19 patients, while struggling to maintain the routine surgical and medical requirements of their communities. Most countries from the Americas, Europe, and Asia either halted elective surgeries or placed heavy limitations on them.1–3 Other countries, such as Japan and Sweden, had less restrictive measures and continued with almost fully normal activity.4,5 The majority of countries that restricted surgical services, besides the acute care surgery (ACS) and trauma surgery, continued with a handful of oncological cases which had a narrow window for a therapeutic operation. The infrastructure of some countries and regions was so completely overwhelmed that they were forced to shut down all surgical services (emergent and elective) due to a limited number of respirators and staff. In Israel, the Ministry of Health ordered all elective surgical services to be suspended, with the exception of time-dependent oncological cases.6
A perfect level of preparedness for an unforeseeable event is never possible, yet lessons learned from past tragedies and patterns from similar events are what guide preparedness efforts and the early stages of response.7 For example, during Hurricane Katrina in the Gulf Coast region of the United States, “alternative-site” primary care and triage facilities were established to buffer the surge toward functioning emergency rooms (ERs) and hospitals.8 In addition, during this natural disaster, mobile surgical units were deployed to provide damage control surgery and ICU level of care.9 During the Ebola epidemic of 2014-2015 in Sierra Leone, surgical admission and operations fell drastically when compared to their preoutbreak numbers, with a large number due to the death of surgeons and the lack of personal protective equipment.10 Israel’s own experience with civilian hospital activity during a military conflict showed a significant decrease in elective surgical procedures while only performing oncologic surgeries and emergent surgery procedures.11
Understanding the patterns of presentation and outcomes of acute surgical illnesses during a disaster is vital in order to establish appropriate and adequate care on a national level. Such knowledge can allow hospitals to adequately prepare for a surge of patients by estimating the number of general surgeons, trauma surgeons, and operating room(OR)/ICU resources that must be allocated.6,12–15
There is paucity of data describing how war, natural disasters, or an epidemic/pandemic16 affect the presentation and treatment of emergent surgical illnesses. Here, we analyze our ACS patient population presenting to an Israeli tertiary hospital during a month of complete lockdown in our country due to the COVID-19 pandemic.
Methods:
All patients who presented to the Tel Aviv Sourasky Medical Center (TASMC) emergency department (ED) and were admitted to the surgical division with an acute surgical pathology were included in this study. Tel Aviv Sourasky Medical Center is a 1500-bed, Level-1 trauma and tertiary care center serving downtown Tel Aviv and surrounding communities. The study period compared patients from March 15 to April 14, 2019 to the corresponding period in 2020, the specific dates of Israel’s full lockdown as ordered by the Ministry of Health, as a result of the COVID-19 pandemic. After receiving approval by our Internal Ethics Committee, retrospective data were obtained from the computerized medical record system. We reviewed patients' demographics, comorbidities, preoperative/preadmission diagnosis, imaging, intraoperative parameters, and postoperative course including complications, length of stay, and morbidity and mortality outcomes. The APACHE-II score17 was used to assess the severity of systemic illness at presentation. Postoperative morbidity was graded according to the Clavien-Dindo (CD) classification,18 and a major complication was defined as ≥ III. A total of 644 patients were admitted from the ED to the surgery department during these 2 periods. Out of this number, 78 patients were excluded, and therefore 566 patients were included in the study cohort. Excluded from this study are patients whose original admission order was canceled, admitted for semi-elective cases, incomplete medical records or unclear diagnosis, those for immediate transplant, nonsurgical diagnosis, and patients being admitted for a complication from a previous elective surgery. Figure 1 shows the outline of the study process. Intraoperative complications were classified according to the Classic Delphi study.19Figure 1.Outline of study process.
Outline of study process.
During the COVID-19 outbreak, the ED triage was based on clinical suspicion of COVID-19-related symptoms. All cases with even mild suspicion of COVID-19 were referred to a separated and isolated ED wing. A trained senior ED resident or attending was responsible for the triage checkpoint at all hours of the day or night. This was not only to maintain a high level of triage quality but also to directly discharge patients who would not benefit from ED workup, to identify critical patients that needed immediate airway intervention, and to immediately obtain samples for COVID-19 polymerase chain reaction (PCR) testing. COVID-19-related placement and triage was conducted based on the National Early Warning Score 2 (NEWS 2 score).20 Patients entering the COVID-19 isolated wing were further stratified as suspected with pending results or as having a diagnosed active COVID-19 infection. Polymerase chain reaction results for all patients were obtained within 6 hours. All personnel in the COVID-19 wing used full personal protective equipment. Senior emergency medicine and internal medicine residents and physicians were responsible for the care of these patients while in the isolation wing. Surgical consultations were provided upon request.
Statistical analysis was performed using the IBM SPSS v.26 statistics data editor. Continuous data were expressed as median values with the corresponding standard deviation. Student’s t-test was used for continuous data, and the Chi-square test was used for categorical data. Sub-analysis and post hoc analysis were performed by mean of intervention (surgical, percutaneous drainage, and endoscopic) as well as by age (above 70). A P-value of <.05 was considered statistically significant.
Results:
Patient Demographics and Clinical Characteristics Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Demographics, Intervention, and Diagnosis.
Abbreviations: ED, emergency department.
Patients Who Had Surgical Intervention.
Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Comparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.
Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Demographics, Intervention, and Diagnosis.
Abbreviations: ED, emergency department.
Patients Who Had Surgical Intervention.
Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Comparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.
Treatment and Outcome Characteristics For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.
For the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.
When analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Elderly Patients Sub-Analysis.
Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Only ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10
10 Patients Originally Triaged as Potential COVID-19 Patients.
For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.
For the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.
When analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Elderly Patients Sub-Analysis.
Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Only ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10
10 Patients Originally Triaged as Potential COVID-19 Patients.
Patient Demographics and Clinical Characteristics:
Overall, 302 surgical patients admitted to the ED during the COVID-19 quarantine compared to 1519 patients in the same time frame of 2019. Of these patients admitted during the COVID-19 pandemic, a higher percentage were hospitalized for further treatment, 69.2% vs 23.5% (P = .003). Table 1 depicts their demographic and clinical characteristics. Median age was comparable in both groups 57 vs 62 years old (P = .33), respectively; the proportion of elderly patients (age >70 years) was also equal, 37% vs 33% (P = .44). Male gender was more common during the COVID-19 quarantine, 60% vs 54%; however, it did not significantly differ (P = .15). As shown in Table 1, the rates of non-Jewish citizensand tourists and immigrants admitted to surgery were significantly reduced during the pandemic, 5.9% vs1% and 6.2% vs 3.4%, respectively (P = .022). During the quarantine time period, overall patients had a higher mean Charlson comorbidity index 1.94 vs 1.33 (P = .004) and a higher APACHE-II score 6.69 vs 5.75 (P = .024). A higher APACHE-II score was also noticed amid the group of patients who underwent surgery during the quarantine as compared with the parallel time period in 2019, 6.15 vs 3.98, respectively (P = .003) (Table 2).Table 1.Demographics, Intervention, and Diagnosis.20192020P-ValueED admission1519302–Hospitalization357 (23.5%)209 (69.2%).03Age (median)56.34 ± 21.0758.07 ± 21.03.35Gender (male/female)193/164126/83.15Ethnicity (Jewish/minorities/tourists and foreigners)87.9%/5.9%/6.2%95.7%/1%/3.4%.022Charlson comorbidity index1.33 ± 2.231.94 ± 2.77.004APACHE-II score5.75 ± 4.346.69 ± 4.19.024Time to hospitalization10.12 ± 5.317.53 ± 4.24<.001Type of interventionSurgery158 (44.3%)72 (34.5%).12Drainage22 (6.2%)22 (10.5%).06Endoscopy39 (10.9%)32 (15.3%).12Angiography2 (.6%)0 (0%).29Radiation1 (.3%)0 (0%).45Differential diagnosis of hospitalized patientsAppendicitis52 (14.6%)26 (12.4%).51Diverticulitis13 (3.6%)11 (5.3%).37Cholecystitis28 (7.8%)16 (7.7%).94Gastrointestinal bleeding48 (13.5%)34 (16.3%).39Hernia15 (4.2%)5 (2.4%).27Bowel obstruction33 (9.2%)31 (14.8%).06Perianal abscess39 (10.9%)15 (7.2%).16Pancreatitis15 (4.2%)12 (5.7%).42Cholangitis25 (7%)7 (3.4%).08Trauma26 (7.3%)12 (5.7%).49Other61 (17.1%)38 (18.2%).76NA2 (.6%)2 (1%).59Abbreviations: ED, emergency department.Table 2.Patients Who Had Surgical Intervention.20192020P-ValueNumber of patients15872.08Age48.9 ± 21.449.36 ± 20.75.87Gender (male/female)87/7144/28.39Charlson comorbidity index.82 ± 1.59.96 ± 1.74.56APACHE-II score3.98 ± 3.756.15 ± 4.64.003Median time to hospitalization9:23 (6:14-13:18)6:56 (4:15-10:02).72Median time to surgical intervention19:01 (10:22-42:25)11:00 (7:27-23:26)<.001Median duration of surgery1:04 (0:29-1:41)1:14 (0:42-2:10)<.001LOS from surgery5.69 ± 10.435.06 ± 6.6.588Presence of attending70 (44.3%)41 (56.9%).031Laparoscopic surgery84 (53.2%)30 (41.7%).011Intraoperative complication (Delphi study)9 (5.7%)7 (9.7%).091PostoperativeComplications (Clavien-Dindo score)15 (3.2%)5 (6.9%).019221 (13.3%)9 (12.5%).0533a1 (.6%)1 (2.8%).023b3 (1.9%)3 (2.8%).04741 (.6%)1 (1.4%).04455 (3.2%)2 (2.8%).053Major complication CD ≥ 310 (6.3%)7 (9.7%).03Number of PRBC during hospitalization.08 .42.42 1.58.013LOS5.69 10.435.06 6.7.65In-hospital mortality5 (3.1%)4 (5.5%).32630-day readmission10 (6.49%)6 (11.1%).27330-day mortality4 (2.53%)4 (5.5%).12Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Demographics, Intervention, and Diagnosis.
Abbreviations: ED, emergency department.
Patients Who Had Surgical Intervention.
Abbreviation: CD, Clavien-Dindo; LOS, length of stay; PRBC, packed red blood cells.
Comparing the two study periods, the specific common diagnoses for what patients were hospitalized for showed no significant difference (Table 1). The five most common diagnoses were the same in both 2019 and 2020—appendicitis, gastrointestinal bleeding, perianal abscess, bowel obstruction, and cholecystitis. Only the most prevalent of these diagnoses was different between the two study periods, with gastrointestinal hemorrhage being the most admitted diagnosis (14.3%) during the COVID-19 period, compared to the appendicitis (12.8% of admissions) during the previous year.
Treatment and Outcome Characteristics:
For the entire cohort, the median time to hospitalization was significantly shorter during the quarantine period 7:53 hours vs 10:12 hours (P = <.001). During the lockdown, 72 (34.5%) had surgery, the majority for appendicitis. The rate of patients who had surgery during April 2019 was greater (44.5% vs 34.5%, P = .12) despite not being statistically significant. During the COVID-19 quarantine, there was also more endoscopic and percutaneous drainage intervention (15.3% vs 10.9%, P = .12 and 10.6% vs 6.2%, P = .06, respectively) (Table 1). In a sub-analysis of patients who underwent percutaneous drainage or endoscopy therapy, there were no significant differences in the APACHE-II score or any other clinical outcomes.
For the patients requiring surgery (Table 2), the time to hospitalization was not significantly shorter (P = .72); however, the median time from ED admission to surgery was significantly shorter during the COVID-19 quarantine month, 19:01 hours (range 10:22-42:25) vs 11:00 hours (7:27-23:26) (P = <.001). Also during this quarantine period, the rate of laparoscopic procedures was lower, 41.7% vs 53.2% (P = .011), an attending surgeon was present in more cases (56.9% vs 44.3%, P = .031), and the median duration of surgery was longer (1:14 hours vs 1:04 hours, P = <.001). The average number of RBC units consumed during surgery and postoperatively was significantly higher during the COVID-19 quarantine period (.42 vs .08, P = .013). During the quarantine time, intraoperative and postoperative complication rates did not significantly differ (9.7% vs 5.7%, P = .091 and 22.8% vs 29.2%, P = .37); however, major complications categorized as CD >3 were more common (9.7% vs 6.3%, P = .03). In any age-group, there were no statistical differences in length of stay, in-hospital or 30-day mortality rates, and readmission amid the groups of patients having surgery.
When analyzing only elderly patients, age>70, they exhibited similar demographics and trends in 2020 to the entire cohort by having similar Charlson comorbidity index scores (P = .044), and a shorter time to hospitalization (P = <.001), yet there was no difference with regard to the APACHE-II score (P = .47). In this elderly subgroup, the 30-day mortality rate was increased in 2020 (3.57% vs 11.3%, P = .045), with no significant change in in-hospital mortality (Table 3).Table 3.Elderly Patients Sub-Analysis.20192020P-ValueN11978Age80.34 ± 6.8879.76 ± 6.87.563Gender (male/female)59/6042/36.559Charlson comorbidity index2.39 ± 2.653.22 ± 3.04.044APACHE-II score9.66 ± 3.6710.07 ± 3.42.466Time to hospitalization10.55 ± 5.417.51 ± 4.34<.001Patients requiring surgery3615.084Time to surgical intervention53.91 ± 88.8237.34 ± 65.67.461Duration of surgery1.89 ± 1.02.07 ± 1.47.154LOS from surgery12.4 ± 17.57.07 ± 7.74.143Presence of attending25 (69.4%)10 (66.7%)1.0Laparoscopic surgery15 (41.7%)6 (40%).97Intraoperative complications3 (8.3%)5 (31.3%).057Major complication CD≥36 (16.7%)5 (31.3%).215Number of PRBC during hospitalizationAll patients.24 ± .72.85 ± 1.53<.001Patients undergoing surgery.11 ± .401.0 ± 1.84.01LOSAll patients11.64 ± 13.66.41 ± 5.18<.001Patients undergoing surgery14.75 ± 17.138.57 ± 7.62.085In-hospital mortality5 (4.2%)6 (7.6%).26930-day readmission16 (13.45%)7 (8.97%).52830-day mortalityAll patients4 (3.4%)7 (9.0%).045Patients undergoing surgery3 (2.5%)3 (3.8%).345Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Elderly Patients Sub-Analysis.
Abbreviation: LOS, length of stay; PRBC, packed red blood cells.
Only ten patients of the entire 2020 cohort were first triaged to the COVID-19 isolation wing of the ED. Of these patients, none were positive for COVID-19, four required emergent surgery, and three percutaneous drainage for intra-abdominal infections (Table 4). For the patients who entered the COVID-19 isolation wing, the average time to hospitalization was 8:00 hours (SD 7:30 hours), which was significantly less when compared to the non-isolated patients in 2020 with a time to hospitalization of 7:30 hours (SD 4:05 hours), P = .027. The average time to nonsurgical intervention and surgical intervention for the patients initially isolated due to COVID-19 protocol was 14:30 hours (SD 9:42 hours) and 31:08 hours (SD 15:15 hours). This was similar to those not isolated during the same period that were 39:25 hours (SD 43:34 hours, P-value .1) and 31:13 hours (SD 67:40 hours, P-value .5). There were no confirmed COVID-19 positive patients who had an acute surgical illness requiring surgery or hospitalization for surgical care.Table 4.10 Patients Originally Triaged as Potential COVID-19 Patients.DiagnosisNumber of PatientsAppendicitis1Diverticulitis1Cholecystitis3Gastrointestinal bleeding0Hernia1Bowel obstruction0Perianal abscess0Pancreatitis1Cholangitis1Trauma1Other (excluded)Intestinal perforationNA (excluded)0Total10
10 Patients Originally Triaged as Potential COVID-19 Patients.
Discussion:
During this period, we have shown that despite a significant decrease in the number of patients presenting to our ER, a majority of them presented with more severe disease when compared to the previous year. In general, the patients undergoing an operation were in worse condition than the patients without operation. Clearly, during this pandemic, or any other natural disaster or military campaign, there is limited ability to curtail the inevitable presentation of acute surgical pathologies or traumatic events. During challenging circumstances, hospitals must be prepared for and must be able to maintain emergent surgical services in order to avoid preventable deaths and mortality. The results of this study have shown the effect of the COVID-19 pandemic on our patient population suffering from acute general surgery illnesses.
There was no difference in the pathologies presenting between the two periods, which is an important statistical finding, yet must be taken in proper context. Depending on the type of catastrophe, the required mobilization of specific medical specialties and strain on the systems will be drastically different. During times of war or natural disasters, hospitals will require a larger ratio of orthopedic surgeons, general surgeons, surgical specialties, OR access, and ICU beds.14 In contrast, pathogen-derived epidemics/pandemics will require more specialists in the fields of internal medicine (ie, infectious disease, pulmonology, intensivist, and hematologist), ICU beds, and potential access to extracorporeal membrane oxygenation.15 As seen with our results, this primarily respiratory pandemic did not significantly change the acute surgical pathologies.
In regard to the younger patient population, there were several observations that were of interest yet not statistically significant. These patients appeared to present later in their course of disease, with a higher rate of complicated pathologies (ie, perforated appendix and gangrenous cholecystitis), postoperative complications, and blood transfusions. These younger patients were therefore more likely able to tolerate their symptoms at home for a longer period and therefore had a delay in diagnosis and treatment. In contrast, the older population, with their lower physiological reserve, appeared less able to delay medical treatment and came to the hospital regardless of fears and inconveniences caused by the pandemic restrictions.
The “fear” of presenting to the ER with severe illness has been documented from this current COVID-19 pandemic21 and from previously published literature during catastrophes. For example, during the SARS epidemic in 2002, Taiwan and Hong Kong showed a decrease in routine care not related to the severe acute respiratory syndrome.22 During this COVID-19 pandemic, one recent study reported the patterns of patients presenting to the ER of a busy level-1 trauma center in America. This group showed a global decrease in arrivals to the ER, along with a decreased proportion of patients presenting with abdominal pain when compared to other systemic complaints before the pandemic.23 This cannot be accurately compared to our data because we were only looking at acute surgical pathologies. Nevertheless, what was reported in this reference might also be a result of avoiding going to the hospital with abdominal pain during a pandemic that involves primarily respiratory symptoms. In Italy, toward the beginning of the pandemic, a series of children, without COVID-19-related illness, were shown to have presented late and in more critical condition, due to the fear of presenting to a hospital or lack of provisional care because of closure of health consults/centers.24 This corresponds with our overall results in general of sicker patients being admitted to the surgical ward.
The country where this study took place, Israel, also presents a unique confounding factor to these data. Our hospital is a public hospital within a socialized system where every citizen is born with universal health care coverage25; therefore, lacking health care is rarely a reason for not seeking medical attention.26 Compared to the rest of the world, our mortality has been comparatively low.27,28 Our “lockdown” measures and monitoring methods were considered aggressive, but the country also progressed in easing the regulations and opening its economy relatively fast. Nevertheless, the cooperation and coordination of our Ministry of Health, security sectors, and private industry played a pivotal role in preventing our hospital infrastructure from being completely overwhelmed. Our results showed a different demographic in terms of ethnicity presenting to the hospital during the COVID-19 pandemic. There was a significant increase in the ratio of Israeli-Jewish to Israeli-non-Jewish patients presenting to the ED and a smaller proportion of minorities, tourist, and foreign workers when compared to 2019. As mentioned above, the TASMC is located in the center of Tel Aviv, where 91% of the population is Jewish and 9% are non-Jewish. This difference might be explained by the lack of transport, hesitancy to travel distances, and fear of larger medical centers because of their treatment of COVID-19 patients.29 Hence, smaller local medical centers were potentially receiving more patients, regardless of appropriateness of level of care.
We found a low incidence of mis-triage of surgical patients into the COVID-19 isolation wing by our ER triage protocol. Out of the 209 patients admitted during this time, only 10 were initially placed in isolation. None of these 10 isolated patients had a positive COVID-19 test result. Despite being placed in triage, the average time to hospitalization, despite being statistically different, was only 30 minutes longer than the non-isolated group. When intervention (surgical and nonsurgical) was needed in the isolated group, there was no increase in time when compared to the non-isolated patients and, on average, even occurred more rapidly. With many surgical pathologies presenting with fever, it was difficult, especially at the beginning of the pandemic, not to over triage these patients and potentially delay intervention for surgical emergencies. This became especially relevant as evidence began to show that a possible presenting symptom of COVID-19 is fever with diarrhea.30 The significant reduction in time to hospitalization during the pandemic may be a confounding effect of both a decrease in volume to the ED and also sicker patients presenting to the ED, which would require quicker diagnosis and appropriate therapy. This decrease in hospitalization time happened despite a reduction in working staff.
Multiple studies have established that this elderly population is at an increased risk to clinically significant COVID-19.31 Our elderly population (>70 years old) during the pandemic had slightly more comorbidities and had a greater 30-day mortality than the previous year. Despite their increased comorbidities, they did not present to the hospital sicker than their counterparts the previous year. This trend might demonstrate the opposite of the “fear” phenomenon mentioned above for younger patients that sicker, elder patients present more easily to the hospital driven by the distress that something is wrong. With the elderly patients who did not undergo surgery, we were able to significantly reduce their hospitalization time. We have emphasized in our department not to neglect surgical pathologies during this time in this vulnerable population.
An interesting finding from the patients undergoing surgery during the pandemic is that they had a higher APACHE score, more complications, more blood given, yet more cases were open (verses laparoscopic), and there was an attending present more often. This seemingly contradicting findings might be a true indicator of how severely ill/complicated the patients were when compared to the previous year. Yet, this finding would need to be compared to other large tertiary centers in order to gather a significant conclusion. There are additional limitations to this study, including the fact that it is coming from a single urban referral center. These data may not be representative of other smaller and/or rural centers. Other limitations include the short study time and not having a cohort of surgical patients who were COVID-19 positive. The short study time was chosen in order to describe the initial effect of the pandemic and the most constricting limitations imposed by the government during the initial lockdown. Therefore, the initial month of true, full lockdown was taken and compared to the previous year. Clearly, during different stages and evolution of the pandemic, societies and medical systems have changed/adapted; therefore, the time when this study was conducted needs to be taken into consideration. There was no (knowingly) COVID-19-positive patient in our cohort and therefore no comparison between the differences in surgical pathology progression between positive and negative surgical patients. As there is more understanding of the COVID-19 pathophysiology, this would be an important patient population to analyze.
In conclusion, we have shown that during this COVID-19 pandemic, the patients presenting were sicker than the patients during the same time frame in the previous year despite there being fewer patients needing surgical admission. We were able to decrease the time until hospitalization and the length of hospitalization on the highly vulnerable elderly population. These data support the need for advocating and educating the public to present to hospitals without delay if there are symptoms of a possible acute surgical illness. Clearly, there are difficult times that may force the population to stay home (natural disasters and war), but fear alone should not delay the presentation of a patient with abdominal pain to the hospital. With well-organized systems and cooperation between governing and health sectors, hospitalization can be streamlined and physician/hospital workforce exposure can be minimized, while not compromising the level of care and treatment needed for acute surgical pathologies. Through understanding the surgical needs of a population during a catastrophic event, the public health sector may more rapidly and precisely distribute needed personnel, essential resources, and allocate certain medical centers as referral points for surgical emergencies.
| Background:
The COVID-19 pandemic has transformed and affected every aspect of health care. Like any catastrophic event, the stress on hospitals to maintain a certain level of function is immense. Acute surgical pathologies cannot be prevented or curtailed; therefore, it is important to understand patterns and outcomes during catastrophes in order to optimize care and organize the health care system.
Methods:
In a single urban tertiary care center, a retrospective study examined the first complete lockdown period of Israel during the COVID-19 pandemic. This was compared to the same time period the previous year.
Results:
During the pandemic, time to hospitalization was significantly decreased. There was also an overall reduction in surgical admissions yet with a higher percentage being hospitalized for further treatment (69.2% vs 23.5%). The patients admitted during this time had a higher APACHE-II score and Charlson comorbidity index score. During the pandemic, time to surgery was decreased, there were less laparoscopic procedures, and more RBC units were used per patient. There were no differences in overall complications, except when sub-analyzed for major complications (9.7% vs 6.3%). There was no significant difference in overall in-house mortality or morbidity. Length of hospitalization was significantly decreased in the elderly population during the pandemic.
Conclusions:
During the COVID-19 pandemic, despite a significantly less number of patients presenting to the hospital, there was a higher percentage of those admitted needing surgical intervention, and they were overall sicker than the previous year. | null | null | 7,876 | 290 | [
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||||
A novel variable number of tandem repeat of the natriuretic peptide precursor B gene's 5'-flanking region is associated with essential hypertension among Japanese females. | 17554401 | Brain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH. | BACKGROUND | The polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants. | METHODS | A novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046). | RESULTS | The 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females. | CONCLUSIONS | [
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] | 1885554 | 1. Introduction | Natriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.
BNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.
The aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH. | 2. Subjects and Methods | Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.
The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.
Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.
Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.
Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.
Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.
Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.
Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.
Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).
Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).
Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.
Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant. | 3. Results | We discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).
The association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).
On multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).
The clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).
The plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.
All subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.5<BMI<25; obese, BMI >25). There was no association between the genotype and the BMI among the groups (Table 5). | null | null | [
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"The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.",
"Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.",
"Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.",
"Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.",
"Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).",
"Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant."
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"Sequencing analysis",
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"Statistical analysis",
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"Natriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.\nBNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.\nThe aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH.",
" Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\nThe EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.\n Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\nPlasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.\n Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\nGenomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.\n Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\nTwo oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.\n Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\nGenotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).\n Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.\nData are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.",
"The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.",
"Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.",
"Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.",
"Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.",
"Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).",
"Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.",
"We discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).\nThe association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).\nOn multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).\nThe clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).\nThe plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.\nAll subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.5<BMI<25; obese, BMI >25). There was no association between the genotype and the BMI among the groups (Table 5).",
"Ogawa et al. reported that the 5'-flanking region of the 1.9 kilo-base pairs in the NPPB gene has a high transcriptional activity. Using the deletion mutant model, the deletion of sequences between -1288 and -1095 reduced transcriptional activity to approximately 30%. These deleted sequences contain a characteristic CT-rich region (-1248 to -1191), followed by an Alu family sequence (-1190 to -934). Thus, these results are consistent with the hypothesis that Alu repeat sequences in the 5'-flanking regions have regulatory roles in NPPB expression 14. Therefore, we assessed the association between mutations or polymorphisms in the 5'-flanking region of NPPB and the presence of EH. In the present study, a novel VNTR was discovered, and a significant association between the VNTR and EH was found in female patients. The present study found that the number of patients with a 16 repeat allele of VNTR was lower in EH women than in NT women. It is well known that the plasma BNP level is higher in EH patients than in NT subjects, since an elevated blood pressure results in a high plasma BNP level, which is one of the protective factors for hypertension 4. We compared the plasma BNP levels of patients with and without the 16 repeat allele and found that there was no significant difference between the two groups. In fact, there were not enough subjects to allow the association studies to be done by gender. Given our results, these limitations should be addressed in future studies. It is possible that factors other than the NPPB gene may have affected the BNP levels, since many factors, including cardiac function and blood pressure, are known to affect human plasma BNP levels. Thus, it is possible that the plasma BNP level is not an accurate reflection of the function of the NPPB gene.\nIn the present study, the overall distribution of the VNTR genotype and the allele frequency were significantly different in females but not in males. Gender-specific susceptibility to EH is an interesting finding, but its importance is still unclear 11. Redfield et al. reported that BNP levels increased with age, and were higher in females than males among subjects with no known cardiovascular or detectable structural heart disease 18. Maffei et al. reported that hormone replacement therapy increased BNP levels in postmenopausal women 19. Although the absolute BNP value was different between these two studies, which used two different assays, the associations of the BNP levels with age and gender were consistent. Furthermore, the BNP level that had the optimal sensitivity and specificity for detecting systolic dysfunction in the overall population increased with age and was higher in women. This underscores the clinical relevance of the relationship of age, gender, and BNP. In both studies that used different assays, the effect of gender on BNP was substantial and independent of other factors 18. Unfortunately, we were not able to obtain samples to measure plasma BNP and ANP levels, due to the difficulty in obtaining written informed consent for blood examinations from subjects not receiving medications.\nIn the Japanese population, it has been reported that plasma BNP levels are positively associated with age, urinary salt excretion, higher blood pressure, a high R-wave voltage in the 12-lead ECG (Code 3-1 or 3-3), and female gender 20,21. On the other hand, Freitag et al., based on multivariate models adjusting for known risk factors, showed that elevated plasma BNP levels were associated with an increased risk of blood pressure progression in males but not in females. However, there was no significant trend of an increasing incidence of hypertension among BNP categories in either males or females. In a community-based sample, higher plasma BNP levels were found to be associated with an increased risk of BP progression in males, but not in females 22. Further studies are needed to resolve these conflicting results.\nSince there are many loci with a high degree of polymorphism in the number of tandemly repeated nucleotide sequence units, VNTR polymorphisms, also called minisatellites, were originally studied for linkage-mapping purposes. VNTRs have a highly polymorphic nature that makes them very useful as markers, both in linkage studies to map disease loci in families and in forensic applications. Recent reports indicate that some VNTR sequences may function as transcriptional or translational regulators, and that they may modify the function of a protein when the tandemly repeated region lies within the coding region of the gene 23. Although no clear effect on transcription has been shown, it has been reported that a VNTR in the second intron of the serotonin transporter gene is associated with susceptibility to major depression 24.\nWe previously determined the structural organization of human natriuretic peptide receptor genes 25-28 and identified an insertion/deletion mutation in the 5'-untranslated region of NPRA 12. The deletion encompasses eight nucleotides and alters the binding sites for the AP2 and zeste transcription factors. Transcriptional activity of the deletion allele was less than 30% that of the wild-type allele. The deletion allele was significantly more common in the EH group than in the NT group. These findings suggest that in Japanese individuals, this deletion in the NPRA gene reduces receptor activity and may confer increased susceptibility for the individual to develop EH or left ventricular hypertrophy (LVH). Animal models with a deletion of this gene develop disorders that resemble the symptoms of subjects with a deleted allele in the 5'-untranslated region of NPRA. We previously isolated a missense mutation of the NPRA gene 29 and a VNTR polymorphism upstream of the NPRC gene; this VNTR influences blood pressure levels in obesity-associated hypertension 30. Since the sampling of the above reports was different from the present experiment, it was impossible to analyze the relationship between systemic natriuretic peptide genes and EH.\nWang et al. reported that obese individuals have low circulating natriuretic peptide levels, which may contribute to their susceptibility to hypertension and hypertension-related disorders. The mechanisms linking obesity to hypertension have not been established, but sodium retention and excessive sympathetic tone are key contributors. Natriuretic peptides are important regulators of sodium homeostasis and neurohormonal activation; this raises the possibility that obese individuals have an impaired natriuretic peptide response 31. Therefore, we examined the relationship between the genotype and BMI and found no significant association.\nIn conclusion, a novel VNTR in the 5'-flanking region of the NPPB gene was discovered. This polymorphism was associated with EH in female subjects. However, this finding does not necessarily imply that there is a relationship between the NPPB gene and EH. Further studies of other polymorphisms are needed to determine whether there is an association between the NPPB gene and EH."
] | [
"intro",
"methods",
null,
null,
null,
null,
null,
null,
"results",
"discussion"
] | [
"Brain natriuretic peptide",
"essential hypertension",
"variable number of tandem repeat",
"association study"
] | 1. Introduction:
Natriuretic peptides constitute a family of three structurally related molecules: atrial natriuretic peptide (ANP) 1, brain natriuretic peptide (BNP) 2, and C-type natriuretic peptide (CNP) 3. ANP and BNP act mainly as cardiac hormones and are produced primarily by the atria and ventricles, respectively, whereas C-type natriuretic peptide is expressed mainly in the brain 4,5.
BNP, which was originally isolated from the porcine brain 6, shows an amino acid sequence homology to ANP. BNP has central and peripheral actions that are similar to those of ANP. The heart has the highest concentrations of BNP, and BNP acts as a cardiac hormone. Intravenous injection of BNP causes a significant decrease in blood pressure 7. Transgenic mice that overexpress BNP have a measurable reduction in blood pressure 8. Mukoyama et al. showed that plasma BNP levels are higher in individuals with essential hypertension (EH) than in normotensive (NT) individuals 7. These findings suggest that the BNP gene is a candidate gene for EH. EH is thought to be a multifactorial disorder; several studies have shown that an association analysis with genetic variants can be used to identify susceptibility genes for EH 9.
The aim of the present study was to identify mutations or polymorphisms in the 5'-flanking region of the NPPB gene and to assess the relationship between variants of the gene and EH.
2. Subjects and Methods:
Subjects The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.
The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.
Biochemical analysis Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.
Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.
Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.
Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.
Sequencing analysis Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.
Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.
Genotyping Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).
Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).
Statistical analysis Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.
Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.
Subjects:
The EH group consisted of 317 patients (mean age, 49.7 ± 7.8 years) with EH diagnosed based on sitting systolic blood pressure (SBP) greater than 160 mmHg and/or diastolic blood pressure (DBP) greater than 100 mmHg measured on three separate occasions within 2 months after the initial BP measurement (Table 1). These patients were not being treated with antihypertensive drugs. All EH subjects had a positive family history of hypertension; patients diagnosed with secondary hypertension were excluded from the study. We also enrolled 262 healthy, normotensive (NT) subjects (mean age, 51.2 ± 10.8 years). None of the NT subjects had a family history of hypertension; all had an SBP less than 130 mmHg and a DBP less than 85 mmHg. A family history of hypertension was defined as having grandparents, uncles, aunts, parents, or siblings who had been diagnosed with hypertension. Written informed consent was obtained from each subject based on a protocol approved by the Ethics Committee of the Nihon University School of Medicine and the Clinical Studies Committee of Nihon University Hospital 10.
Biochemical analysis:
Plasma total cholesterol concentrations, as well as serum creatinine and uric acid concentrations, were measured using standard methods in the Clinical Laboratory Department of Nihon University Hospital 11. Plasma BNP levels were measured in NT subjects and EH patients. A total of 76 subjects (43 in the NT group and 33 in the EH group) who did or did not have the 16 repeat allele were selected randomly. Plasma BNP levels were measured using a highly sensitive immunoradiometric assay (Shiono RIA BNP assay kit, Shionogi Co., Ltd., Tokyo, Japan) as described previously 12.
Polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP):
Genomic DNA was extracted from peripheral blood leukocytes using a standard method 13. To screen for mutations, two oligonucleotide primers (sense, 5'- AAGGAGGCACTGGGAGAGGGGAAAT -3' (bases -1323 to -1299 from the major transcriptional initiation site) and antisense, 5'-AATTAGCTGGGCATGGTGGCAGGCG-3' (bases -1075 to -1051)) that recognize part of the 5'-flanking region of the NPPB gene were designed, since this region has been reported to be a major promoter region (Fig. 1A) 14. PCR-SSCP was done (GenePhor System; Amersham Biosciences Corp, Piscataway, NJ, USA) 15. PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Branchburg, NJ, USA) with the following amplification conditions: initial denaturation at 96°C for 3 min followed by 35 cycles of 98.5°C for 25 s, 65°C for 30 s, 68°C for 30 s, and a final extension of 68°C for 10 min. PCR products were separated by electrophoresis on 10% precast polyacrylamide gels (Amersham Biosciences Corp) at 5°C for 80 min and then subjected to silver staining (Dai-ichi Kagaku, Tokyo, Japan). The electrophoresis parameters were set according to the manufacturer's protocol.
Sequencing analysis:
Two oligonucleotides (sense, 5'-AAGGAGGCACTGGGAGAGGGGAAAT-3' (bases -1323 to -1299) and antisense, 5'- CCCCACCAAGCCAACACAGGATGGA -3' (bases -919 to-895) were used to amplify a 429-bp product from genomic DNA (Fig. 1A). The PCR products were purified using a Microcon 100 column (U.S. Amicon Inc. Beverly, MA,USA). The resulting products were ligated to pCR2.1TM vectors and cloned (TA Cloning Kit, Invitrogen, San Diego, CA, USA). The ligation of the products was confirmed by direct DNA sequencing (ABI PRISM 310 Genetic Analyzer) 16.
Genotyping:
Genotyping was done using fragment analysis, and the sequencing primers were used. PCR amplification consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 98.5°C for 25 sec, 63°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. Then, the PCR products were analyzed using an automatic electrophoresis system (Agilent 2100 bioanalyzer systemTM; Agilent Technologies, Waldbronn, Germany).
Statistical analysis:
Data are presented as the mean ± SD. The Hardy-Weinberg equilibrium was assessed by doing chi-square (χ2) analysis. Differences in the clinical data between the EH and NT groups were assessed by analysis of variance (ANOVA). The distributions of the genotypes or alleles between EH patients and NT subjects were tested using a two-sided Fisher's exact test. Multiple logistic regression analyses were done to assess the contribution of confounders (gender, BMI) 17. A value of P < 0.05 was considered statistically significant.
3. Results:
We discovered a novel variable number of tandem repeat (VNTR) polymorphism consisting of a 11-nucleotide repeat of 4 base pairs (bp) in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) (GenBank accession number AB265677). This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC (Fig. 1A). There were 8 alleles of this VNTR polymorphism, ranging from 9-repeat to 19-repeat (Fig. 1B).
The association study showed that the 11-repeat allele was most frequent in the NT group (88.2%). The 16-repeat allele was second most frequent in the NT group (10.5%). Furthermore, in the NT group, the observed and expected genotypes were in good agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Of note, the overall distribution of genotypes in females was significantly different between the EH and NT groups (p=0.039); the frequency of the 16-repeat allele was significantly lower in the EH group (6.5%) than in the NT group (12.2%, p=0.046) (Table 2).
On multiple logistic regression analysis, a significant association between allele 16 (p=0.034) and female gender was noted, even after adjustment for confounding factors; the calculated odds ratio was 1.18 (95%CI: 1.07-1.20).
The clinical data of each genotype were assessed. There were no significant differences in SBP and DBP levels, or in the pulse of subjects with or without the 16 repeat allele (Table 3).
The plasma BNP level was significantly higher in the EH group than in the NT group (p=0.0203). The plasma BNP level in each genotype with or without the 16 repeat allele was determined (Table 4); there were no significant differences among the groups. It was impossible to perform this analysis in EH females, as none of them had the 16 repeat allele.
All subjects were classified into 3 groups based on their BMI levels (lean, BMI<18.5; normal, 18.5<BMI<25; obese, BMI >25). There was no association between the genotype and the BMI among the groups (Table 5).
4. Discussion:
Ogawa et al. reported that the 5'-flanking region of the 1.9 kilo-base pairs in the NPPB gene has a high transcriptional activity. Using the deletion mutant model, the deletion of sequences between -1288 and -1095 reduced transcriptional activity to approximately 30%. These deleted sequences contain a characteristic CT-rich region (-1248 to -1191), followed by an Alu family sequence (-1190 to -934). Thus, these results are consistent with the hypothesis that Alu repeat sequences in the 5'-flanking regions have regulatory roles in NPPB expression 14. Therefore, we assessed the association between mutations or polymorphisms in the 5'-flanking region of NPPB and the presence of EH. In the present study, a novel VNTR was discovered, and a significant association between the VNTR and EH was found in female patients. The present study found that the number of patients with a 16 repeat allele of VNTR was lower in EH women than in NT women. It is well known that the plasma BNP level is higher in EH patients than in NT subjects, since an elevated blood pressure results in a high plasma BNP level, which is one of the protective factors for hypertension 4. We compared the plasma BNP levels of patients with and without the 16 repeat allele and found that there was no significant difference between the two groups. In fact, there were not enough subjects to allow the association studies to be done by gender. Given our results, these limitations should be addressed in future studies. It is possible that factors other than the NPPB gene may have affected the BNP levels, since many factors, including cardiac function and blood pressure, are known to affect human plasma BNP levels. Thus, it is possible that the plasma BNP level is not an accurate reflection of the function of the NPPB gene.
In the present study, the overall distribution of the VNTR genotype and the allele frequency were significantly different in females but not in males. Gender-specific susceptibility to EH is an interesting finding, but its importance is still unclear 11. Redfield et al. reported that BNP levels increased with age, and were higher in females than males among subjects with no known cardiovascular or detectable structural heart disease 18. Maffei et al. reported that hormone replacement therapy increased BNP levels in postmenopausal women 19. Although the absolute BNP value was different between these two studies, which used two different assays, the associations of the BNP levels with age and gender were consistent. Furthermore, the BNP level that had the optimal sensitivity and specificity for detecting systolic dysfunction in the overall population increased with age and was higher in women. This underscores the clinical relevance of the relationship of age, gender, and BNP. In both studies that used different assays, the effect of gender on BNP was substantial and independent of other factors 18. Unfortunately, we were not able to obtain samples to measure plasma BNP and ANP levels, due to the difficulty in obtaining written informed consent for blood examinations from subjects not receiving medications.
In the Japanese population, it has been reported that plasma BNP levels are positively associated with age, urinary salt excretion, higher blood pressure, a high R-wave voltage in the 12-lead ECG (Code 3-1 or 3-3), and female gender 20,21. On the other hand, Freitag et al., based on multivariate models adjusting for known risk factors, showed that elevated plasma BNP levels were associated with an increased risk of blood pressure progression in males but not in females. However, there was no significant trend of an increasing incidence of hypertension among BNP categories in either males or females. In a community-based sample, higher plasma BNP levels were found to be associated with an increased risk of BP progression in males, but not in females 22. Further studies are needed to resolve these conflicting results.
Since there are many loci with a high degree of polymorphism in the number of tandemly repeated nucleotide sequence units, VNTR polymorphisms, also called minisatellites, were originally studied for linkage-mapping purposes. VNTRs have a highly polymorphic nature that makes them very useful as markers, both in linkage studies to map disease loci in families and in forensic applications. Recent reports indicate that some VNTR sequences may function as transcriptional or translational regulators, and that they may modify the function of a protein when the tandemly repeated region lies within the coding region of the gene 23. Although no clear effect on transcription has been shown, it has been reported that a VNTR in the second intron of the serotonin transporter gene is associated with susceptibility to major depression 24.
We previously determined the structural organization of human natriuretic peptide receptor genes 25-28 and identified an insertion/deletion mutation in the 5'-untranslated region of NPRA 12. The deletion encompasses eight nucleotides and alters the binding sites for the AP2 and zeste transcription factors. Transcriptional activity of the deletion allele was less than 30% that of the wild-type allele. The deletion allele was significantly more common in the EH group than in the NT group. These findings suggest that in Japanese individuals, this deletion in the NPRA gene reduces receptor activity and may confer increased susceptibility for the individual to develop EH or left ventricular hypertrophy (LVH). Animal models with a deletion of this gene develop disorders that resemble the symptoms of subjects with a deleted allele in the 5'-untranslated region of NPRA. We previously isolated a missense mutation of the NPRA gene 29 and a VNTR polymorphism upstream of the NPRC gene; this VNTR influences blood pressure levels in obesity-associated hypertension 30. Since the sampling of the above reports was different from the present experiment, it was impossible to analyze the relationship between systemic natriuretic peptide genes and EH.
Wang et al. reported that obese individuals have low circulating natriuretic peptide levels, which may contribute to their susceptibility to hypertension and hypertension-related disorders. The mechanisms linking obesity to hypertension have not been established, but sodium retention and excessive sympathetic tone are key contributors. Natriuretic peptides are important regulators of sodium homeostasis and neurohormonal activation; this raises the possibility that obese individuals have an impaired natriuretic peptide response 31. Therefore, we examined the relationship between the genotype and BMI and found no significant association.
In conclusion, a novel VNTR in the 5'-flanking region of the NPPB gene was discovered. This polymorphism was associated with EH in female subjects. However, this finding does not necessarily imply that there is a relationship between the NPPB gene and EH. Further studies of other polymorphisms are needed to determine whether there is an association between the NPPB gene and EH.
| Background:
Brain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH.
Methods:
The polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants.
Results:
A novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046).
Conclusions:
The 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females. | null | null | 4,569 | 319 | [
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] | null | null | [CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY] | [CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY] | [CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY] | null | [CONTENT] Brain natriuretic peptide | essential hypertension | variable number of tandem repeat | association study [SUMMARY] | null | [CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY] | [CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY] | [CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY] | null | [CONTENT] 5' Flanking Region | Adult | Asian People | Base Sequence | Body Mass Index | Female | Gene Frequency | Genotype | Humans | Hypertension | Japan | Linkage Disequilibrium | Logistic Models | Middle Aged | Minisatellite Repeats | Molecular Sequence Data | Natriuretic Peptide, Brain | Phenotype | Polymerase Chain Reaction | Polymorphism, Genetic | Polymorphism, Single-Stranded Conformational | Sequence Analysis, DNA [SUMMARY] | null | [CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY] | [CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY] | [CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY] | null | [CONTENT] natriuretic peptide genes | atrial natriuretic peptide | bnp acts cardiac | brain natriuretic peptide | peptide bnp type [SUMMARY] | null | [CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY] | [CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY] | [CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY] | null | [CONTENT] eh | bnp | subjects | nt | pcr | hypertension | plasma | levels | patients | gene [SUMMARY] | null | [CONTENT] bnp | natriuretic | natriuretic peptide | anp | peptide | brain | gene | eh | variants | mainly [SUMMARY] | [CONTENT] pcr | min | subjects | eh | mmhg | hypertension | products | measured | bases | usa [SUMMARY] | [CONTENT] repeat | allele | group | repeat allele | 16 | 16 repeat | 16 repeat allele | bmi | nt group | table [SUMMARY] | null | [CONTENT] bnp | eh | pcr | min | nt | subjects | hypertension | repeat | products | plasma [SUMMARY] | null | [CONTENT] ||| BNP ||| 5'-flanking | NPPB [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] 5'-flanking ||| 4 | TTTC ||| 8 | 9 | 19 ||| 317 | 262 ||| 11 | 88.2% | 16 | second | 10.5% ||| Hardy-Weinberg ||| EH ||| 16 | 6.5% | 12.2% [SUMMARY] | null | [CONTENT] ||| BNP ||| 5'-flanking | NPPB ||| 5'-flanking ||| 4 | TTTC ||| 8 | 9 | 19 ||| 317 | 262 ||| 11 | 88.2% | 16 | second | 10.5% ||| Hardy-Weinberg ||| EH ||| 16 | 6.5% | 12.2% ||| 16 | 5'-flanking | NPPB [SUMMARY] | null |
||||
Vitamin D deficiency and visceral adipose tissue in early pregnant women. | 34215200 | We aimed to assess the correlation between vitamin D serum level and visceral fat tissue during early pregnancy. | BACKGROUND | This cross-sectional study was performed in Pernambuco, Brazil. 190 low risk pregnant women (8-16 gestational weeks) were eligible. Visceral adipose tissue was measured by ultrasonography following the technique described by Armellini. The 25(OH) D in serum was determined through chemiluminescence. The Spearman correlation test was applied to evaluate the correlation between vitamin D serum level and VAT, considering p < 0.05 to be significant. | METHODS | Vitamin D insufficiency was present in 129 (67.8 %) of subjects. Pregnant women with or without vitamin D deficiency did not differ in age, gestational age, nutritional status and visceral adipose tissue. No correlation between visceral adipose tissue and 25(OH) D was observed: - 0.057 (p = 0.435). | RESULTS | Maternal visceral adipose tissue and vitamin D serum level are not correlated during pregnancy. | CONCLUSIONS | [
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"Female",
"Gestational Age",
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"Intra-Abdominal Fat",
"Nutritional Status",
"Pregnancy",
"Pregnant Women",
"Prenatal Nutritional Physiological Phenomena",
"Vitamin D",
"Vitamin D Deficiency",
"Young Adult"
] | 8252319 | Background | Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].
It is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].
In pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy. | null | null | Results | 190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.
Table 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343
Characteristics of pregnant women studied according to the vitamin D status
Vitamin D (30ng/mL)
< 30 (n = 129)
Median ± SD (Average)
≥ 30 (n = 61)
Median ± SD (Average)
Table 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero
Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index
(1)Statistically different from zero | Conclusions | Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings. | [
"Background",
"Methods",
"Statistical analyses"
] | [
"Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.",
"This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.",
"Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25."
] | [
null,
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"Conclusions"
] | [
"Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].\nIt is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].\nIn pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.",
"This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.\nPregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.\nThe 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].\nStatistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.\nData were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.",
"Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.\nThe choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.",
"190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.\n\nTable 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343\nCharacteristics of pregnant women studied according to the vitamin D status\nVitamin D (30ng/mL)\n< 30 (n = 129)\nMedian ± SD (Average)\n≥ 30 (n = 61)\nMedian ± SD (Average)\n\nTable 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero\nSpearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index\n(1)Statistically different from zero",
"In the present study we did not find association between serum VD values and visceral fat in pregnant women. For the best of our knowledge, it is the first time that this association is assessed during pregnancy. Some studies have described an inverse association between VDD and visceral fat in non-pregnancy populations [24, 14]. However, among the various physiological changes that occur during pregnancy, the redistribution of adipose tissue is one of them. Although not yet properly studied, it is admitted that an increase in visceral adipose tissue occurs, but the specific function of visceral adipose in pregnancy is still unknown. Thus, it seems likely that visceral adipose tissue has somewhat distinct functions in pregnancy and our findings cannot be compared with non-pregnant populations.\nRecently, Carreras-Badosa et al. found that maternal serum VD was inversely associated with visceral fat and in their offspring at the age of 5–6 years [25]. However, diet and lifestyle habits were not studied in both mothers and their children, and this, rather than maternal VD status could explain the relationship between lower maternal serum VD and offspring adiposity.\nAn increased risk of VDD has been described worldwide in obese individual. The underlying explanations are not clear. Receptors of VD are expressed in adipose tissue and 25(OH) D could modulate adipogenesis through VD receptors-dependent inhibition of specific components [26]. Other explanation is based on experimental findings that deficiency of VD can increases lipogenesis upregulating adipocyte calcium signaling and improving the secretion of parathyroid hormone [27]. However, the role of VD in adipogenesis or other functions of VAT during pregnancy is still unknown.\nPregnancy VDD has been described with a prevalence of 18–84 % [9]. In the present study, the prevalence of VDD was high (68 %). This value was similar to other Brazilian studies and higher than that described in countries such as Spain and United States [28]. Brazil is a tropical country with continental dimensions and abundant sunshine. The municipality of Petrolina is located at approximately − 9°, which favors the synthesis of VD. However, there is a paradox in countries with low solar incidence and with a lower frequency of VDD when compared with countries with higher solar incidence, certainly because of protection measures that prevent its synthesis and bioavailability [29].\nIn the multicenter HAPO study, increased maternal BMI was associated with lower maternal 25(OH) D levels [30]. However, BMI is not an accurate measure of fat tissue, especially during pregnancy. Besides, the deposition of adipose tissue preferably occurs in two distinct anatomical places, VAT and subcutaneous adipose tissue, and BMI does not differentiate them. We determine VAT through ultrasound and this method has been shown to be safe, effective, simple and reproducible [31].\nOur study has some limitations. At first, a cross-sectional design was performed and we could not determine the causal relationship between VD and VAT. Second, other possible important variables confounding the VD status, like sun light exposition, dietary habits and possible seasonal variations in VD, were not evaluated in our study. However, despite these limitations our study has strengths. The present study is the first to assess the correlation of VD with VAT thickness in pregnant women. A large sample was studied and specific methods to measure VD and VAT were used.",
"Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings."
] | [
null,
null,
null,
"results",
"discussion",
"conclusion"
] | [
"Visceral adipose tissue",
"Vitamin D",
"Pregnancy",
"Ultrasound",
"Obesity"
] | Background:
Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].
It is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].
In pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.
Methods:
This cross-sectional study was performed at the Hospital Dom Malan located in Petrolina (9°S and 40°W), Pernambuco state, Northeast of Brazil. Hospital Dom Malan registers about 6 000 births per year. This region has an average ultraviolet index of 13 during most of the year.
Pregnant women aged 13–39 years and 8–16 weeks of gestational age were eligible. Exclusion criteria were twin pregnancy, diabetes mellitus, previous gestational diabetes or preeclampsia, fetal malformations, maternal mental disease. After screening, 190 pregnant women were included. All participants answered a questionnaire regarding socio-demographic and clinical information were taken from the medical record. Weight and height were measured according to the standardized methodology. Body mass index (BMI) was calculated by the formula: weight (kg) / height (m2) and Atalah classification was used [21]. VAT was measured by ultrasonography performed by a qualified specialist. A Philips D7 ultrasound device, equipped with a 3.0 to 7.0 MHz convex transducer, multifrequency (Bothell, WA/USA), was used. VAT was determined following the technique described by Armellini [22]: the convex transducer positioned immediately above the umbilical scar, the distance in centimeters being measured between the inner edge of the rectus abdominis muscle, at the point of its insertion in the alba line, and the anterior wall of the abdominal aorta.
The 25(OH) D in serum was determined through chemiluminescence, using the Atellica EVA-Siemens device (Erlangen/Germany) with a result expressed in nanograms per Milliliter. Insufficiency and deficiency values were respectively between 20 and 30 ng/mL and < 20 ng/mL, according to the values adopted by the Endocrine Society Practice Guidelines [23].
Statistical analyses Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.
The choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.
Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.
The choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.
Statistical analyses:
Data were analyzed descriptively through absolute and percentage frequencies in the categorical variables and the measures: mean, standard deviation (mean ± SD), and median to numerical variables. To assess a significant association between two categorical variables, Pearson’s chi-square test including the likelihood ratio test and confidence interval (CI) was used. For the comparison between categories concerning the numerical variables, the Mann–Whitney test was used. To assess the degree of the relationship between two numerical variables, Spearman’s correlation coefficient and the specific Student’s t-test for the null correlation hypothesis were obtained.
The choice of the Mann–Whitney test and Spearman correlation was due to the absence of normality, which was verified by the Shapiro–Wilk test. The margin of error used in deciding the statistical tests was 5 and 95 % CI. The data were entered into the EXCEL spreadsheet, and the program used to obtain the statistical calculations was IMB SPSS in version 25.
Results:
190 pregnant women were studied. The mean age was 26 ± 5.76 years and pregnant women were included in the study with a 12.3 ± 2.5 weeks of gestation. The BMI ranged from 16.6 to 47.0 kg/m² (25.7 ± 4.9 kg/m²); 57 (30 %) were overweight and 27 (14.2 %) obese. 129 (67.8 %) of pregnant women had vitamin D insuficiency, i.e., serum value of 25 (OH) D < 30 ng/mL. The thickness of the VAT varied between 0.9 and 6.1 cm (2.9 ± 0.9 cm). Pregnant women with or without VDD did not differ in age, gestational age, nutritional status and VAT (Table 1). The Spearman value correlation between VAT and 25(OH) D was – 0.057, (P value = 0.435). Table 2 presents Spearman correlation between VAT, 25(OH) D and age, weight and BMI.
Table 1Characteristics of pregnant women studied according to the vitamin D statusVitamin D (30ng/mL)Variable< 30≥ 30TotalP valueOR (CI 95 %)n%n%n%TOTAL129100.061100.0190100.0Age range (years)P = 0.331 13 a 19118.51118.02211.61.00 20 a 243930.21626.25528.92.44 (0.88–6.75) 25 a 294434.11626.26031.62.75 (1.00–7.57) 30 a 342317.81321.33618.91.77 (0.60–5.20) 35 a 42129.358.2178.92.40 (0.63–9.14)Gestational age (weeks)P = 0.306 < 101612.446.62010.51.72 (0.52–5.68) 10–125542.63252.58745.80.74 (0.39–1.41) > 125845.02541.08343.71.00Visceral adipose tissue (cm)P = 0.340 < Percentile 25 (2.31)3023.31829.54825.31.00 > Percentile 25 (2.31) to 50 (2.80)3627.91321.34925.81.66 (0.70–3.94) > Percentile 50 (2.80) to 75 (3.50)3023.31931.14925.80.95 (0.42–2.15) > Percentile 75 (3.50)3325.61118.04423.21.80 (0.73–4.42)ATALAHP = 0.838 Low weight129.358.2178.91.00 Normal weight5945.73252.59147.90.77 (0.25–2.37) Overweight3930.21727.95629.50.96 (0.29–3.14) Obesity1914.7711.52613.71.13 (0.29–4.39)VariableVitamin D (30ng/mL)< 30 (n = 129)Median ± SD (Average)≥ 30 (n = 61)Median ± SD (Average)P valuePre gestational weight65.03 ± 13.08 (64.00)62.07 ± 11.93 (58.00)P = 0.108Weight66.33 ± 13.32 (63.70)63.73 ± 12.25 (61.30)P = 0.196Body Mass Index25.96 ± 5.07 (24.96)25.27 ± 4.69 (24.88)P = 0.343
Characteristics of pregnant women studied according to the vitamin D status
Vitamin D (30ng/mL)
< 30 (n = 129)
Median ± SD (Average)
≥ 30 (n = 61)
Median ± SD (Average)
Table 2Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass indexVariableVisceral adipose tissue25(OH)DAge0,299 (< 0,001)a-0,117 (0,108)Weight0,525 (< 0,001)a-0,111 (0,126)Body Mass Index0,574 (< 0,001)a-0,130 (0,074)(1)Statistically different from zero
Spearman correlation between visceral adipose, 25(OH) D and age, weight and body mass index
(1)Statistically different from zero
Discussion:
In the present study we did not find association between serum VD values and visceral fat in pregnant women. For the best of our knowledge, it is the first time that this association is assessed during pregnancy. Some studies have described an inverse association between VDD and visceral fat in non-pregnancy populations [24, 14]. However, among the various physiological changes that occur during pregnancy, the redistribution of adipose tissue is one of them. Although not yet properly studied, it is admitted that an increase in visceral adipose tissue occurs, but the specific function of visceral adipose in pregnancy is still unknown. Thus, it seems likely that visceral adipose tissue has somewhat distinct functions in pregnancy and our findings cannot be compared with non-pregnant populations.
Recently, Carreras-Badosa et al. found that maternal serum VD was inversely associated with visceral fat and in their offspring at the age of 5–6 years [25]. However, diet and lifestyle habits were not studied in both mothers and their children, and this, rather than maternal VD status could explain the relationship between lower maternal serum VD and offspring adiposity.
An increased risk of VDD has been described worldwide in obese individual. The underlying explanations are not clear. Receptors of VD are expressed in adipose tissue and 25(OH) D could modulate adipogenesis through VD receptors-dependent inhibition of specific components [26]. Other explanation is based on experimental findings that deficiency of VD can increases lipogenesis upregulating adipocyte calcium signaling and improving the secretion of parathyroid hormone [27]. However, the role of VD in adipogenesis or other functions of VAT during pregnancy is still unknown.
Pregnancy VDD has been described with a prevalence of 18–84 % [9]. In the present study, the prevalence of VDD was high (68 %). This value was similar to other Brazilian studies and higher than that described in countries such as Spain and United States [28]. Brazil is a tropical country with continental dimensions and abundant sunshine. The municipality of Petrolina is located at approximately − 9°, which favors the synthesis of VD. However, there is a paradox in countries with low solar incidence and with a lower frequency of VDD when compared with countries with higher solar incidence, certainly because of protection measures that prevent its synthesis and bioavailability [29].
In the multicenter HAPO study, increased maternal BMI was associated with lower maternal 25(OH) D levels [30]. However, BMI is not an accurate measure of fat tissue, especially during pregnancy. Besides, the deposition of adipose tissue preferably occurs in two distinct anatomical places, VAT and subcutaneous adipose tissue, and BMI does not differentiate them. We determine VAT through ultrasound and this method has been shown to be safe, effective, simple and reproducible [31].
Our study has some limitations. At first, a cross-sectional design was performed and we could not determine the causal relationship between VD and VAT. Second, other possible important variables confounding the VD status, like sun light exposition, dietary habits and possible seasonal variations in VD, were not evaluated in our study. However, despite these limitations our study has strengths. The present study is the first to assess the correlation of VD with VAT thickness in pregnant women. A large sample was studied and specific methods to measure VD and VAT were used.
Conclusions:
Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings.
| Background:
We aimed to assess the correlation between vitamin D serum level and visceral fat tissue during early pregnancy.
Methods:
This cross-sectional study was performed in Pernambuco, Brazil. 190 low risk pregnant women (8-16 gestational weeks) were eligible. Visceral adipose tissue was measured by ultrasonography following the technique described by Armellini. The 25(OH) D in serum was determined through chemiluminescence. The Spearman correlation test was applied to evaluate the correlation between vitamin D serum level and VAT, considering p < 0.05 to be significant.
Results:
Vitamin D insufficiency was present in 129 (67.8 %) of subjects. Pregnant women with or without vitamin D deficiency did not differ in age, gestational age, nutritional status and visceral adipose tissue. No correlation between visceral adipose tissue and 25(OH) D was observed: - 0.057 (p = 0.435).
Conclusions:
Maternal visceral adipose tissue and vitamin D serum level are not correlated during pregnancy. | Background:
Vitamin D deficiency (VDD) is a major global public health problem in all age groups, even in developed countries and regions with adequate ultraviolet radiation. Exposure to sunlight is the main source of vitamin D (VD) and factors such as season, time of day, latitude, skin phototype, sun exposure duration, type of clothing and the use of sunscreens may influence its synthesis [1–3]. Brazil, a tropical/subtropical country with elevated ultraviolet radiation has a very high prevalence of VDD [4]. VD has many actions in the body including the musculoskeletal system, calcium homoeostasis and immune system [5]. Recently, a link between VD and chronic non-communicable diseases has emerged including insulin resistance, diabetes and cardiovascular disease [6–8]. These chronic diseases are also commonly associated with obesity [9].
It is believed that VDD is associated with obesity, and adipose tissue may be responsible for its lower bioavailability. Visceral Adipose Tissue (VAT) easily absorbs VD by chemical affinity. Therefore, volumetric dilution has been proposed as the main mechanism to elucidate its low levels in obesity [10–12]. Two large epidemiological studies have shown that the thickness of VAT correlated inversely with the serum concentration of 25OHD in the adult population [11, 13]. Hao et al. found an inversely association between vitamin level and VAT in Chinese men [14]. Zhang et al. also reported a significant negative association between VAT and VDD in men and pre-menopausal women but not in post-menopausal women [15]. Batista et al. found that excess visceral adiposity, hypertriglyceridemia and high low-density lipoprotein cholesterol levels were strong predictors of hypovitaminosis D [16]. A meta-analysis of randomized clinical trials reported positive effects of VD supplementation on fat mass [17].
In pregnant women, VDD is also an important health problem with a prevalence ranging from 5 to 84 % [12, 18]. Besides, pregnancy is associated with a VAT increased and it has been linked to insulin resistance and hyperglycemia [19, 20]. However, we did not find in our search studies evaluating the association between VDD and VAT in pregnant women. We aimed to assess the correlation between VD serum level and VAT during early pregnancy.
Conclusions:
Our data demonstrated that maternal visceral adiposity and low concentrations of VD were not associated during pregnancy. However, as this was the first study to assess the association between VAT and VD in pregnant women, further studies are needed to confirm these findings. | Background:
We aimed to assess the correlation between vitamin D serum level and visceral fat tissue during early pregnancy.
Methods:
This cross-sectional study was performed in Pernambuco, Brazil. 190 low risk pregnant women (8-16 gestational weeks) were eligible. Visceral adipose tissue was measured by ultrasonography following the technique described by Armellini. The 25(OH) D in serum was determined through chemiluminescence. The Spearman correlation test was applied to evaluate the correlation between vitamin D serum level and VAT, considering p < 0.05 to be significant.
Results:
Vitamin D insufficiency was present in 129 (67.8 %) of subjects. Pregnant women with or without vitamin D deficiency did not differ in age, gestational age, nutritional status and visceral adipose tissue. No correlation between visceral adipose tissue and 25(OH) D was observed: - 0.057 (p = 0.435).
Conclusions:
Maternal visceral adipose tissue and vitamin D serum level are not correlated during pregnancy. | 2,629 | 192 | [
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"vitamin vd factors",
"bioavailability visceral adipose"
] | null | [CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity [SUMMARY] | null | [CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity [SUMMARY] | [CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity [SUMMARY] | [CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity [SUMMARY] | [CONTENT] Visceral adipose tissue | Vitamin D | Pregnancy | Ultrasound | Obesity [SUMMARY] | [CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY] | null | [CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Body Mass Index | Brazil | Cross-Sectional Studies | Female | Gestational Age | Humans | Intra-Abdominal Fat | Nutritional Status | Pregnancy | Pregnant Women | Prenatal Nutritional Physiological Phenomena | Vitamin D | Vitamin D Deficiency | Young Adult [SUMMARY] | [CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY] | null | [CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY] | [CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY] | [CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY] | [CONTENT] vitamin deficiency vdd | adipose tissue vat | modulate adipogenesis vd | vitamin vd factors | bioavailability visceral adipose [SUMMARY] | [CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY] | null | [CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY] | [CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY] | [CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY] | [CONTENT] vd | vat | 25 | test | variables | correlation | women | pregnant | adipose | pregnant women [SUMMARY] | [CONTENT] vd | vdd | vat | obesity | vitamin | associated | women | diseases | insulin resistance | health [SUMMARY] | null | [CONTENT] 25 | 30 | 00 | 80 | 50 | oh | age | sd average | percentile | 75 [SUMMARY] | [CONTENT] vd | vd associated pregnancy study | associated pregnancy study assess | maternal visceral | maternal visceral adiposity | pregnant women studies | pregnant women studies needed | maternal visceral adiposity low | confirm findings | women studies needed confirm [SUMMARY] | [CONTENT] vd | test | variables | vat | 25 | numerical | numerical variables | pregnancy | women | vdd [SUMMARY] | [CONTENT] vd | test | variables | vat | 25 | numerical | numerical variables | pregnancy | women | vdd [SUMMARY] | [CONTENT] [SUMMARY] | null | [CONTENT] Vitamin D | 129 | 67.8 % ||| ||| 25(OH | 0.057 | 0.435 [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] ||| Pernambuco | Brazil ||| 190 | 8 ||| Armellini ||| 25(OH ||| ||| Spearman | VAT | 0.05 ||| ||| Vitamin D | 129 | 67.8 % ||| ||| 25(OH | 0.057 | 0.435 ||| [SUMMARY] | [CONTENT] ||| Pernambuco | Brazil ||| 190 | 8 ||| Armellini ||| 25(OH ||| ||| Spearman | VAT | 0.05 ||| ||| Vitamin D | 129 | 67.8 % ||| ||| 25(OH | 0.057 | 0.435 ||| [SUMMARY] |
|||||
The Relationship Between Thyroid Antibodies and Vitamin D Level in Primary Hypothyroidism. | 33424090 | Vitamin D deficiency is a global health problem. Its deficiency has been reported to be associated with different autoimmune diseases. | INTRODUCTION | A total number of 150 individuals were enrolled in this study. They were divided into the fallowing groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease. Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibodies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). | METHODS | Serum levels of 25 (OH) vitamin D recorded a highly significant difference between the studies groups (20,76±6,31 ng/ml in group I vs. 24,37±9,05ng/ml in group II vs. 24,57±6,45ng/ml in group III, p<0,01). Moreover, there was a highly significant difference between patients with AITD and patients without AITD (20,76±6,31ng/ml vs. 24,37±9,05ng/ml, respectively; p<0,01). The concentration of anti-TPO and anti-TG antibodies were statistically significant higher in patients with vitamin D deficiency (p< 0,001). Serum TSH were significantly higher in group I (p< 0,001). | RESULTS | Significantly low levels of vitamin D were documented in patients with AITD that were related to the presence of anti-thyroid antibodies and higher level thyroid-stimulation hormone (TSH), suggesting the involvement of vitamin D in the pathogenesis of AITD and the advisability of supplementation. | CONCLUSION | [
"Adult",
"Aged",
"Autoantibodies",
"Female",
"Healthy Volunteers",
"Humans",
"Hypothyroidism",
"Immunoglobulins, Thyroid-Stimulating",
"Male",
"Middle Aged",
"Vitamin D",
"Vitamin D Deficiency"
] | 7780811 | INTRODUCTION | Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8). | METHODS | The study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.
Anti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.
The statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05). | null | null | CONCLUSION | Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD. | [
"INTRODUCTION",
"AIM",
"RESULTS",
"DISCUSSION",
"CONCLUSION"
] | [
"Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).",
"The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.",
"This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).",
"Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.",
"Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD."
] | [
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"AIM",
"METHODS",
"RESULTS",
"DISCUSSION",
"CONCLUSION"
] | [
"Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).",
"The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.",
"The study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.\nAnti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.\nThe statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05).",
"This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations. \nTotal amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.\nSerum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).\nRegarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).\nThe post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).\nRegarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)\nRegarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).\nMoreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).\nThe Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).",
"Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).\nMajority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).\nIn our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.\nThis is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.\nAlso, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.\nMoreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.\nOur study showed a significant negative correlation between vitamin D and TSH level (p=0.009).\nAn experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.\nHowever, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.",
"Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD."
] | [
null,
null,
"methods",
null,
null,
null
] | [
"autoimmune thyroid disease",
"vitamin D",
"thyroid autoantibodies",
"thyroid-stimulation hormone"
] | INTRODUCTION:
Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).
AIM:
The aim of this study was to evaluate the relation between vitamin D level, thyroid-stimulation hormone (TSH) and thyroid antibodies in primary hypothyroidism.
METHODS:
The study is of a retrospective-prospective character, and it included a total of 150 individuals and conducted at the Radiology and Nuclear Medicine Clinic, Department for Thyroid Diseases, University Clinical Centre Tuzla, in period between January 2018 and December 2019. Participants were divided into the following groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibidies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG). The biochemical parameters were assayed in the Department of Thyroid Diseases attached to our clinic. Thyroid-stimulating hormone (TSH) were measured with a fluoroimmunometric assay (DELFIA) on the machine Wallac delfia fluorometer. TSH levels between 0,63-4,19mIU/L were regarded normal.
Anti-TPO and anti-TG were tested by radioimmunoassay (RIA). The measuring of the serum anti-TPO and anti-TG concentration was performed on Wallac Wizard 1470 automatic gamma counter. Positive anti-TPO, and anti-TG were defined as a value greater than > 60 IU/ml. Elevated serum levels of thyroid autoantibodies were used for diagnosis of AITD. Electro-chemiluminescence binding assay (ECLIA) was used for vitamin D ( total 25 hydroxy vitamin D) on the mashine Cobas e 411 Rosche. Vitamin D deficiency is defined as a 25 (OH) vitamin D below 20 ng/ml and vitamin D insufficiency as 25(OH) vitamin D of 21-29 ng/ml. Levels of 25(OH) vitamin D > 30 ng/ml are considered to be optimal.
The statistical analysis was conducted with SPSS version 23.0 for Windows. The descriptive analysis was applied for all date processing. In the statistic data processing, the fallowing were applied: Tukey’s post hoc test, Spearman rank correlation coefficient. The level of check importance was set on 5% (p < 0,05).
RESULTS:
This study enrolled 150 participants who were subdivided into three groups: group I included 50 patients recently diagnosed with AITD (evidence by elevated anti-TPO, and anti-TG serum levels), group II included 50 patients without autoimmune thyroid disease (non-AITD). Group III included 50 apparently healthy participans representing a control group with normal clinical examination and were not complaining of either any chronic medical diseases, or history of thyroid diseases or any chronic illness that may interfere with results to be obtained. Also, these participants were not on any sort of vitamin D supplementations.
Total amount of 150 participants were included in our research, aging 11 to 83, and 89,33% were women.
Serum levels of 25 (OH) vitamin D recorded a significant difference between the studied groups (20.76±6.31 ng/ml in group I vs. 24.37±9.05 ng/ml in group II vs. 24.57±6.45ng/ml in group III, p<0.05).
Regarding vitamin D sufficiency, it was revealed that 25 (OH) vitamin D was deficient in 68%, insufficient in 12% and sufficient in 20% of the group I vs. deficient in 38%, insufficient in 28% and sufficient 34% of the group II. On the contrary, 25 (OH) vitamin D was deficient in only 24%, insufficient in 62%, and sufficient in 14% of control group, as illustrated in (Table 1).
The post-hoc Tukey test was used to determine the existence of significant differences in the analyzed parameters between patients with AITD with non-AITD, and each of them with control group. Age distribution showed statistical difference between with non-AITD patients and control group. Moreover, there was a highly significant difference between patients with AITD and patients with non- AITD regarding TSH (7.57±8.68 mU/l in patients with AITD vs. 2.82±1.60 mU/l in patients non AITD, p<0.05) and between patients with AITD and control group ( 7.57±8.68 mU/l vs. 2.53±0.87 mU/l), but no significant statistical difference between patient with non-AITD and control group regarding TSH (2.82±1.60 mU/l vs. 2.53±0.87 mU/l).
Regarding anti-TPO level and anti-TG level, there was a significant difference between patients with AITD and patients with non-AITD (1715.58± 969.79 IU/ml; 293.47±429.50 IU/ml vs. 25.31±10.51 IU/ml; 16.56±7.4IU/ml and between patients with AITD and control group (1715.58±969.79 IU/ml; 293.47± 429.50IU/ml vs. 45.37±66.85 IU/ml; 20.92±14.15 IU/ml), but no significant difference between patients with non-AITD and control group (25.31±10.51 IU/ml; 16.56± 7.4 IU/ml vs. 45.37± 66.85 IU/ml; 20.92±14.15 IU/ml)
Regarding 25 (OH) vitamin D level, there was a significant difference between patients with AITD and patients with non AITD (20.76±6.31 vs. 24.37±9.05 ng/ml, respectively, p<0,05), as well as between patients with AITD and control group (20.76±6.31 vs. 24.57±6.45 ng/ml, respectively, p<0.05), but no significant difference between patients with non-AITD and control group (24.37±9.05 ng/ml vs. 24.57±6.45 ng/ml), as illustrated in (Table 2).
Moreover, vitamin D deficiency was more frequent in patients with AITD (68%) versus in patients without AITD (38%).
The Spearman test was used to investigate correlation of nonparametric variables including vitamin D, TSH and autoantibodies. The results revealed there was a significant negative correlation between the vitamin D with TSH level (p=0.009, r=-0.212) and between vitamin D with anti-TG level (p=0.000, r=-0.328) and anti-TPO level (p=0.006, r=-0.225), as show in (Table 3).
DISCUSSION:
Thyroid diseases are among the most common endocrine abnormalities, and AITDs are perhaps the most prevalent autoimmune diseases (9, 10). AITD are relatively common organ-specific autoimmune disorders that cause diseases ranging in severity from hypothyroidism to hyperthyroidism (11). As an immune modulator, vitamin D is involved in the onset and development of AITD (7, 12).
Majority of the participants in our study were females (90%). This finding was similar to that of Mackawy et al. (13). He stated that serum vitamin D levels were significantly more decreased in females than males. This was in accordance with our finding. Although several authors have reported that vitamin D levels did not differ significantly between males and females (13, 14, 15).
In our study, there was a highly significant difference regarding 25 (OH) vitamin D level among the studied groups; 25(OH) vitamin D level was 20.76±6.31 ng/ml in patients with AITD versus 24.37±9.05 ng/ml in patients with non-AITD versus 24.57±6.45 ng/ml in control group. Regarding vitamin D status, it was deficient in 68% in patients with AITD, 38% in patients with non-AITD, whereas it was deficient in 24% in control group.
This is in agreement with Friedman study (16) who explained the low levels of vitamin D in patients with hypothyroidism by two mechanisms. First, it may be owing to poor absorption of vitamin D from the intestine. Second, the body may not activate vitamin D properly. Importantly, both vitamin D and thyroid hormone bind to similar receptors called steroid hormone receptors. A different gene in the vitamin D receptor was shown to predispose people to AITD.
Also, Kivity et al. (17) reported that the prevalence of vitamin D deficiency was significantly higher in 50 patients with AITD compared with 98 healthy individuals (72% vs. 30,6%, respectively, p<0.001). Vitamin D deficiency was also found to be correlated with the presence of anti-thyroid antibodies (p=0.01), suggesting the involvement of vitamin D in the pathogenesis of AITD.
Moreover, we found that 68% of patients with AITD had vitamin D deficiency, whereas only 38% of patients without AITD had vitamin D deficiency. In our study, we found that serum TSH was significantly higher in patients with AITD than patients without AITD. However , a study Shin et al (18) did not find any significant difference. This could be due to a different sample population.
Our study showed a significant negative correlation between vitamin D and TSH level (p=0.009).
An experimental study by Byron Richards (18) studied, that was showed a lack of vitamin D leading to the possibility of increased thyroid -stimulating hormone, so the significant (p<0,05) negative correlation between vitamin D and TSH indicates the correlation between hypothyroidism and vitamin D.
However, we are documented a statistically negative correlation between vitamin D levels and thyroid antibodies, this result comes in agreement with Hosny, et al. (18), who found a negative relation between serum 25 (OH) vitamin D and anti-TPO and anti-TG. As well as, Khare, et al. (19), noted that the amount of serum 25(OH)D did not differ significantly between positive TPO-Ab and negative TPO-Ab subjects, which is in accordance with our results. Also, Yasmeh, et al. (20), have found no association with anti-TPO positivity and a poor inverse correlation between the levels of Vitamin D and anti-TPO has been found, which is contradictory to our findings. In other studies, several researchers found that the incidence of 25(OH)D, deficiency among TPO-Ab positive was significantly higher that in TPO-Ab negative hypothyreoid patients (7, 21). The contradictory and different results of the study are partly due to inter-assay and inter-laboratory variability in 25 (OH)D levels of Vitamin D, seasonal differences in 25(OH) blood samples.
CONCLUSION:
Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD.
| Background:
Vitamin D deficiency is a global health problem. Its deficiency has been reported to be associated with different autoimmune diseases.
Methods:
A total number of 150 individuals were enrolled in this study. They were divided into the fallowing groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease. Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibodies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG).
Results:
Serum levels of 25 (OH) vitamin D recorded a highly significant difference between the studies groups (20,76±6,31 ng/ml in group I vs. 24,37±9,05ng/ml in group II vs. 24,57±6,45ng/ml in group III, p<0,01). Moreover, there was a highly significant difference between patients with AITD and patients without AITD (20,76±6,31ng/ml vs. 24,37±9,05ng/ml, respectively; p<0,01). The concentration of anti-TPO and anti-TG antibodies were statistically significant higher in patients with vitamin D deficiency (p< 0,001). Serum TSH were significantly higher in group I (p< 0,001).
Conclusions:
Significantly low levels of vitamin D were documented in patients with AITD that were related to the presence of anti-thyroid antibodies and higher level thyroid-stimulation hormone (TSH), suggesting the involvement of vitamin D in the pathogenesis of AITD and the advisability of supplementation. | INTRODUCTION:
Vitamin D deficiency is a global health problem. Prevalence of vitamin D deficiency or insufficiency is over a billion worldwide (1). The role of vitamin D has been evolving since the time of its discovery in the early 20th century from being a simple vitamin D to a steroid prohormone (2). Vitamin D deficiency has been shown to be associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and type 1 diabetes, and that vitamin D supplementation prevents the onset and/or development of these autoimmune diseases (3). Autoimmune thyroid diseases (AITD), including Hashimoto’s (HT) and Graves’s (GD), are the most common organ-specific autoimmune disorders (4). These AITD are polygenic diseases resulting form a combination of genetic predisposition (thyroid-specific genes and immune-modulating genes) and environmental triggers ( iodine, selenium, drugs, irradiation, smoking, infections, stress, etc), characterized by lymphocystic infiltration into the thyroid gland and production of thyroid-specific autoantibodies (4, 5). Both vitamin D and thyroid hormone bind to the steroid hormone receptors. Moreover, vitamin D mediates its effect by binding to vitamin D receptor (VDR), and activation of VDR-responsive genes. VDR gene polymorphism was found in association with autoimmune thyroid diseases (AITD) (6). Few studies were conducted to find any significant association between the levels of vitamin D and hypothyroidism and it’s pathogenesis but yielded conflicting results. Kivity et al. in 2011 documented significantly low levels of 25(OH) vitamin D with autoimmune thyroid disease, whereas a study by Goswami et al. showed a weak association between 25(OH) vitamin D levels and thyroid peroxidase antibody (TPO-At) titers (7, 8).
CONCLUSION:
Significantly lower levels of vitamin D were documented in patients with AITD. Deficiency of vitamin D was linked to the presence of thyroid antibodies and abnormal thyroid functions. Further studies are required to determine whether vitamin D deficiency is the causal factor or the consequence of primary hypothyroidism, and to provide insight into the efficacy and safely of vitamin D as a therapeutic tool for AITD. | Background:
Vitamin D deficiency is a global health problem. Its deficiency has been reported to be associated with different autoimmune diseases.
Methods:
A total number of 150 individuals were enrolled in this study. They were divided into the fallowing groups: group I included 50 patients with autoimmune thyroid disease (AITD), group II included 50 patients without autoimmune thyroid disease. Group III included 50 apparently healthy participants representing a control group. All participants underwent a detailed clinical examination and laboratory tests including, 25 (OH) vitamin D, thyroid-stimulating hormone (TSH) and thyroid autoantibodies assessment, including anti-thyroid peroxidase antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG).
Results:
Serum levels of 25 (OH) vitamin D recorded a highly significant difference between the studies groups (20,76±6,31 ng/ml in group I vs. 24,37±9,05ng/ml in group II vs. 24,57±6,45ng/ml in group III, p<0,01). Moreover, there was a highly significant difference between patients with AITD and patients without AITD (20,76±6,31ng/ml vs. 24,37±9,05ng/ml, respectively; p<0,01). The concentration of anti-TPO and anti-TG antibodies were statistically significant higher in patients with vitamin D deficiency (p< 0,001). Serum TSH were significantly higher in group I (p< 0,001).
Conclusions:
Significantly low levels of vitamin D were documented in patients with AITD that were related to the presence of anti-thyroid antibodies and higher level thyroid-stimulation hormone (TSH), suggesting the involvement of vitamin D in the pathogenesis of AITD and the advisability of supplementation. | 2,368 | 311 | [
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|||||
Activation of pro-oncogenic pathways in colorectal hyperplastic polyps. | 24209454 | In contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP. | BACKGROUND | We retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients. | METHODS | Mean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression. | RESULTS | HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential. | CONCLUSIONS | [
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] | 3829387 | Background | Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas
[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations
[3-5].
The hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%
[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma
[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.
[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP
[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis
[12].
PG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion
[13]. In colorectal cancers, gastrin gene expression is up-regulated
[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps
[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established
[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium
[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways
[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer
[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression. | Methods | Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic
[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table
1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.
Clinical and histological features of hyperplastic polyps
Progastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.
We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic
[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table
1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.
Clinical and histological features of hyperplastic polyps
Progastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.
Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized
[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho
[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work
[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.
Using the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).
Using the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.
For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized
[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho
[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work
[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.
Using the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).
Using the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.
Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.
Spearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.
All tests were two-sided and statistical significance was set at a p value of 0.05.
***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11
[34].
Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.
Spearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.
All tests were two-sided and statistical significance was set at a p value of 0.05.
***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11
[34]. | Results | Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table
1. To take into account the heterogeneity of PG staining in HP observed in Figure
1, PG expression was analyzed in Table
1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.
Distribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.
Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table
1. To take into account the heterogeneity of PG staining in HP observed in Figure
1, PG expression was analyzed in Table
1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.
Distribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.
Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of PG-positive cells in these different sample tissues are reported in Figure
4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.
Progastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.
Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of PG-positive cells in these different sample tissues are reported in Figure
4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.
Progastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.
The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure
4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).
Interestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table
2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table
2).
Expression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps
SD: standard deviation.
(a)Cuzick test for trend across ordered groups.
(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.
Expressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.
Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure
4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).
Interestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table
2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table
2).
Expression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps
SD: standard deviation.
(a)Cuzick test for trend across ordered groups.
(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.
Expressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.
The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3.
The percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure
4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).
As observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.
3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table
2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).
Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3.
The percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure
4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).
As observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.
3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table
2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116). | Conclusion | HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.
Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Written informed consent was obtained from the patient for the publication of this report and any accompanying images. | [
"Background",
"Patients and data collection",
"Immunohistochemistry",
"Statistical analysis",
"Clinical and histological characteristics",
"Expression of progastrin in normal mucosa and colonic neoplasms",
"The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression",
"The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression",
"Consent",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.",
"We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.",
"For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.",
"Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].",
"Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.",
"Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.",
"Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.",
"Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).",
"Written informed consent was obtained from the patient for the publication of this report and any accompanying images.",
"The author(s) declare that they have no competing interests.",
"CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Patients and data collection",
"Immunohistochemistry",
"Statistical analysis",
"Results",
"Clinical and histological characteristics",
"Expression of progastrin in normal mucosa and colonic neoplasms",
"The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression",
"The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression",
"Discussion",
"Conclusion",
"Consent",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas\n[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations\n[3-5].\nThe hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%\n[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma\n[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.\n[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP\n[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis\n[12].\nPG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion\n[13]. In colorectal cancers, gastrin gene expression is up-regulated\n[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps\n[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established\n[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium\n[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways\n[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer\n[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.",
" Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\nWe examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.\n Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\nFor immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.\n Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].\nUnivariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].",
"We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic\n[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table \n1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.\nClinical and histological features of hyperplastic polyps\nProgastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.",
"For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized\n[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho\n[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work\n[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.\nUsing the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).\nUsing the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.",
"Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.\nSpearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.\nAll tests were two-sided and statistical significance was set at a p value of 0.05.\n***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11\n[34].",
" Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\nClinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.\n Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\nRepresentative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.\n The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\nRepresentative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.\n The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).\nRepresentative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).",
"Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table \n1. To take into account the heterogeneity of PG staining in HP observed in Figure \n1, PG expression was analyzed in Table \n1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.\nDistribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.",
"Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of PG-positive cells in these different sample tissues are reported in Figure \n4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.\nExpression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.\nProgastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.",
"Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure \n4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).\nInterestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table \n2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table \n2).\nExpression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps\nSD: standard deviation.\n(a)Cuzick test for trend across ordered groups.\n(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.\nExpressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.",
"Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures \n2 and\n3.\nThe percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure \n4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).\nAs observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.\n3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table \n2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).",
"In the present study, we demonstrated a significant increase in the activation of the pro-oncogenic pathway, ERK1/2, in HP as compared to normal tissue. More interestingly, we showed a significant correlation between ERK pathway activation in HP and the expression of PG that is recognized as a growth factor for colonic epithelial cells. ERK activation was significantly higher in lesions with strong PG expression. Activation of this signaling pathway by PG has been previously reported in normal colonic epithelial cells from a transgenic mouse model overexpressing PG and has been linked to an increased risk of developing preneoplastic lesions in the colonic epithelium\n[20]. Therefore HP overexpressing PG that have a high activation of the ERK pathway might reflect less latent lesions.\nThe PG gene has been previously shown to be a target of two pro-oncogenic pathways frequently activated in colorectal cancer: APC/β-catenin and K-ras\n[35-37]. APC deletions or β-catenin mutations have not been reported in HP and we recently published that this pathway is not activated in HP with PG overexpression\n[12]. Therefore, it is unlikely that this pathway is involved in the expression of PG in these lesions. In contrast, KRAS mutations have been observed in thirty-seven percent of HP\n[5] and might lead to the increase in PG expression and ERK activation observed in the present study. However we cannot exclude an additional mechanism leading to PG expression in HP since in our study, nearly to sixty percent of HP presented an overexpression of PG or p-ERK. In a recent publication, Bongers et al.\n[38] have reported the activation of the EGFR pathway in seventy percent of HP from a small cohort of 27 samples. Interestingly, EGFR ligands have been shown to be potent regulators of the progastrin gene and an EGF response element has been identified on the progastrin promoter\n[39,40]. Therefore the EGFR pathway activated in HP might also contribute to PG overexpression and ERK activation independently of K-ras.\nProgastrin is clearly recognized as an autocrine growth factor for colorectal cancer cells and blocking PG expression has been shown to inhibit cellular growth in vitro and in vivo on tumor xenografts\n[23,41,42]. It is probable that an autocrine mechanism occurs in HP producing PG and leading to ERK activation. However a recent publication from Duckworth et al.\n[43] suggests that an indirect mechanism might be also proposed. These authors have shown that PG is capable to activate colonic fibroblasts leading to growth factors secretion that in turn stimulate colonic epithelial cells. These results therefore suggest that PG produced by HP might also activate the ERK pathway in colonic epithelial cells via a dialogue with the fibroblasts present in the stroma.\nThe identity of the receptor mediating the PG effects on colonic epithelial cells or fibroblasts remains an important point of debate. Several publications have shown that the receptor specific for the mature form of gastrin, the CCK-2 receptor, is not involved in the PG effect on fibroblasts or colon cancer cells\n[18,43,44]. In contrast the data from Jin et al.\n[45] suggest a role of this receptor in the proliferative effects of PG in vivo, although the nature of the interaction between PG and the CCK2 receptor in this study remains to be identified. Other studies have shown a role of ferric ions, Annexin A2 or glycosaminoglycans in the binding of PG to cell surface\n[20,46,47]. However, the cell surface protein that directly binds PG remained to be identified.\nIn contrast to what we observed for the ERK pathway, STAT3 activation was not significantly different between HP and normal colon. In addition we did not observe a significant correlation with PG expression. We previously demonstrated an association between STAT3 activation and PG in vivo, in transgenic mice overexpressing the prohormone\n[20]. STAT3 activation by PG might required high level of progastrin expression, as found in adenomas or adenocarcinomas.\nPreviously we demonstrated that PG expression in HP may predict occurrence of metachronous adenomas\n[12]. Including additional biomarkers might improve the specificity of such a test. P-ERK might be an interesting factor since this pro-oncogenic factor is overexpressed in a subset of PG positive HP.",
"HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.\n Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.\nWritten informed consent was obtained from the patient for the publication of this report and any accompanying images.",
"Written informed consent was obtained from the patient for the publication of this report and any accompanying images.",
"The author(s) declare that they have no competing interests.",
"CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2407/13/531/prepub\n"
] | [
null,
"methods",
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null,
null
] | [
"Hyperplastic polyps",
"Colorectal",
"Progastrin",
"ERK",
"STAT3",
"Pro-oncogenic pathways"
] | Background:
Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas
[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations
[3-5].
The hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%
[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma
[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.
[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP
[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis
[12].
PG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion
[13]. In colorectal cancers, gastrin gene expression is up-regulated
[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps
[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established
[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium
[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways
[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer
[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.
Methods:
Patients and data collection We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic
[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table
1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.
Clinical and histological features of hyperplastic polyps
Progastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.
We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic
[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table
1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.
Clinical and histological features of hyperplastic polyps
Progastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.
Immunohistochemistry For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized
[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho
[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work
[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.
Using the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).
Using the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.
For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized
[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho
[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work
[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.
Using the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).
Using the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.
Statistical analysis Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.
Spearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.
All tests were two-sided and statistical significance was set at a p value of 0.05.
***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11
[34].
Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.
Spearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.
All tests were two-sided and statistical significance was set at a p value of 0.05.
***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11
[34].
Patients and data collection:
We examined 48 cases of HP (from 48 different patients) diagnosed in the pathology department of Rangueil Hospital (Toulouse, France) in 2008. We excluded patients with a history of familial adenomatous polyposis or hyperplastic polyposis and HP which display criteria of sessile serrated adenomas according to Torlakovic
[9,31]. All polyps measured less than 1 cm with an average diameter of 3 mm. For comparison, we also selected 12 normal colonic tissue specimens from resected non-complicated diverticular disease and 15 adenomas (10 low, and 5 high grade dysplasia adenomas). Clinical data (age, gender, site, size, number of HP at diagnosis, history of colorectal adenoma, or adenocarcinoma and the presence of synchronous adenoma or adenocarcinoma were collected for all 48 patients (Table
1). Approval of an institutional research ethics committee (Medical University of Toulouse) was obtained in accordance with the precepts of the Helsinki Declaration.
Clinical and histological features of hyperplastic polyps
Progastrin expression was analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”. Chi 2 tests (for categorical variables) and Kruskal-Wallis test (for continuous variables) were performed to compare clinical and immune-histological features between progastrin expression groups. 95% CI: Binomial exact 95- confidence interval was calculated for each percentage, if no other mention. SD: standard deviation a: Fischer exact test.
Immunohistochemistry:
For immunohistochemistry on the formaldehyde-fixed, paraffin embedded tissues, antigen retrieval was performed on dewaxed sections by water-bath heating slides in 10 mM Tris-EDTA buffer (pH9) (Cliniscience, Nanterre, France). After peroxidase and serum blocking, primary antibodies was applied overnight. We then, used the Dako Envision + System-HRP according to manufacturer protocol (Cliniscience). Specific primary polyclonal antibody against PG used for immunohistochemistry (dilution: 1:1000) was previously characterized
[12,32]. Primary monoclonal antibodies against tyrosine705-phospho-STAT3 (pY-STAT3) and threonin202/tyrosin204-phospho-ERK-1/2 (pERK1/2) (dilution 1:400) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). PG antibodies were provided by the University of Melbourne, Department of Surgery (Victoria, Australia). Colonic tissue sections known to be positive for PG, p-ERK and p-STAT3 were used as positive controls. For negative controls the primary antibody was omitted. In addition, the anti-PG antibody was incubated with the immunizing peptide that abolished the staining reaction. Analysis of the whole polyp section was performed. Staining for PG (cytoplasmic), p-ERK (cytoplasmic) and p-STAT3 (nuclear) were measured by percentage of stained epithelial cells in the whole polyp. All specimens were examined in a double blinded fashion by two pathologists trained to identify the pathological features of colonic cancer. The coefficient of concordance correlation, c-rho
[33] was calculated in order to determine inter-rater agreement for immunohistochemistry staining. Because the inter-rater agreement was excellent (c-rho = 0.99 for progastrin and pY-STAT3; and c-rho = 0.98 for p-ERK staining), percentages were reported as the average results between the two readers. As defined in our previous work
[12], progastrin staining was also recorded as no/low, moderate or high expression. The “normality” threshold of progastrin expression (low expression) was determined using the 95th percentile of percentage of stained cells in normal colonic tissue (<10%). Moderate expression of progastrin was defined as staining in 10% to 50% of epithelial cells and high expression as staining in more than 50% of epithelial cells.
Using the 95th percentile of percentage of p-ERK stained epithelial cells in normal mucosa, we defined p-ERK overexpression as staining in more than 15% of epithelial cells. Moderate expression (defined as staining in 15% to 50% of epithelial cells) was distinguished from high expression (staining in more than 50% of epithelial cells).
Using the 95th percentile of percentage of pY-STAT3 stained epithelial cells in normal mucosa, we defined pY-STAT3 overexpression as staining in more than 5% of epithelial cells. High expression was defined as staining in more than 50% of epithelial cells.
Statistical analysis:
Univariate analysis was conducted to compare clinical and immunohistochemistry findings between the different study groups using the Chi2 test or Fisher exact test (when required) for categorical variables and the nonparametric rank tests (Wilcoxon-Mann–Whitney or Kruskal_Wallis) or Cuzick nonparametric test for trend across ordered groups for quantitative variables.
Spearman nonparametric correlation test was used to assess the correlations between the expression of progastrin and p-ERK or pY-STAT3.
All tests were two-sided and statistical significance was set at a p value of 0.05.
***p < 0.001; **0.001 < p < 0.01; *0.01 < p < 0.05. Analyses were performed using the statistical software, STATA v11
[34].
Results:
Clinical and histological characteristics Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table
1. To take into account the heterogeneity of PG staining in HP observed in Figure
1, PG expression was analyzed in Table
1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.
Distribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.
Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table
1. To take into account the heterogeneity of PG staining in HP observed in Figure
1, PG expression was analyzed in Table
1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.
Distribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.
Expression of progastrin in normal mucosa and colonic neoplasms Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of PG-positive cells in these different sample tissues are reported in Figure
4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.
Progastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.
Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of PG-positive cells in these different sample tissues are reported in Figure
4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.
Progastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.
The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure
4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).
Interestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table
2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table
2).
Expression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps
SD: standard deviation.
(a)Cuzick test for trend across ordered groups.
(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.
Expressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.
Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure
4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).
Interestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table
2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table
2).
Expression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps
SD: standard deviation.
(a)Cuzick test for trend across ordered groups.
(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.
Expressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.
The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3.
The percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure
4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).
As observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.
3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table
2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).
Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3.
The percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure
4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).
As observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.
3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table
2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).
Clinical and histological characteristics:
Clinical and histological features of patients and their polyps as well as PG staining in HP epithelial cells are shown in Table
1. To take into account the heterogeneity of PG staining in HP observed in Figure
1, PG expression was analyzed in Table
1 as a 3 classes variable as detailed in “Methods”. In our sample, no/low expression of progastrin was observed in 44% of the HP (95% CI: 29%-59%), moderate expression in 25% (14%-40%) and strong expression in 31% (19%-46%). Although an increased prevalence of HP with high PG expression can be observed in women, PG expression was not significantly correlated to the following variables: age, history of adenoma or carcinoma, synchronous adenoma or carcinoma, localization and HP histological features.
Distribution of percentage of progastrin stained epithelial cells in HP. No/low expression of progastrin was determined using the 95th percentile of percentage of stained cells in normal colonic mucosa (< 10%). Among HP overexpressing the prohormone (> 10%), moderate expression (10%-50%) and high expression (> 50) were distinguished accordingly to the bimodal distribution of progastrin expression.
Expression of progastrin in normal mucosa and colonic neoplasms:
Representative pictures of PG staining, obtained with the anti-PG antibody in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of PG-positive cells in these different sample tissues are reported in Figure
4A. Mean percentage of PG positive cells observed in the 48 HP was significantly higher than in normal colon (respectively 31% and 3% p = 0.0021). In low and high grade tubular adenomas, percentages of PG positive cells were also higher compared to normal colon (respectively, 87%, p = 0.0001 and 85%, p = 0.0014). No significant difference was observed between low and high grade adenomas. HP showed an intermediate expression of PG between normal mucosa and colonic adenomas.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in hyperplastic polyps. (A, B) Progastrin staining in HP: A) no/weak staining B) high progastrin staining. (C, D) p-ERK1/2 staining in HP: C) no/weak staining D) high p-ERK1/2 staining. (E, F) pY-STAT3 staining in HP: E) no/weak staining F) high pY-STAT3 staining.
Expression of Progastrin p-ERK1/2 and pY-STAT3 in normal colonic mucosa and adenomas. (A-C) Progastrin staining: A) Negative staining in normal colon B) high progastrin staining in low grade and C) high grade adenomas. (D-F) p-ERK1/2 staining: D) Negative staining E) high p-ERK1/2 staining in low grade and F) high grade adenomas. (G-I) pY-STAT3 staining: G) Negative staining in normal colon H) high pY-STAT3 staining in low grade and I) high grade adenomas.
Progastrin, p-ERK1/2, and pY-STAT3 expression in colonic tissues. Percentage of (A) progastrin (B) p-ERK1/2 (C) pY-STAT3 positive cells for normal colon, Hyperplastic polyps (HP), low grade dysplasia tubular adenomas and high grade dysplasia tubular adenomas. Quantifications are presented as means ± S.E.M.
The ERK pathway in normal colonic mucosa, HP and adenomas and its relationship with progastrin expression:
Representative pictures of p-ERK1/2 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3. The percentages of pERK1/2-positive cells in these different sample tissues are reported in Figure
4B. Mean percentage of p-ERK positive epithelial cells in normal colonic tissues reached 7%. In HP, the activation of ERK was significantly higher, with 33% of stained epithelial cells (p = 0.0008). In low grade and high grade adenomas, mean percentages of stained cells were similar (51%) and significantly higher than in normal mucosa (respectively, p = 0.0002 and p = 0.0105).
Interestingly, in HP, ERK activation was significantly higher in lesions with strong PG expression (53% of stained cells) as compared to no/low or moderate PG expression (respectively 29% and 22%, p = 0.015) (Table
2). As described in “methods” we also defined 3 groups for p-ERK expression. 65% of HP presented an overexpression of p-ERK, including 37% of moderate expression and 28% of high expression. Analysis of the increase in percentage across the different expression groups showed that the expression of p-ERK and PG was significantly correlated (p = 0.008) (Table
2).
Expression of progastrin, p-ERK1/2 and pY-STAT3 in hyperplastic polyps
SD: standard deviation.
(a)Cuzick test for trend across ordered groups.
(b)Spearman nonparametric correlation test was used for comparison of ordered qualitative variable.
Expressions of PG, .P-ERK and PY-STAT3 were analyzed as a 3 classes variable: no/low expression, moderate expression and high expression as detailed in “Methods”.
The STAT3 pathway in normal colonic mucosa HP and adenomas and its relationship with progastrin expression:
Representative pictures of pY-STAT3 staining in normal colon, HP, low grade and high grade adenomas are shown in Figures
2 and
3.
The percentages of pY-STAT3-positive cells in these different sample tissues are reported in Figure
4C. Mean percentage of pY-STAT3-positive cells in normal colonic mucosa was only 1%. In HP the percentage of stained cells was 10% but was not significantly higher than in normal colonic mucosa. In low grade and high grade adenomas, mean percentages of stained cells were respectively 38% and 31% and significantly higher than in normal mucosa (respectively, p = 0.0014 and p = 0.0041).
As observed for PG and P-ERK no significant difference in PY-STAT3 staining was observed between low and high grade adenomas.
3 groups for pY-STAT3 expression were defined as described in “Methods”. 23% of HP presented an overexpression of pY-STAT3, including 14% with moderate expression and 9% with high expression (Table
2). In HP, mean percentage of pY-STAT3 staining was not associated to the different classes of PG staining (p = 0.297) and no correlation between these two factors was observed (p = 0.3116).
Discussion:
In the present study, we demonstrated a significant increase in the activation of the pro-oncogenic pathway, ERK1/2, in HP as compared to normal tissue. More interestingly, we showed a significant correlation between ERK pathway activation in HP and the expression of PG that is recognized as a growth factor for colonic epithelial cells. ERK activation was significantly higher in lesions with strong PG expression. Activation of this signaling pathway by PG has been previously reported in normal colonic epithelial cells from a transgenic mouse model overexpressing PG and has been linked to an increased risk of developing preneoplastic lesions in the colonic epithelium
[20]. Therefore HP overexpressing PG that have a high activation of the ERK pathway might reflect less latent lesions.
The PG gene has been previously shown to be a target of two pro-oncogenic pathways frequently activated in colorectal cancer: APC/β-catenin and K-ras
[35-37]. APC deletions or β-catenin mutations have not been reported in HP and we recently published that this pathway is not activated in HP with PG overexpression
[12]. Therefore, it is unlikely that this pathway is involved in the expression of PG in these lesions. In contrast, KRAS mutations have been observed in thirty-seven percent of HP
[5] and might lead to the increase in PG expression and ERK activation observed in the present study. However we cannot exclude an additional mechanism leading to PG expression in HP since in our study, nearly to sixty percent of HP presented an overexpression of PG or p-ERK. In a recent publication, Bongers et al.
[38] have reported the activation of the EGFR pathway in seventy percent of HP from a small cohort of 27 samples. Interestingly, EGFR ligands have been shown to be potent regulators of the progastrin gene and an EGF response element has been identified on the progastrin promoter
[39,40]. Therefore the EGFR pathway activated in HP might also contribute to PG overexpression and ERK activation independently of K-ras.
Progastrin is clearly recognized as an autocrine growth factor for colorectal cancer cells and blocking PG expression has been shown to inhibit cellular growth in vitro and in vivo on tumor xenografts
[23,41,42]. It is probable that an autocrine mechanism occurs in HP producing PG and leading to ERK activation. However a recent publication from Duckworth et al.
[43] suggests that an indirect mechanism might be also proposed. These authors have shown that PG is capable to activate colonic fibroblasts leading to growth factors secretion that in turn stimulate colonic epithelial cells. These results therefore suggest that PG produced by HP might also activate the ERK pathway in colonic epithelial cells via a dialogue with the fibroblasts present in the stroma.
The identity of the receptor mediating the PG effects on colonic epithelial cells or fibroblasts remains an important point of debate. Several publications have shown that the receptor specific for the mature form of gastrin, the CCK-2 receptor, is not involved in the PG effect on fibroblasts or colon cancer cells
[18,43,44]. In contrast the data from Jin et al.
[45] suggest a role of this receptor in the proliferative effects of PG in vivo, although the nature of the interaction between PG and the CCK2 receptor in this study remains to be identified. Other studies have shown a role of ferric ions, Annexin A2 or glycosaminoglycans in the binding of PG to cell surface
[20,46,47]. However, the cell surface protein that directly binds PG remained to be identified.
In contrast to what we observed for the ERK pathway, STAT3 activation was not significantly different between HP and normal colon. In addition we did not observe a significant correlation with PG expression. We previously demonstrated an association between STAT3 activation and PG in vivo, in transgenic mice overexpressing the prohormone
[20]. STAT3 activation by PG might required high level of progastrin expression, as found in adenomas or adenocarcinomas.
Previously we demonstrated that PG expression in HP may predict occurrence of metachronous adenomas
[12]. Including additional biomarkers might improve the specificity of such a test. P-ERK might be an interesting factor since this pro-oncogenic factor is overexpressed in a subset of PG positive HP.
Conclusion:
HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.
Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Consent:
Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Competing interests:
The author(s) declare that they have no competing interests.
Authors’ contributions:
CD1 contributed to study conception and design, acquisition analysis and interpretation of the data, statistical analysis, drafting and revision of the manuscript. CB1, JP1 contributed to study design, acquisition and analysis of the data, and revision of the manuscript. MBD2 and ECJM1 contributed to interpretation of the data, and revision of the manuscript, CS1 contributed to study conception and design, data interpretation, drafting and revision of the manuscript, study supervision. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2407/13/531/prepub
| Background:
In contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP.
Methods:
We retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients.
Results:
Mean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression.
Conclusions:
HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential. | Background:
Among colorectal polyps, the serrated adenomas represent a heterogeneous group of lesions including, sessile serrated adenomas (SSA) and traditional serrated adenomas (TSA). They are associated with a significant cancer risk and represent neoplastic precursor lesion of serrated adenocarcinomas
[1,2]. Both SSA and TSA have a high frequency of DNA methylation. However, SSA have been linked to adenocarcinomas with microsatellite instability (MSI) with a positive immunostaining for Cytokeratin 7 (CK7) and mostly localized in the proximal colon. In contrast TSA are essentially microsatellite stable or show low level of MSI and lead to serrated adenocarcinomas in the distal colon with a positive immunostaining for CK7 and CK20. In addition, SSA are frequently BRAF-mutated whereas TSA show a high frequency of K-ras mutations
[3-5].
The hyperplastic polyps (HP) are the most frequently occurring lesions in the colon with prevalence in western populations of 10% to 35%
[6]. HP are usually considered as innocuous lesions with no malignant potential. However, large HP (size > 10 mm) and the presence of multiple HP (number > 5) in hyperplastic polyposis syndrome have been clearly associated with colorectal adenomas or adenocarcinoma
[7-10]. However, some authors have proposed that a subset of HP may be associated to an increased risk to develop adenomas. Huang et al.
[11] found that patients with HP on initial colonoscopic examination have an increased incidence of colorectal adenomas on follow-up colonoscopy. In addition, we recently published a retrospective study in which 41% of patients, without history of colorectal pathology, presenting initial true HP (<10 mm) with no SSA or TSA features, subsequently developed adenomas after resection of these HP
[12]. Interestingly HP from patients who developed adenomas overexpressed the prohormone progastrin (PG) which is recognized as a growth factor, playing an important role in colon carcinogenesis
[12].
PG is the precursor of the amidated gastrin. This hormone is mainly produced by antral G cells of the stomach and is known as a potent stimulant of gastric acid secretion
[13]. In colorectal cancers, gastrin gene expression is up-regulated
[14]. However, in these tumors, gastrin is incompletely maturated and gastrin precursors, particularly PG, are mainly secreted. High concentrations of PG are found in colon tumors and in blood of patients with colorectal cancer. PG is also expressed in adenomatous polyps
[15-17]. In contrast, this hormone precursor is absent from the healthy intestinal epithelium. The proliferative effects of PG on normal and cancerous colorectal cells in vitro and in vivo have been clearly established
[18-25]. In addition transgenic mice overexpressing progastrin present an increased proliferative index in colonic mucosa. They also have an increased risk of developing preneoplastic lesions in colonic epithelium
[22]. These effects are mediated through the activation of signaling pathways such as the extracellular signal-regulated kinase (ERK) and the signal transducer and activator of transcription 3 (STAT3) pathways
[20]. These pathways that transduce extracellular signals to the nucleus and regulate gene transcription are known to be activated in many human cancers, including colorectal cancer
[20,26-30]. They have been shown to regulate cell functions involved in carcinogenesis, such as cell proliferation, survival or migration. In the present study, to better characterize at the molecular level the subset of HP that may be associated with a risk to develop colonic neoplasm, we assessed the activation of the ERK and STAT3 pathways in HP and we analyzed the correlation with PG expression.
Conclusion:
HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential.
Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Written informed consent was obtained from the patient for the publication of this report and any accompanying images. | Background:
In contrast to sessile serrated adenomas and traditional serrated adenomas which are associated with a significant cancer risk, the role of hyperplastic polyps (HP) in colorectal carcinogenesis as well as the molecular mechanisms underlying their development remain controversial and still need to be clarified. Several reports suggest that a subset of HP may represent precursor lesions of some colorectal cancers. However, biomarkers are needed to identify the subset of HP that may have a malignant potential. The hormone precursor, progastrin (PG) has been involved in colon carcinogenesis and is known to activate pro-oncogenic pathways such as the ERK or the STAT3 pathway. We therefore analyzed PG expression and the activation of these signaling factors in HP.
Methods:
We retrospectively analyzed PG expression as well as the phosphorylation of ERK and STAT3 by immunohistochemistry in HP from 48 patients.
Results:
Mean percentages of epithelial cells positive for PG or phospho-ERK were respectively, 31% and 33% in HP and were significantly higher in these lesions compared to normal colon (3%, p=0.0021 and 7%, p=0.0008, respectively). We found a significant correlation between PG and phospho-ERK expression in HP with ERK activation significantly stronger in lesions with high progastrin expression (p=0.015). In contrast, STAT3 was not significantly activated in HP compared to normal colon and we did not observe a significant correlation with PG expression.
Conclusions:
HP overexpressing PG that have the highest activation of the ERK pathway might reflect less latent lesions that might have a malignant potential. | 8,620 | 293 | [
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] | [CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY] | [CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY] | [CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY] | [CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY] | [CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY] | [CONTENT] Hyperplastic polyps | Colorectal | Progastrin | ERK | STAT3 | Pro-oncogenic pathways [SUMMARY] | [CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY] | [CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY] | [CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY] | [CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY] | [CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY] | [CONTENT] Adenoma | Adult | Aged | Aged, 80 and over | Colonic Polyps | Extracellular Signal-Regulated MAP Kinases | Female | Gastrins | Humans | Hyperplasia | Intestinal Mucosa | Male | Middle Aged | Oncogene Proteins | Protein Precursors | Retrospective Studies | Risk Factors | STAT3 Transcription Factor | Signal Transduction [SUMMARY] | [CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY] | [CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY] | [CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY] | [CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY] | [CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY] | [CONTENT] colonic neoplasms representative | serrated adenomas ssa | features colonic cancer | adenomatous polyposis hyperplastic | adenocarcinomas microsatellite instability [SUMMARY] | [CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY] | [CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY] | [CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY] | [CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY] | [CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY] | [CONTENT] expression | staining | hp | pg | high | cells | stat3 | progastrin | normal | adenomas [SUMMARY] | [CONTENT] colorectal | hp | tsa | ssa | adenomas | serrated | pathways | gastrin | risk | pg [SUMMARY] | [CONTENT] staining | expression | epithelial | epithelial cells | cells | 50 epithelial cells | 50 epithelial | defined | stat3 | test [SUMMARY] | [CONTENT] staining | expression | grade | high | hp | py stat3 | py | stat3 | adenomas | normal [SUMMARY] | [CONTENT] consent | accompanying images | patient publication report | informed consent obtained patient | informed consent obtained | informed consent | informed | consent obtained | consent obtained patient | consent obtained patient publication [SUMMARY] | [CONTENT] expression | staining | hp | pg | cells | high | stat3 | grade | py stat3 | py [SUMMARY] | [CONTENT] expression | staining | hp | pg | cells | high | stat3 | grade | py stat3 | py [SUMMARY] | [CONTENT] ||| HP ||| HP ||| ERK | STAT3 ||| HP [SUMMARY] | [CONTENT] ERK | STAT3 | HP | 48 [SUMMARY] | [CONTENT] PG | 31% | 33% | HP | 3% | p=0.0021 | 7% ||| ERK ||| STAT3 | HP [SUMMARY] | [CONTENT] ERK [SUMMARY] | [CONTENT] ||| HP ||| HP ||| ERK | STAT3 ||| HP ||| ERK | STAT3 | HP | 48 ||| PG | 31% | 33% | HP | 3% | p=0.0021 | 7% ||| ERK ||| STAT3 | HP ||| ERK [SUMMARY] | [CONTENT] ||| HP ||| HP ||| ERK | STAT3 ||| HP ||| ERK | STAT3 | HP | 48 ||| PG | 31% | 33% | HP | 3% | p=0.0021 | 7% ||| ERK ||| STAT3 | HP ||| ERK [SUMMARY] |
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Effect of the interleukin 10 polymorphisms on interleukin 10 production and visceral hypersensitivity in Chinese patients with diarrhea-predominant irritable bowel syndrome. | 31205078 | Irritable bowel syndrome (IBS), a functional gastrointestinal disorder, is characterized by cytokine imbalance. Previously, decreased plasma interleukin 10 (IL-10) level was reported in patients with IBS, which may be due to genetic polymorphisms. However, there are no reports correlating the IL-10 polymorphisms with IL-10 production in patients with IBS. This study aimed to analyze the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with diarrhea-predominant IBS (IBS-D). | BACKGROUND | Two IL-10 single nucleotide polymorphisms (rs1800871 and rs1800896) were detected in 120 patients with IBS-D and 144 healthy controls (HC) using SNaPshot. IBS symptom severity score, Bristol scale, hospital anxiety, and depressive scale (HADS) were used to evaluate the clinical symptoms, as well as the psychological status and visceral sensitivity of the subjects. IL-10 levels in the plasma and peripheral blood mononuclear cell (PBMC) culture supernatant were measured using enzyme-linked immunosorbent assay, while those in ileal and colonic mucosal biopsies were measured using immunohistochemistry. | METHODS | The frequency of rs1800896 C allele was significantly lower in the patients with IBS-D than that in the HC (odds ratio: 0.49, 95% confidence interval: 0.27-0.92, P = 0.0240). The IL-10 levels in the plasma (P = 0.0030) and PBMC culture supernatant (P = 0.0500) of the CT genotype subjects were significantly higher than those in the TT genotype subjects. The CT genotype subjects exhibited a higher pain threshold in the rectal distention test than the TT genotype subjects. Moreover, IL-10 rs1800871 GG genotype subjects showed an increase in the HADS score compared to other genotype subjects. | RESULTS | IL-10 rs1800896 C allele is correlated with higher IL-10 levels in the plasma and the PBMC culture supernatant, which is associated with a higher pain threshold in the Chinese patients with IBS-D. This study provides an explicit relationship of IL-10 polymorphisms with IL-10 production, which might help in understanding the pathogenesis of IBS-D. | CONCLUSIONS | [
"Adolescent",
"Adult",
"Aged",
"Diarrhea",
"Genotype",
"Humans",
"Interleukin-10",
"Irritable Bowel Syndrome",
"Middle Aged",
"Peripheral Blood Stem Cells",
"Polymorphism, Single Nucleotide",
"Young Adult"
] | 6616227 | Introduction | Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]
IL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.
Therefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D. | Methods | Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.
Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.
This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.
Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.
SNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.
DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.
SNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.
Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.
The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.
Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.
Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.
Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.
Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.
Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.
Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed. | Results | Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.
Clinical characteristics for patients with IBS-D and HC.
A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.
Clinical characteristics for patients with IBS-D and HC.
Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.
Genotyping of IL-10 gene polymorphisms.
Genotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.
IL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.
The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.
Genotyping of IL-10 gene polymorphisms.
Genotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.
IL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.
Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.
Concentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.
IL-10 levels in different genotype of IL-10 polymorphisms.
IL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.
The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.
Concentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.
IL-10 levels in different genotype of IL-10 polymorphisms.
IL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.
Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.
Association of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.
The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.
Association of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10. | null | null | [
"Ethical approval",
"Study design and participants recruitment",
"Cytokine gene polymorphisms evaluated through SNaPshot",
"Isolation and culture of peripheral blood mononuclear cells",
"Enzyme-linked immunosorbent assay",
"Immunohistochemistry staining",
"Statistical analysis",
"Clinical characteristics of the subjects",
"Genotyping of IL-10 gene polymorphisms",
"Measurements for the IL-10 level",
"Association of IL-10 polymorphisms and clinical symptoms",
"Acknowledgements",
"Funding",
"Conflicts of interest"
] | [
"The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.",
"This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.",
"DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.",
"The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.",
"Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.",
"Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.",
"Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.",
"A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.",
"The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.",
"The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.",
"The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.",
"The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.",
"The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).",
"None."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Ethical approval",
"Study design and participants recruitment",
"Cytokine gene polymorphisms evaluated through SNaPshot",
"Isolation and culture of peripheral blood mononuclear cells",
"Enzyme-linked immunosorbent assay",
"Immunohistochemistry staining",
"Statistical analysis",
"Results",
"Clinical characteristics of the subjects",
"Genotyping of IL-10 gene polymorphisms",
"Measurements for the IL-10 level",
"Association of IL-10 polymorphisms and clinical symptoms",
"Discussion",
"Acknowledgements",
"Funding",
"Conflicts of interest"
] | [
"Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]\nIL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.\nTherefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D.",
" Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\nThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.\n Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\nThis study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.\n Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\nDNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.\n Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\nThe 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.\n Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\nSystematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.\n Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\nImmunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.\n Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.\nData were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.",
"The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.",
"This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.",
"DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.\nSNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.",
"The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.",
"Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.",
"Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.",
"Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.",
" Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\nA total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.\n Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\nThe genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.\n Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\nThe levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.\n Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.\nThe correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.",
"A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.\nClinical characteristics for patients with IBS-D and HC.",
"The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.\nGenotyping of IL-10 gene polymorphisms.\nGenotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.\nIL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.",
"The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.\nConcentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.\nIL-10 levels in different genotype of IL-10 polymorphisms.\nIL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.",
"The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.\nAssociation of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.",
"Patients with IBS-D present a particularly VH with a looser stool consistency, which may result by chronic systemic and mucosal inflammation. IL-10 plays an important role in the anti-inflammation response and is considered to be a potent suppressor of T lymphocytes or macrophages and their derived effector molecules, such as proinflammatory cytokines (IL-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemotactic protein 1, macrophage inflammatory protein 1α).[18] It is speculated that IL-10 gene polymorphisms might be involved in the IL-10 production.[19] Our study suggested that the carriers with IL-10 rs1800896 C allele have a lower risk for developing IBS-D, which may be associated with higher production of IL-10.\nThe gene encoding IL-10 is located on the human chromosome 1q31–1q32. The two polymorphisms analyzed in this study were −1082 A/G and −819 C/T, which are located in the gene promoter region. According to the single nucleotide polymorphism database (http://www.ncbi.nlm.nih.gov/SNP/), they are annotated as rs1800896 (T > C) and rs1800871 (A > G), which represents the polymorphism in the complementary strands. Qin et al[13] investigated that the IL-10 rs1800871 polymorphism was associated with a decreased risk for developing IBS in the eastern population. Moreover, other studies have confirmed that the C allele of IL-10 rs1800896 was strongly associated with the decreased risk for developing IBS in the western population.[7] However, there are studies which have reported no correlation between IL-10 polymorphisms and the risk for developing IBS.[12,20] In this study, the frequency of IL-10 rs1800896 C allele was significantly (OR: 0.49, 95% CI: 0.27–0.92, P = 0.0240) lower in the patients with IBS-D and was associated with a decreased risk for developing IBS-D. While the polymorphism rs1800871 presented no association with developing IBS, which investigated in patients with IBS-D and the healthy subjects.\nIL-10 was initially described as a T helper 2-type cytokine and was reported to be expressed in various cells of the adaptive immune system as well as the cells of the innate immune system. Patients with IBS exhibit cytokine imbalance characterized by decreased levels of IL-10 and increased levels of TNF-α.[1,7] The lower levels of IL-10 can be a predictor for IBS development.[21] The T allele of IL-10 rs1800896 has been reported to be associated with lower production of IL-10, while the C allele is associated with higher IL-10 production.[16,22] The C allele is associated with IL-10 production in the low stimulated cell culture.[23] In addition, there are very few studies that report a direct correlation between IL-10 polymorphisms and IL-10 production. Our data confirmed that the subjects carrying C allele of IL-10 rs1800896 exhibited a higher IL-10 concentration in the plasma and the PBMC culture supernatant. Additionally, we also demonstrated that the subjects with CT genotype were less sensitive in the rectal distention test than the subjects with TT genotype. Earlier studies demonstrated that IL-10 and other inflammatory cytokine concentration in the PBMC correlated with the severity of symptoms in IBS-D, including the intensity and frequency of painful events and motility-associated symptoms.[24] Some researchers hypothesize that IBS is on a spectrum with inflammation bowel disease (IBD), because they have largely overlapping pathophysiological mechanisms and clinical symptoms.[25,26] For example, the abdominal pain and diarrhea are predominant symptoms for patients with IBS or IBD, and post-inflammatory abnormalities are important in IBD and IBS. The subjects without C allele are more likely to develop IBS after intestinal infection.[3,27] Additionally, the polymorphisms of IL-10 gene can exert a protective effect on patients with IBD, ulcerative colitis or Crohn disease.[28,29]\nOur finding was intriguing as the IL-10 rs1800871 polymorphism exhibited a marginally positive correlation with the HADS score. The development of depressive disorder is considered to be associated with the activation of systemic inflammation as well.[30,31] It has been reported that the genotypes IL-10 rs1800871 and IL-10 rs1800896 decrease the risk for developing depression and are correlated with the Hamilton depression rating scale.[32] Globally, patients with IBS have a high comorbidity rate for depression or anxiety.[33] Our study supports the correlation between high comorbidity in IBS and mental disorder in an aspect of genetics polymorphism.\nTo the best of our knowledge, this study took the lead in studying the correlates of IL-10 gene polymorphisms and the expression level of IL-10. However, this study has some limitations. Only two polymorphisms for IL-10 were analyzed in this study. There may be other SNPs in IL-10 corresponding to rs1800896 and/or rs1800871, which may contribute to IL-10 expression. On the other hand, further studies are required for evaluating the transcriptional and epigenetic effects in IL-10 polymorphism.\nIn summary, the IL-10 rs1800896 polymorphism has a correlation with a higher concentration of IL-10 in both the plasma and the PBMC culture supernatant of Chinese patients with IBS-D. Additionally, this SNP is also associated with a higher visceral pain threshold. This study demonstrated a correlation between the IL-10 polymorphisms and IL-10 production, which might help in understanding the pathogenesis of IBS-D.",
"The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.",
"The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).",
"None."
] | [
"intro",
"methods",
null,
null,
null,
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
null,
null,
null
] | [
"Irritable bowel syndrome",
"Interleukin 10",
"Polymorphisms",
"Visceral hypersensitivity",
"Depression"
] | Introduction:
Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder characterized by the presence of abdominal pain, bloating, and altered bowel habits. According to the Rome IV criteria, there are four subtypes of IBS: constipation-predominant IBS, diarrhea-predominant IBS (IBS-D), diarrhea and constipation mixed IBS, and unsubtyped IBS. The pathophysiological mechanisms underlying IBS are unclear. The abnormalities of motility, visceral hypersensitivity (VH), gut microbial alteration, and psychological stress contribute to the clinical symptoms of IBS. Additionally, systemic and mucosal immune activation play an important role in IBS. Previous studies have confirmed that patients with IBS exhibited an imbalanced cytokine profile.[1,2] The infection after an acute gastroenteritis is a major trigger for IBS development, resulting in post-infectious IBS.[3,4] The plasma concentration of interleukin 6 (IL-6) and IL-8 tends to increase and that of interferon-γ tends to decrease in the patients with IBS. IL-10, an anti-inflammatory cytokine, is very important in the immune activation of the patients with IBS. However, there are conflicting reports on the role of IL-10 in IBS.[1,5,6] Some studies have reported that the IL-10 in plasma decreased in patients with IBS,[7,8] while others reported that there was no difference when compared to that in the healthy participants.[1,9] Several lines of evidences have demonstrated that there was a decrease in the level of IL-10 mRNA in the intestinal mucosa of patients with IBS.[10,11]
IL-10 is synthesized in the immune cells such as T and B lymphocytes, monocytes, macrophages, and mast cells. The cytokine gene polymorphisms have been suggested to influence the cytokine production. The single nucleotide polymorphisms (SNPs) of IL-10 gene, such as IL-10-1082 G/A (rs1800896) and IL-10-819 C/T (rs1800871), are both associated with IBS.[12–15]IL-10 rs1800896 polymorphism is associated with the enhanced production of IL-10 cytokine in vitro and is more prevalent in the healthy subjects.[16] Earlier studies have demonstrated that rs1800896 and rs1800871 polymorphisms of IL-10 were both correlated with the risk of developing IBS-D, which indicated the genetic susceptibility of patients with IBS-D.[17] However, most of the studies were conducted on Western populations and with limited sample size. Additionally, the correlation between IL-10 gene polymorphisms and IL-10 production is not yet defined.
Therefore, this study analyzed the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with IBS-D.
Methods:
Ethical approval The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.
Study design and participants recruitment This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.
This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.
Cytokine gene polymorphisms evaluated through SNaPshot DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.
SNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.
DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.
SNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.
Isolation and culture of peripheral blood mononuclear cells The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.
The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.
Enzyme-linked immunosorbent assay Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.
Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.
Immunohistochemistry staining Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.
Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.
Statistical analysis Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.
Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.
Ethical approval:
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Health Science Center (No. 2013-12). All the participants provided written informed consent.
Study design and participants recruitment:
This study was conducted from 2013 to 2018. We recruited IBS-D patients according to Rome III criteria from the Outpatients of Gastroenterology Department of Peking University Third Hospital sequentially, aging from 18 to 65 years old. Healthy volunteers (aging from 18 to 65 years) were recruited from community. The participants were excluded if they met the following exclusion criteria: (1) histories of antibiotics, probiotics/prebiotics, or psychotropic medications intake during the previous 4 weeks; (2) systemic or gastrointestinal diseases, such as diabetes mellitus and inflammatory bowel disease; (3) current infectious diseases of the respiratory, digestive, or urinary system; (4) a history of abdominal surgery. Gastrointestinal symptom severity, daily bowel movement frequency and consistency were evaluated by IBS symptom severity scores and Bristol stool form scale. Visceral sensitivity was assessed by rectal distension test using BAROSTAT (Distender Series II; G&J Electronics, Ontario, Canada). Sensory thresholds for initial sensory, initial defecation, and defecation urgency were calculated for each individual by the lowest pressures. Psychological status was evaluated by hospital anxiety and depressive scale (HADS). All participants underwent colonoscopy or had colonoscopy/barium enema performed in the past 6 months to rule out organic colonic diseases. Participants underwent colonoscopy in Peking University Third Hospital with biopsies in distal ileal and sigmoid colonic mucosa. Each participant was routine to investigate a hemogram, plasma chemistry profile, blood test for hepatic virus B and C and HIV, stool microscopy and occult blood testing, liver function tests. Peripheral blood was collected for further analysis.
Cytokine gene polymorphisms evaluated through SNaPshot:
DNA was isolated from cells of approximate 4 mL of the peripheral blood following a phenol/chloroform protocol. Each DNA sample was quantified twice using the DNA quantification NanoDrop (Thermo Scientific, Waltham, MA, USA). Samples were only accepted if the average DNA concentration was at least 0.25 ng/mL and the coefficient of variation between the two rounds of quantification was smaller than 0.1.
SNaPshot was used to genotype the IL-10 polymorphisms including rs1800896 and rs1800871. The amplification primers of the candidate SNPs as followed. IL-10 rs1800896: Forward, 5′-ACACTACTAAGGCTTCTTTGGGA-3′; Reverse, 5′-TACAAGGGTACACCAGTGC(C/T)A-3′. IL-10 rs1800871: Forward, 5′-AAGGTTTCATTCTATGTGCTGG-3′; Reverse, 5′-GTAAGAGTAGTCTGCACT TGCTG-3′. Genomic DNA was diluted to a concentration of 10 ng/μL before identification of genetic mutations. A multiplex SNaPshot assay (ABI PRISM, Foster City, CA, USA) was employed to determine the genotypes. First, 10 ng of genomic DNA was added to a 10 μL polymerase chain reaction (PCR) mixture containing 20 μmol dNTPs (Promega, Madison, WI, USA), 0.5 U of FastStart Taq DNA polymerase (Kapa Biosystems, Woburn, MA, USA), 1 μL 10× PCR buffer with MgCl2 (15 mmol/L), and amplification primers at a terminal concentration of 0.1 μmol/L. The thermal cycler conditions of multiplex PCR amplification were as follows: initial denaturation at 94°C for 5 min and amplification for 10 cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by 30 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 10 min. Subsequently, the PCR products were examined by electrophoresis in a 2.5% agarose gel. Then, we purified the PCR products using a mix of 5.4 U of Exonuclease I (NEB, Beverly, MA, USA) and 1.33 U of shrimp alkaline phosphatase (Fermentas, Lithuania) incubated at 37°C for 60 min followed by 85°C for 20 min. Subsequently, the multiplex SNaPshot sequencing reactions were performed in a final volume of 5 μL containing 2 μL of purified multiple PCR products, 1 μL of SNaPshot Multiplex Mix, 0.4 μL of 10× sequencing buffer (ABI, Los Angeles, CA, USA), and 3 μL of SNaPshot sequencing primers. The thermal cycler conditions were an initial denaturation followed by 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Then, the depuration of product was performed with 1 U of CIP at 37°C for 60 min and 75°C for 15 min. Finally, the SNaPshot products (1 μL) were genotyped in the ABI 3730 Genetic Analyzer platform before they were mixed with 8.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (ABI). Data were analyzed by GeneMapper 4.0 (ABI). In order to guarantee the quality of the data, approximately 3% of the samples were randomly selected and regenotyped by direct sequencing.
Isolation and culture of peripheral blood mononuclear cells:
The 4 mL of peripheral blood sample was collected in ethylene diamine tetraacetic acid vials and plasma was collected. Ficoll Histopaque was used for peripheral blood mononuclear cells (PBMCs) isolation according to the protocol.[13] PBMCs were harvested and counted with a hemocytometer. Cell viability was assessed by trypan blue staining following which they were resuspended at 1 × 106 cells/mL in complete media. Cells were transferred to plates and incubated, non-stimulated, for 72 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free supernatants were stored frozen at −80°C and analyzed for cytokine levels in batches.
Enzyme-linked immunosorbent assay:
Systematic inflammatory tone was assessed by measuring in both plasma and supernatant of PBMCs culturing through enzyme-linked immunosorbent assay (ELISA; EBioscience (Barcelona, Spain), Human IL-10 Platinum ELISA Kit). All samples and standards were assayed in two duplicates. To start with the measurement, each well of the 96-well plate was pre-wet with 200 μL assay buffer, then covered with a foil plate sealer and incubated 10 min at room temperature on a shaker. A volume of 25 μL of standard, wash buffer (served as the blank) or sample and 25 μL microparticles was added to each well and incubated at 4°C overnight. The liquid in each well was removed and wells were washed with 200 μL wash buffer for two times. After wash buffer was removed thoroughly, 25 μL biotin antibody was added to each well and incubated at room temperature for 2 h. The liquid in wells was removed and wells were washed with 200 μL wash buffer for two times again. Afterwards, 25 μL streptavidin-phycoerythrin was added to each well and incubated at room temperature for 30 min followed by twice wash with the Wash Buffer. A volume of 150 μL wash buffer was added to each well to resuspend to microparticles and incubated for 5 min on the shaker. Then, the plate was placed into Luminex 200 to measure median fluorescence intensity of standards and samples.
Immunohistochemistry staining:
Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.
Statistical analysis:
Data were expressed as mean ± standard deviation or median (q25, q75) depending on data distribution. Comparisons of parametric data with normal distribution between two groups were performed by Student t test. Comparisons of no-normality data between two groups were performed by Mann-Whitney U test. Non-parametric data were compared by Chi-square test if the theoretical value was more than 5, otherwise, the Fisher exact test was used. A P < 0.05 was considered statistically significant. Both allele and genotype models (allele model; dominant model; recessive model; homozygote model; heterozygote [model]) were used. Genetic association analyses and odds ratio (OR) calculations were performed for minor alleles based on genotypes using IBM SPSS version 30.0 (IBM Corp., Armonk, NY, USA) and PLINK 1.0.7 (http://pngu.mgh.harvard.edu/purcell/plink), Hardy-Weinberg equilibrium (HWE) determination and Pearson correlation analysis were also performed.
Results:
Clinical characteristics of the subjects A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.
Clinical characteristics for patients with IBS-D and HC.
A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.
Clinical characteristics for patients with IBS-D and HC.
Genotyping of IL-10 gene polymorphisms The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.
Genotyping of IL-10 gene polymorphisms.
Genotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.
IL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.
The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.
Genotyping of IL-10 gene polymorphisms.
Genotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.
IL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.
Measurements for the IL-10 level The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.
Concentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.
IL-10 levels in different genotype of IL-10 polymorphisms.
IL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.
The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.
Concentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.
IL-10 levels in different genotype of IL-10 polymorphisms.
IL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.
Association of IL-10 polymorphisms and clinical symptoms The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.
Association of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.
The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.
Association of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.
Clinical characteristics of the subjects:
A total of 264 participants aged between 18 and 65 years old were enrolled in this study. Based on the Rome III criteria, we confirmed that there were 120 patients with IBS-D (IBS group). Totally 144 healthy volunteers (healthy controls [HC] group) without previous or current gastrointestinal symptoms and infection were recruited during the same time. The clinical characteristics such as the gender, age, and body mass index were similar between the IBS and HC groups [Table 1]. The IBS group exhibited a higher HADS score than the HC group (HC vs. IBS: 6.50 [2.00, 12.00] vs. 7.25 [11.50, 15.00]). The scores for abdominal pain, abdominal bloating, dissatisfaction with bowel habits and life disturbance were significantly higher in the IBS group than those in the HC group (all P < 0.05) [Table 1]. Moreover, the IBS group had a significantly looser stool consistency, based on the Bristol scale, than the HC group (P = 0.001). A total of 78 participants underwent rectal distention test (52 in the IBS group and 26 in the HC group). The IBS group exhibited a significantly lower visceral pain threshold in the initial sensory, initial defecation, and defecation urgency than the HC group (all P < 0.05) [Table 1], indicating that patients with IBS-D were more sensitive in the visceral nociception.
Clinical characteristics for patients with IBS-D and HC.
Genotyping of IL-10 gene polymorphisms:
The genotype distribution of all studied polymorphisms in the HC group were consistent with the HWE (IL-10 rs1800871: P = 0.5400; IL-10 rs1800896: P = 0.500). The detective rate for rs1800871 and rs1800896 was 99.52% and 99.76%, respectively [Table 2]. The genotype of these two SNPs is shown in Figure 1. There was no correlation between rs1800871 and the risk for developing IBS-D in the allele or any other genotype model [Table 3]. The frequency of rs1800896 C allele was significantly lower in the IBS group than that in the HC group (OR: 0.49, 95% confidence interval [CI]: 0.27–0.92, P = 0.024). In genotype analysis, the frequency of rs1800896 CC + CT genotype in the IBS group was significantly lower (OR: 0.51, 95% CI: 0.27–0.99, P = 0.0450) than that in the HC group in the dominant model.
Genotyping of IL-10 gene polymorphisms.
Genotyping of IL-10 rs1800871 (A) and rs1800896 (B). G: guanine; C: cytosine; T: thymine; A: adenine. IL-10: Interleukin 10.
IL-10 gene polymorphisms analysis of IBS-D and HC in allele model and genotype model.
Measurements for the IL-10 level:
The levels of IL-10 in the plasma and the PBMC culture supernatant of 82 subjects in the IBS group and 38 subjects in the HC group were detected using the ELISA. The expression of IL-10 in the intestinal biopsy was detected in 52 subjects of the IBS group and 26 subjects of the HC group. There was no difference in the IL-10 concentration in the plasma, PBMC culture supernatant, and ileum or colon mucosa between the IBS and HC groups [Figure 2]. Then, the IL-10 level was compared among the different genotypes of IL-10 rs1800871 and IL-10 rs1800896. As shown in Table 4, there was no difference in the IL-10 level among the AA, GA, and GG genotypes of IL-10 rs1800871. As for IL-10 rs1800896, the subjects with CT genotype exhibited a significantly higher IL-10 concentration in the plasma (TT vs. CT: 1.37 [0.74, 2.02] vs. 1.82 [1.29, 3.22], P = 0.003) and the PBMC culture supernatant (TT vs. CT: 31.69 [12.09, 76.87] vs. 55.87 [23.65, 104.80], P = 0.050) than the TT genotype subjects [Table 4 and Figure 3]. The expression of IL-10 in the ileum and that in the colon was not statistically different.
Concentrations of IL-10 in plasma (A), PBMC culture supernatant (B) and its expression in ileum (C) and colon (D) mucosa between IBS and HC groups (Scale bar = 100 μm). PBMC: Peripheral blood mononuclear cells; HC: Healthy controls; IBS: Irritable bowel syndrome; IL-10: Interleukin 10.
IL-10 levels in different genotype of IL-10 polymorphisms.
IL-10 levels in plasma (A) and PBMC culture supernatant (B) when comparing IL-10 rs1800896 TT genotype with CT genotype; ∗P ≤ 0.05, Mann-Whitney U test. PBMC: Peripheral blood mononuclear cells; IL-10: Interleukin 10.
Association of IL-10 polymorphisms and clinical symptoms:
The correlation between IL-10 polymorphisms (rs1800871 and rs1800896) and clinical symptoms was evaluated. IL-10 rs1800871 was significantly correlated to the HADS score (R = 0.234, P = 0.023). The subjects with GG genotype had a higher HADS score than the subjects with AA or AG genotype [Figure 4A]. There was a significant positive correlation between IL-10 rs1800896 polymorphism and the pain threshold for initial defecation (R = 0.310, P = 0.007) and the defecation urgency (R = 0.298, P = 0.010). The subjects with CT genotype presented a significantly higher pain threshold of initial defecation (P = 0.007) and defecation urgency (P = 0.010) than the subjects with TT genotype [Figure 4B and 4C]. There was no correlation between the IL-10 polymorphisms and other clinical symptoms.
Association of IL-10 rs1800871 genotype and HADS scores (A); association of IL-10 rs1800896 genotype and initial defecation (B) or defecation urgency threshold (C); ∗P < 0.05. HADS: Hospital anxiety and depression scale; IL-10: Interleukin 10.
Discussion:
Patients with IBS-D present a particularly VH with a looser stool consistency, which may result by chronic systemic and mucosal inflammation. IL-10 plays an important role in the anti-inflammation response and is considered to be a potent suppressor of T lymphocytes or macrophages and their derived effector molecules, such as proinflammatory cytokines (IL-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemotactic protein 1, macrophage inflammatory protein 1α).[18] It is speculated that IL-10 gene polymorphisms might be involved in the IL-10 production.[19] Our study suggested that the carriers with IL-10 rs1800896 C allele have a lower risk for developing IBS-D, which may be associated with higher production of IL-10.
The gene encoding IL-10 is located on the human chromosome 1q31–1q32. The two polymorphisms analyzed in this study were −1082 A/G and −819 C/T, which are located in the gene promoter region. According to the single nucleotide polymorphism database (http://www.ncbi.nlm.nih.gov/SNP/), they are annotated as rs1800896 (T > C) and rs1800871 (A > G), which represents the polymorphism in the complementary strands. Qin et al[13] investigated that the IL-10 rs1800871 polymorphism was associated with a decreased risk for developing IBS in the eastern population. Moreover, other studies have confirmed that the C allele of IL-10 rs1800896 was strongly associated with the decreased risk for developing IBS in the western population.[7] However, there are studies which have reported no correlation between IL-10 polymorphisms and the risk for developing IBS.[12,20] In this study, the frequency of IL-10 rs1800896 C allele was significantly (OR: 0.49, 95% CI: 0.27–0.92, P = 0.0240) lower in the patients with IBS-D and was associated with a decreased risk for developing IBS-D. While the polymorphism rs1800871 presented no association with developing IBS, which investigated in patients with IBS-D and the healthy subjects.
IL-10 was initially described as a T helper 2-type cytokine and was reported to be expressed in various cells of the adaptive immune system as well as the cells of the innate immune system. Patients with IBS exhibit cytokine imbalance characterized by decreased levels of IL-10 and increased levels of TNF-α.[1,7] The lower levels of IL-10 can be a predictor for IBS development.[21] The T allele of IL-10 rs1800896 has been reported to be associated with lower production of IL-10, while the C allele is associated with higher IL-10 production.[16,22] The C allele is associated with IL-10 production in the low stimulated cell culture.[23] In addition, there are very few studies that report a direct correlation between IL-10 polymorphisms and IL-10 production. Our data confirmed that the subjects carrying C allele of IL-10 rs1800896 exhibited a higher IL-10 concentration in the plasma and the PBMC culture supernatant. Additionally, we also demonstrated that the subjects with CT genotype were less sensitive in the rectal distention test than the subjects with TT genotype. Earlier studies demonstrated that IL-10 and other inflammatory cytokine concentration in the PBMC correlated with the severity of symptoms in IBS-D, including the intensity and frequency of painful events and motility-associated symptoms.[24] Some researchers hypothesize that IBS is on a spectrum with inflammation bowel disease (IBD), because they have largely overlapping pathophysiological mechanisms and clinical symptoms.[25,26] For example, the abdominal pain and diarrhea are predominant symptoms for patients with IBS or IBD, and post-inflammatory abnormalities are important in IBD and IBS. The subjects without C allele are more likely to develop IBS after intestinal infection.[3,27] Additionally, the polymorphisms of IL-10 gene can exert a protective effect on patients with IBD, ulcerative colitis or Crohn disease.[28,29]
Our finding was intriguing as the IL-10 rs1800871 polymorphism exhibited a marginally positive correlation with the HADS score. The development of depressive disorder is considered to be associated with the activation of systemic inflammation as well.[30,31] It has been reported that the genotypes IL-10 rs1800871 and IL-10 rs1800896 decrease the risk for developing depression and are correlated with the Hamilton depression rating scale.[32] Globally, patients with IBS have a high comorbidity rate for depression or anxiety.[33] Our study supports the correlation between high comorbidity in IBS and mental disorder in an aspect of genetics polymorphism.
To the best of our knowledge, this study took the lead in studying the correlates of IL-10 gene polymorphisms and the expression level of IL-10. However, this study has some limitations. Only two polymorphisms for IL-10 were analyzed in this study. There may be other SNPs in IL-10 corresponding to rs1800896 and/or rs1800871, which may contribute to IL-10 expression. On the other hand, further studies are required for evaluating the transcriptional and epigenetic effects in IL-10 polymorphism.
In summary, the IL-10 rs1800896 polymorphism has a correlation with a higher concentration of IL-10 in both the plasma and the PBMC culture supernatant of Chinese patients with IBS-D. Additionally, this SNP is also associated with a higher visceral pain threshold. This study demonstrated a correlation between the IL-10 polymorphisms and IL-10 production, which might help in understanding the pathogenesis of IBS-D.
Acknowledgements:
The authors would like to thank all the patients and healthy volunteers. We thank Dr. Han-Yan Yu for technical support who from Medical Research Center of Peking University Third Hospital. And we thank Dr. Yi-Xuan Liu, Hui Wei, and Lu Zhang for their contribution to the study.
Funding:
The studies were supported by grants from the National Natural Science Foundation of China (No. 81670491) and National “Twelfth Five-Year” Plan for Science and Technology of China (No. 2012BAI06B02).
Conflicts of interest:
None.
| Background:
Irritable bowel syndrome (IBS), a functional gastrointestinal disorder, is characterized by cytokine imbalance. Previously, decreased plasma interleukin 10 (IL-10) level was reported in patients with IBS, which may be due to genetic polymorphisms. However, there are no reports correlating the IL-10 polymorphisms with IL-10 production in patients with IBS. This study aimed to analyze the effect of IL-10 polymorphisms on IL-10 production and its correlation with the clinical symptoms in Chinese patients with diarrhea-predominant IBS (IBS-D).
Methods:
Two IL-10 single nucleotide polymorphisms (rs1800871 and rs1800896) were detected in 120 patients with IBS-D and 144 healthy controls (HC) using SNaPshot. IBS symptom severity score, Bristol scale, hospital anxiety, and depressive scale (HADS) were used to evaluate the clinical symptoms, as well as the psychological status and visceral sensitivity of the subjects. IL-10 levels in the plasma and peripheral blood mononuclear cell (PBMC) culture supernatant were measured using enzyme-linked immunosorbent assay, while those in ileal and colonic mucosal biopsies were measured using immunohistochemistry.
Results:
The frequency of rs1800896 C allele was significantly lower in the patients with IBS-D than that in the HC (odds ratio: 0.49, 95% confidence interval: 0.27-0.92, P = 0.0240). The IL-10 levels in the plasma (P = 0.0030) and PBMC culture supernatant (P = 0.0500) of the CT genotype subjects were significantly higher than those in the TT genotype subjects. The CT genotype subjects exhibited a higher pain threshold in the rectal distention test than the TT genotype subjects. Moreover, IL-10 rs1800871 GG genotype subjects showed an increase in the HADS score compared to other genotype subjects.
Conclusions:
IL-10 rs1800896 C allele is correlated with higher IL-10 levels in the plasma and the PBMC culture supernatant, which is associated with a higher pain threshold in the Chinese patients with IBS-D. This study provides an explicit relationship of IL-10 polymorphisms with IL-10 production, which might help in understanding the pathogenesis of IBS-D. | null | null | 10,183 | 396 | [
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] | null | null | [CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY] | [CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY] | [CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY] | null | [CONTENT] Irritable bowel syndrome | Interleukin 10 | Polymorphisms | Visceral hypersensitivity | Depression [SUMMARY] | null | [CONTENT] Adolescent | Adult | Aged | Diarrhea | Genotype | Humans | Interleukin-10 | Irritable Bowel Syndrome | Middle Aged | Peripheral Blood Stem Cells | Polymorphism, Single Nucleotide | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Diarrhea | Genotype | Humans | Interleukin-10 | Irritable Bowel Syndrome | Middle Aged | Peripheral Blood Stem Cells | Polymorphism, Single Nucleotide | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Aged | Diarrhea | Genotype | Humans | Interleukin-10 | Irritable Bowel Syndrome | Middle Aged | Peripheral Blood Stem Cells | Polymorphism, Single Nucleotide | Young Adult [SUMMARY] | null | [CONTENT] Adolescent | Adult | Aged | Diarrhea | Genotype | Humans | Interleukin-10 | Irritable Bowel Syndrome | Middle Aged | Peripheral Blood Stem Cells | Polymorphism, Single Nucleotide | Young Adult [SUMMARY] | null | [CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY] | [CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY] | [CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY] | null | [CONTENT] ibs symptom severity | activation patients ibs | ibs pathophysiological mechanisms | ibs spectrum inflammation | ibs exhibit cytokine [SUMMARY] | null | [CONTENT] 10 | il | il 10 | ibs | group | genotype | μl | hc | rs1800896 | rs1800871 [SUMMARY] | [CONTENT] 10 | il | il 10 | ibs | group | genotype | μl | hc | rs1800896 | rs1800871 [SUMMARY] | [CONTENT] 10 | il | il 10 | ibs | group | genotype | μl | hc | rs1800896 | rs1800871 [SUMMARY] | null | [CONTENT] 10 | il | il 10 | ibs | group | genotype | μl | hc | rs1800896 | rs1800871 [SUMMARY] | null | [CONTENT] ibs | il | 10 | il 10 | patients ibs | patients | cytokine | production | studies | polymorphisms [SUMMARY] | [CONTENT] μl | 10 | 30 | min | buffer | dna | wash | usa | snapshot | incubated [SUMMARY] | [CONTENT] 10 | il 10 | il | group | hc | ibs | genotype | hc group | ibs group | subjects [SUMMARY] | null | [CONTENT] 10 | il | il 10 | ibs | group | μl | hc | genotype | rs1800896 | hc group [SUMMARY] | null | [CONTENT] ||| 10 | IBS ||| IL-10 | IBS ||| IL-10 | IL-10 | Chinese [SUMMARY] | [CONTENT] Two | IL-10 | rs1800871 | rs1800896 | 120 | 144 ||| Bristol | HADS ||| [SUMMARY] | [CONTENT] HC | 0.49 | 95% | 0.27 | 0.0240 ||| IL-10 | 0.0030 | 0.0500 | CT | TT ||| CT | TT ||| IL-10 | rs1800871 GG | HADS [SUMMARY] | null | [CONTENT] ||| 10 | IBS ||| IL-10 | IBS ||| IL-10 | IL-10 | Chinese ||| Two | IL-10 | rs1800871 | rs1800896 | 120 | 144 ||| Bristol | HADS ||| ||| HC | 0.49 | 95% | 0.27 | 0.0240 ||| IL-10 | 0.0030 | 0.0500 | CT | TT ||| CT | TT ||| IL-10 | rs1800871 GG | HADS ||| Chinese | IL-10 [SUMMARY] | null |
||||
Prevalence and pattern of sexual assault in Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria. | 28154687 | Sexual violence is an important public health problem of growing concern all over the world. This study was conducted to determine the prevalence and pattern of sexual assault managed in Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria. | INTRODUCTION | It was a retrospective study that looked into cases of sexual assault admitted into the hospital between January 2010 and December 2014. Information on patients' biodata, and relevant details on the cases were extracted from the patients' case files and analyzed. | METHODS | Out of the 5317 gynecological admissions during the period under study, 45 (0.84%) were cases of sexual assault. Of these, only 34 case files were available for data extraction. The patients' ages ranged from 2 to 37 years (mean = 12.6 + 8.3). About two thirds (61.8%) of those affected were young children (aged 12 years and below). In majority of cases (70.6%) the assault was penetrative, and in most of the cases (91.2%) only a single assailant was involved. In close to two thirds of cases, the assailant was either an acquaintance (38.2%) or a family member (20.6%). Although law enforcement agents were informed in majority (58.8%) of cases, arrests were made in less than half (41.2%). | RESULTS | Although the prevalence of sexual assault in this study appears to be low, a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children's movement, and stringent laws should be enacted and enforced to curb this heinous act. | CONCLUSION | [
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"Young Adult"
] | 5267856 | Introduction | Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].
In the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area. | Methods | This was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital. | Results | Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.
Socio-demographic profile of patients
There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.
Socio-demographic profile of patients
Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.
Place and type of assault
Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.
Place and type of assault
Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.
The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.
Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).
Mode of presentation and type of injury sustained
Time interval between occurrence of assault and presentation
The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).
Mode of presentation and type of injury sustained
Time interval between occurrence of assault and presentation
Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).
Use of weapons in assault
No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).
Use of weapons in assault
Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).
Profile of assailants
In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).
Profile of assailants
Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).
Involvement of law enforcement agents
Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).
Involvement of law enforcement agents | Conclusion | Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.
What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.
Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half. | [
"Prevalence of sexual assault and socio-demographic profile of patients",
"Place and type of assault",
"Mode of presentation and type of injury sustained",
"Time interval between occurrence of assault and presentation",
"Use of weapon in assault",
"Profile of assailants",
"Involvement of law enforcement agents",
"What is known about this topic",
"What this study adds"
] | [
"There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients",
"Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault",
"The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.",
"The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation",
"No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault",
"In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants",
"Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents",
"Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.",
"Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Introduction",
"Methods",
"Results",
"Prevalence of sexual assault and socio-demographic profile of patients",
"Place and type of assault",
"Mode of presentation and type of injury sustained",
"Time interval between occurrence of assault and presentation",
"Use of weapon in assault",
"Profile of assailants",
"Involvement of law enforcement agents",
"Discussion",
"Conclusion",
"What is known about this topic",
"What this study adds"
] | [
"Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].\nIn the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.",
"This was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital.",
" Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\nThere were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients\n Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\nMajority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault\n Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\nThe most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.\n Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\nThe time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation\n Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\nNo weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault\n Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\nIn majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants\n Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents\nAlthough reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents",
"There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.\nSocio-demographic profile of patients",
"Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.\nPlace and type of assault",
"The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.",
"The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).\nMode of presentation and type of injury sustained\nTime interval between occurrence of assault and presentation",
"No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).\nUse of weapons in assault",
"In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).\nProfile of assailants",
"Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).\nInvolvement of law enforcement agents",
"Although the prevalence of sexual assault was relatively low (0.84%) in this study, it confirms the existence of the phenomenon in Sokoto, Nigeria. This figure probably represents the tip of the iceberg in terms of its burden in the study area, if the low prevalence of sexual assault reported in health facilities based studies [15, 18, 21] is juxtaposed with the very high prevalence reported in community based studies [14, 16, 17] across Nigeria. It is important to note that the prevalence of sexual assault recorded in this study is much higher than that obtained in a study in Zaria, also located in north western Nigeria that reported a prevalence of 0.06% [21]. While studies conducted in Lagos and Ile-lfe that reported prevalence of 0.76% [15] and 0.69% [8] respectively, compare well with the finding in this study, other health facilities based studies by Ashimi et al, in a rural tertiary health facility in north western Nigeria [11] and Daru et al, in Jos [28] reported higher prevalence of 3.0% and 5.6% respectively. These findings show that sexual assault is incontrovertibly a problem in Sokoto, just like other parts of Nigeria. About two-thirds (61.8%) of the victims in this study were young children aged 12 years and below. This is in agreement with the findings in studies by Bhattacharyya et al [29] and Lakew [30] in which teenagers accounted for majority of cases. The assault took place in either the victims or assailants homes in more than half of cases (54%), and in two-thirds of cases the assailants were either acquaintances (38.2%) or family members (20.6%). These findings are in concordance with the findings in studies conducted in Benin [31] and Osogbo [19]. This is not surprising considering the vulnerability of the age group involved. Most of the victims were children, who were possibly left in the care of these family members or neighbors in the absence of their parents.\nVaginal bleeding due to laceration was the commonest mode of presentation seen in this study (29.4%). This is similar to the finding in a study conducted in Birnin-Kudu, North-West Nigeria, where vaginal laceration constituted 24% of cases [11]. A possible explanation for these findings is that the two studies were conducted within the same geopolitical zone of Nigeria, perhaps with similar socio-cultural factors. However, in contrast to the finding in this study where only a fifth (20.5%) of victims presented with no demonstrable injury, majority (70.8%) of the cases in the Birni-Kudu study had no demonstrable injury. Majority of the victims in this study (61.7%) presented within 8 to 16 hours of the assault. This is in contrast to the findings from Lagos [15] and Benin City [31], where 64.5% and 47% respectively presented late. Only a few (14.7%) victims were assaulted with weapons in this study, this could be due to the fact that they were mostly young children, and were less likely to struggle with the assailants to warrant the use of weapons. Also, majority of the victims (91.2%) were assaulted by only one assailant, who was also known to them in majority of cases. This is similar to the finding in other studies [8, 32] that reported that majority of victims were assaulted by person(s) known to them. One of the most interesting findings of this study is the fact that majority of cases (58.8%) were reported to law enforcement agents, even though arrests were made in less than half (41.2%) of cases. This may not be unconnected with the fact that most of the victims were young children and their parents could have taken the decision to report the incident on their behalf, unlike adult victims that would have concealed the incident and suffer in silence due to fear of stigmatization, rejection by the society and safety concerns. This finding agrees with the findings in other studies [20, 21] that reported higher likelihood of arrests by law enforcement agents in juvenile assaults.",
"Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.\n What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\nSexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.\n What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.\nSexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.",
"Sexual assault is prevalent across Nigeria, similar to the situation across the world;\nMajority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;\nMajority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.",
"Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;\nVictims were predominantly young children aged 12 years and below;\nWhereas, law enforcement agents were informed in most of the cases, arrests were made in less than half."
] | [
"intro",
"methods",
"results",
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null,
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"discussion",
"conclusion",
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] | [
"Prevalence",
"pattern",
"sexual assault",
"UDUTH",
"Sokoto"
] | Introduction:
Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].
In the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.
Methods:
This was a retrospective study based on data extracted from the case files of patients seen at the Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, North-western Nigeria, between January 2012 and December 2014. The hospital is a referral centre that caters for Sokoto, Zamfara and Kebbi states. The hospital offers preventive, promotive and curative services. Case files of all patients presenting with a history of sexual assault were reviewed. A designed proforma was used to extract information on patient’s bio data, type of assault, mode of presentation, type(s) of injury sustained, weapon(s) of assault, relationship of offender to the victim, and involvement of law enforcement agents. Data cleansing was done, and then analyzed using IBM SPSS version 20 statistical software package, and the results were presented as descriptive and inferential statistics. All levels of statistical significance were set at p < 0.05. Ethical approval was obtained from the Ethical Committee of the hospital.
Results:
Prevalence of sexual assault and socio-demographic profile of patients There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.
Socio-demographic profile of patients
There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.
Socio-demographic profile of patients
Place and type of assault Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.
Place and type of assault
Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.
Place and type of assault
Mode of presentation and type of injury sustained The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.
The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.
Time interval between occurrence of assault and presentation The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).
Mode of presentation and type of injury sustained
Time interval between occurrence of assault and presentation
The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).
Mode of presentation and type of injury sustained
Time interval between occurrence of assault and presentation
Use of weapon in assault No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).
Use of weapons in assault
No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).
Use of weapons in assault
Profile of assailants In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).
Profile of assailants
In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).
Profile of assailants
Involvement of law enforcement agents Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).
Involvement of law enforcement agents
Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).
Involvement of law enforcement agents
Prevalence of sexual assault and socio-demographic profile of patients:
There were 5317 gynecological cases seen during the period under review out of which 45 were cases of sexual assault, giving a prevalence of 0.84%. Out of the 45 cases of sexual assault seen at the hospital in the period under review, only 34 case files were available for data extraction. Almost all, 33 (97.1%) of the 34 patients were females. The patients’ ages ranged from 2 to 37 years (Mean = 12.6 ± 8.3). A larger proportion of victims (41.2%) aged 5 to 12 years; and about a fifth of victims (20.6%) were preschool children aged 4 years and below. Young children aged 12 years and below therefore accounted for majority (61.8%) of victims as shown in Table 1.
Socio-demographic profile of patients
Place and type of assault:
Majority of the assaults took place either in the homes (38.2%) or neighborhood (14.7%) of the victims or assaulters. Four (11.8%) of the 34 victims were assaulted in the class room. There was penetration of the vagina in majority, 24 (70.6%) of the 34 patients (mostly penile or fingering by the assailants) as shown in Table 2. Vaginal penetration was significantly more prevalent among victims in the 5-12 years age group (92.9%), while fondling was more prevalent among those aged 4 years and below (85.7%), Fisher’s Exact χ2 = 12.942, p = 0.002.
Place and type of assault
Mode of presentation and type of injury sustained:
The most common mode of presentation was vaginal bleeding (50.0%), while the most common injury sustained was vaginal laceration (some of which necessitated examination under anesthesia and suturing of the laceration in order to arrest the bleeding). One death was recorded (a 9 year old girl brought in dead with her throat cut, and with bleeding from major vessels). Other modes of presentation and types of injury sustained are as shown in Table 3.
Time interval between occurrence of assault and presentation:
The time interval between occurrence of assault and presentation at the hospital ranged from less than 1 hour to 168 hours (median = 11 hours). Although, majority of the victims (44.1%) presented at the hospital within 8 hours following the assault, about a fifth (20.9%) of victims presented more than 24 hours after the assault (Table 4).
Mode of presentation and type of injury sustained
Time interval between occurrence of assault and presentation
Use of weapon in assault:
No weapon was used to subdue the victims by the assailants in most, 15 (44.1%) of the 34 cases seen. Fourteen (41.1%) were threatened verbally, while weapons were used in the remaining 5 (14.7%) cases, of which knife was used in 4 and gun was used in the remaining one (Table 5). Although no weapon was used in cases of victims aged 4 years and below, there was no significant association between age of victim and use of weapon (Fisher's Exact χ2 = 13.618, p = 0.623).
Use of weapons in assault
Profile of assailants:
In majority, 31 (91.2%) of the 34 cases of assault, only 1 assailant was involved. In more than half, 20 (58.8%) of cases the assaulters were known to the victim (either acquaintance or family member). The occupation of most of the assailants was not known in most cases (Table 6).
Profile of assailants
Involvement of law enforcement agents:
Although reports were made to the law enforcement agents in majority 20 (58.8%) of the 34 cases, the assailants were arrested in less than half 14 (41.7%) of the cases (Table 7).
Involvement of law enforcement agents
Discussion:
Although the prevalence of sexual assault was relatively low (0.84%) in this study, it confirms the existence of the phenomenon in Sokoto, Nigeria. This figure probably represents the tip of the iceberg in terms of its burden in the study area, if the low prevalence of sexual assault reported in health facilities based studies [15, 18, 21] is juxtaposed with the very high prevalence reported in community based studies [14, 16, 17] across Nigeria. It is important to note that the prevalence of sexual assault recorded in this study is much higher than that obtained in a study in Zaria, also located in north western Nigeria that reported a prevalence of 0.06% [21]. While studies conducted in Lagos and Ile-lfe that reported prevalence of 0.76% [15] and 0.69% [8] respectively, compare well with the finding in this study, other health facilities based studies by Ashimi et al, in a rural tertiary health facility in north western Nigeria [11] and Daru et al, in Jos [28] reported higher prevalence of 3.0% and 5.6% respectively. These findings show that sexual assault is incontrovertibly a problem in Sokoto, just like other parts of Nigeria. About two-thirds (61.8%) of the victims in this study were young children aged 12 years and below. This is in agreement with the findings in studies by Bhattacharyya et al [29] and Lakew [30] in which teenagers accounted for majority of cases. The assault took place in either the victims or assailants homes in more than half of cases (54%), and in two-thirds of cases the assailants were either acquaintances (38.2%) or family members (20.6%). These findings are in concordance with the findings in studies conducted in Benin [31] and Osogbo [19]. This is not surprising considering the vulnerability of the age group involved. Most of the victims were children, who were possibly left in the care of these family members or neighbors in the absence of their parents.
Vaginal bleeding due to laceration was the commonest mode of presentation seen in this study (29.4%). This is similar to the finding in a study conducted in Birnin-Kudu, North-West Nigeria, where vaginal laceration constituted 24% of cases [11]. A possible explanation for these findings is that the two studies were conducted within the same geopolitical zone of Nigeria, perhaps with similar socio-cultural factors. However, in contrast to the finding in this study where only a fifth (20.5%) of victims presented with no demonstrable injury, majority (70.8%) of the cases in the Birni-Kudu study had no demonstrable injury. Majority of the victims in this study (61.7%) presented within 8 to 16 hours of the assault. This is in contrast to the findings from Lagos [15] and Benin City [31], where 64.5% and 47% respectively presented late. Only a few (14.7%) victims were assaulted with weapons in this study, this could be due to the fact that they were mostly young children, and were less likely to struggle with the assailants to warrant the use of weapons. Also, majority of the victims (91.2%) were assaulted by only one assailant, who was also known to them in majority of cases. This is similar to the finding in other studies [8, 32] that reported that majority of victims were assaulted by person(s) known to them. One of the most interesting findings of this study is the fact that majority of cases (58.8%) were reported to law enforcement agents, even though arrests were made in less than half (41.2%) of cases. This may not be unconnected with the fact that most of the victims were young children and their parents could have taken the decision to report the incident on their behalf, unlike adult victims that would have concealed the incident and suffer in silence due to fear of stigmatization, rejection by the society and safety concerns. This finding agrees with the findings in other studies [20, 21] that reported higher likelihood of arrests by law enforcement agents in juvenile assaults.
Conclusion:
Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.
What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.
Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.
What is known about this topic:
Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
What this study adds:
Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.
| Background:
Sexual violence is an important public health problem of growing concern all over the world. This study was conducted to determine the prevalence and pattern of sexual assault managed in Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria.
Methods:
It was a retrospective study that looked into cases of sexual assault admitted into the hospital between January 2010 and December 2014. Information on patients' biodata, and relevant details on the cases were extracted from the patients' case files and analyzed.
Results:
Out of the 5317 gynecological admissions during the period under study, 45 (0.84%) were cases of sexual assault. Of these, only 34 case files were available for data extraction. The patients' ages ranged from 2 to 37 years (mean = 12.6 + 8.3). About two thirds (61.8%) of those affected were young children (aged 12 years and below). In majority of cases (70.6%) the assault was penetrative, and in most of the cases (91.2%) only a single assailant was involved. In close to two thirds of cases, the assailant was either an acquaintance (38.2%) or a family member (20.6%). Although law enforcement agents were informed in majority (58.8%) of cases, arrests were made in less than half (41.2%).
Conclusions:
Although the prevalence of sexual assault in this study appears to be low, a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children's movement, and stringent laws should be enacted and enforced to curb this heinous act. | Introduction:
Sexual violence is an important public health problem of growing concern all over the world. Sexual abuse is an unwanted sexual activity in which the perpetrator uses force, threats or takes advantage of victim not able to give consent. When the force used is immediate, of short duration or infrequent it is termed as sexual assault. The offender is referred to as sexual abuser, or molester [1]. The term also covers any behavior by an adult towards a child to stimulate either the adult or the child sexually. When the victim is younger than age of consent, it is referred to as child sexual abuse. Both sexes are affected and most victims and perpetrators know each other. The assailants usually range from family members to acquaintances to strangers [2–5]. Sexual abuse is associated with significant morbidity and mortality. Various types of injuries are seen as a result of physical force such as severe multiple bruises in uncommon sites, vaginal and anal lacerations and perforations have been reported [6–10]. The victim is also exposed to sexually-transmitted disease, psychological trauma and risk of unwanted pregnancy [11, 12]. Although the trauma of the assault heals with time, it leaves long term psychological and medical problems behind [13]. Younger juvenile victims are more likely than older victims to be sexually abused [14, 15]. The abuse may involve use of objects, forcible fondling, and forcible sodomy. Forcible rape has been shown to be more likely to involve a single victim than any other sexual assault. Personal weapons such as hands, fist, and legs are commonly used during rape however other weapons such as knife and gun have been noted to be used. Records have shown that in some cases no weapon was used (only verbal threats), but the use of weapons is more likely when the victim is older [16, 17]. The time of the day when sexual abuse occurs is related primarily to the age of the victim. For adult victims sexual assault is more likely to occur after midnight while the pattern in juvenile assault is said to be earlier in the day (during or after school hours) [18, 19]. Nearly all the offenders reported to the law enforcement agents were males. However, female molesters commonly assault children under the age of 6 years. Juvenile assaults were more likely to result in arrests by law enforcement agents [20, 21].
In the United States, nearly 1 in 5 (18.3%) women and 1 in 75 (1.4%) men reported experiencing rape at some time in their lives [22]. Similar findings were obtained in studies across Africa. In a study in the Democratic Republic of Congo, 16% of women reported having sex against their will [23]. In another study among female university students in Ethiopia, 14.3% reported having experienced completed rape since being admitted into the university [24]. Also, in a study in South Africa, 24.9% of young men reported having raped a female previously [25]. In Nigeria, whereas relatively low prevalence of sexual assault was obtained in most of the health facility based studies ranging from 0.06% in Zaria [21], 0.76% in Lagos [15], 2.1% in Calabar [18], to 5.2% of all gynecological emergencies in Ile-Ife [8], a very high prevalence of sexual assuault was reported in community based studies across the country ranging from 14% among out of school adolescents in an urban slum in Lagos [16], 69.9% among juvenile female street hawkers from two urban towns in Anambra state [17] to 78.5% among employed girls in Maiduguri [14]. Despite the legal provisions of life jail with or without canning for sexual assaulters in Nigeria [26], the high prevalence of sexual assault in community based studies across the country may not be unconnected with the fact that most cases of rape are unreported by the victims out of fear of stigmatization, rejection by the society, and safety concerns, coupled with the fact that even for cases that are reported, the perpetrators are rarely prosecuted. Other forces believed to be responsible for the high prevalence of sexual assault in Nigerian include the enduring culture of male dominance, female social and economic disempowerment, and lack of synergy in civil society initiatives [27]. A study like this has never been conducted in Sokoto, the prevalence of sexual assault in Sokoto is therefore unknown despite the fact that sexual assaults exist in this locality. This study was therefore conducted to determine the prevalence and pattern of sexual assault seen at the Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, with a view to making recommendations towards addressing the problem in the study area.
Conclusion:
Although the prevalence of sexual assault in this study appears to be low, it confirms the existence of the phenomenon in Sokoto, Nigeria, and a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children’s movements, and stringent laws should be enacted and enforced to curb this heinous act.
What is known about this topic Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
Sexual assault is prevalent across Nigeria, similar to the situation across the world;
Majority of victims are often aged less than 18 years and the assaults are mostly perpetrated by persons known to them;
Majority of cases go unreported due to fear of stigmatization, rejection by the society and safety concerns.
What this study adds Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half.
Sexual assault exists in Sokoto, Nigeria, even though the prevalence appears to be low;
Victims were predominantly young children aged 12 years and below;
Whereas, law enforcement agents were informed in most of the cases, arrests were made in less than half. | Background:
Sexual violence is an important public health problem of growing concern all over the world. This study was conducted to determine the prevalence and pattern of sexual assault managed in Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria.
Methods:
It was a retrospective study that looked into cases of sexual assault admitted into the hospital between January 2010 and December 2014. Information on patients' biodata, and relevant details on the cases were extracted from the patients' case files and analyzed.
Results:
Out of the 5317 gynecological admissions during the period under study, 45 (0.84%) were cases of sexual assault. Of these, only 34 case files were available for data extraction. The patients' ages ranged from 2 to 37 years (mean = 12.6 + 8.3). About two thirds (61.8%) of those affected were young children (aged 12 years and below). In majority of cases (70.6%) the assault was penetrative, and in most of the cases (91.2%) only a single assailant was involved. In close to two thirds of cases, the assailant was either an acquaintance (38.2%) or a family member (20.6%). Although law enforcement agents were informed in majority (58.8%) of cases, arrests were made in less than half (41.2%).
Conclusions:
Although the prevalence of sexual assault in this study appears to be low, a major cause for concern is the fact that those affected were predominantly young children. Parents should be more vigilant in monitoring their children's movement, and stringent laws should be enacted and enforced to curb this heinous act. | 4,541 | 316 | [
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] | [CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY] | [CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY] | [CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY] | [CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY] | [CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY] | [CONTENT] Prevalence | pattern | sexual assault | UDUTH | Sokoto [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | Age Distribution | Child | Child Abuse, Sexual | Child, Preschool | Crime Victims | Female | Hospitalization | Hospitals, University | Humans | Male | Nigeria | Prevalence | Rape | Retrospective Studies | Sex Offenses | Young Adult [SUMMARY] | [CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY] | [CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY] | [CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY] | [CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY] | [CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY] | [CONTENT] victim sexual assault | concerns sexual assault | sexual assault study | sexual abuse occurs | child sexual abuse [SUMMARY] | [CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY] | [CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY] | [CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY] | [CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY] | [CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY] | [CONTENT] assault | victims | cases | sexual | majority | years | sexual assault | study | aged | prevalence [SUMMARY] | [CONTENT] sexual | reported | abuse | female | rape | likely | study | assault | sexual assault | sexual abuse [SUMMARY] | [CONTENT] hospital | data | files patients | statistical | ethical | case files patients | case files | case | files | patients [SUMMARY] | [CONTENT] cases | victims | assault | 34 | presentation | table | years | patients | assailants | majority [SUMMARY] | [CONTENT] nigeria | predominantly young | appears low | appears | predominantly young children | predominantly | sexual assault | sexual | children | sokoto nigeria [SUMMARY] | [CONTENT] cases | victims | assault | majority | sexual | years | 34 | sexual assault | aged | table [SUMMARY] | [CONTENT] cases | victims | assault | majority | sexual | years | 34 | sexual assault | aged | table [SUMMARY] | [CONTENT] ||| Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria [SUMMARY] | [CONTENT] between January 2010 and December 2014 ||| [SUMMARY] | [CONTENT] 5317 | 45 | 0.84% ||| only 34 ||| 2 to 37 years | 12.6 + 8.3 ||| About two thirds | 61.8% | 12 years ||| 70.6% | 91.2% ||| close to two thirds | 38.2% | 20.6% ||| 58.8% | less than half | 41.2% [SUMMARY] | [CONTENT] ||| [SUMMARY] | [CONTENT] ||| Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria ||| between January 2010 and December 2014 ||| ||| 5317 | 45 | 0.84% ||| only 34 ||| 2 to 37 years | 12.6 + 8.3 ||| About two thirds | 61.8% | 12 years ||| 70.6% | 91.2% ||| close to two thirds | 38.2% | 20.6% ||| 58.8% | less than half | 41.2% ||| ||| [SUMMARY] | [CONTENT] ||| Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria ||| between January 2010 and December 2014 ||| ||| 5317 | 45 | 0.84% ||| only 34 ||| 2 to 37 years | 12.6 + 8.3 ||| About two thirds | 61.8% | 12 years ||| 70.6% | 91.2% ||| close to two thirds | 38.2% | 20.6% ||| 58.8% | less than half | 41.2% ||| ||| [SUMMARY] |
||||||
Association between Epstein-Barr Virus Gene Polymorphism and Breast Cancer Risk among Egyptian Females. | 35225477 | Epstein-Barr virus (EBV) has been implicated in the development of breast cancer (BC) since 1995. It is classified into A/B genotypes, C/D subtypes, and F/f variants according to variations in its genome. | BACKGROUND | Three hundred and sixty-two participants (142 BC patients and 220 controls) were enrolled in this study. All participants were screened for EBV infection by determination of viral-capsid-IgG antibodies in their sera. EBNA-1 gene was detected by PCR in tumor biopsies of seropositive patients and in peripheral blood mononuclear cells of controls. A/B genotyping of EBV was performed by nested-PCR targeting the EBNA-2 gene. C/D subtypes and F/f variants were identified by Restriction fragment length polymorphism at BamHI-I W1/I1 and BamHI-F regions of EBV genome, respectively. | METHODS | Among 362 participants, 300(82.9%) were EBV-seropositive, including 120/142(84.5%) of the BC patients and 180/220(81.8 %) of the controls. EBNA-1 gene was positive in 54(45%) of seropositive BC patients and in 38(21.1%) of seropositive controls. There was a significant association of EBNA-1 gene with breast cancer (OR=3.05, 95%CI=1.84-5.07). Moreover, EBNA-1 gene positivity was significantly associated with the more aggressive tumors. Genotype-A and prototype-F were predominant among patients (90.4%, 100%, respectively) as well as among controls (91.7%, 100%, respectively) with no statistical significant association with BC risk. However, subtype-D was significantly more frequent in patients (95.6%) than in controls (64.7%) and was significantly associated with a higher BC risk as compared to subtype-C (OR=11.7, 95%CI=2.4-57.08). Subtype-D was significantly associated with higher grades tumors (100% among grade III), with progesteron receptor-negative tumors and with HER2-positive tumors (100% for each). The combined genotypes that significantly associated with BC risk were ADF (OR=4.9) and BDF (OR=5.5). | RESULTS | Subtype-D of EBV could be the only EBV type implicated in BC development among Egyptian females and associated more with poor prognosis. | CONCLUSIONS | [
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] | 9272632 | Introduction | Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).
EBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).
The presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).
EBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015).
EBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007).
Diagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).
Breast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015).
In 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).
To date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC. | null | null | Results |
Detection of anti-VCA IgG of EBV by ELISA
Screening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.
Detection of EBV DNA by PCR
EBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.
Genotyping of EBV
Analysis of A/B types of EBV
Successful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.
Analysis of C/D subtypes of EBV
Amplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.
Analysis of F/f variants of EBV
The BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.
Combined Genotypes of EBV
A comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.
Association between EBV DNA positivity and tumor characteristics
Among 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4.
Association between EBV genotypes and tumor characteristics
A/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.
Target Genes and Their Primer Sequence
bp, base pair; F, forward; R, reverse.
Frequency of EBV Infection in Breast Cancer Patients and Controls
EBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer.
Flowchart Indicating the Design and Results of the Present Study
EBV Typing in Breast Cancer Patients and Controls
EBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions
Association between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients
*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2
Association between Specific EBV Genotypes and Tumor Features
IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth | null | null | [
"Author Contribution Statement"
] | [
"Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript."
] | [
null
] | [
"Introduction",
"Materials and Methods",
"Results",
"Discussion",
"Author Contribution Statement"
] | [
"Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).\nEBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).\nThe presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).\nEBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015). \nEBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007). \nDiagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).\nBreast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015). \nIn 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).\nTo date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC.",
"\nStudy subjects \n\nThis case-control study included 362 female participants; 142 breast cancer (BC) patients and 220 healthy controls. All patients and controls were recruited from the Oncology Center, Faculty of Medicine, Mansoura University (OCMU) in Egypt through the period from January 2019 to December 2020. The ages of all patients and controls were matched (p >0.05). Patients’ ages ranged from 37 to 79 years old with a median age of 54 years. The diagnosis of BC in all enrolled patients was based on clinical examination, radiological findings, and histopathological examination of tissue biopsy. Control subjects were selected from women who attended OCMU for annual mammography as part of a breast checkup and we excluded those with a history of BC and those with fibroadenomas.\n\nSample collection\n\nFresh tumor tissue and blood specimen were obtained from BC patients, whereas only blood specimen was obtained from the control group. The tissue sample from each tumor was divided into two parts; one part was fixed in formaldehyde and delivered to OCMU’s pathology laboratory for histopathological examination, while the other part was stored at -80°C to be used for PCR. The blood specimen was obtained via venipuncture and divided into two tubes; a plain tube for separation of serum and an EDTA tube for separation of peripheral blood mononuclear cells (PBMCs), both of which were kept at –80°C until use.\n\nStudy design \n\nA Flowchart showing the workflow of this study is presented in Figure 1. All included BC patients and controls were screened for EBV infection by the detection of anti-viral capsid antigen (VCA) IgG antibodies in their sera. Then EBV seropositive subjects were investigated for the presence of EBV DNA by PCR amplification of the EBNA-1 gene; either in tumor biopsy of BC patients or in PBMCs of controls. Thereafter, A/B genotypes, C/D subtypes and F/f variants of EBV were identified in all EBV-DNA positive specimens by analysis of three regions in the EBV genome (EBNA-2 gene, BamHI-I W1/I1 and BamHI-F regions, respectively).\n\nHistopathological examination \n\nDiagnosis of BC and determination of its histological type was achieved in OCMU’s pathology laboratory by examination of hematoxylin-eosin (H-E) stained slides prepared from tumor biopsies. Only tumor biopsies with cancerous cells of more than 60% were included in the study. Grading of the tumors was performed according to the Scarff Bloom Richardson classification (Bloom and Richardson, 1957). Detection of hormonal receptor status [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)] of all included tumor biopsies was done by immunohistochemical staining of slides cut from tissue microarray prepared blocks as described previously (Ahmed and Yussif, 2016).\n\nDetection of EBV-viral capsid antigen (VCA) IgG antibodies\n\nScreening of all included BC patients and controls for EBV infection was performed by detection of anti-VCA IgG antibodies by ELISA (Bio-Rad Medical Diagnostics, GmbH, Germany) following the manufacturer guidelines.\n\nPolymerase chain reaction (PCR) amplification of EBNA-1 gene\n\nDetection of EBV DNA was performed by PCR amplification of EBNA-1 gene. DNA was extracted from all specimens with serologically confirmed EBV infection (120 tumor biopsies from BC patients and 180 PMNCs from controls) using the QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s tissue and blood protocols. Then EBV DNA was detected by PCR amplification of the EBNA-1 gene using specific primers listed in Table 1 according to the previously described PCR protocol (Hashimoto et al., 1995).\n\nDetection of A/B genotypes of EBV \n\nNested PCR that targets EBNA-2 gene was used for genotyping of EBV into genotype-A and genotype-B (Ayee et al., 2020). In the first round of PCR amplification, a common region of EBNA-2 was amplified using EBNA-2A and EBNA-2B as forward and reverse primers, respectively. A PCR reaction was performed in a total volume of 12.5ul and the final concentrations of each component were as follows; 1X One TaqR Quick-Load 2X master mix with standard buffer (Bio Labs, UK), 0.5µM from each of the forward and reverse primers, and 0.5µg/µL of the extracted DNA. Amplification was achieved in a thermal cycler under the following conditions; 2min at 94°C for initial denaturation, then followed by 35 cycles of amplification (each cycle consisted of 60s at 94°C, 90s at 52°C and 4min at 72°C), followed by a cycle of 10min at 72°C for final extension.\nIn the 2nd round of nested PCR, the reaction volume was 12.5ul and the components were the same as in the 1st round except that 0.5ul of the amplified product from the 1st round was used as a template and three primers were used; EBNA-2C as a forward primer (common to genotype-A and genotype-B), EBNA-2D and EBNA-2E as reverse primers specific for genotype-A and genotype-B, respectively. The cycling conditions were as follows: An initial cycle of heat denaturation for 2min at 94°C, 35 amplification cycles (30s at 94°C, 60s at 52°C, and 2 min at 72°C), and a final extension cycle for 10min at 72°C. The resultant amplicon was visualized by electrophoresis on an agarose gel with 2% ethidium bromide. Samples were identified by the presence of their respective bands (250 bp for genotype-A and 300 bp for genotype-B).\n\nDetection of C/D subtypes and F/f variants of EBV \n\nRestriction fragment length polymorphism (RFLP) was used for classification of the EBV into C/D subtypes and F/f variants according to the existence of an extra restriction enzyme site at the BamHI-I W1/I1 and BamHI-F regions of the EBV genome, respectively. PCR was performed, followed by RFLP as previously described (Henry et al., 2001).\nIn brief, the reaction conditions for PCR amplification of each region were similar except for the use of forward and reverse primers specific for each region. The total reaction volume was 25ul consisting of 22.0µl of PCR master mix (1x), 1µl of each forward and reverse primer (0.5μM), and 1µl of DNA template. PCR cycles included an initial step of denaturation at 94°C for 5min followed by 40 amplification cycles; denaturation at 94ºC for 30s, annealing at 55°C for 30s and extension at 72°C for 1min , thereafter a step of final extension for 10min at 72°C. After PCR, the enzymatic digestion of the amplified product was performed in a total reaction volume of 20ul that contained 10µl of the amplified products, 2µl of reaction buffer, 1.5ul of distilled water, and 1.5µl of BamHI endonucleases (10U/ul) (Promega, Madison). This mixture was incubated at 37ºC for 2hs then followed by agarose gel (2%) electrophoresis of enzyme-digested BamHI-I W1/I1 region and BamHI-F region for determination of C/D subtypes and F/f variants, respectively. The product size of BamHI W1/I1 region after enzyme digestion is either 206 bp with subtype-C or 139bp and 67bp with subtype-D. For the products of enzyme digested BamHI F region, the presence of one band (199 bp) identified an F-variant whereas the presence of two bands (128bp and 71bp) indicated an f-variant. \n\nStatistical analysis \n\nThe Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The qualitative data were expressed as frequencies and percentages. The differences in distribution of EBV types between tissue biopsies of BC patients and sera of the control group were compared by a univarite binary logistic regression test with the calculation of odds ratio (OR) and 95% confidence intervals (CIs). The difference between groups was considered statistically significant if the p-value was less than 0.05.",
"\nDetection of anti-VCA IgG of EBV by ELISA\n\nScreening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.\n\nDetection of EBV DNA by PCR \n\nEBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.\n\nGenotyping of EBV \n\n\nAnalysis of A/B types of EBV \n\nSuccessful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.\n\nAnalysis of C/D subtypes of EBV \n\nAmplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.\n\nAnalysis of F/f variants of EBV \n\nThe BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.\n\nCombined Genotypes of EBV\n\nA comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.\n\nAssociation between EBV DNA positivity and tumor characteristics\n\nAmong 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4. \n\nAssociation between EBV genotypes and tumor characteristics\n\nA/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.\nTarget Genes and Their Primer Sequence\nbp, base pair; F, forward; R, reverse.\nFrequency of EBV Infection in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer. \nFlowchart Indicating the Design and Results of the Present Study\nEBV Typing in Breast Cancer Patients and Controls\nEBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions\nAssociation between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients\n*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2 \nAssociation between Specific EBV Genotypes and Tumor Features\nIDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth",
"The presence of different polymorphisms in certain regions of the EBV genome resulted in the presence of different genotypes, subtypes, and variants of EBV (Ayadi et al., 2007). However, little is known about how EBV genetic diversity may affect EBV-associated disease (Palser et al., 2015). Moreover, up to date, there is a controversy whether these EBV variants are geographically related, or disease-associated. In 1995, the association of EBV with BC was reported but no study has been conducted in Egypt since then to investigate if there is a specific association of BC with certain types of EBV or if it is simply a geographical distribution. \nIn this study, the seroprevalence of EBV was 84.5% in BC patients and was 81.8% in the control group with no statistical significant difference. This is consistent with the high prevalence of EBV in the world. It is estimated that more than 90% of the world’s population is EBV-seropositive (Tzellos and Farrell, 2012). Smatti et al., (2017) reported that the EBV seroprevalence rate among healthy blood donors was 100% in Egyptians and Pakistanis, 98.3% in Indians, 96.8% in Syrians, and 97.8% in Qataris.\nAlthough this study detected a high seroprevalence of EBV among all study participants, the latent infection with EBV (as detected by molecular detection of the EBNA-1 gene) was significantly higher in BC tumor biopsies (45%) as compared to PMNCs of the control group (21.1%). This is in line with two previous Egyptian studies that detected EBV infection in 35.3% and 25% of breast carcinomas (Mohamed et al., 2007; Fawzy et al., 2008). Additionally, using PCR as a method of EBV detection, the prevalence of EBV infection among BC patients in various regions of the world was about 10–50% (Perkins et al., 2006; Lorenzetti et al., 2010). Other studies, on the other hand, failed to detect EBV among BC patients (Lespagnard et al., 1995; Kijima et al., 2001). This discrepancy in the results could be attributed to a variety of factors, such as geographical variation, differences in EBV infection detection methods, specimen type, use of different genes in the EBV genome for PCR, and the inclusion of different histological types of BC. However, studies that used biopsy specimens and used nested PCR as a method for detection showed less heterogeneity (Farahmand et al., 2019).\nIn this case-control study, EBV-DNA positivity increased the risk of BC development by about threefold (OR = 3.05). This is similar to two recent meta-analysis that reported that the risk of BC association with EBV infection is 3.84 and 4.74 folds higher as compared to controls (Bae and Kim, 2016; Farahmand et al., 2019). \nIn the current study, in an attempt to clarify if there is an association between BC and specific types of EBV in Egypt, we detected the distribution frequency of different EBV types obtained from tumor tissues of BC women and compared them with those obtained from PMNCs of healthy non-BC females.\nRegarding the distribution of A/B genotypes of EBV among the studied BC patients and healthy controls, we found that most of the EBV detected in both BC tumor biopsies and PBMNCs of the controls were of genotype-A (90.9% and 91.7%, respectively, p= 0.8). The high prevalence of genotype-A among the participants of this study is consistent with its geographical distribution. Genotype-A is the most prevalent genotype of EBV in the world, predominantly in the United States, France, Germany, North Africa, Turky, and Asia. Whereas, genotype of EBV predominates more in Central Africa and New Guinea (Ibrahim et al., 2010). In this study, the distribution difference of genotype-A and gentype-B was statistically insignificant between BC patients and the control group. As a result, it appears that neither genotype-A nor genotype-B has a preferential association with BC in Egypt. In the same context, the association of A/B genotypes of EBV with other malignant diseases such as NPC seems to be variable worldwide. Tamura et al (1993) reported no association of A/B genotypes of EBV with NPC in Japan, as they were predominant in patients as well as in general populations. Klemenc et al., (2006) and Ayadi et al., (2007), reported a high incidence of genotype-A in Slovenian and Tunisian patients with NPC, respectively. Ayee et al., (2020) identified genotype-B as the virulent genotype in Ghana and associated it with the likelihood of NPC development in Ghanaian patients.\nRegarding the polymorphism in the BamHI-I W1/I1 region. This study demonstrated that the frequency of subtype-D EBV is significantly higher in BC tumor biopsies (95.6%) as compared to controls (64.7%) (OR =11.7, 95%CI = 2.4-57.1, P =0.002). Similarly, Zhang et al., (2017) found a significant association between subtype-D and the risk of BC among Chinese populations (OR = 2.86), suggesting that subtype-D may play a role in the pathogenesis of BC. It is worth noting that the association of EBV subtype-D with other malignant diseases has previously been demonstrated; Abdel Hamid et al., (1992) discovered that subtype-D is detected more frequently in Egyptian NPC than subtype-C. Similarly, it was reported that EBV of subtype-D was associated with NPC in each of Slovenian patients (62.5%), Tunisian population (98%) and Northern China (32.2%) (Cui et al., 2011). Moreover, subtype-D was found to be associated not only with NPC but also with gastric carcinoma in Iranian population (90%), in Southern Tunisian population (100%), in Latin American population (85%) (Corvalan et al., 2006; Abdirad et al., 2007; BenAyed-Guerfali et al., 2011). \nThe genetic evidence that C/D subtypes of EBV could be related to the development of tumors is that some EBV genes, such as BamH1-A Rightward Frame-1 (BARF1) and Latent Membrane Protein-2A are present near the C/D locus. These genes are involved in transformation and immortalization during the tumorigenic process (Corvalan et al., 2006). However, the precise mechanisms need more exploration.\nRegarding the polymorphism at the BamHI-F region, we detected that EBV from all BC tumor biopsies and control PMNCs was of prototype-F. Therefore, the association of prototype-F with Egyptian BC is not likely. It was proposed that f-variant is only a geographic polymorphism because the EBV that was detected in Egyptian, Algerian, and Tunisian NPCs was of prototype-F, whereas the EBV that was detected in Asian NPCs was of variant-f (Cui et al., 2011). However, Zhou et al., (2001) reported that variant-f is found only in Chinese NPCs and not in Chinese HD patients. Therefore, the hypothesis that the f-variant is a geographically restricted polymorphism needs further investigation. \nIn the present study, ADF was the most prevalent combination of EBV genotypes among BC patients as well as among control subjects. Likewise, Klemenc et al., (2006) found that ADF is the most predominant combination of EBV genotypes in Slovenian patients with NPC as well as in the healthy population. Additionally, the risk of BC was significantly higher with each of ADF (OR=4.92) and BDF (OR=5.54) combinations and not significantly increased with BCF (P=0.217). Therefore, our findings could point to the implication of specific combinations of EBV genotypes in the development of BC in Egypt. Consequently, our results are not consistent with the hypothesis suggested by Khanim et al (1996) that EBV strains are geographically determined rather than disease-associated. \nAs regards the association between EBV and the characteristics of BC. EBV was detected more frequently in BC of larger size, higher stage, and higher grade. These findings are similar to that made previously by Murray et al., (2003) and Mohamed et al., (2007) who found that EBV is significantly associated with the more aggressive tumors. Other studies, however, debate our findings as they did not find any statistically significant association between EBV positivity and either grade, stage, or metastasis of the tumor (Richardson et al., 2015; Ahmed and Yussif, 2016; El-Naby et al., 2017).\nIn this study, there was no demonstrable significant association between EBV positivity and the steroid-hormone receptor. This agrees with many previous studies (Tiwawech et al., 2008; Ahmed and Yussif, 2016; Bae and Kim, 2016; Preciado et al., 2005). On the contrary, other investigators have shown that EBV is significantly associated with steroid hormone receptor-negative tumors (Bonnet et al., 1999; Murray et al., 2003; El-Naby et al., 2017). \nWe found that 75% of EBV-DNA positive BC biopsies are also reactive to HER2 expression. This is close to the result reported by Mohamed et al., (2007) who reported that 55.9% of DNA positive cases were HER2 positive. This finding correlates with the recognition that EBV could transform epithelial cells of the breast into malignancy via stimulating the HER2/HER3 signalling pathways (Szostek et al., 2009). Furthermore, our findings point to some extent that subtype-D of EBV could be the only EBV type that might be implicated in the development of BC with poor prognosis because it was the only type that showed more association with tumor of higher grade and with progesterone receptor-free tumors (p= 0.04). \nIn Conclusion, to the best of our knowledge, this is the first study in Egypt that provides baseline data on the prevalence of different EBV types in BC patients as well as in healthy controls. Overall, this study found that the D-subtype of EBV is BC-associated. Whereas, A/B genotypes and F/f variants of EBV are not associated with BC. Additionally, specific combinations of EBV genotypes (ADF and BDF) were BC- associated. Moreover, our findings indicated that EBV could have an impact on the histological criteria of BC and thus could have a role in prognosis and in optimizing the therapeutic strategies for BC. Nevertheless, our results are considered a platform for any future studies with a larger number of patients with BC in Egypt as well as in other parts of the world to investigate whether variations in the distribution of different types of EBV are geographically related or BC related. ",
"Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript."
] | [
"intro",
"materials|methods",
"results",
"discussion",
null
] | [
"Epstein",
"Barr virus",
"breast",
"cancer",
"RFLP",
"genotyping",
"Egypt"
] | Introduction:
Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, is a Herpes virus that belongs to the Herpesviridae family. It is highly widespread, affecting more than 90% of the world’s population (Dowd et al., 2013).
EBV genome is 172,000 base pairs long and although it can code for more than 85 proteins, the well-known ones are few; Six EBV nuclear antigens (EBNAs 1, 2, 3A, 3B, and 3C, and EBNA-LP); three latent membrane proteins (LMPs 1, 2A, 2B), also known as latent genes , and small noncoding EBV-encoded RNAs (EBERs 1 and 2) (Kalla and Hammerschmidt, 2012).
The presence of polymorphisms in several regions of the EBV genome define different EBV types and variants, which have been shown to have a distinctive geographical distribution (Ayadi et al., 2006).
EBV is classified into two genotypes: genotype-A and genotype -B (sometimes known as type-1 and type-2). EBV Genotypes-A and-B were initially distinguished based on changes in the EBNA2 sequence ,which only shows 70% of gene homology and 54% of protein homology between genotype-A and genotype -B (Farrell, 2015). Although Palser and his colleagues observed that differences between genotype-A and genotype -B could be caused by variations in the sequences of EBNA2 and EBNA3A, EBNA3B, and EBNA3C genes, they found that the most frequent variance between genotype-A and genotype -B is the differences in the EBNA2 gene (Palser et al., 2015).
EBV BamHI-F and BamHI-W/ Bam HI-I boundary regions in the EBV genome have been identified as BamHI endonuclease restriction sites. Therefore, restriction fragment polymorphism (REFLP) analysis would help to further classify each of genotype-A and genotype-B into C/D subtypes and into F/f variants based on the presence or lack of BamHI restriction sites in the previously mentioned regions. Subtype-C is distinguished by the absence of a BamHI restriction site between the BamHI-W and BamHI-I fragments, while subtype-D has an extra BamHI site in the same region. In the BamHI-F fragments, prototype-F has no BamHI site, whereas variant-f has an extra BamHI site (Ayadi et al., 2007).
Diagnosis of EBV infection depends on a variety of tests, including nonspecific tests like the monospot test for heterophile antibodies, serological tests for detection of EBV- specific antibodies and molecular assays for detection of nucleic acid (Hess, 2004). Viral capsid antigen (VCA)-IgG, VCA-IgM, and EBNA-1 IgG are frequently required for the detection of EBV antibodies. VCA-IgG antibodies are superior to VCA-IgM in diagnosing past EBV infections. Furthermore, approximately 5–10% of healthy people infected with EBV never produce EBNA-IgG and this percentage is higher in immunodeficient people. Therefore, VCA-IgG antibodies are the most accurate single test for detection of past infection with EBV (Smatti et al., 2018). EBNA1 gene is constantly present in all EBV-infected cells. As a result, it is used for molecular detection of EBV infection (Sun et al., 2015).
Breast cancer (BC) is the most frequently diagnosed malignancy as well as the leading cause of cancer death among women, worldwide. In accordance to WHO estimates, BC would be diagnosed in 2.1 million women every year. According to reports in 2018, over 627,000 women died from the disease, accounting for almost 15% of all cancer-related fatalities among women, worldwide (Farahmand et al., 2019). BC is a complex disease with an aetiology that is poorly understood. Many traditional factors were identifid as risk factors for BC; family history, early menarche, late menopause, obesity and nulliparity (Kaminska et al., 2015).
In 1995, Labrecque and his colleagues were the first to identify the association between EBV and BC, and since then, many studies have confirmed the role of EBV in the development of BC (Labrecque et al., 1995). Recently, there has been an increasing interest to specify the association between different types of EBV and malignant diseases (Neves et al., 2017). However, the majority of these studies were focused on EBV-associated nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma, and they proposed that the pathological effects of EBV might vary according to the type of EBV strain (Corvalan et al., 2006; Chen et al., 2010).
To date, most of the researches that studied the relationship between EBV and breast carcinoma were interested in investigating whether or not there is an association between EBV infection and the development of BC, and none of these studies determined the association between different types of EBV and BC. Therefore, we investigated the variations in the distribution of different types, subtypes, and variants of EBV between BC women and healthy controls in Egypt and to clarify the relationship between specific EBV types and the histopathological features of BC.
Materials and Methods:
Study subjects
This case-control study included 362 female participants; 142 breast cancer (BC) patients and 220 healthy controls. All patients and controls were recruited from the Oncology Center, Faculty of Medicine, Mansoura University (OCMU) in Egypt through the period from January 2019 to December 2020. The ages of all patients and controls were matched (p >0.05). Patients’ ages ranged from 37 to 79 years old with a median age of 54 years. The diagnosis of BC in all enrolled patients was based on clinical examination, radiological findings, and histopathological examination of tissue biopsy. Control subjects were selected from women who attended OCMU for annual mammography as part of a breast checkup and we excluded those with a history of BC and those with fibroadenomas.
Sample collection
Fresh tumor tissue and blood specimen were obtained from BC patients, whereas only blood specimen was obtained from the control group. The tissue sample from each tumor was divided into two parts; one part was fixed in formaldehyde and delivered to OCMU’s pathology laboratory for histopathological examination, while the other part was stored at -80°C to be used for PCR. The blood specimen was obtained via venipuncture and divided into two tubes; a plain tube for separation of serum and an EDTA tube for separation of peripheral blood mononuclear cells (PBMCs), both of which were kept at –80°C until use.
Study design
A Flowchart showing the workflow of this study is presented in Figure 1. All included BC patients and controls were screened for EBV infection by the detection of anti-viral capsid antigen (VCA) IgG antibodies in their sera. Then EBV seropositive subjects were investigated for the presence of EBV DNA by PCR amplification of the EBNA-1 gene; either in tumor biopsy of BC patients or in PBMCs of controls. Thereafter, A/B genotypes, C/D subtypes and F/f variants of EBV were identified in all EBV-DNA positive specimens by analysis of three regions in the EBV genome (EBNA-2 gene, BamHI-I W1/I1 and BamHI-F regions, respectively).
Histopathological examination
Diagnosis of BC and determination of its histological type was achieved in OCMU’s pathology laboratory by examination of hematoxylin-eosin (H-E) stained slides prepared from tumor biopsies. Only tumor biopsies with cancerous cells of more than 60% were included in the study. Grading of the tumors was performed according to the Scarff Bloom Richardson classification (Bloom and Richardson, 1957). Detection of hormonal receptor status [estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2)] of all included tumor biopsies was done by immunohistochemical staining of slides cut from tissue microarray prepared blocks as described previously (Ahmed and Yussif, 2016).
Detection of EBV-viral capsid antigen (VCA) IgG antibodies
Screening of all included BC patients and controls for EBV infection was performed by detection of anti-VCA IgG antibodies by ELISA (Bio-Rad Medical Diagnostics, GmbH, Germany) following the manufacturer guidelines.
Polymerase chain reaction (PCR) amplification of EBNA-1 gene
Detection of EBV DNA was performed by PCR amplification of EBNA-1 gene. DNA was extracted from all specimens with serologically confirmed EBV infection (120 tumor biopsies from BC patients and 180 PMNCs from controls) using the QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s tissue and blood protocols. Then EBV DNA was detected by PCR amplification of the EBNA-1 gene using specific primers listed in Table 1 according to the previously described PCR protocol (Hashimoto et al., 1995).
Detection of A/B genotypes of EBV
Nested PCR that targets EBNA-2 gene was used for genotyping of EBV into genotype-A and genotype-B (Ayee et al., 2020). In the first round of PCR amplification, a common region of EBNA-2 was amplified using EBNA-2A and EBNA-2B as forward and reverse primers, respectively. A PCR reaction was performed in a total volume of 12.5ul and the final concentrations of each component were as follows; 1X One TaqR Quick-Load 2X master mix with standard buffer (Bio Labs, UK), 0.5µM from each of the forward and reverse primers, and 0.5µg/µL of the extracted DNA. Amplification was achieved in a thermal cycler under the following conditions; 2min at 94°C for initial denaturation, then followed by 35 cycles of amplification (each cycle consisted of 60s at 94°C, 90s at 52°C and 4min at 72°C), followed by a cycle of 10min at 72°C for final extension.
In the 2nd round of nested PCR, the reaction volume was 12.5ul and the components were the same as in the 1st round except that 0.5ul of the amplified product from the 1st round was used as a template and three primers were used; EBNA-2C as a forward primer (common to genotype-A and genotype-B), EBNA-2D and EBNA-2E as reverse primers specific for genotype-A and genotype-B, respectively. The cycling conditions were as follows: An initial cycle of heat denaturation for 2min at 94°C, 35 amplification cycles (30s at 94°C, 60s at 52°C, and 2 min at 72°C), and a final extension cycle for 10min at 72°C. The resultant amplicon was visualized by electrophoresis on an agarose gel with 2% ethidium bromide. Samples were identified by the presence of their respective bands (250 bp for genotype-A and 300 bp for genotype-B).
Detection of C/D subtypes and F/f variants of EBV
Restriction fragment length polymorphism (RFLP) was used for classification of the EBV into C/D subtypes and F/f variants according to the existence of an extra restriction enzyme site at the BamHI-I W1/I1 and BamHI-F regions of the EBV genome, respectively. PCR was performed, followed by RFLP as previously described (Henry et al., 2001).
In brief, the reaction conditions for PCR amplification of each region were similar except for the use of forward and reverse primers specific for each region. The total reaction volume was 25ul consisting of 22.0µl of PCR master mix (1x), 1µl of each forward and reverse primer (0.5μM), and 1µl of DNA template. PCR cycles included an initial step of denaturation at 94°C for 5min followed by 40 amplification cycles; denaturation at 94ºC for 30s, annealing at 55°C for 30s and extension at 72°C for 1min , thereafter a step of final extension for 10min at 72°C. After PCR, the enzymatic digestion of the amplified product was performed in a total reaction volume of 20ul that contained 10µl of the amplified products, 2µl of reaction buffer, 1.5ul of distilled water, and 1.5µl of BamHI endonucleases (10U/ul) (Promega, Madison). This mixture was incubated at 37ºC for 2hs then followed by agarose gel (2%) electrophoresis of enzyme-digested BamHI-I W1/I1 region and BamHI-F region for determination of C/D subtypes and F/f variants, respectively. The product size of BamHI W1/I1 region after enzyme digestion is either 206 bp with subtype-C or 139bp and 67bp with subtype-D. For the products of enzyme digested BamHI F region, the presence of one band (199 bp) identified an F-variant whereas the presence of two bands (128bp and 71bp) indicated an f-variant.
Statistical analysis
The Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The qualitative data were expressed as frequencies and percentages. The differences in distribution of EBV types between tissue biopsies of BC patients and sera of the control group were compared by a univarite binary logistic regression test with the calculation of odds ratio (OR) and 95% confidence intervals (CIs). The difference between groups was considered statistically significant if the p-value was less than 0.05.
Results:
Detection of anti-VCA IgG of EBV by ELISA
Screening of 142 BC patients and 220 healthy females for past exposure to EBV infection revealed that 120/142 (84.5%) of the cases and 180/220 (81.8%) of the controls were seropositive for anti-VCA IgG with no statistically significant difference between them (p=0.5), Table 2.
Detection of EBV DNA by PCR
EBV-DNA was detected in 54/120(45%) of BC tissue biopsies and in 38/180 (21.1%) of control PBMCs. There was a significant association between BC and EBV-DNA positivity (OR=3.05, 95%CI: 1.84-5.07, p<0.001), Table 2.
Genotyping of EBV
Analysis of A/B types of EBV
Successful amplification of the EBNA-2 gene was observed in 52/54 (96.3%) of BC patients and in 36/38 (94.7%) of controls. Nested PCR of the EBNA-2 gene revealed that genotype-A EBV was more frequently detected than genotype-B EBV in both BC patients (90.4% vs. 9.6%) and healthy control groups (91.7% vs. 8.3%). There was no statistically significant difference in the distribution of genotypes-A and -B between BC patients and the control group (p=0.8), Table 3.
Analysis of C/D subtypes of EBV
Amplification of the BamHI W1/I1 boundary region was successful in 45/54 (83.3%) of BC patients and in 34/38 (89.5%) of controls. Among 45 BC patients with successfully amplified BamHI W1/I1 region, subtype-D was detected more frequently than subtype-C (95.6% vs. 4.4%). Similarly, in the control group, subtype-D was more frequent than subtype-C (64.7% vs. 35.3%). There was a statistically significant difference in the distribution of subtypes-D and-C between BC patients and the control group (p=0.002). Subtype-D EBV was significantly more frequently associated with tissue biopsies from breast tumors (95.6%) than with PMNCs from controls (64.7%) (OR=11.7, 95%CI= 2.4-57.1), Table 3.
Analysis of F/f variants of EBV
The BamHI-F region was amplified successfully in 48/54 (88.9%) of BC tumor biopsies and in36/38 (94.7%) of controls. The successfully amplified EBV from all BC patients and controls was of F-variant, Table 3.
Combined Genotypes of EBV
A comparison of the combined genotypes of EBV between BC patients and controls revealed that the most predominant combination of EBV genotypes among BC patients as well as among the control group was ADF (84.6% vs. 62.5%, respectively). In BC patients, BDF was the second most frequent combined genotype (10.3%), followed by ACF and BCF (2.6% for each). Whereas in the control group, the second most frequent combined genotype was ACF (28.1%), followed by BDF (6.2%) and BCF (3.1%). The distribution of the EBV genotype combination between BC patients and controls was statistically significant (p=0.022). The combined genotypes that significantly associated with BC risk were ADF (OR= 4.9, 95%CI= 1.61-14.99) and BDF (OR= 5.5, 95%CI= 1.25-24.41), Table 3.
Association between EBV DNA positivity and tumor characteristics
Among 120 seropositive BC patients, there was a significant association between detection of EBV-DNA (EBNA-1 gene) and each of the large tumor size and higher tumor stage (P = 0.001 for each). Also, positivity of EBV-DNA was significantly associated with carcinomas of infiltrating ductal type (75%), and with tumors of high histological grade (71.4%, 50%, and 14.3% for grades III, II, and I, respectively). Moreover, the proportion of EBV DNA positive tumors was significantly higher in metastatic tumors (p=0.001). On the other hand, there was no observed association between EBV-DNA detection in BC tissues and steroid hormone receptors. However, EBV-DNA was detected significantly more frequently in HER2-positve tumors than in HER2-negative tumors (75% vs. 25%, p= 0.001), Table 4.
Association between EBV genotypes and tumor characteristics
A/B genotypes of EBV did not show significant associations with any of tumor characteristics. Similarly, no association of statistical significance was observed between the C/D subtypes of EBV and either tumor size, tumor stage, tumor histology, presence of metastasis, or oestrogen receptor. However, the C/D subtypes of EBV from BC biopsies showed significant associations with tumor grade, progesterone receptor, and HER2- positive tumors. The proportion of subtype D positive BC biopsies increased significantly with tumour grade, from 86.7% for grade II to 100% for grade III. Although the D-subtype of EBV was detected more frequently in progesterone receptor-negative tumors (100%) than in progesterone receptor-positive tumors (86.7%), it was significantly more frequent in HER2-positive tumors than HER2- negative tumors (100% vs. 83.3%, p= 0.01), Table 5.
Target Genes and Their Primer Sequence
bp, base pair; F, forward; R, reverse.
Frequency of EBV Infection in Breast Cancer Patients and Controls
EBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; BC, breast cancer.
Flowchart Indicating the Design and Results of the Present Study
EBV Typing in Breast Cancer Patients and Controls
EBV, Epstein-Barr virus; VCA, Viral capsid antigen; EBNA, Epstein Barr virus nuclear antigen; NA, not amplified gene; BC, breast cancer; a, Combined types of EBV were calculated among 39 cases and 32 controls who were successfully typed for all 3 investigated regions
Association between EBV-DNA Positivity and Character of Tumors among 120 EBV Seropositive BC Patients
*Total number of EBV DNA-positive in this study was 54 of 120 seropositive positive BC patients; EBV, Epstein-Barr virus; IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth factor receptor-2
Association between Specific EBV Genotypes and Tumor Features
IDC, Infiltrating ductal carcinoma; ILC, Invasive lobular carcinoma; PR, Progesterone receptor; ER, Estrogen receptor; HER2, Human epidermal growth
Discussion:
The presence of different polymorphisms in certain regions of the EBV genome resulted in the presence of different genotypes, subtypes, and variants of EBV (Ayadi et al., 2007). However, little is known about how EBV genetic diversity may affect EBV-associated disease (Palser et al., 2015). Moreover, up to date, there is a controversy whether these EBV variants are geographically related, or disease-associated. In 1995, the association of EBV with BC was reported but no study has been conducted in Egypt since then to investigate if there is a specific association of BC with certain types of EBV or if it is simply a geographical distribution.
In this study, the seroprevalence of EBV was 84.5% in BC patients and was 81.8% in the control group with no statistical significant difference. This is consistent with the high prevalence of EBV in the world. It is estimated that more than 90% of the world’s population is EBV-seropositive (Tzellos and Farrell, 2012). Smatti et al., (2017) reported that the EBV seroprevalence rate among healthy blood donors was 100% in Egyptians and Pakistanis, 98.3% in Indians, 96.8% in Syrians, and 97.8% in Qataris.
Although this study detected a high seroprevalence of EBV among all study participants, the latent infection with EBV (as detected by molecular detection of the EBNA-1 gene) was significantly higher in BC tumor biopsies (45%) as compared to PMNCs of the control group (21.1%). This is in line with two previous Egyptian studies that detected EBV infection in 35.3% and 25% of breast carcinomas (Mohamed et al., 2007; Fawzy et al., 2008). Additionally, using PCR as a method of EBV detection, the prevalence of EBV infection among BC patients in various regions of the world was about 10–50% (Perkins et al., 2006; Lorenzetti et al., 2010). Other studies, on the other hand, failed to detect EBV among BC patients (Lespagnard et al., 1995; Kijima et al., 2001). This discrepancy in the results could be attributed to a variety of factors, such as geographical variation, differences in EBV infection detection methods, specimen type, use of different genes in the EBV genome for PCR, and the inclusion of different histological types of BC. However, studies that used biopsy specimens and used nested PCR as a method for detection showed less heterogeneity (Farahmand et al., 2019).
In this case-control study, EBV-DNA positivity increased the risk of BC development by about threefold (OR = 3.05). This is similar to two recent meta-analysis that reported that the risk of BC association with EBV infection is 3.84 and 4.74 folds higher as compared to controls (Bae and Kim, 2016; Farahmand et al., 2019).
In the current study, in an attempt to clarify if there is an association between BC and specific types of EBV in Egypt, we detected the distribution frequency of different EBV types obtained from tumor tissues of BC women and compared them with those obtained from PMNCs of healthy non-BC females.
Regarding the distribution of A/B genotypes of EBV among the studied BC patients and healthy controls, we found that most of the EBV detected in both BC tumor biopsies and PBMNCs of the controls were of genotype-A (90.9% and 91.7%, respectively, p= 0.8). The high prevalence of genotype-A among the participants of this study is consistent with its geographical distribution. Genotype-A is the most prevalent genotype of EBV in the world, predominantly in the United States, France, Germany, North Africa, Turky, and Asia. Whereas, genotype of EBV predominates more in Central Africa and New Guinea (Ibrahim et al., 2010). In this study, the distribution difference of genotype-A and gentype-B was statistically insignificant between BC patients and the control group. As a result, it appears that neither genotype-A nor genotype-B has a preferential association with BC in Egypt. In the same context, the association of A/B genotypes of EBV with other malignant diseases such as NPC seems to be variable worldwide. Tamura et al (1993) reported no association of A/B genotypes of EBV with NPC in Japan, as they were predominant in patients as well as in general populations. Klemenc et al., (2006) and Ayadi et al., (2007), reported a high incidence of genotype-A in Slovenian and Tunisian patients with NPC, respectively. Ayee et al., (2020) identified genotype-B as the virulent genotype in Ghana and associated it with the likelihood of NPC development in Ghanaian patients.
Regarding the polymorphism in the BamHI-I W1/I1 region. This study demonstrated that the frequency of subtype-D EBV is significantly higher in BC tumor biopsies (95.6%) as compared to controls (64.7%) (OR =11.7, 95%CI = 2.4-57.1, P =0.002). Similarly, Zhang et al., (2017) found a significant association between subtype-D and the risk of BC among Chinese populations (OR = 2.86), suggesting that subtype-D may play a role in the pathogenesis of BC. It is worth noting that the association of EBV subtype-D with other malignant diseases has previously been demonstrated; Abdel Hamid et al., (1992) discovered that subtype-D is detected more frequently in Egyptian NPC than subtype-C. Similarly, it was reported that EBV of subtype-D was associated with NPC in each of Slovenian patients (62.5%), Tunisian population (98%) and Northern China (32.2%) (Cui et al., 2011). Moreover, subtype-D was found to be associated not only with NPC but also with gastric carcinoma in Iranian population (90%), in Southern Tunisian population (100%), in Latin American population (85%) (Corvalan et al., 2006; Abdirad et al., 2007; BenAyed-Guerfali et al., 2011).
The genetic evidence that C/D subtypes of EBV could be related to the development of tumors is that some EBV genes, such as BamH1-A Rightward Frame-1 (BARF1) and Latent Membrane Protein-2A are present near the C/D locus. These genes are involved in transformation and immortalization during the tumorigenic process (Corvalan et al., 2006). However, the precise mechanisms need more exploration.
Regarding the polymorphism at the BamHI-F region, we detected that EBV from all BC tumor biopsies and control PMNCs was of prototype-F. Therefore, the association of prototype-F with Egyptian BC is not likely. It was proposed that f-variant is only a geographic polymorphism because the EBV that was detected in Egyptian, Algerian, and Tunisian NPCs was of prototype-F, whereas the EBV that was detected in Asian NPCs was of variant-f (Cui et al., 2011). However, Zhou et al., (2001) reported that variant-f is found only in Chinese NPCs and not in Chinese HD patients. Therefore, the hypothesis that the f-variant is a geographically restricted polymorphism needs further investigation.
In the present study, ADF was the most prevalent combination of EBV genotypes among BC patients as well as among control subjects. Likewise, Klemenc et al., (2006) found that ADF is the most predominant combination of EBV genotypes in Slovenian patients with NPC as well as in the healthy population. Additionally, the risk of BC was significantly higher with each of ADF (OR=4.92) and BDF (OR=5.54) combinations and not significantly increased with BCF (P=0.217). Therefore, our findings could point to the implication of specific combinations of EBV genotypes in the development of BC in Egypt. Consequently, our results are not consistent with the hypothesis suggested by Khanim et al (1996) that EBV strains are geographically determined rather than disease-associated.
As regards the association between EBV and the characteristics of BC. EBV was detected more frequently in BC of larger size, higher stage, and higher grade. These findings are similar to that made previously by Murray et al., (2003) and Mohamed et al., (2007) who found that EBV is significantly associated with the more aggressive tumors. Other studies, however, debate our findings as they did not find any statistically significant association between EBV positivity and either grade, stage, or metastasis of the tumor (Richardson et al., 2015; Ahmed and Yussif, 2016; El-Naby et al., 2017).
In this study, there was no demonstrable significant association between EBV positivity and the steroid-hormone receptor. This agrees with many previous studies (Tiwawech et al., 2008; Ahmed and Yussif, 2016; Bae and Kim, 2016; Preciado et al., 2005). On the contrary, other investigators have shown that EBV is significantly associated with steroid hormone receptor-negative tumors (Bonnet et al., 1999; Murray et al., 2003; El-Naby et al., 2017).
We found that 75% of EBV-DNA positive BC biopsies are also reactive to HER2 expression. This is close to the result reported by Mohamed et al., (2007) who reported that 55.9% of DNA positive cases were HER2 positive. This finding correlates with the recognition that EBV could transform epithelial cells of the breast into malignancy via stimulating the HER2/HER3 signalling pathways (Szostek et al., 2009). Furthermore, our findings point to some extent that subtype-D of EBV could be the only EBV type that might be implicated in the development of BC with poor prognosis because it was the only type that showed more association with tumor of higher grade and with progesterone receptor-free tumors (p= 0.04).
In Conclusion, to the best of our knowledge, this is the first study in Egypt that provides baseline data on the prevalence of different EBV types in BC patients as well as in healthy controls. Overall, this study found that the D-subtype of EBV is BC-associated. Whereas, A/B genotypes and F/f variants of EBV are not associated with BC. Additionally, specific combinations of EBV genotypes (ADF and BDF) were BC- associated. Moreover, our findings indicated that EBV could have an impact on the histological criteria of BC and thus could have a role in prognosis and in optimizing the therapeutic strategies for BC. Nevertheless, our results are considered a platform for any future studies with a larger number of patients with BC in Egypt as well as in other parts of the world to investigate whether variations in the distribution of different types of EBV are geographically related or BC related.
Author Contribution Statement:
Mashaly M: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP), wrote the original draft of the manuscript, revised statistical analysis and interpreted the obtained results. Ghorab D: performed the histopathological part of the study for breast cancer diagnosis. Hegazy M, Abdelkhalek M and Gaballah K: Contributed to the clinical examination of all participants and collection of the study samples and data. Elzehery R: Contributes to the concept and design of the study, performed the laboratory work concerned with EBV diagnosis (ELISA, PCR, REFLP). All authors read and revised the manuscript.
| Background:
Epstein-Barr virus (EBV) has been implicated in the development of breast cancer (BC) since 1995. It is classified into A/B genotypes, C/D subtypes, and F/f variants according to variations in its genome.
Methods:
Three hundred and sixty-two participants (142 BC patients and 220 controls) were enrolled in this study. All participants were screened for EBV infection by determination of viral-capsid-IgG antibodies in their sera. EBNA-1 gene was detected by PCR in tumor biopsies of seropositive patients and in peripheral blood mononuclear cells of controls. A/B genotyping of EBV was performed by nested-PCR targeting the EBNA-2 gene. C/D subtypes and F/f variants were identified by Restriction fragment length polymorphism at BamHI-I W1/I1 and BamHI-F regions of EBV genome, respectively.
Results:
Among 362 participants, 300(82.9%) were EBV-seropositive, including 120/142(84.5%) of the BC patients and 180/220(81.8 %) of the controls. EBNA-1 gene was positive in 54(45%) of seropositive BC patients and in 38(21.1%) of seropositive controls. There was a significant association of EBNA-1 gene with breast cancer (OR=3.05, 95%CI=1.84-5.07). Moreover, EBNA-1 gene positivity was significantly associated with the more aggressive tumors. Genotype-A and prototype-F were predominant among patients (90.4%, 100%, respectively) as well as among controls (91.7%, 100%, respectively) with no statistical significant association with BC risk. However, subtype-D was significantly more frequent in patients (95.6%) than in controls (64.7%) and was significantly associated with a higher BC risk as compared to subtype-C (OR=11.7, 95%CI=2.4-57.08). Subtype-D was significantly associated with higher grades tumors (100% among grade III), with progesteron receptor-negative tumors and with HER2-positive tumors (100% for each). The combined genotypes that significantly associated with BC risk were ADF (OR=4.9) and BDF (OR=5.5).
Conclusions:
Subtype-D of EBV could be the only EBV type implicated in BC development among Egyptian females and associated more with poor prognosis. | null | null | 5,997 | 430 | [
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] | null | null | null | [CONTENT] Epstein | Barr virus | breast | cancer | RFLP | genotyping | Egypt [SUMMARY] | null | [CONTENT] Epstein | Barr virus | breast | cancer | RFLP | genotyping | Egypt [SUMMARY] | null | [CONTENT] Epstein | Barr virus | breast | cancer | RFLP | genotyping | Egypt [SUMMARY] | null | [CONTENT] Adult | Aged | Breast Neoplasms | Case-Control Studies | Egypt | Epstein-Barr Virus Infections | Epstein-Barr Virus Nuclear Antigens | Female | Genotype | Herpesvirus 4, Human | Humans | Leukocytes, Mononuclear | Middle Aged | Polymerase Chain Reaction | Polymorphism, Restriction Fragment Length | Prognosis | Viral Proteins [SUMMARY] | null | [CONTENT] Adult | Aged | Breast Neoplasms | Case-Control Studies | Egypt | Epstein-Barr Virus Infections | Epstein-Barr Virus Nuclear Antigens | Female | Genotype | Herpesvirus 4, Human | Humans | Leukocytes, Mononuclear | Middle Aged | Polymerase Chain Reaction | Polymorphism, Restriction Fragment Length | Prognosis | Viral Proteins [SUMMARY] | null | [CONTENT] Adult | Aged | Breast Neoplasms | Case-Control Studies | Egypt | Epstein-Barr Virus Infections | Epstein-Barr Virus Nuclear Antigens | Female | Genotype | Herpesvirus 4, Human | Humans | Leukocytes, Mononuclear | Middle Aged | Polymerase Chain Reaction | Polymorphism, Restriction Fragment Length | Prognosis | Viral Proteins [SUMMARY] | null | [CONTENT] revealed genotype ebv | ebv genome identified | genes ebv genome | genotypes ebv studied | different genes ebv [SUMMARY] | null | [CONTENT] revealed genotype ebv | ebv genome identified | genes ebv genome | genotypes ebv studied | different genes ebv [SUMMARY] | null | [CONTENT] revealed genotype ebv | ebv genome identified | genes ebv genome | genotypes ebv studied | different genes ebv [SUMMARY] | null | [CONTENT] ebv | bc | patients | genotype | bc patients | association | tumor | bamhi | study | controls [SUMMARY] | null | [CONTENT] ebv | bc | patients | genotype | bc patients | association | tumor | bamhi | study | controls [SUMMARY] | null | [CONTENT] ebv | bc | patients | genotype | bc patients | association | tumor | bamhi | study | controls [SUMMARY] | null | [CONTENT] ebv | bamhi | genotype | bc | antibodies | vca | women | igg | genotype genotype | different [SUMMARY] | null | [CONTENT] ebv | bc | patients | bc patients | tumors | tumor | dna | ebv dna | vs | table [SUMMARY] | null | [CONTENT] ebv | bc | patients | genotype | bc patients | study | association | bamhi | tumor | pcr [SUMMARY] | null | [CONTENT] Epstein-Barr | EBV | BC | 1995 ||| F/ [SUMMARY] | null | [CONTENT] 362 | 300(82.9% | EBV | 120/142(84.5% | BC | 180/220(81.8 % ||| 54(45% | BC | 38(21.1% ||| OR=3.05 | 95%CI=1.84-5.07 ||| ||| Genotype-A | 90.4% | 100% | 91.7% | 100% | BC ||| 95.6% | 64.7% | BC | OR=11.7 | 95%CI=2.4-57.08 ||| 100% | 100% ||| BC | BDF [SUMMARY] | null | [CONTENT] Epstein-Barr | EBV | BC | 1995 ||| F/ ||| Three hundred and sixty-two | 142 | BC | 220 ||| EBV ||| PCR ||| EBV ||| F/ | Restriction | EBV ||| ||| 362 | 300(82.9% | EBV | 120/142(84.5% | BC | 180/220(81.8 % ||| 54(45% | BC | 38(21.1% ||| OR=3.05 | 95%CI=1.84-5.07 ||| ||| Genotype-A | 90.4% | 100% | 91.7% | 100% | BC ||| 95.6% | 64.7% | BC | OR=11.7 | 95%CI=2.4-57.08 ||| 100% | 100% ||| BC | BDF ||| EBV | EBV | BC | Egyptian [SUMMARY] | null |
|||
Evaluation of Three Multiplex Real-time Reverse Transcription PCR Assays for Simultaneous Detection of SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus in Nasopharyngeal Swabs. | 34904407 | In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. | BACKGROUND | A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. | METHODS | Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. | RESULTS | The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era. | CONCLUSION | [
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] | 8668494 | INTRODUCTION | The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10
SARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).
Since the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.
Molecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS). | METHODS | Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.
To detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.
All kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.
PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.
To detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.
All kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.
Specimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.
Four hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.
We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.
Four hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.
Sample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.
A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.
Analytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).
Cross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.
The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).
Cross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.
Evaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.
The RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.
Positive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).
Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.
The RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.
Positive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).
Ethics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).
This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262). | RESULTS | Analytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.
PCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.
In the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.
In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.
PCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.
In the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.
Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.
PPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.
All specimens were derived from coronavirus disease 2019 confirmed cases.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.
There were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.
Five samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.
RSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.
Finally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.
Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.
PPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.
All specimens were derived from coronavirus disease 2019 confirmed cases.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.
There were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.
Five samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.
RSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.
Finally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples. | null | null | [
"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV",
"Specimen collection",
"Sample processing and PCR",
"Analytical performance: limit of detection (LoD) and cross-reactivity",
"Evaluation of the clinical performance of the three molecular diagnostic kits",
"Ethics statement",
"Analytical performance",
"Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV"
] | [
"PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.",
"We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.",
"A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.",
"The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.",
"Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).",
"This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).",
"In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.",
"Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples."
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV",
"Specimen collection",
"Sample processing and PCR",
"Analytical performance: limit of detection (LoD) and cross-reactivity",
"Evaluation of the clinical performance of the three molecular diagnostic kits",
"Ethics statement",
"RESULTS",
"Analytical performance",
"Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV",
"DISCUSSION"
] | [
"The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10\nSARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).\nSince the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.\nMolecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS).",
"Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nPowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.\nSpecimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nWe selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.\nSample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nA 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.\nAnalytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nThe LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.\nEvaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nReference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).\nEthics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).\nThis study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).",
"PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.\nTo detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.\nAll kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.",
"We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.\nFour hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.",
"A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.",
"The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).\nCross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.",
"Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.\nThe RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.\nPositive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).",
"This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).",
"Analytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nIn comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.\nClinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.\nCompared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.",
"In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.\nPCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.\nIn the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).\nRT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.",
"Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.\nPPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.\nAll specimens were derived from coronavirus disease 2019 confirmed cases.\nSARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.\nThere were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.\nFive samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.\nRSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.\nFinally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.",
"In the evaluation of analytical sensitivity, the LoD is dependent on reference material kits, extraction methods, PCR machines, and other experimental conditions. Therefore, the LoDs reported in our study may not correspond with practical LoDs in the real world. In the evaluation of cross-reactivity of the three assays, all three kits did not present cross-reactivity with common respiratory viruses. Especially, for SARS-CoV-2, three assays showed no reactive with coronavirus 229E, OC43, and NL63.\nAll three kits had 100% NPA in SARS-CoV-2 results, but PPAs in SARS-CoV-2 results ranged from 92.8% to 95.9%; among 97 SARS-CoV-2 positive samples, 10 (10.3%) produced discrepant results among the three assays. All three kits had 100% PPA in influenza A/B results. In contrast to 100% NPA in influenza B results in all three kits, NPAs in influenza A results ranged from 99.7% to 100.0% due to two false influenza A-positive reactions. According to the LoD testing of AdvanSure and Allplex kits and RSV sequencing results, Allplex detected RSV more sensitively than AdvanSure.\nMolecular diagnostic kits for detecting SARS-CoV-2 have shifted from single SARS-CoV-2 detection to multi-detection of SARS-CoV-2 and other common respiratory pathogens. Nörz et al.23 adapted a laboratory-developed multiplex RT-PCR assay for the simultaneous detection of SARS-CoV-2 and influenza A/B on a fully automated high-throughput system and demonstrated analytical performance comparable with that of currently available commercial tests. Sensitivities of SARS-CoV-2, influenza A, and influenza B in the multiplex assay were 98.1%, 97.67%, and 100%, respectively.23 Xpert Xpress SARS-CoV-2/Flu-RSV (Xpert 4-in-1 assay) was launched with Emergency Use Authorization of the United States Food and Drug Administration in September 2020 and demonstrated a performance highly comparable with those of Xpert SARS-CoV-2 and Xpert Flu/RSV assays.24 PPAs and NPAs of SARS-CoV-2, influenza A, influenza B, and RSV in Xpert -in-1 assay were 98.48%/100%, 100%/100%, 100%/99.54%, and 100%/100%, respectively.24\nA case of co-infection of SARS-CoV-2 and other respiratory viruses was absent in the present study. SARS-CoV-2-positive specimens were collected between September 2020 and November 2020. In Korea, RSV and influenza outbreak seasons classically begin in autumn and winter, respectively.25 Although the collection period overlapped with RSV outbreak season, due to COVID-19-related quarantine and improved hygiene, transmission rates of influenza and RSV were lower.26 A US research team reported that 20.7% of specimens positive for SARS-CoV-2 were positive for one or more additional pathogens, compared with 26.7% negative for SARS-CoV-2.27 The most commonly co-infected pathogens were rhinovirus/enterovirus followed by RSV and non-SARS-CoV-2 Coronaviridae.27 A single-centered retrospective study was performed in Wuhan, China, between January 12 and February 21, 2020 during the initial COVID-19 outbreak.28 Unexpectedly, among COVID-19 patients, only 42.7% (131/307) of patients were singly positive for SARS-CoV-2; 57.3% (176/307) of COVID-19 patients were also positive for influenza viruses including influenza A (49.8%) and influenza B (7.5%).28 Another single-centered retrospective study conducted in Lukou District, China showed that 14.1% (11/78) of COVID-19 patients were co-infected with other respiratory pathogens including RSV (3/11, 36.3%) and influenza B (1/11, 9.1%).14 In contrast, a Korean research team demonstrated co-infection cases of SARS-CoV-2 and influenza virus or RSV were absent.29 Using the Allplex RV-Essential Assay (Seegene) detecting seven respiratory viruses including adenovirus, influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, RSV, and human rhinovirus, the co-infection rate in COVID-19 patients was 2.2%.29 The most frequently detected virus was adenovirus, followed by human rhinovirus and metapneumovirus.29 However, comprehensive PCR testing to detect respiratory viruses other than influenza virus and RSV was not performed in SARS-CoV-2-positive specimens in the present study.\nPowerChek, STANDARD M, and Allplex kits each specified different cycle conditions for rRT-PCR. PowerChek did not require any pre-amplification cycles during PCR. In the PCR protocol of STANDARD M, five pre-amplification cycles preceded the amplification cycles, and the time and temperature conditions of both cycles were the same. Allplex had three pre-amplification cycles with different time and temperature conditions from those of the amplification cycles. Volume of template RNA per test and the number of pre-amplification cycles and amplification cycles of the three multi-target kits were compared with corresponding single SARS-CoV-2 detection kits: PowerChek 2019-nCoV Real-time PCR (Kogene Biotech), STANDARD M nCoV Real-Time Detection (SD BioSensor), and Allplex 2019-nCoV (Seegene).17 KogeneBiotech and SD BioSensor had the same volume of template RNA per test and number of pre-amplification and amplification cycles in each pair of single- and multi-target assays. However, the multi-target assay of Allplex required 10 μL of template RNA compared with 8 μL in the single-target assay. Allplex single-target assay performed 45 amplification cycles without pre-amplification but the multi-target assay executed 3 pre-amplification and 42 amplification cycles. Allplex multi-target assay was thought to be designed to increase sensitivity than the single-target assay by increasing the volume of template RNA.\nThis study had some limitations. First, as previously mentioned, the evaluation of the LoD of the kits may not have reflected the practical analytical sensitivity because the measured LoD may have been affected by extraction method, type of PCR machine, etc. Second, for SARS-CoV-2, cross-reactivity test with other coronaviruses except 229E, OC43, and NL63 was not performed. Third, although the AdvanSure kit was treated as a comparator kit in this study, a true reference method was absent. Indeed, the AdvanSure kit was less sensitive for RSV compared with the Allplex kit. Finally, because multiplex PCR used to detect respiratory viruses other than influenza A/B and RSV was not performed in SARS-CoV-2-positive samples, co-infection rates of other respiratory viruses and SARS-CoV-2 could not be estimated.\nIn conclusion, the overall performance of these three multiplex rRT-PCR assays for concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. In the COVID-19 pandemic era, all three kits are suitable for the prompt differential diagnosis of COVID-19, influenza, and RSV infection in patients with respiratory symptoms."
] | [
"intro",
"methods",
null,
null,
null,
null,
null,
null,
"results",
null,
null,
"discussion"
] | [
"SARS-CoV-2",
"Influenza A Virus",
"Influenza B Virus",
"Respiratory Syncytial Viruses",
"Multiplex Polymerase Chain Reaction"
] | INTRODUCTION:
The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health crisis caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1 The COVID-19 pandemic began in December 2019 in Wuhan City, China, and has lasted for more than a year worldwide.2 Clinical presentation of COVID-19 ranges from asymptomatic cases and mild illness to critical conditions featuring acute respiratory distress syndrome.3 In Korea, patients less than 10 years and over 70 years are less frequently infected with COVID-19, and those aged between 20–59 years are more commonly infected.4 COVID-19 disease severity is associated with increased age.5 Influenza virus (Flu) and respiratory syncytial virus (RSV) are common respiratory pathogens that cause seasonal epidemics.67 Influenza infection tend to be less severe, typically resulting in uncomplicated upper respiratory tract illness, but, in rare cases, influenza infection can induce a complicated disease with severe viral pneumonia.8 Children are most likely to be infected with influenza, and people aged 65 years and older are least likely to become ill from influenza.9 RSV is recognized as the most common cause of bronchiolitis and pneumonia in children younger than 1 year. RSV infection can produce common cold-like symptoms but may be severe in infants and young children. In older adults, RSV usually causes no symptoms or upper respiratory infection but may lead to severe lower respiratory infection.10
SARS-CoV-2, influenza, and RSV infections should be managed in different ways. COVID-19 management requires strict quarantine and isolation strategies that are not applied to influenza and RSV infection management. Among these three infective diseases, influenza is the only effectively treatable condition; influenza can be treated with the drug, oseltamivir (Tamiflu).
Since the clinical presentations of SARS-CoV-2, influenza, and RSV infections have overlapping symptoms, differential diagnosis of these diseases is challenging. Specifically, rapid diagnosis is needed in elderly patients because SARS-CoV-2, influenza, and RSV infections result in substantial morbidity and mortality in these patients.611 In the COVID-19 pandemic era, clinicians may encounter a co-infection of SARS-CoV-2 with influenza or RSV.121314 According to a Nature survey, many researchers expect SARS-CoV-2 to become endemic.15 In the future, SARS-CoV-2, influenza, and RSV are expected to be the most common respiratory viruses infected in humans. Therefore, multiplex detection of SARS-CoV-2, influenza, and RSV will be essential and commonly used.
Molecular testing is the gold standard for the detection of many viruses, and real-time reverse transcription polymerase chain reaction (rRT-PCR) is most widely used molecular method for qualitative diagnostic testing.16 Numerous rRT-PCR kits that solely detect SARS-CoV-2 have been developed and commercialized worldwide, including in Korea.171819 Seegene has the highest market share in the SARS-CoV-2 single molecular diagnostic kit market in Korea, followed by KogeneBiotech and SD BioSensor. The United States Food and Drug Administration approved the use of SARS-CoV-2 molecular diagnostic kits produced by these three companies under Emergency Use Authorization in April 2020.20 Recently, these three manufacturers have developed molecular diagnostic kits for concurrent detection of SARS-CoV-2, influenza A/B, and RSV. The kits produced by Seegene and KogeneBiotech were approved for use by the Ministry of Food and Drug Safety in Korea in January 2021 and November 2020, respectively, although the kit produced by SD BioSensor has not yet been approved (last reviewed on October 20, 2021). In this study, we evaluated the analytical and clinical performance of these three multiplex rRT-PCR assays for simultaneous detection of SARS-CoV-2, influenza A/B, and RSV in nasopharyngeal swabs (NPS).
METHODS:
Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.
To detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.
All kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.
PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.
To detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.
All kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.
Specimen collection We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.
Four hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.
We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.
Four hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.
Sample processing and PCR A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.
A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.
Analytical performance: limit of detection (LoD) and cross-reactivity The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).
Cross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.
The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).
Cross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.
Evaluation of the clinical performance of the three molecular diagnostic kits Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.
The RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.
Positive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).
Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.
The RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.
Positive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).
Ethics statement This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).
This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).
Three molecular diagnostic kits for simultaneous detection of SARS-CoV-2 and influenza A/B and/or RSV:
PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech, Seoul, Korea) and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor, Osong, Korea) are multiplex kits for the detection of SARS-CoV-2 and influenza A/B. Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) is a multiplex kit for the detection of SARS-CoV-2, influenza A/B, and RSV.
To detect SARS-CoV-2, PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech), and STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor) target the envelope (E) and open reading frame 1ab (ORF1ab) coding regions of the SARS-CoV-2 genome, whereas Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene, Seoul, Korea) targets the nucleocapsid (N), RNA-dependent RNA polymerase (RdRp), and spike (S) coding regions. According to the World Health Organization guideline,21 rRT-PCR results are interpreted as positive when all target genes are detected. If no target genes are detected, the test is interpreted as negative. The cases where the target genes are partially detected are considered inconclusive. For the detection of influenza A, PowerChek, STANDARD M, and Allplex target the matrix (M) genes. For the detection of influenza B, PowerChek, STANDARD M, and Allplex target the nucleoprotein (NP), nonstructural (NS) 1, and NS genes, respectively. Allplex targets the matrix (M) gene for the detection of RSV. All kits are based on one-step rRT-PCR in one tube. Specifications of the three assays are described in Table 1.
All kits are one-step RT-PCR kits. Pre-amplification was not counted for Ct.
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, SC2 = SARS-CoV-2, IC = internal control, exo IC = exogenous internal control, endo IC = endogenous internal control, Ct = cycle threshold.
Specimen collection:
We selected 103 SARS-CoV-2 positive and 201 SARS-CoV-2 negative specimens that were collected from September 2020 to November 2020 and reported by STANDARD™ M new Coronavirus (nCoV) Real-Time Detection kit (STANDARD M nCoV; SD BioSensor). The specimens were nasopharyngeal and/or oropharyngeal swabs from patients. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays and STANDARD M nCoV. Re-testing using STANDARD M nCoV revealed that the results of six SARS-CoV-2 positive samples were changed from positive to inconclusive or negative, and the results of the remaining 97 SARS-CoV-2 positive samples were unchanged. We excluded the six specimens and finally included 97 SARS-CoV-2-positive and 201 SARS-CoV-2-negative specimens. All 97 SARS-CoV-2-positive samples were acquired from confirmed cases.
Four hundred and three respiratory virus-positive NPS samples tested by AdvanSure™ respiratory viruses (RV) real-time RT-PCR assay (AdvanSure; LG Life Sciences, Seoul, Korea) were collected from December 2017 to November 2020. A substantial portion of the respiratory virus-positive NPS samples tested by the AdvanSure diagnostic kit was acquired before the COVID-19 pandemic. Aliquots of samples were stored at −80°C. We retrieved the aliquots and thawed them for testing using the three assays. When the results of the three assays were different from the previously reported results of the AdvanSure kit, the corresponding aliquots were tested with the AdvanSure kit again. We used re-tested AdvanSure results if tested again; if not, the previously reported results were used for analysis. All specimens harbored one or more respiratory viruses including 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive samples.
Sample processing and PCR:
A 200 μL volume of the patient specimen was extracted using the EMAG system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions, with a nucleic acid elution volume of 50 μL. All real-time PCR analyses were performed using the CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). PCR reactions were performed in a total volume of 20 μL (15 μL PCR reaction mixture and 5 μL template RNA), a total volume of 30 μL (20 μL PCR reaction mixture and 10 μL template RNA), and a total volume of 20 μL (9 μL PCR reaction mixture and 11 μL template RNA) using the PowerChek, STANDARD M, and Allplex kits, respectively.
Analytical performance: limit of detection (LoD) and cross-reactivity:
The LoD was determined using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare, Milford, MA, USA). AccuPlex virus products contained non-replicative recombinant viruses including full genomes of SARS-CoV-2 and influenza A/B and partial genome of RSV (NC_001803, 1..4380; 8460..15191). We obtained seven concentrations (5,000, 1,667, 500, 167, 50, 17, and 5 copies/mL) by serial dilution from reference non-replicative recombinant viruses. The first two concentrations (5,000 and 1,667 copies/mL) were tested with eight replicates each. The other concentrations were tested with 24 replicates each. LoD was defined as the input copy number per 1 mL with a 95% probability of a positive PCR and was calculated by probit regression analysis using International Business Machines (IBM) Statistical Package for the Social Sciences software package (version 25; IBM, Armonk, NY, USA) with 95% confidence intervals (CIs).
Cross-reactivity was verified using the 403 clinical samples containing typical respiratory viruses. Cross-reactivity was defined as the ability to generate a false-positive in similar viruses during RT-PCR analysis. Reference results were acquired using the AdvanSure diagnostic kits. Among the 403 respiratory virus-positive NPS samples, 47 samples tested positive for multiple respiratory pathogens. Since all three diagnostic kits were not regarded as cross-reactive to the 47 samples, each sample was regarded as multiple samples harboring different viruses.
Evaluation of the clinical performance of the three molecular diagnostic kits:
Reference results for positive and negative agreements were defined as follows: With regards to SARS-CoV-2, positive samples were defined as the sum of positive samples for all three kits and confirmed cases with discrepant results between three kits. Negative samples were defined as samples that tested negative for all three kits. In terms of influenza A/B, positive or negative samples were defined when two or three kits showed positive or negative results, respectively. When one kit produced discrepant results with the other two kits, the result of this kit was regarded as false.
The RSV results of the Allplex diagnostic kit were compared with those of the AdvanSure comparator kit. The discordant results between the three kits and the AdvanSure comparator kit might be due to differences in sensitivity. Therefore, we estimated LoDs for influenza A/B and RSV in AdvanSure using the AccuPlex™ SARS-CoV-2, Flu A/B, and RSV reference material kits (SeraCare). RT-PCR and gene sequencing to identify RSV were performed on samples that produced discrepant results between Allplex and AdvanSure.
Positive percent agreements (PPAs) and negative percent agreements (NPAs) with 95% CIs were calculated using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Cohen’s kappa values with 95% CIs were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/kappa1/) (GraphPad).
Ethics statement:
This study was approved by the Institutional Review Board of the Seoul National University Boramae Medical Center and the requirement for informed consent was waived (IRB No. 20-2020-262).
RESULTS:
Analytical performance In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.
PCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.
In the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.
In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.
PCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.
In the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.
Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.
PPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.
All specimens were derived from coronavirus disease 2019 confirmed cases.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.
There were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.
Five samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.
RSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.
Finally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.
Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.
PPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.
All specimens were derived from coronavirus disease 2019 confirmed cases.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.
There were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.
Five samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.
RSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.
Finally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.
Analytical performance:
In comparison of the LoD for the ORF1ab or RdRp genes of SARS-CoV-2, the three kits showed no significant difference in detecting specific SARS-CoV-2 genes due to overlapping 95% CIs (Table 2). The LoDs for influenza A M gene were not significantly different between the three assays. In comparison of the LoD for the NP, NS1, or NS genes of influenza B, the PowerChek had a lower LoD than the STANDARD M and Allplex kits.
PCR = polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, LoD = limit of detection, CI = confidence interval, RSV = respiratory syncytial virus.
In the evaluation of cross-reactivity in the three kits, all kits were not cross-reactive with other common human respiratory viruses such as common cold viruses (coronavirus 2229E, OC43, and NL63), adenovirus, bocavirus, metapneumovirus, parainfluenza virus, and rhinovirus (Table 3).
RT-PCR = reverse transcription polymerase chain reaction, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, RSV = respiratory syncytial virus, Flu = influenza virus, SC2 = SARS-CoV-2.
Clinical performance for the detection of SARS-CoV-2, influenza A/B, and RSV:
Compared with the reference results derived from the combinations of the three kits’ results, the three kits had greater than 92% PPA and NPA in the detection of SARS-CoV-2 and influenza A/B. Kappa values of the three kits indicated perfect agreements in the detection of SARS-CoV-2 and influenza A/B (Table 4). Furthermore, there were no significant differences in PPA, NPA, and kappa values among the three assays in both SARS-CoV-2 and influenza A/B detection. Discordant SARS-CoV-2 results were found in 10 specimens (Table 5). All the 10 specimens were acquired from confirmed COVID-19 patients. Additional SARS-CoV-2 data of the three kits were described in Supplementary Tables 1 and 2.
PPA and NPA were presented as % with 95% confidence intervals and Kappa with 95% confidence intervals.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, PPA = positive percent agreement, NPA = negative percent agreement.
All specimens were derived from coronavirus disease 2019 confirmed cases.
SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2, Ct = cycle threshold, Interpr. = interpretation, P = positive, I = inconclusive, N = negative.
There were two influenza A false-positive results; the STANDARD M and Allplex kits each produced a false-positive result in two different samples, although both assays had a NPA of 99.7% (Table 5, Supplementary Table 3). In the first sample, PowerChek and Allplex kits did not detect influenza A, but the STANDARD M did with a cycle threshold (Ct) value of 34.0. In the second sample, PowerChek and STANDARD M only detected influenza B, but the Allplex kit additionally detected influenza A with a Ct value of 34.0.
Five samples that tested negative for RSV using the AdvanSure comparator kit tested positive using the Allplex kit (Table 6, Supplementary Table 3). In the LoD test of the AdvanSure kit, the LoDs for influenza A, influenza B, and RSV were as follows: 250,172.6 copies/mL (95% CI, 23,630.6–4.74×1013; P = 0.988), 747.9 copies/mL (95% CI, 481.0–1,585.1; P = 0.807), and more than 5,000 copies/mL, respectively. A previous study reported that LoD of RSV in the AdvanSure kit was 2,920 copies/mL22 which was higher than that of the Allplex kit. RT-PCR and gene sequencing confirmed that among these five samples four were identified as RSV-positive, and their RSV Ct values in Allplex ranged from 34.4 to 35.9. However, one sample was not identified as RSV-positive, despite producing an RSV Ct value of 36.7 using the Allplex kit.
RSV = respiratory syncytial virus, RT-PCR = reverse transcription polymerase chain reaction, NA = not applicable, PPA = positive percent agreement, CI = confidence interval, NPA = negative percent agreement.
Finally, influenza- or RSV-positive cases did not occur in the SARS-CoV-2-positive samples, and SARS-CoV-2-positive cases did not in the influenza A/B-positive, RSV-positive, or other respiratory viruses-positive samples.
DISCUSSION:
In the evaluation of analytical sensitivity, the LoD is dependent on reference material kits, extraction methods, PCR machines, and other experimental conditions. Therefore, the LoDs reported in our study may not correspond with practical LoDs in the real world. In the evaluation of cross-reactivity of the three assays, all three kits did not present cross-reactivity with common respiratory viruses. Especially, for SARS-CoV-2, three assays showed no reactive with coronavirus 229E, OC43, and NL63.
All three kits had 100% NPA in SARS-CoV-2 results, but PPAs in SARS-CoV-2 results ranged from 92.8% to 95.9%; among 97 SARS-CoV-2 positive samples, 10 (10.3%) produced discrepant results among the three assays. All three kits had 100% PPA in influenza A/B results. In contrast to 100% NPA in influenza B results in all three kits, NPAs in influenza A results ranged from 99.7% to 100.0% due to two false influenza A-positive reactions. According to the LoD testing of AdvanSure and Allplex kits and RSV sequencing results, Allplex detected RSV more sensitively than AdvanSure.
Molecular diagnostic kits for detecting SARS-CoV-2 have shifted from single SARS-CoV-2 detection to multi-detection of SARS-CoV-2 and other common respiratory pathogens. Nörz et al.23 adapted a laboratory-developed multiplex RT-PCR assay for the simultaneous detection of SARS-CoV-2 and influenza A/B on a fully automated high-throughput system and demonstrated analytical performance comparable with that of currently available commercial tests. Sensitivities of SARS-CoV-2, influenza A, and influenza B in the multiplex assay were 98.1%, 97.67%, and 100%, respectively.23 Xpert Xpress SARS-CoV-2/Flu-RSV (Xpert 4-in-1 assay) was launched with Emergency Use Authorization of the United States Food and Drug Administration in September 2020 and demonstrated a performance highly comparable with those of Xpert SARS-CoV-2 and Xpert Flu/RSV assays.24 PPAs and NPAs of SARS-CoV-2, influenza A, influenza B, and RSV in Xpert -in-1 assay were 98.48%/100%, 100%/100%, 100%/99.54%, and 100%/100%, respectively.24
A case of co-infection of SARS-CoV-2 and other respiratory viruses was absent in the present study. SARS-CoV-2-positive specimens were collected between September 2020 and November 2020. In Korea, RSV and influenza outbreak seasons classically begin in autumn and winter, respectively.25 Although the collection period overlapped with RSV outbreak season, due to COVID-19-related quarantine and improved hygiene, transmission rates of influenza and RSV were lower.26 A US research team reported that 20.7% of specimens positive for SARS-CoV-2 were positive for one or more additional pathogens, compared with 26.7% negative for SARS-CoV-2.27 The most commonly co-infected pathogens were rhinovirus/enterovirus followed by RSV and non-SARS-CoV-2 Coronaviridae.27 A single-centered retrospective study was performed in Wuhan, China, between January 12 and February 21, 2020 during the initial COVID-19 outbreak.28 Unexpectedly, among COVID-19 patients, only 42.7% (131/307) of patients were singly positive for SARS-CoV-2; 57.3% (176/307) of COVID-19 patients were also positive for influenza viruses including influenza A (49.8%) and influenza B (7.5%).28 Another single-centered retrospective study conducted in Lukou District, China showed that 14.1% (11/78) of COVID-19 patients were co-infected with other respiratory pathogens including RSV (3/11, 36.3%) and influenza B (1/11, 9.1%).14 In contrast, a Korean research team demonstrated co-infection cases of SARS-CoV-2 and influenza virus or RSV were absent.29 Using the Allplex RV-Essential Assay (Seegene) detecting seven respiratory viruses including adenovirus, influenza A virus, influenza B virus, metapneumovirus, parainfluenza virus, RSV, and human rhinovirus, the co-infection rate in COVID-19 patients was 2.2%.29 The most frequently detected virus was adenovirus, followed by human rhinovirus and metapneumovirus.29 However, comprehensive PCR testing to detect respiratory viruses other than influenza virus and RSV was not performed in SARS-CoV-2-positive specimens in the present study.
PowerChek, STANDARD M, and Allplex kits each specified different cycle conditions for rRT-PCR. PowerChek did not require any pre-amplification cycles during PCR. In the PCR protocol of STANDARD M, five pre-amplification cycles preceded the amplification cycles, and the time and temperature conditions of both cycles were the same. Allplex had three pre-amplification cycles with different time and temperature conditions from those of the amplification cycles. Volume of template RNA per test and the number of pre-amplification cycles and amplification cycles of the three multi-target kits were compared with corresponding single SARS-CoV-2 detection kits: PowerChek 2019-nCoV Real-time PCR (Kogene Biotech), STANDARD M nCoV Real-Time Detection (SD BioSensor), and Allplex 2019-nCoV (Seegene).17 KogeneBiotech and SD BioSensor had the same volume of template RNA per test and number of pre-amplification and amplification cycles in each pair of single- and multi-target assays. However, the multi-target assay of Allplex required 10 μL of template RNA compared with 8 μL in the single-target assay. Allplex single-target assay performed 45 amplification cycles without pre-amplification but the multi-target assay executed 3 pre-amplification and 42 amplification cycles. Allplex multi-target assay was thought to be designed to increase sensitivity than the single-target assay by increasing the volume of template RNA.
This study had some limitations. First, as previously mentioned, the evaluation of the LoD of the kits may not have reflected the practical analytical sensitivity because the measured LoD may have been affected by extraction method, type of PCR machine, etc. Second, for SARS-CoV-2, cross-reactivity test with other coronaviruses except 229E, OC43, and NL63 was not performed. Third, although the AdvanSure kit was treated as a comparator kit in this study, a true reference method was absent. Indeed, the AdvanSure kit was less sensitive for RSV compared with the Allplex kit. Finally, because multiplex PCR used to detect respiratory viruses other than influenza A/B and RSV was not performed in SARS-CoV-2-positive samples, co-infection rates of other respiratory viruses and SARS-CoV-2 could not be estimated.
In conclusion, the overall performance of these three multiplex rRT-PCR assays for concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. In the COVID-19 pandemic era, all three kits are suitable for the prompt differential diagnosis of COVID-19, influenza, and RSV infection in patients with respiratory symptoms.
| Background:
In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits.
Methods:
A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV.
Results:
Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure.
Conclusions:
The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era. | null | null | 9,168 | 389 | [
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] | null | null | [CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY] | [CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY] | [CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY] | null | [CONTENT] SARS-CoV-2 | Influenza A Virus | Influenza B Virus | Respiratory Syncytial Viruses | Multiplex Polymerase Chain Reaction [SUMMARY] | null | [CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY] | [CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY] | [CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY] | null | [CONTENT] COVID-19 | Cross Reactions | Humans | Influenza A virus | Influenza B virus | Influenza, Human | Limit of Detection | Multiplex Polymerase Chain Reaction | Nasopharynx | Nucleocapsid Proteins | Polyproteins | RNA, Viral | Reagent Kits, Diagnostic | Republic of Korea | Respiratory Syncytial Virus Infections | Respiratory Syncytial Virus, Human | SARS-CoV-2 | Viral Matrix Proteins | Viral Proteins [SUMMARY] | null | [CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY] | [CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY] | [CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY] | null | [CONTENT] covid 19 pandemic | coronavirus disease | respiratory viruses influenza | age influenza virus | covid 19 disease [SUMMARY] | null | [CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY] | [CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY] | [CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY] | null | [CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | respiratory | samples | pcr [SUMMARY] | null | [CONTENT] influenza | infection | cov | sars cov | sars | rsv | influenza rsv | sars cov influenza rsv | cov influenza rsv | cov influenza [SUMMARY] | [CONTENT] positive | samples | sars cov | cov | sars | μl | results | kit | kits | pcr [SUMMARY] | [CONTENT] positive | influenza | cov | sars cov | sars | rsv | table | npa | kits | respiratory [SUMMARY] | null | [CONTENT] cov | sars cov | sars | positive | influenza | rsv | kits | samples | respiratory | kit [SUMMARY] | null | [CONTENT] 2019 | COVID-19 | 2 | RSV ||| Three | transcription | Korea ||| PowerChek | Influenza | Multiplex Real-time | PCR Kit | PowerChek | KogeneBiotech | STANDARD | STANDARD M | SD BioSensor | Allplex | Allplex | Seegene ||| [SUMMARY] | [CONTENT] ||| Ninety-seven | 201 | 71 | 50 | 78 | RSV | 207 | the three assays ||| AdvanSure | AdvanSure | LG Life Sciences | RSV [SUMMARY] | [CONTENT] three ||| three ||| three | 92% | ≥ 0.95 | RSV [SUMMARY] | null | [CONTENT] 2019 | COVID-19 | 2 | RSV ||| Three | transcription | Korea ||| PowerChek | Influenza | Multiplex Real-time | PCR Kit | PowerChek | KogeneBiotech | STANDARD | STANDARD M | SD BioSensor | Allplex | Allplex | Seegene ||| ||| ||| Ninety-seven | 201 | 71 | 50 | 78 | RSV | 207 | the three assays ||| AdvanSure | AdvanSure | LG Life Sciences | RSV ||| three ||| three ||| three | 92% | ≥ 0.95 | RSV ||| three | RSV ||| COVID-19 | influenza | RSV | COVID-19 [SUMMARY] | null |
||||
Regulation of pentraxin-3 by antioxidants. | 19864306 | Pentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis. | BACKGROUND | Human endothelial cells were cultured with lipopolysaccharide 2 microg ml(-1), peptidoglycan G 20 microg ml(-1), tumour necrosis factor (TNF) alpha 10 ng ml(-1), interleukin-1 (IL-1) beta 20 ng ml(-1), or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 microM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers. | METHODS | PTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml(-1) compared with 6 (1-12) ng ml(-1), P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNFalpha plus IL-1beta. PTX3 concentrations were lower in cells co-treated with antioxidants (all P<0.05), associated with lower nuclear factor kappaB expression for NAC and trolox (P<0.05). | RESULTS | PTX3 expression is down-regulated in vitro by antioxidants. Plasma levels of PTX3 are elevated in sepsis but seem to be unrelated to markers of oxidant stress or antioxidant capacity. | CONCLUSIONS | [
"APACHE",
"Adult",
"Aged",
"Aged, 80 and over",
"Antioxidants",
"Biomarkers",
"C-Reactive Protein",
"Cells, Cultured",
"Endothelium, Vascular",
"Female",
"Humans",
"Inflammation Mediators",
"Lipid Peroxides",
"Male",
"Middle Aged",
"Oxidative Stress",
"Pilot Projects",
"Sepsis",
"Serum Amyloid P-Component",
"Up-Regulation",
"Young Adult"
] | 2777941 | null | null | Methods | Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.
In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.
In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.
Yeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.
Endothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.
All measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.
All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.
Yeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.
Endothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.
All measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.
Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.
CRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).
PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.
CRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).
Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.
NFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.
Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.
NFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.
Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.
Total lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.
Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.
Total lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.
Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.
For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant. | Results | Fifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.
(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.
Characteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct
To assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.
Plasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.
We also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.
The effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).
Nuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).
The effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001). | null | null | [
"Patient study",
"In vitro study",
"Pentraxin-3 and CRP measurement",
"Nuclear factor κB",
"Total antioxidant capacity and lipid peroxides",
"Data analysis",
"Funding"
] | [
"In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.",
"All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.",
"PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).",
"Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.",
"Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.",
"For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.",
"A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust."
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"Methods",
"Patient study",
"In vitro study",
"Pentraxin-3 and CRP measurement",
"Nuclear factor κB",
"Total antioxidant capacity and lipid peroxides",
"Data analysis",
"Results",
"Discussion",
"Funding"
] | [
" Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\nIn this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.\n In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\nAll reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.\n Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\nPTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).\n Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\nNuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.\n Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\nTotal antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.\n Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.\nFor the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.",
"In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.",
"All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.\nYeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.\nEndothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.\nAll measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.",
"PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.\nCRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).",
"Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.\nNFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.",
"Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.\nTotal lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.",
"For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.",
"Fifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.\n(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.\nCharacteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct\nTo assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.\nPlasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.\nWe also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.\nThe effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).\nNuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).\nThe effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001).",
"We showed that PTX3 was up-regulated in human endothelial cells in response to bacterial cell proteins, heat-killed Candida cells, or cytokines, with the greatest response occurring with a combination of TNFα plus IL-1β. Endothelial PTX3 response to an inflammatory stimulus was significantly reduced by treatment with NAC, trolox, or idebenone, and was associated with lower nuclear NFκB expression in NAC and trolox-treated cells. Idebenone had no effect on NFκB expression. We also showed that circulating PTX3 concentrations are higher in patients with sepsis compared with healthy subjects and are higher in female patients than males. PTX3 was also related to APACHE II score quartile. In addition, total antioxidant capacity was low and lipid hydroperoxide levels were elevated in the patients with sepsis, indicating oxidative stress. However, PTX3 concentrations in patients with sepsis were not related to antioxidant capacity, despite the regulation of PTX3 expression by antioxidants in vitro.\nPTX3 is a member of the pentraxin superfamily, a family of proteins characterized by a multimeric, usually pentameric structure.12 CRP, the prototype of the pentraxin family, is an acute-phase protein produced in the liver in response to inflammatory signals such as IL-6. PTX3 is the prototype of the long pentraxin family and has some similarities with the short pentraxins but differs with respect to cellular source, and ligand recognition. PTX3 is produced in response by a variety of cell types, including macrophages and monocytes, dendritic cells, fibroblasts, epithelial, and endothelial cells, in response to inflammatory signals such as TNFα, IL-1β, microbial proteins including LPS, lipoarabinomannans, and PepG, but not IL-6.1–5\nWe found that exposure of endothelial cells to LPS, PepG, TNFα, or IL-1β resulted in PTX3 up-regulation, with an additive response when cells were treated with both TNFα and IL-1β, as previously reported.3–57 We also found that a ratio of between 1 and 10 heat-killed Candida cells to one endothelial cell resulted in an up-regulation of PTX3 expression by endothelial cells. Infection of mice with intra-cerebroventricular injection of live C. albicans was previously shown to result in up-regulation of PTX3 in the brain,19 but PTX3 expression by endothelial cells in response to C. albicans has not been described. Endothelial cells phagocytose live Candida cells, leading to secretion of cytokines including TNFα and IL-1β.20–23 The up-regulation of PTX3 expression found in our study may be related to TNFα and IL-1α produced as a result of phagocytosis of heat-killed Candida cells by the endothelial cells, although it has been reported that Candida cells killed with sodium periodate, although phagocytosed, are unable to elicit this cytokine response.2021 However, in another study, heat-killed Candida cells were able to stimulate monocytes to release pro-inflammatory cytokines,24 suggesting that the means of killing the cells may be relevant.\nPTX3 blood levels are usually <10 ng ml−1 in healthy subjects and increase rapidly during inflammation and infection, leading to investigation of the potential of PTX3 as an early biomarker for sepsis. In a heterogeneous group of critically ill patients ranging from those with no infection to those with septic shock, it was reported that PTX3 levels were higher in the more severely ill patients.25 We also found that PTX3 levels were related to severity of illness as assessed by APACHE II score. The APACHE scores were not presented in that report,25 making further comparisons difficult. PTX3 has also been found to be increased in patients with acute respiratory distress syndrome.26 In a recent study in children with meningococcal disease, high PTX3 and low CRP concentrations at admission discriminated between the presence and the absence of shock.27 PTX3 did not correlate with paediatric risk of mortality, whereas CRP correlated negatively. PTX3 blood levels increase more rapidly than CRP and only loose2526 or no27 correlations are found between circulating levels of PTX3 and CRP. PTX3 has also been shown to be up-regulated in patients with dengue virus infection.28\nMarked oxidative stress has been consistently reported in patients with sepsis8202930 and acts as a trigger for up-regulation of many inflammatory responses. The human PTX3 proximal promoter contains binding sites for several transcription factors, including activator protein-1 (AP-1), NFκB, and nuclear factor-IL-6. The NFκB proximal site is essential for induction by IL-1β and TNFα,67 and PTX3 was shown to be regulated by NFκB.31 NFκB is redox-sensitive32 and has been shown to be involved in regulation of inflammatory responses in patients with sepsis: we and others have found elevated activation of NFκB linked to increased mortality3334 and inhibitable by NAC treatment, resulting in down-regulation of inflammatory mediators.35 We therefore investigated whether antioxidants were able to down-regulate PTX3 expression by endothelial cells in vitro. We found that NAC, trolox, and idebenone treatment all resulted in lower PTX3 levels on exposure to an inflammatory stimulus. In the case of NAC and trolox, this was accompanied by lower nuclear expression of NFκB. In a study using macrophages, the antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced PTX3 expression in murine macrophages via an NFκB-dependent mechanism.31 However, there are other redox-sensitive transcription factors besides NFκB, such as AP-1,36 which may also be important for regulation of PTX3. Exogenous and endogenous antioxidants have been shown to be effective in blocking activation of NFκB either directly or indirectly, including NAC and trolox.3536 Although co-enzyme Q10 can inhibit NFκB,37 we can find no reports of the effect of idebenone on transcription, and so the mechanism of the down-regulation of PTX3 expression by idebenone remains unknown.\nOxidative stress can be defined as an imbalance between the production of reactive oxygen species and their removal by protective antioxidants. We measured lipid hydroperoxide levels and total antioxidant capacity in our patients with sepsis. Lipid peroxidation is the oxidative degradation of lipids and has been reported previously to be elevated in critically ill patients.8101230 Total antioxidant capacity reflects the ability of plasma to resist oxidative damage in vitro and has been widely used to assess the antioxidant status of patients with sepsis.8929 We found that lipid hydroperoxides were increased and total antioxidant capacity was decreased in our patients. The classical view of lipid peroxidation products is that they are essentially harmful, inducing and propagating chronic inflammatory reactions. However, it has been suggested that lipid peroxide breakdown products may promote cellular defence mechanisms,3839 such as induction of signalling pathways, resulting in up-regulation of anti-inflammatory mediators, and inhibition of signalling pathways which control pro-inflammatory mediator expression. Thus, the role of lipid hydroperoxides in inflammatory responses in sepsis is unclear. We found that neither lipid hydroperoxides nor antioxidant capacity was related to PTX3 levels, despite the effect of antioxidants on PTX3 expression in vitro. Although this was a small pilot study, there was a range of PTX values and oxidative stress was apparent, and so we would have expected some evidence of a relationship. These results may reflect the complex nature of the role of redox state involved in regulation of inflammatory pathways in vivo.\nOther factors also influence PTX3 levels. In a study of more than 2000 healthy Japanese subjects, plasma PTX3 levels were reported to be lower in men than in women, higher in older age groups, and inversely correlated with triglycerides.40 We also found that PTX3 levels in patients with sepsis were highest in the female patients, but the skewed age range of patients with sepsis did not allow assessment of any age. The previous study of critically ill patients25 did not report sex distribution. It remains unclear as to the importance of the contribution of these confounding factors.\nIn conclusion, we report that PTX3 expression in endothelial cells can be up-regulated by a variety of inflammatory mediators, including bacterial cell proteins, cytokines, and C. albicans and is down-regulated by NAC and trolox through an NFκB-mediated process. PTX3 levels are increased in patients with sepsis, and related to APACHE II score. Although we found evidence of oxidative stress in our patients, the regulation of PTX3 by antioxidants in vitro was not reflected clinically in this pilot study. Further work is needed to clarify regulatory mechanisms for PTX3 and its use as a biomarker in sepsis.",
"A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust."
] | [
"methods",
null,
null,
null,
null,
null,
null,
"results",
"discussion",
null
] | [
"immune response",
"infection, bacterial",
"infection, fungal",
"metabolism, protein, acute phase"
] | Methods:
Patient study In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.
In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.
In vitro study All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.
Yeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.
Endothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.
All measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.
All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.
Yeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.
Endothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.
All measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.
Pentraxin-3 and CRP measurement PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.
CRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).
PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.
CRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).
Nuclear factor κB Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.
NFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.
Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.
NFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.
Total antioxidant capacity and lipid peroxides Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.
Total lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.
Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.
Total lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.
Data analysis For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.
For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.
Patient study:
In this pilot study, 20 consecutive patients were recruited from the intensive care unit (ICU) within 24 h of fulfilling the criteria for sepsis, after local ethical approval and obtaining written informed consent from the patient or assent from a close relative. The criteria used were those recommended by the Consensus Meeting of the American Thoracic Society and the American Society of Critical Care Medicine,15 namely clinical suspicion of infection plus two of the following: tachycardia (>100 beats min−1), tachypnoea (>20 bpm or ventilated), or leucocyte count <4 or >12×109 litre−1. Patients <16 yr old, who were pregnant or lactating, HIV positive, on steroid or immunosuppression therapy, who had any form of cancer or autoimmune disease or who were taking statins, were excluded. Five patients were subsequently excluded; two were found subsequently not to have sepsis and another three were taking statins. Heparinized blood was obtained from an indwelling cannula and immediately centrifuged and the plasma stored at −80°C for PTX3 analysis. Acute physiological and chronic health evaluation (APACHE) II score was also recorded. Blood samples were also obtained with consent from 11 healthy laboratory and research staff (age range 25–50 yr) using heparinized vacutainers and samples were treated as for patients.
In vitro study:
All reagents were obtained from Sigma Aldrich Ltd, Poole, Dorset, UK, unless stated otherwise. The human umbilical vein endothelial cell line HUVEC-C was used (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in 6-well plates as we have previously described15 in Dulbecco's Modified Eagle's Medium (Lonza Wokingham Ltd, Berkshire, UK) containing heat-inactivated fetal calf serum 10%, gentamicin 50 µg ml−1, and amphotericin B 250 µg ml−1.
Yeast cells of the human pathogenic fungus C. albicans wild-type derivative strain NGY152 were used.16 Candida albicans was grown in Sabouraud broth overnight at 30°C with shaking. The overnight culture was diluted 1:100 in fresh broth, grown until spectrophotometry showed an absorbance of 0.5 at 600 nm, then harvested. The pellet was washed three times in phosphate-buffered saline (PBS) and resuspended to a final concentration of 1×108 yeast cells ml−1 in PBS. The cells were heat-killed by incubation at 56°C for 2 h.
Endothelial cells were cultured for 24 h at 37°C in the presence of lipopolysaccharide (LPS) 2 µg ml−1 plus peptidoglycan G (PepG) 20 µg ml−1, tumour necrosis factor α (TNFα, PeproTech EC Ltd, London, UK) 10 ng ml−1, interleukin-1β (IL-1β, PeproTech) 20 ng ml−1, TNFα and IL-1β combined, or killed C. albicans cells at a multiplicity of infection (MOI) of 1–10, along with the antioxidants 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, a water-soluble vitamin E analogue, 100 mM), N-acetylcysteine (NAC, a glutathione precursor, 25 mM) or 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone, a water-soluble co-enzyme Q10 analogue, 1 µM, a kind gift from Dr M.P. Murphy, MRC-Dunn Nutrition Unit, Cambridge, UK), or appropriate solvent controls. Culture supernatants were stored at –80°C for subsequent PTX3 measurement. In separate experiments, endothelial cells were treated with IL-1β and TNFα plus antioxidants as above and nuclear extracts were prepared for NFκB assay.
All measurements on cells were corrected for viable cell number which was determined using acid phosphatase activity.17 The precision of this assay (% coefficient of variation) was 1.06%.
Pentraxin-3 and CRP measurement:
PTX3 expression from plasma or culture supernatants was measured by enzyme immunoassay (R&D Systems Europe Ltd, Abingdon, Oxon, UK). Briefly, 96-well plates were coated with anti-PTX3 monoclonal antibody and incubated overnight at 4°C. After incubation, plates were washed with PBS containing Tween 20 0.05% and blocked with skim milk powder 10% for 1 h at 37°C. Plates were washed again and recombinant human PTX3 as a calibration standard, or plasma or culture supernatant was added to each well. After 2 h incubation at 37°C, plates were washed and biotinylated anti-PTX3 polyclonal antibody was added for a further hour at 37°C followed by 40 min incubation with streptavidin–horseradish peroxidase (HRP) then a chromogen substrate. The reaction was stopped using sulphuric acid and quantified spectrophotometrically. The precision of this assay (% coefficient of variation) was 2.9%.
CRP was measured colorimetrically using a Siemens ADVIA 2400 autoanalyzer (Siemens Diagnostics, Tarrytown, NY, USA).
Nuclear factor κB:
Nuclear extraction was performed using the NucBuster kit (Novagen, Merk Chemicals, Nottingham, UK) according to the manufacturer's instructions. Briefly, cells were suspended in NucBuster extraction reagent 1 and vortexed for 15 s, incubated on ice for 5 min and vortexed again. Nuclei were sedimented by centrifugation at 13 000g at 4°C, for 5 min, the supernatant was removed and the nuclei pellet was resuspended in Nucbuster extraction reagent 2 containing protease inhibitor cocktail and dithiothreitol 1.28 mM. The samples were then vortexed, incubated on ice, and centrifuged as before. The supernatant (nuclear extract) was then used in the NFκB assay.
NFκB was measured using the NoShift transcription factor immunoassay kit (Novagen, Merk Chemicals). First, nuclear extract was incubated on ice for 30 min with binding buffer containing poly(dI-dC), salmon sperm DNA, and wild-type DNA (biotinylated, 10 pmol ml−1). After incubation, the reaction mixture was made up to 100 µl with binding buffer before being transferred to a streptavidin-coated 96-well plate and incubated at 37°C for 1 h. The plate was then washed, detection antibody added, and incubated again at 37°C for 1 h. The plate was again washed before the addition of goat-anti-mouse IgG HRP conjugate for 30 min before being washed five times. Chromogen substrate was then added to the wells and incubated at room temperature until colour developed. This reaction was stopped with the addition of hydrochloric acid, quantified spectrophotometrically, and expressed in terms of nuclear protein content, measured using the Bradford reagent. The precision of this assay (% coefficient of variation) was 1.4%.
Total antioxidant capacity and lipid peroxides:
Total antioxidant capacity was measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA) based on the ability of plasma antioxidants to inhibit oxidation of 2,2′-azino-di-[3-ethylbenzothiaolinesulphonate] (ABTS) to the ABTS radical by metmyoglobin.18 The capacity of plasma to prevent oxidation of ABTS is compared with that of trolox and is quantified as molar trolox equivalents. The precision of this assay (% coefficient of variation) was 3.4%.
Total lipid hydroperoxides were measured using a spectrophotometric technique.12 Lipid hydroperoxides are extracted into chloroform, which eliminates interference by hydrogen peroxide or endogenous ferric ions in the sample. The precision of this assay (% coefficient of variation) was 0.63%.
Data analysis:
For the in vitro study, four replicate independent experiments in triplicate were performed. No assumptions were made about the distribution of data which were analysed using the Kruskal–Wallis with Mann–Whitney post hoc testing and Bonferroni's correction as appropriate. Patients' plasma PTX3 and total antioxidant capacity were compared with those of healthy subjects using the Mann–Whitney U-test. All data are presented as median (range). A P-value of <0.05 was considered to be significant.
Results:
Fifteen patients were included (six females and nine males, aged 22–85 yr) (Table 1). The median (range) APACHE II score was 21 (9–32). PTX3 levels in plasma were significantly higher in patients with sepsis than in healthy people [26 (1–202) ng ml−1 compared with 6 (1–12) ng ml−1, P=0.01, Fig. 1a]. PTX3 levels were also significantly lower in men than in women [16.7 (1–42) ng ml−1 compared with 55 (24–202) ng ml−1, P<0.003, Fig. 1a]. When PTX3 levels in patients were plotted according to APACHE II score quartile, PTX3 levels were related to APACHE score quartile (Fig. 1b). Median (range) CRP was 164 (20–370) mg ml−1 and was unrelated to APACHE II score quartile.
(a) Plasma PTX3 concentrations in 15 patients with sepsis and 11 healthy subjects. Squares are females, circles are males. (b) Data sets compared using Mann–Whitney U-test. Plasma PTX3 concentrations according to APACHE II score quartile in 15 patients with sepsis.
Characteristics of patients with sepsis. UTI, urinary tract infection; CBD, common bile duct
To assess oxidative stress, we measured total plasma antioxidant capacity and plasma lipid hydroperoxides. Antioxidant capacity was significantly lower in patients with sepsis than healthy controls [0.99 (0.1–1.7) mM compared with 2.2 (1.3–3.3) mM, P=0.01, Fig. 2]. Lipid hydroperoxides were below the limit of detection in plasma from all healthy subjects. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3–10.6) nM. There was no relationship between PTX3 and either antioxidant capacity or lipid hydroperoxides. Total antioxidant capacity and lipid hydroperoxide were not different between males and females.
Plasma total antioxidant capacity in 15 patients with sepsis and 11 healthy subjects. Data sets compared using Mann–Whitney U-test.
We also measured PTX3 secretion from human endothelial cells in vitro. In the absence of an inflammatory stimulus, endothelial cells produced detectable basal levels of PTX3 (Fig. 3). When we exposed cells to either LPS plus PepG, TNFα, IL-1β, TNFα plus IL-1β, or C. albicans, PTX3 levels in culture medium were significantly higher than in untreated cells (Fig. 3). There was no difference in PTX3 expression in endothelial cells exposed to C. albicans between 1 and 10 MOI (data not shown). PTX3 expression was greatest in cells exposed to both TNFα and IL-1α and was ∼10-fold higher than in control cells (P<0.001, Fig. 3). When we treated cells concurrently with antioxidants, PTX3 levels were lower, independent of the cell stimulus, than without antioxidants.
The effect of different inflammatory stimuli on PTX3 expression by human endothelial cells in vitro. Note the different scales on the two graphs. Data are from four replicate experiments conducted in triplicate. LPS, lipopolysaccharide (2 µg ml−1); PepG, peptidoglycan G (20 µg ml−1); TNFα, tumour necrosis factor α (10 ng ml−1); IL-1β, interleukin-1β (20 ng ml−1); C. albicans, heat-killed Candida albicans cells (MOI=3).
Nuclear expression of NFκB was higher in cells treated with TNFα and IL-1β than controls (P<0.001, Fig. 4), and was lower in cells also treated with trolox or NAC, but not idebenone, compared with those treated with TNFα plus IL-1β alone (Fig. 4).
The effect of antioxidants on PTX3 and NFκB expression in human endothelial cells treated with TNFα and IL-1β. Data are from four replicate experiments conducted in triplicate and were analysed by the Kruskal–Wallis with Mann–Whitney U post hoc testing. *Significantly lower than cells without antioxidant (P<0.001).
Discussion:
We showed that PTX3 was up-regulated in human endothelial cells in response to bacterial cell proteins, heat-killed Candida cells, or cytokines, with the greatest response occurring with a combination of TNFα plus IL-1β. Endothelial PTX3 response to an inflammatory stimulus was significantly reduced by treatment with NAC, trolox, or idebenone, and was associated with lower nuclear NFκB expression in NAC and trolox-treated cells. Idebenone had no effect on NFκB expression. We also showed that circulating PTX3 concentrations are higher in patients with sepsis compared with healthy subjects and are higher in female patients than males. PTX3 was also related to APACHE II score quartile. In addition, total antioxidant capacity was low and lipid hydroperoxide levels were elevated in the patients with sepsis, indicating oxidative stress. However, PTX3 concentrations in patients with sepsis were not related to antioxidant capacity, despite the regulation of PTX3 expression by antioxidants in vitro.
PTX3 is a member of the pentraxin superfamily, a family of proteins characterized by a multimeric, usually pentameric structure.12 CRP, the prototype of the pentraxin family, is an acute-phase protein produced in the liver in response to inflammatory signals such as IL-6. PTX3 is the prototype of the long pentraxin family and has some similarities with the short pentraxins but differs with respect to cellular source, and ligand recognition. PTX3 is produced in response by a variety of cell types, including macrophages and monocytes, dendritic cells, fibroblasts, epithelial, and endothelial cells, in response to inflammatory signals such as TNFα, IL-1β, microbial proteins including LPS, lipoarabinomannans, and PepG, but not IL-6.1–5
We found that exposure of endothelial cells to LPS, PepG, TNFα, or IL-1β resulted in PTX3 up-regulation, with an additive response when cells were treated with both TNFα and IL-1β, as previously reported.3–57 We also found that a ratio of between 1 and 10 heat-killed Candida cells to one endothelial cell resulted in an up-regulation of PTX3 expression by endothelial cells. Infection of mice with intra-cerebroventricular injection of live C. albicans was previously shown to result in up-regulation of PTX3 in the brain,19 but PTX3 expression by endothelial cells in response to C. albicans has not been described. Endothelial cells phagocytose live Candida cells, leading to secretion of cytokines including TNFα and IL-1β.20–23 The up-regulation of PTX3 expression found in our study may be related to TNFα and IL-1α produced as a result of phagocytosis of heat-killed Candida cells by the endothelial cells, although it has been reported that Candida cells killed with sodium periodate, although phagocytosed, are unable to elicit this cytokine response.2021 However, in another study, heat-killed Candida cells were able to stimulate monocytes to release pro-inflammatory cytokines,24 suggesting that the means of killing the cells may be relevant.
PTX3 blood levels are usually <10 ng ml−1 in healthy subjects and increase rapidly during inflammation and infection, leading to investigation of the potential of PTX3 as an early biomarker for sepsis. In a heterogeneous group of critically ill patients ranging from those with no infection to those with septic shock, it was reported that PTX3 levels were higher in the more severely ill patients.25 We also found that PTX3 levels were related to severity of illness as assessed by APACHE II score. The APACHE scores were not presented in that report,25 making further comparisons difficult. PTX3 has also been found to be increased in patients with acute respiratory distress syndrome.26 In a recent study in children with meningococcal disease, high PTX3 and low CRP concentrations at admission discriminated between the presence and the absence of shock.27 PTX3 did not correlate with paediatric risk of mortality, whereas CRP correlated negatively. PTX3 blood levels increase more rapidly than CRP and only loose2526 or no27 correlations are found between circulating levels of PTX3 and CRP. PTX3 has also been shown to be up-regulated in patients with dengue virus infection.28
Marked oxidative stress has been consistently reported in patients with sepsis8202930 and acts as a trigger for up-regulation of many inflammatory responses. The human PTX3 proximal promoter contains binding sites for several transcription factors, including activator protein-1 (AP-1), NFκB, and nuclear factor-IL-6. The NFκB proximal site is essential for induction by IL-1β and TNFα,67 and PTX3 was shown to be regulated by NFκB.31 NFκB is redox-sensitive32 and has been shown to be involved in regulation of inflammatory responses in patients with sepsis: we and others have found elevated activation of NFκB linked to increased mortality3334 and inhibitable by NAC treatment, resulting in down-regulation of inflammatory mediators.35 We therefore investigated whether antioxidants were able to down-regulate PTX3 expression by endothelial cells in vitro. We found that NAC, trolox, and idebenone treatment all resulted in lower PTX3 levels on exposure to an inflammatory stimulus. In the case of NAC and trolox, this was accompanied by lower nuclear expression of NFκB. In a study using macrophages, the antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced PTX3 expression in murine macrophages via an NFκB-dependent mechanism.31 However, there are other redox-sensitive transcription factors besides NFκB, such as AP-1,36 which may also be important for regulation of PTX3. Exogenous and endogenous antioxidants have been shown to be effective in blocking activation of NFκB either directly or indirectly, including NAC and trolox.3536 Although co-enzyme Q10 can inhibit NFκB,37 we can find no reports of the effect of idebenone on transcription, and so the mechanism of the down-regulation of PTX3 expression by idebenone remains unknown.
Oxidative stress can be defined as an imbalance between the production of reactive oxygen species and their removal by protective antioxidants. We measured lipid hydroperoxide levels and total antioxidant capacity in our patients with sepsis. Lipid peroxidation is the oxidative degradation of lipids and has been reported previously to be elevated in critically ill patients.8101230 Total antioxidant capacity reflects the ability of plasma to resist oxidative damage in vitro and has been widely used to assess the antioxidant status of patients with sepsis.8929 We found that lipid hydroperoxides were increased and total antioxidant capacity was decreased in our patients. The classical view of lipid peroxidation products is that they are essentially harmful, inducing and propagating chronic inflammatory reactions. However, it has been suggested that lipid peroxide breakdown products may promote cellular defence mechanisms,3839 such as induction of signalling pathways, resulting in up-regulation of anti-inflammatory mediators, and inhibition of signalling pathways which control pro-inflammatory mediator expression. Thus, the role of lipid hydroperoxides in inflammatory responses in sepsis is unclear. We found that neither lipid hydroperoxides nor antioxidant capacity was related to PTX3 levels, despite the effect of antioxidants on PTX3 expression in vitro. Although this was a small pilot study, there was a range of PTX values and oxidative stress was apparent, and so we would have expected some evidence of a relationship. These results may reflect the complex nature of the role of redox state involved in regulation of inflammatory pathways in vivo.
Other factors also influence PTX3 levels. In a study of more than 2000 healthy Japanese subjects, plasma PTX3 levels were reported to be lower in men than in women, higher in older age groups, and inversely correlated with triglycerides.40 We also found that PTX3 levels in patients with sepsis were highest in the female patients, but the skewed age range of patients with sepsis did not allow assessment of any age. The previous study of critically ill patients25 did not report sex distribution. It remains unclear as to the importance of the contribution of these confounding factors.
In conclusion, we report that PTX3 expression in endothelial cells can be up-regulated by a variety of inflammatory mediators, including bacterial cell proteins, cytokines, and C. albicans and is down-regulated by NAC and trolox through an NFκB-mediated process. PTX3 levels are increased in patients with sepsis, and related to APACHE II score. Although we found evidence of oxidative stress in our patients, the regulation of PTX3 by antioxidants in vitro was not reflected clinically in this pilot study. Further work is needed to clarify regulatory mechanisms for PTX3 and its use as a biomarker in sepsis.
Funding:
A.L.H. was an Anaesthetic Research Society vacation scholar. N.R.W. and H.F.G. are funded by the Medical Research Council, the British Journal of Anaesthesia, and the Intensive Care Society. N.A.R.G. acknowledges funding from the Wellcome Trust.
| Background:
Pentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis.
Methods:
Human endothelial cells were cultured with lipopolysaccharide 2 microg ml(-1), peptidoglycan G 20 microg ml(-1), tumour necrosis factor (TNF) alpha 10 ng ml(-1), interleukin-1 (IL-1) beta 20 ng ml(-1), or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 microM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers.
Results:
PTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml(-1) compared with 6 (1-12) ng ml(-1), P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNFalpha plus IL-1beta. PTX3 concentrations were lower in cells co-treated with antioxidants (all P<0.05), associated with lower nuclear factor kappaB expression for NAC and trolox (P<0.05).
Conclusions:
PTX3 expression is down-regulated in vitro by antioxidants. Plasma levels of PTX3 are elevated in sepsis but seem to be unrelated to markers of oxidant stress or antioxidant capacity. | null | null | 6,611 | 369 | [
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|||
Evaluation of Muscle Mass and Stiffness with Limb Ultrasound in COVID-19 Survivors. | 35250860 | acute illnesses, like COVID-19, can act as a catabolic stimulus on muscles. So far, no study has evaluated muscle mass and quality through limb ultrasound in post-COVID-19 patients. | BACKGROUND | cross sectional observational study, including patients seen one month after hospital discharge for SARS-CoV-2 pneumonia. The patients underwent a multidimensional evaluation. Moreover, we performed dominant medial gastrocnemius ultrasound (US) to characterize their muscle mass and quality. | METHODS | two hundred fifty-nine individuals (median age 67, 59.8% males) were included in the study. COVID-19 survivors with reduced muscle strength had a lower muscle US thickness (1.6 versus 1.73 cm, p =0.02) and a higher muscle stiffness (87 versus 76.3, p = 0.004) compared to patients with normal muscle strength. Also, patients with reduced Short Physical Performance Battery (SPPB) scores had a lower muscle US thickness (1.3 versus 1.71 cm, p = 0.01) and a higher muscle stiffness (104.9 versus 81.07, p = 0.04) compared to individuals with normal SPPB scores. The finding of increased muscle stiffness was also confirmed in patients with a pathological value (≥ 4) at the sarcopenia screening tool SARC-F (103.0 versus 79.55, p < 0.001). Muscle stiffness emerged as a significant predictor of probable sarcopenia (adjusted OR 1.02, 95% C.I. 1.002 - 1.04, p = 0.03). The optimal ultrasound cut-offs for probable sarcopenia were 1.51 cm for muscle thickness (p= 0.017) and 73.95 for muscle stiffness (p = 0.004). | RESULTS | we described muscle ultrasound characteristics in post COVID-19 patients. Muscle ultrasound could be an innovative tool to assess muscle mass and quality in this population. Our preliminary findings need to be confirmed by future studies comparing muscle ultrasound with already validated techniques for measuring muscle mass and quality. | DISCUSSION | [
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] | 8892603 | Background | Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).
Acute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).
Acute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).
Skeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.
Finally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia. | null | null | Results | Two hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study.
Table 1
shows the main baseline characteristics of the study population.
Table 2
illustrates the main characteristics of their COVID-19 hospitalization.
Table 3
provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.
Baseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.
*Hand grip strength < 27 kg in men; < 16 kg in woman.
SARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).
Main characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.
*Hand grip strength < 27 kg in men; < 16 kg in woman.
SARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).
Muscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.
*Hand grip strength < 27 kg in men; < 16 kg in woman.
**data on muscle stiffness were available for only 152 patients.
SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).
The comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in
Tables 1
–
3
. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in
Figure 1
.
Number of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.
The patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04).
Figure 2
illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.
Comparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.
Osteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).
The patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.
The patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).
Table 4
shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.
Spearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.
MNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).
We detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).
Pennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).
At the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.
Cut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in
Figures 1S–3S
. | null | null | [
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"Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.",
"This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).",
"The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA)."
] | [
null,
null,
null
] | [
"Background",
"Material and Methods",
"Statistical Analyses",
"Results",
"Discussion",
"Data Availability Statement",
"Ethics Statement",
"Author Contributions",
"Funding",
"Conflict of Interest",
"Publisher’s Note"
] | [
"Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).\nAcute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).\nAcute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).\nSkeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.\nFinally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.",
"This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.\nDuring the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.\nFinally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.\nBy limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.\nDuring muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.\nImages were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.\n Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).\nThe baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).",
"The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).\nWe assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.\nProbable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.\nFinally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.\nAll statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).",
"Two hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study. \nTable 1\n shows the main baseline characteristics of the study population. \nTable 2\n illustrates the main characteristics of their COVID-19 hospitalization. \nTable 3\n provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.\nBaseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).\nMain characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\nSARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nMuscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.\n*Hand grip strength < 27 kg in men; < 16 kg in woman.\n**data on muscle stiffness were available for only 152 patients.\nSARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).\nThe comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in \nTables 1\n–\n3\n. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in \nFigure 1\n.\nNumber of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.\nThe patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04). \nFigure 2\n illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.\nComparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.\nOsteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).\nThe patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.\nThe patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).\n\n\nTable 4\n shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.\nSpearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.\nMNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).\nWe detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).\nPennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).\nAt the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.\nCut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in \nFigures 1S–3S\n.",
"In this observational study, we found that one month after hospital discharge, COVID-19 survivors with reduced muscle function displayed low muscle mass and increased muscle stiffness at the ultrasound evaluation of the dominant medial gastrocnemius as compared with those with normal muscle function. The finding of increased muscle stiffness was confirmed in patients with a pathological SARC-F score too. Moreover, we detected a significant correlation between muscle ultrasound parameters and age, nutritional status and muscle performance. Finally, we detected an association between muscle stiffness and probable sarcopenia and with the ROC analyses we identified the cut-offs of the muscle ultrasound parameters, associated with probable sarcopenia.\nOur findings refer to preliminary data, collected one month after hospital discharge. The 3 and 6 months follow ups of the patients of this study are ongoing. Indeed, it is of paramount importance to continue follow up visits over time, because it has been reported that musculoskeletal symptoms can persist 3 and 6 months after hospitalization in COVID-19 survivors (45). Assessing whether these symptoms are underpinned by alteration of muscle function, mass and quality will allow the characterization of the COVID-19 disease on muscles, and the long-term effects of acute sarcopenia, that are presently unknown (46).\nOur results on the negative correlation between both muscle thickness and age, and pennation angle and age are in line with the typical architectural remodelling of ageing (47) characterized by decreased muscle size, and reduced pennation angles (48). Indeed, muscle thickness and pennation angle are characteristics of the muscle architecture, that are strongly related one to the other, as previously demonstrated by Kubo (49), and as confirmed in our study.\nDetecting changes in muscle architecture is of extreme importance, since these alterations have an impact on the mechanical behaviour of muscles (50). Our study confirmed the presence of a significant correlation between muscle ultrasound characteristics and measures of muscle function. Moreover, we found a significant correlation between muscle ultrasound aspects and nutritional status evaluated with the MNA-SF. Malnutrition, particularly when disease-associated, is known to be associated with alterations in body composition and reductions in fat free mass (51). Both the European Society of Clinical Nutrition and Metabolism (52) and the Global Leadership Initiative on Malnutrition (53) guidelines recommend the evaluation of fat free mass as a diagnostic criterion for malnutrition. Therefore, the association of a malnutrition screening tool with impaired measures of muscle mass and quality is not surprising. Muscle ultrasound is a simple and non-expensive tool to assess skeletal muscle characteristics, and could be a valuable instrument for the screening of malnutrition. Our finding is in line with the study by Mateos-Angulo et al. (54) that detected an association between MNA-SF and muscle thickness, measured with ultrasonography, in istitutionalized older adults.\nCompared to the study of Minetto et al. (55), the median values of muscle thickness were higher in our population (1.7, IQR 1.44 - 1.93 cm in the total sample of our study versus 1.42 ± 0.03 cm in men and 1.23 ± 0. 28 cm in women in the study of Minetto). However, Minetto et al. considered a small sample (44 people) of institutionalized pre-frail and frail older adults (mean age 79.2 ± 8.3 years) whereas our study included 259 community dwelling people with a median age of 67 years (IQR 56 – 75). Our data on muscle thickness are more in line with the findings of Kubo et al. (49) who detected a mean muscle thickness of 1.93 ± 0.27 cm in community dwelling men (mean age 69.5 ± 4.2 years) and of 1.77 ± 0.23 cm in community dwelling women (mean age 68.0 ± 5.3 years).\nDifferently from Kubo, the median values of the pennation angle, were higher in our sample (22.4° versus 16.5° in men and 15.6° in women). Anyway, the measurement of the pennation angle is strongly influenced by the pressure that the sonographer exerts on the muscle, and further data are needed to define normal and pathological values of this parameter in older people.\nOur study showed that muscle stiffness is augmented in post COVID-19 patients with reduced muscle function and pathological SARC-F score, as compared with those who had normal values of muscle function and SARC-F. Since we measured muscle stiffness with muscles in a resting condition, our finding refers to passive muscle stiffness. Passive muscle stiffness is an important characteristic, since it regulates the interactions between body and environment. When muscle stiffness is too elevated, the energy of the body-environment interactions can be transmitted to the tissues, causing an injury (56). For example, in people with an elevated muscle stiffness, there is a higher risk of muscle damage after eccentric exercise (57, 58).\nPassive muscle stiffness is influenced by collagen deposition, inflammation and swelling (59–61). Previous studies showed that the amount of collagen, of advanced glycation end-products and of collagen cross-linking in connective tissue, increase with ageing (62, 63). Indeed, we found a significant correlation between muscle stiffness and age. In addition to increasing muscle stiffness [as demonstrated in aged (64) and sarcopenic muscles (65)], the alterations of the extracellular matrix may also favour muscle mass decrease. The alterations in muscle extracellular matrix can alter the regenerative potential of the myogenic progenitor cells (66). However, the exact relation between muscle stiffness and aging has not been clearly elucidated so far. While some studies demonstrated higher muscle stiffness in older people (67–70), others detected opposite results (71). Our findings are in line with the first ones.\nIn this study we identified possible muscle ultrasound parameters cut-offs, for probable sarcopenia. Muscle ultrasound is a non-invasive, little expensive and low time-consuming technique. As such, it could potentially be considered an optimal screening test for probable sarcopenia. In our study, the muscle thickness cut off for probable sarcopenia had a high specificity (76%), but a low sensitivity (41%). It is known that highly specific screening tests unlikely yield false positive results (72). Therefore, people with a pathologic muscle thickness would likely have probable sarcopenia. On the contrary, the ultrasound cut-off for muscle stiffness had a high sensitivity (77%) but a low specificity (48%). Highly sensitive screening tests unlikely generate false negative outcomes (72). Thus, people with a normal muscle stiffness would not have probable sarcopenia.\nUnfortunately, the AUC of the ROC curves were < 0.7. These results indicate that muscle ultrasound has a low accuracy in detecting probable sarcopenia, compared to the gold standard hand grip test. Anyway, these results refer to a preliminary and reduced sample, and could be improved by future wider studies. Moreover, muscle ultrasound could be used as a complementary technique to hand grip test to assess the morphologic characteristics of skeletal muscle in patients with probable sarcopenia.\nOur study has the merit of having described for the first-time muscle mass and characteristics of post COVID-19 patients with the use of limb muscle ultrasound. Description of muscle ultrasound parameters of post COVID-19 patients with impaired muscle function and pathological SARC-F score is important, since no accepted definition of muscle quality exists so far. Characterizing the changes of muscle architecture through a non-invasive and easy to use tool as echography would provide information to better define muscle quality. Finally, we identified possible cut-off values of the muscle ultrasound parameters suggestive of the risk of sarcopenia in post COVID-19 patients. It could be speculated that muscle ultrasonography may detect subjects slowly recovering from COVID-19, and with potentially negative long-term sequelae.\nSome limitations of this study deserve to be mentioned: the relatively limited sample size, the fact that some patients with dementia (in the absence of their care-givers) could have improperly answered to some questions of the SARC-F, the missing information on muscle stiffness for 107 patients, and the dependency on the ability of the operator for the evaluation of muscle mass and quality. Due to the lack of measures of muscle mass/function prior to SARS-CoV-2 infection or during hospitalization, we could not specifically address the impact of COVID-19 on skeletal muscle. However, the main aim of our study was to characterize muscle mass and quality by muscle ultrasound in a population prone to skeletal muscle impairment (19, 20), and to assess the association of ultrasound parameters with established tools for the assessment of the risk of sarcopenia. Further studies are needed to assess whether our findings can be generalized to patient populations other than COVID-19 survivors. Finally, an important limit is that we did not compare ultrasound muscle characteristics against reference methods for measuring fat free mass, such as dual-energy X-ray absorptiometry, CT or MRI. In the future, wider, multicenter studies will help better define the role of ultrasound for the evaluation of muscle quantity and quality, and correlate these data to relevant clinical outcomes.\nIn conclusion, we showed that muscle ultrasound parameters have a significant correlation with age, nutritional status and muscle performance in COVID-19 survivors. Although our findings need to be confirmed by studies comparing muscle ultrasound against validated techniques for measuring muscle mass and quality, our study suggests, for the first time, that muscle ultrasound could be an innovative tool to assess muscle mass and quality in COVID-19 survivors.",
"The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.",
"The studies involving human participants were reviewed and approved by San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). The patients/participants provided their written informed consent to participate in this study.",
"All authors made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted.",
"This study was financially supported by Ministero della Salute, Italy, and by COVID-19 donations.",
"The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.",
"All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."
] | [
null,
null,
null,
"results",
"discussion",
"data-availability",
"ethics-statement",
"author-contributions",
"funding-information",
"COI-statement",
"disclaimer"
] | [
"muscle",
"ultrasound",
"sarcopenia",
"COVID - 19",
"muscle mass",
"muscle quality"
] | Background:
Sarcopenia is a progressive and generalized skeletal muscle disorder, characterized by an accelerated loss of muscle mass and function, which is associated with an increased likelihood of developing adverse outcomes (1–6). Decline in muscle mass is not homogenous across different body anatomic regions, as sarcopenia occurs earlier in the lower limbs (7). Moreover, muscle quality, which is precociously impaired in sarcopenia (2), has an important impact on muscle function (8) and clinical outcomes (9), independently from muscle mass reduction. Muscle quality is determined by micro- and macroscopic changes in muscle architecture and composition (8, 10). In research settings, magnetic resonance imaging (MRI) and computed tomography (CT) have been used to study muscle quality, by assessing fat infiltration into muscle, and evaluating muscle attenuation (11, 12). On the other hand, muscle quality has also been defined in functional terms, as the muscle strength delivered per unit of muscle mass (13, 14) or volume (15). Because of its effects on muscle performance and clinical outcomes, muscle quality should always be considered in the assessment of sarcopenic subjects. Unfortunately, there has not been universal consensus yet on which method should be used for the evaluation of muscle quality in the routine clinical practice (1).
Acute illnesses, like COVID-19, can act as a catabolic stimulus on muscles (16, 17). Indeed, weight loss has been reported to be pronounced in COVID-19 patients (17, 18) who are at high risk of developing acute sarcopenia (19, 20). However, the degree of muscle mass and functional loss depends on multiple factors: preexisting conditions (i.e. age, frailty, comorbidities), the degree of inflammatory response to the SARS-CoV-2 infection, anorexia, inadequate protein supply, and physical inactivity during the active phase of the COVID-19 disease (21). Old, comorbid and frail patients are at higher risk of developing acute sarcopenia, even in the presence of a mild COVID-19 disease. Anyway, acute sarcopenia can occur in previously robust individuals too (16, 22, 23).
Acute sarcopenia augments patients’ vulnerability to stressors (24, 25), increasing their risk of developing adverse outcomes. Moreover, acute sarcopenia can evolve into chronic sarcopenia (26), a condition closely related to frailty (24).
Skeletal muscle ultrasound is an accurate imaging technique (27) for evaluating muscle architecture, and for quantifying muscle mass, as demonstrated by its comparison with direct anatomical assessment on cadavers (28, 29) and MRI studies (30). However, its role in diagnosing sarcopenia is just speculative. None of the current definitions of sarcopenia includes muscle echography in its diagnostic algorithms (1, 31, 32), and the analysis of current medical literature highlights the absence of a standardized method for performing muscle ultrasonography, to detect sarcopenia in clinical practice (33). In addition, normative values for defining lower limb ultrasound quantity and quality are lacking.
Finally, no study has evaluated muscle mass in post COVID-19 patients, through limb ultrasound, so far. The main objective of our study was evaluating muscle mass and quality through lower limb ultrasound in a cohort of COVID-19 survivors. As secondary objectives we performed i) a correlation of the muscle ultrasound parameters with validated measures of muscle function, nutritional status and inflammatory indexes during hospital stay, ii) an assessment of the association between muscle ultrasound parameters and probable sarcopenia and iii) a definition of ultrasound parameters cut-off values associated with probable sarcopenia.
Material and Methods:
This was a cross sectional observational study. We evaluated patients attending a dedicated post-COVID-19 outpatient clinic, who were previously hospitalized for SARS-CoV-2 pneumonia in the Internal Medicine Departments of the San Raffaele University Hospital, Milan, Italy (34). The data presented in this study were collected in the visits that took place one month after hospital discharges, from the 15th April 2021 till the 15th July 2021. We are still collecting data on three- and six-months follow-ups. The present study was part of the COVID-BioB study (NCT04318366), which aimed at characterizing hospitalized COVID-19 patients, through the prospective collection of several demographic, anthropometric, clinical and laboratory variables, as previously described (35). The COVID-BioB study protocol was approved by the San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). A convenience sample size was used due to the setting of the COVID-19 pandemic.
During the follow-up visits, the patients underwent a multidimensional evaluation, consisting in: medical history, including self-reported weight loss during hospitalization, physical examination, anthropometric measurements to calculate the body mass index (BMI), before hospital stay, and one month after hospital discharge, screening for sarcopenia through the Strength, Assistance with walking, Rising from a chair, Climbing stairs, and Falls (SARC-F) questionnaire (36), assessment of muscle strength through the hand grip strength test (37), evaluation of muscle performance with the Short Physical Performance Battery (SPPB) test (38) and screening for malnutrition with the Mini Nutritional Assessment Short Form (MNA-SF) questionnaire (39). Patients suffering from dementia were generally helped by their care-givers in the compilation of the questionnaires.
Finally, all patients underwent muscle ultrasound of the dominant medial gastrocnemius, to assess muscle mass and quality. We chose to evaluate gastrocnemius muscle, because it has a pennate architecture, thus allowing the assessment of pennation angle. Pennation angle is the angle formed at the fiber insertions into deep aponeurosis in pennate muscles and it is strongly correlated to muscle mass. Pennation angle was automatically calculated by the ultrasound software after the sonographer had manually identified the angle formed between muscle fiber insertions and deep aponeurosis.
By limb ultrasound, muscle quality can be assessed either with the determination of muscle echogenicity (40, 41), or with muscle stiffness (42). However, there is no consensus on which of these parameters should be preferred, in the assessment of muscle quality by limb ultrasound. Therefore, since our radiologists had more experience in the evaluation of muscle stiffness, we chose to investigate this aspect of muscle quality.
During muscle ultrasound, the patients laid prone on the examination couch, with the foot positioned perpendicularly to the tibia outside the couch. The ultrasound examinations were performed by two trained sonographers (SD and MC). To improve acoustic coupling, abundant water-soluble transmission gel was used on the linear array probe (7-10 MHz, General Electric model), using B-mode. The probe was set perpendicularly to the dermal surface, to get images, including both superficial and deep aponeurosis, and with an orientation coinciding with that of the muscle fascicles between the aponeuroses. Images were obtained along the mid-sagittal line of the medial gastrocnemius at the mid-distance between its proximal and distal tendon insertions (43, 44). Depth was initially set at 30 mm, then it was modified during the examination (range: 30-60 mm) to visualize the entire muscle thickness. Resting Euclidean distance between the internal borders of the superficial and deep aponeuroses (i.e. muscle thickness) was assessed in three points of the muscle, equally spaced along the image, and a mean value was calculated. In addition, the angle between the fascicle and the deep aponeurosis (i.e. pennation angle) was calculated.
Images were stored as DICOM files, and transferred to a computer for processing. Muscle stiffness was measured by means of an AGFA Enterprise Imaging program. The size of regions of interest (ROI), to estimate the stiffness index (SI), was set between 0.2 and 0.3 cm². ROI were measured in three points of the medial gastrocnemius, equally spaced along the images. Then a mean value of the three SI obtained, was calculated.
Statistical Analyses The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).
We assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.
Probable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.
Finally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.
All statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).
The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).
We assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.
Probable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.
Finally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.
All statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).
Statistical Analyses:
The baseline characteristics of the study population, the main aspects of the COVID-19 hospitalization, muscle and nutritional parameters one month after hospital discharge were described through descriptive statistics. Continuous variables were presented as mean and standard deviation (SD), when normally distributed, or with median and interquartile range (IQR), when data had a skewed distribution. Dichotomous variables were presented as number (N) and percentage (%). In addition, we performed a comparison of the distribution of categorical and continuous variables among patients with reduced (SPPB ≤8) versus normal (SPPB >8) muscle performance, reduced (Hand Grip Strength < 27 kg in men or < 16 kg in women) versus normal (Hand Grip Strength ≥ 27 kg in men or ≥ 16 kg in women) muscle strength and pathological (SARC-F ≥ 4) or normal (SARC-F < 4) values of the sarcopenia screening tool. Comparisons were made with the chi-squared test for categorical variables, and with the Mann-Whitney U test, for continuous variables. Cut-off values for muscle performance and strength were chosen, according to the European Working Group on Sarcopenia Guidelines (1) and to literature data (36).
We assessed the correlations between muscle ultrasound characteristics (muscle thickness, pennation angle and muscle stiffness), measures of muscle function (SPPB and Hand Grip Strength), nutritional status (MNA-SF), age, SARC-F, inflammatory indexes [highest C Reactive Protein (CRP) and number of days with CRP above the upper normal limit, highest ferritin during hospital stay, highest white blood cells during hospital stay (WBC)] and length of hospital stay through Spearman correlations.
Probable sarcopenia was defined as a reduced muscle strength at the hand grip test, accordingly to the European Working Group on Sarcopenia Guidelines (1). Binary logistic regression analyses were used, to assess the association between muscle ultrasound parameters, and probable sarcopenia. Unadjusted and stepwise adjusted models were performed. Collinearity tests were run before performing the adjusted model; collinear variables were excluded from the multivariable model, and just one proxy of the severity of SARS-CoV-2 infection (Non-Invasive Mechanical Ventilation) and of clinical complexity before hospital admission (number of chronic therapies) were inserted in the model, in addition to the significant predictors at the univariable analyses.
Finally, we performed ROC analyses, to identify optimal cut-off values of muscle ultrasound characteristics (thickness, stiffness and pennation angle) associated with probable sarcopenia. Reduced muscle strength (defined as a Hand Grip Strength < 27 kg in men or < 16 kg in women) was used as state variable. The Area under the curve (AUC) was calculated as a summary of diagnostic accuracy. Maximum value of the Youden’s index was used for selecting the optimum cut-off points.
All statistical analyses were performed with SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).
Results:
Two hundred and fifty-nine patients seen at a dedicated post-COVID-19 outpatient clinic, were included in the study.
Table 1
shows the main baseline characteristics of the study population.
Table 2
illustrates the main characteristics of their COVID-19 hospitalization.
Table 3
provides information on patients’ muscle and nutritional characteristics, one month after hospital discharge.
Baseline characteristics of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool,muscle strength and performance.
*Hand grip strength < 27 kg in men; < 16 kg in woman.
SARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; CKD, Chronic Kidney Disease; TIA, Transient Ischemic attack; COPD, Chronic Obstructive Pulmonary Disease. Bold = statistically significant (p < 0.05).
Main characteristics of the COVID-19 hospitalization of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.
*Hand grip strength < 27 kg in men; < 16 kg in woman.
SARC-F,Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; ICU, Intensive Care Unit; NIV, Non Invasive Ventilation; CRP, C Reactive Protein; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).
Muscle and nutritional characteristics one month after hospital discharge of the study population and their comparison among groups with pathologic and normal values of a sarcopenia screening tool, muscle strength and performance.
*Hand grip strength < 27 kg in men; < 16 kg in woman.
**data on muscle stiffness were available for only 152 patients.
SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery; IQR, Inter Quartile Range; BMI, Body Mass Index; MNA-SF, Mini Nutritional Assessment Short Form. Bold = statistically significant (p < 0.05).
The comparisons between patients with pathological versus normal values of muscle function and of SARC-F are shown in
Tables 1
–
3
. Many patients presented an overlap of pathological tests as illustrated by the Venn Diagram in
Figure 1
.
Number of people with a pathologic value of a sarcopenia screening tool, hand grip strength and muscle performance test. SARC-F, Screening tool for sarcopenia; SPPB, Short Physical Performance Battery.
The patients with an impaired muscle function or with a pathological SARC-F score were older and more often females, compared to individuals with a normal muscle function. They had a lower weight and a higher burden of chronic therapies, both before hospital admission and one month after hospital discharge. Furthermore, their muscle stiffness was higher. Pennation angle and weight loss during hospital stay did not differ. Muscle thickness was significantly lower in patients with reduced versus those with normal muscle strength (1.6 versus 1.73 cm, p = 0.02). This finding was confirmed in patients with reduced versus those with normal muscle performance (1.3 versus 1.71 cm, p = 0.01). Muscle stiffness was higher in patients with reduced muscle strength compared to patients with normal muscle strength (87 versus 76.3, p = 0.004). Also in patients with reduced muscle performance muscle stiffness was higher compared to patients with normal muscle performance (104.9 versus 81.07, p = 0.04).
Figure 2
illustrates the muscle US images of a patient with reduced muscle strength (i.e. probable sarcopenia) and of a patient with normal muscle strength.
Comparison of the limb ultrasound images of a patient with (A) and without probable sarcopenia (B). (A) Muscle thickness: 1.54 cm, muscle stiffness 127 (B) Muscle thickness: 1.81 cm, muscle stiffness 46.8. RED DOTTED ARROW: muscle thickness; ORANGE ARROW: muscle deep aponeurosis; BLU CIRCLE LINE: pennation angle; GREEN line: muscle fascicle length.
Osteoporosis (p < 0.001), dementia (p = 0.005) and vitamin D deficiency (p = 0.008) were more prevalent in people with a high SARC-F score, as compared to those with normal values. Moreover, the patients with a high SARC-F score had higher serum ferritin during hospital stay (p = 0.03), and worse nutritional status, one month after hospital discharge (MNA-SF: 8 versus 9, p = 0.007).
The patients with probable sarcopenia were more frequently affected by hypertension (p= 0.04), diabetes (p= 0.008), stroke or previous transient ischemic attack (p= 0.047), chronic obstructive pulmonary disease (COPD) (p = 0.04), neurological diseases, different from dementia (p = 0.01) and vitamin D deficiency (p = 0.03). Moreover, admission to the Intensive Care Unit was more frequent (p= 0.04), and WBC (p = 0.01) and days of C Reactive Protein (CRP) above the upper normal limit during hospitalization (p < 0.001) were higher in patients with probable sarcopenia.
The patients with a pathological muscle performance were more comorbid than patients with normal muscle performance. In particular, they were more frequently affected by hypertension (p= 0.03), ischemic heart disease (p=0.03), arrhythmia (p = 0.003), COPD (p= 0.01), chronic anaemia (p = 0.03), dementia (p = 0.006) and other neurological (p = 0.002) and psychiatric diseases (p = 0.048). In addition, during hospitalization, they had CRP levels above the upper normal limit, for a longer time (p = 0.03).
Table 4
shows the results of the Spearman correlations among muscle ultrasound characteristics, and the measures of muscle function and nutritional status, age, inflammatory indexes, and length of hospital stay.
Spearman correlations among muscle ultrasound characteristics, measures of muscle function and nutritional status, age, inflammatory indexes and length of hospital stay.
MNA-SF, Mini Nutritional Assessment-Short Form; SPPB, Short Physical Performance Battery; SARC-F, Screening tool for sarcopenia; WBC, White Blood Cells. Bold = statistically significant (p < 0.05).
We detected significant direct correlations, between muscle thickness and pennation angle (p 0.46, p < 0.001), MNA-SF (p 0.16, p = 0.01), grip strength (p 0.32, p < 0.001), SPPB (p 0.2, p = 0.001) and ferritin (p 0.16, p = 0.01) and an inverse correlation of muscle thickness with SARC-F (p -0.23, p < 0.001) and with age (p -0.35, p < 0.001).
Pennation angle had a direct correlation with nutritional status (p 0.17, p = 0.008), and with muscle function (Hand Grip Strength: p 0.16, p = 0.01; SPPB: p 0.13, p = 0.03) and an inverse correlation with SARC-F (p - 0.19, p = 0.005) and with age (p -0.21, p = 0.001). Instead, muscle stiffness showed a direct correlation with age (p 0.22, p = 0.007) and with SARC-F (p 0.3, p < 0.001). Muscle stiffness had an inverse correlation with grip strength (p -0.26, p = 0.001), and with MNA-SF (p -0.26, p = 0.002).
At the univariable binary logistic regression model age (OR 1.07, 95% C.I. 1.04 – 1.09, p < 0.001), the number of chronic therapies at hospital admission (OR 1.24, 95% C.I. 1.13 – 1.35, p < 0.001), length of hospital stay (OR 1.07, 95% C.I. 1.04 – 1.1, p < 0.001), ICU stay (2.61, 95% C.I. 1.03 – 6.63, p = 0.04), NIV use during hospital stay (OR 2.09, 95% C.I. 1.18 – 3.70, p = 0.01), days of CRP above the upper normal limit (OR 1.08, 95% C.I. 1.04 – 1.11, p < 0.001) and muscle stiffness (OR 1.02, 95% C.I. 1.01 – 1.04, p = 0.003) resulted significant predictors of probable sarcopenia. Muscle stiffness (adjusted OR 1.02, 95% C.I. 1.002 – 1.04, p = 0.03) confirmed to be a significant predictor of probable sarcopenia, in the stepwise multivariable model adjusted for age, sex, NIV, and number of chronic therapies.
Cut-offs for probable sarcopenia identified by the ROC analyses were as follows: 1.51 cm for muscle thickness (AUC 0.59, 95% C.I. 0.52 - 0.66 p = 0.017, sensitivity 41%, specificity 76%) and of 73.95 for muscle stiffness (AUC 0.64, 95% C.I. 0.55 – 0.73, p = 0.004, sensitivity 77%, specificity 48%). The results for pennation angle were not statistically significant (AUC 0.55, 95% C.I. 0.48 – 0.62, p = 0.15). ROC curves are illustrated in
Figures 1S–3S
.
Discussion:
In this observational study, we found that one month after hospital discharge, COVID-19 survivors with reduced muscle function displayed low muscle mass and increased muscle stiffness at the ultrasound evaluation of the dominant medial gastrocnemius as compared with those with normal muscle function. The finding of increased muscle stiffness was confirmed in patients with a pathological SARC-F score too. Moreover, we detected a significant correlation between muscle ultrasound parameters and age, nutritional status and muscle performance. Finally, we detected an association between muscle stiffness and probable sarcopenia and with the ROC analyses we identified the cut-offs of the muscle ultrasound parameters, associated with probable sarcopenia.
Our findings refer to preliminary data, collected one month after hospital discharge. The 3 and 6 months follow ups of the patients of this study are ongoing. Indeed, it is of paramount importance to continue follow up visits over time, because it has been reported that musculoskeletal symptoms can persist 3 and 6 months after hospitalization in COVID-19 survivors (45). Assessing whether these symptoms are underpinned by alteration of muscle function, mass and quality will allow the characterization of the COVID-19 disease on muscles, and the long-term effects of acute sarcopenia, that are presently unknown (46).
Our results on the negative correlation between both muscle thickness and age, and pennation angle and age are in line with the typical architectural remodelling of ageing (47) characterized by decreased muscle size, and reduced pennation angles (48). Indeed, muscle thickness and pennation angle are characteristics of the muscle architecture, that are strongly related one to the other, as previously demonstrated by Kubo (49), and as confirmed in our study.
Detecting changes in muscle architecture is of extreme importance, since these alterations have an impact on the mechanical behaviour of muscles (50). Our study confirmed the presence of a significant correlation between muscle ultrasound characteristics and measures of muscle function. Moreover, we found a significant correlation between muscle ultrasound aspects and nutritional status evaluated with the MNA-SF. Malnutrition, particularly when disease-associated, is known to be associated with alterations in body composition and reductions in fat free mass (51). Both the European Society of Clinical Nutrition and Metabolism (52) and the Global Leadership Initiative on Malnutrition (53) guidelines recommend the evaluation of fat free mass as a diagnostic criterion for malnutrition. Therefore, the association of a malnutrition screening tool with impaired measures of muscle mass and quality is not surprising. Muscle ultrasound is a simple and non-expensive tool to assess skeletal muscle characteristics, and could be a valuable instrument for the screening of malnutrition. Our finding is in line with the study by Mateos-Angulo et al. (54) that detected an association between MNA-SF and muscle thickness, measured with ultrasonography, in istitutionalized older adults.
Compared to the study of Minetto et al. (55), the median values of muscle thickness were higher in our population (1.7, IQR 1.44 - 1.93 cm in the total sample of our study versus 1.42 ± 0.03 cm in men and 1.23 ± 0. 28 cm in women in the study of Minetto). However, Minetto et al. considered a small sample (44 people) of institutionalized pre-frail and frail older adults (mean age 79.2 ± 8.3 years) whereas our study included 259 community dwelling people with a median age of 67 years (IQR 56 – 75). Our data on muscle thickness are more in line with the findings of Kubo et al. (49) who detected a mean muscle thickness of 1.93 ± 0.27 cm in community dwelling men (mean age 69.5 ± 4.2 years) and of 1.77 ± 0.23 cm in community dwelling women (mean age 68.0 ± 5.3 years).
Differently from Kubo, the median values of the pennation angle, were higher in our sample (22.4° versus 16.5° in men and 15.6° in women). Anyway, the measurement of the pennation angle is strongly influenced by the pressure that the sonographer exerts on the muscle, and further data are needed to define normal and pathological values of this parameter in older people.
Our study showed that muscle stiffness is augmented in post COVID-19 patients with reduced muscle function and pathological SARC-F score, as compared with those who had normal values of muscle function and SARC-F. Since we measured muscle stiffness with muscles in a resting condition, our finding refers to passive muscle stiffness. Passive muscle stiffness is an important characteristic, since it regulates the interactions between body and environment. When muscle stiffness is too elevated, the energy of the body-environment interactions can be transmitted to the tissues, causing an injury (56). For example, in people with an elevated muscle stiffness, there is a higher risk of muscle damage after eccentric exercise (57, 58).
Passive muscle stiffness is influenced by collagen deposition, inflammation and swelling (59–61). Previous studies showed that the amount of collagen, of advanced glycation end-products and of collagen cross-linking in connective tissue, increase with ageing (62, 63). Indeed, we found a significant correlation between muscle stiffness and age. In addition to increasing muscle stiffness [as demonstrated in aged (64) and sarcopenic muscles (65)], the alterations of the extracellular matrix may also favour muscle mass decrease. The alterations in muscle extracellular matrix can alter the regenerative potential of the myogenic progenitor cells (66). However, the exact relation between muscle stiffness and aging has not been clearly elucidated so far. While some studies demonstrated higher muscle stiffness in older people (67–70), others detected opposite results (71). Our findings are in line with the first ones.
In this study we identified possible muscle ultrasound parameters cut-offs, for probable sarcopenia. Muscle ultrasound is a non-invasive, little expensive and low time-consuming technique. As such, it could potentially be considered an optimal screening test for probable sarcopenia. In our study, the muscle thickness cut off for probable sarcopenia had a high specificity (76%), but a low sensitivity (41%). It is known that highly specific screening tests unlikely yield false positive results (72). Therefore, people with a pathologic muscle thickness would likely have probable sarcopenia. On the contrary, the ultrasound cut-off for muscle stiffness had a high sensitivity (77%) but a low specificity (48%). Highly sensitive screening tests unlikely generate false negative outcomes (72). Thus, people with a normal muscle stiffness would not have probable sarcopenia.
Unfortunately, the AUC of the ROC curves were < 0.7. These results indicate that muscle ultrasound has a low accuracy in detecting probable sarcopenia, compared to the gold standard hand grip test. Anyway, these results refer to a preliminary and reduced sample, and could be improved by future wider studies. Moreover, muscle ultrasound could be used as a complementary technique to hand grip test to assess the morphologic characteristics of skeletal muscle in patients with probable sarcopenia.
Our study has the merit of having described for the first-time muscle mass and characteristics of post COVID-19 patients with the use of limb muscle ultrasound. Description of muscle ultrasound parameters of post COVID-19 patients with impaired muscle function and pathological SARC-F score is important, since no accepted definition of muscle quality exists so far. Characterizing the changes of muscle architecture through a non-invasive and easy to use tool as echography would provide information to better define muscle quality. Finally, we identified possible cut-off values of the muscle ultrasound parameters suggestive of the risk of sarcopenia in post COVID-19 patients. It could be speculated that muscle ultrasonography may detect subjects slowly recovering from COVID-19, and with potentially negative long-term sequelae.
Some limitations of this study deserve to be mentioned: the relatively limited sample size, the fact that some patients with dementia (in the absence of their care-givers) could have improperly answered to some questions of the SARC-F, the missing information on muscle stiffness for 107 patients, and the dependency on the ability of the operator for the evaluation of muscle mass and quality. Due to the lack of measures of muscle mass/function prior to SARS-CoV-2 infection or during hospitalization, we could not specifically address the impact of COVID-19 on skeletal muscle. However, the main aim of our study was to characterize muscle mass and quality by muscle ultrasound in a population prone to skeletal muscle impairment (19, 20), and to assess the association of ultrasound parameters with established tools for the assessment of the risk of sarcopenia. Further studies are needed to assess whether our findings can be generalized to patient populations other than COVID-19 survivors. Finally, an important limit is that we did not compare ultrasound muscle characteristics against reference methods for measuring fat free mass, such as dual-energy X-ray absorptiometry, CT or MRI. In the future, wider, multicenter studies will help better define the role of ultrasound for the evaluation of muscle quantity and quality, and correlate these data to relevant clinical outcomes.
In conclusion, we showed that muscle ultrasound parameters have a significant correlation with age, nutritional status and muscle performance in COVID-19 survivors. Although our findings need to be confirmed by studies comparing muscle ultrasound against validated techniques for measuring muscle mass and quality, our study suggests, for the first time, that muscle ultrasound could be an innovative tool to assess muscle mass and quality in COVID-19 survivors.
Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics Statement:
The studies involving human participants were reviewed and approved by San Raffaele University Hospital Ethics Committee (protocol no. 34/int/2020). The patients/participants provided their written informed consent to participate in this study.
Author Contributions:
All authors made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted.
Funding:
This study was financially supported by Ministero della Salute, Italy, and by COVID-19 donations.
Conflict of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s Note:
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
| Background:
acute illnesses, like COVID-19, can act as a catabolic stimulus on muscles. So far, no study has evaluated muscle mass and quality through limb ultrasound in post-COVID-19 patients.
Methods:
cross sectional observational study, including patients seen one month after hospital discharge for SARS-CoV-2 pneumonia. The patients underwent a multidimensional evaluation. Moreover, we performed dominant medial gastrocnemius ultrasound (US) to characterize their muscle mass and quality.
Results:
two hundred fifty-nine individuals (median age 67, 59.8% males) were included in the study. COVID-19 survivors with reduced muscle strength had a lower muscle US thickness (1.6 versus 1.73 cm, p =0.02) and a higher muscle stiffness (87 versus 76.3, p = 0.004) compared to patients with normal muscle strength. Also, patients with reduced Short Physical Performance Battery (SPPB) scores had a lower muscle US thickness (1.3 versus 1.71 cm, p = 0.01) and a higher muscle stiffness (104.9 versus 81.07, p = 0.04) compared to individuals with normal SPPB scores. The finding of increased muscle stiffness was also confirmed in patients with a pathological value (≥ 4) at the sarcopenia screening tool SARC-F (103.0 versus 79.55, p < 0.001). Muscle stiffness emerged as a significant predictor of probable sarcopenia (adjusted OR 1.02, 95% C.I. 1.002 - 1.04, p = 0.03). The optimal ultrasound cut-offs for probable sarcopenia were 1.51 cm for muscle thickness (p= 0.017) and 73.95 for muscle stiffness (p = 0.004).
Conclusions:
we described muscle ultrasound characteristics in post COVID-19 patients. Muscle ultrasound could be an innovative tool to assess muscle mass and quality in this population. Our preliminary findings need to be confirmed by future studies comparing muscle ultrasound with already validated techniques for measuring muscle mass and quality. | null | null | 7,069 | 356 | [
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"sarcopenic muscles",
"sarcopenia muscle stiffness",
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"muscle quality determined",
"sarcopenia reduced muscle"
] | null | null | null | [CONTENT] muscle | ultrasound | sarcopenia | COVID - 19 | muscle mass | muscle quality [SUMMARY] | null | [CONTENT] muscle | ultrasound | sarcopenia | COVID - 19 | muscle mass | muscle quality [SUMMARY] | null | [CONTENT] muscle | ultrasound | sarcopenia | COVID - 19 | muscle mass | muscle quality [SUMMARY] | null | [CONTENT] Aged | Aged, 80 and over | COVID-19 | Cross-Sectional Studies | Extremities | Female | Humans | Italy | Male | Middle Aged | Muscle Strength | Muscle, Skeletal | Muscular Diseases | Organ Size | SARS-CoV-2 | Sarcopenia | Survivors | Ultrasonography [SUMMARY] | null | [CONTENT] Aged | Aged, 80 and over | COVID-19 | Cross-Sectional Studies | Extremities | Female | Humans | Italy | Male | Middle Aged | Muscle Strength | Muscle, Skeletal | Muscular Diseases | Organ Size | SARS-CoV-2 | Sarcopenia | Survivors | Ultrasonography [SUMMARY] | null | [CONTENT] Aged | Aged, 80 and over | COVID-19 | Cross-Sectional Studies | Extremities | Female | Humans | Italy | Male | Middle Aged | Muscle Strength | Muscle, Skeletal | Muscular Diseases | Organ Size | SARS-CoV-2 | Sarcopenia | Survivors | Ultrasonography [SUMMARY] | null | [CONTENT] sarcopenic muscles | sarcopenia muscle stiffness | muscle quality assessing | muscle quality determined | sarcopenia reduced muscle [SUMMARY] | null | [CONTENT] sarcopenic muscles | sarcopenia muscle stiffness | muscle quality assessing | muscle quality determined | sarcopenia reduced muscle [SUMMARY] | null | [CONTENT] sarcopenic muscles | sarcopenia muscle stiffness | muscle quality assessing | muscle quality determined | sarcopenia reduced muscle [SUMMARY] | null | [CONTENT] muscle | sarcopenia | ultrasound | strength | patients | stiffness | hospital | study | muscle stiffness | muscle ultrasound [SUMMARY] | null | [CONTENT] muscle | sarcopenia | ultrasound | strength | patients | stiffness | hospital | study | muscle stiffness | muscle ultrasound [SUMMARY] | null | [CONTENT] muscle | sarcopenia | ultrasound | strength | patients | stiffness | hospital | study | muscle stiffness | muscle ultrasound [SUMMARY] | null | [CONTENT] muscle | sarcopenia | muscle mass | quality | mass | acute | muscle quality | acute sarcopenia | ultrasound | developing [SUMMARY] | null | [CONTENT] muscle | 001 | 95 | strength | normal | 04 | sarcopenia | patients | 01 | performance [SUMMARY] | null | [CONTENT] muscle | sarcopenia | strength | ultrasound | article | authors | study | patients | hospital | 19 [SUMMARY] | null | [CONTENT] COVID-19 ||| [SUMMARY] | null | [CONTENT] two hundred fifty-nine | age 67 | 59.8% ||| COVID-19 | US | 1.6 | 1.73 cm | 0.02 | 87 | 76.3 | 0.004 ||| Short Physical Performance Battery | SPPB | US | 1.3 | 1.71 cm | 0.01 | 104.9 | 81.07 | 0.04 | SPPB ||| ≥ | 4 | 103.0 | 79.55 | p < 0.001 ||| 1.02 | 95% | C.I. | 1.002 - 1.04 | 0.03 ||| 1.51 cm | 0.017 | 73.95 | 0.004 [SUMMARY] | null | [CONTENT] COVID-19 ||| ||| one month ||| ||| US ||| two hundred fifty-nine | age 67 | 59.8% ||| COVID-19 | US | 1.6 | 1.73 cm | 0.02 | 87 | 76.3 | 0.004 ||| Short Physical Performance Battery | SPPB | US | 1.3 | 1.71 cm | 0.01 | 104.9 | 81.07 | 0.04 | SPPB ||| ≥ | 4 | 103.0 | 79.55 | p < 0.001 ||| 1.02 | 95% | C.I. | 1.002 - 1.04 | 0.03 ||| 1.51 cm | 0.017 | 73.95 | 0.004 ||| COVID-19 ||| ||| [SUMMARY] | null |
|||
Medical residents reflect on their prejudices toward poverty: a photovoice training project. | 25551370 | Clinicians face challenges in delivering care to socioeconomically disadvantaged patients. While both the public and academic sectors recognize the importance of addressing social inequities in healthcare, there is room for improvement in the training of family physicians, who report being ill-equipped to provide care that is responsive to the living conditions of these patients. This study explored: (i) residents' perceptions and experience in relation to providing care for socioeconomically disadvantaged patients, and (ii) how participating in a photovoice study helped them uncover and examine some of their prejudices and assumptions about poverty. | BACKGROUND | We conducted a participatory photovoice study. Participants were four family medicine residents, two medical supervisors, and two researchers. Residents attended six photovoice meetings at which they discussed photos they had taken. In collaboration with the researchers, the participants defined the research questions, took photos, and participated in data analysis and results dissemination. Meetings were recorded and transcribed for analysis, which consisted of coding, peer debriefing, thematic analysis, and interpretation. | METHODS | The medical residents uncovered and examined their own prejudices and misconceptions about poverty. They reported feeling unprepared to provide care to socioeconomically disadvantaged patients. Supported by medical supervisors and researchers, the residents underwent a three-phase reflexive process of: (1) engaging reflexively, (2) break(ing) through, and (3) taking action. The results indicated that medical residents subsequently felt encouraged to adopt a care approach that helped them overcome the social distance between themselves and their socioeconomically disadvantaged patients. | RESULTS | This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients. | CONCLUSIONS | [
"Attitude of Health Personnel",
"Canada",
"Family Practice",
"Humans",
"Internship and Residency",
"Poverty",
"Prejudice",
"Students, Medical"
] | 4323214 | Background | Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].
Clinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].
Physicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].
Hence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].
Photovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].
This participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty. | null | null | Results | The medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.
Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.
Defining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
The discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.
The photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.
The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.
Defining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
The discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.
The photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.
Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.
It was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.
The first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
The professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.
The changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.
It was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.
The first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
The professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.
The changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.
Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents. | Conclusions | This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients. | [
"Study design",
"Recruitment and participants",
"Design and procedure",
"Data analysis",
"Phase1: engaging reflexively with poverty",
"Phase 2: break(ing)through",
"Phase 3: taking action"
] | [
"This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].",
"The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.",
"We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.",
"All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.",
"The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.",
"Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.",
"Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents."
] | [
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Study design",
"Recruitment and participants",
"Design and procedure",
"Data analysis",
"Results",
"Phase1: engaging reflexively with poverty",
"Phase 2: break(ing)through",
"Phase 3: taking action",
"Discussion",
"Conclusions"
] | [
"Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].\nClinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].\nPhysicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].\nHence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].\nPhotovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].\nThis participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty.",
" Study design This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\nThis study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].\n Recruitment and participants The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\nThe first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.\n Design and procedure We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\nWe conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.\n Data analysis All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.\nAll meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.",
"This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].",
"The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.\nFour family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.",
"We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.\nIn the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.\nThe second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.\nIn the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.\nThe researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.\nAt the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.",
"All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.",
"The medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.\n Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\nThe process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.\n Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nZigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\n Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.\nBeing reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.",
"The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.\nDefining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThis is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.\nThe discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.\nThe photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.",
"Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.\nIt was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.\nThe first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\n… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.\nThe professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.\nThe changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.\nThere is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.",
"Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.",
"This study explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons. It also examined how residents’ involvement in a photovoice project helped them uncover their prejudices and assumptions concerning poverty. In this project, residents underwent a three-phase learning process of: (1) engaging reflexively; (2) break(ing)through; and (3) taking action. Sharing experiences involved lengthy conversations in which participants responded to photos presented by others, made connections with other participants’ comments, and highlighted differences and similarities. In fact, most “individual” photos became “group” photos as they were appropriated by each of the participants, who were stimulated by the photos to reflect on their own concerns.\nAs in other photovoice projects, this research generated a reflexive process that led to sustained critical consciousness and constituted “transformative learning” [27,28]. As noted by Sandars [29], the medical residents reported undergoing transformations in their perceptions of themselves and of the world triggered by a process of introspection during which feelings of powerlessness, sadness, shame, and anger emerged.\nWe believe the medical residents’ experience with the photovoice project encouraged them to take poverty into serious consideration in their practice. The entire photovoice project, encompassing three phases, was a learning process for all participants. Residents’ concrete actions in direct response to the project could be seen as evidence that it was an effective teaching tool. As reported by Carlson, Engebretson, and Chamberlain [17], photovoice can be a means for expanding social consciousness and triggering social change, because its use fosters both reflection and action.\nWe acknowledge that the research was limited by the small number of participants. Small sample size in photovoice research initiatives is not uncommon because of the time requirements and the nature of the participants’ involvement. For example, in their oft-cited article on African-American mothers, Killion & Wang [30] had five participants. Another limitation of our study relates to the fact that the medical supervisors fulfilled multiple roles with the medical residents. A posteriori we considered it to be an asset for our data collection, because the supervisors contributed valuable input to the photovoice meetings. However, we observed that power relations could arise among the participants because the supervisors were involved in the residents’ evaluations.\nBy involving both medical residents and supervisors in this photovoice study, we made one of the first attempts in a medical academic milieu to involve healthcare professionals in identifying ways to improve physicians’ competencies in delivering care to socioeconomically disadvantaged patients. The study and its outcomes also generated momentum for changes to the family medicine curriculum at a teaching university. Thus, to improve the knowledge and skills of future primary care doctors and incorporate a “social competence” component into residency programs, we recommend developing an experiential learning approach by giving residents opportunities to be involved in underserved community activities and in reflexive discussion with medical supervisors.\nIn conclusion, photovoice appears to be a promising and innovative teaching approach in medical education, especially for medical residents. More specifically, our participatory research project helped future physicians become aware of their prejudices and motivated them to acquire skills for delivering healthcare to socioeconomically disadvantaged patients. Photovoice can be an effective tool for raising health professionals’ awareness of socioeconomic realities of their patients.",
"This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients."
] | [
"introduction",
"materials|methods",
null,
null,
null,
null,
"results",
null,
null,
null,
"discussion",
"conclusions"
] | [
"Healthcare disparities",
"Poverty",
"Participatory action research",
"Photovoice",
"Residents",
"Education",
"Healthcare professionals",
"Medicine"
] | Background:
Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].
Clinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].
Physicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].
Hence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].
Photovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].
This participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty.
Methods:
Study design This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].
This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].
Recruitment and participants The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.
Four family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.
The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.
Four family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.
Design and procedure We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.
In the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.
The second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.
In the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.
The researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.
At the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.
We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.
In the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.
The second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.
In the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.
The researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.
At the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.
Data analysis All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.
All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.
Study design:
This study was part of a larger research program aimed at identifying family physicians’ competencies in providing care to socioeconomically disadvantaged patients in Quebec, Canada. Photovoice was first developed by Wang and Burris [22] in the 1990s among women in China’s Yunnan Province. The methodology has been applied to various populations since then, including women, children, immigrants, and the elderly, in different parts of the world, including the United States and Canada. There are several examples of projects that use photography to enhance dialogue and social change in the healthcare field [23]. According to Wang and Burris [24] reflexive discussion of photographs facilitates a critical dialogue about specific issues of concern. Photovoice is a participatory action research approach because it is aimed at generating concrete changes that may involve policy-makers and larger sectors of the population [22].
Recruitment and participants:
The first author of the present article is a sociologist and qualitative health researcher who supervises medical residents in their scientific research training. Medical supervisors in the university’s academic primary care unit invited the researcher to present her research projects and interests to medical residents. She briefly presented her research program to the cohort of medical residents (approximately 25 residents) and invited them to explore, under her supervision, a specific research question regarding family medicine practice and poverty.
Four family medicine residents expressed interest in participating in a research project with her and her postdoctoral fellow. The two researchers proposed to collaborate with the medical residents and their clinician supervisors on a project using photovoice. The four medical residents and two medical supervisors (one physician and one psychologist) were then recruited and agreed to participate on a voluntary basis. The medical residents were relatively similar to their counterparts in terms of ethnic and socio-economic background: they were born in Quebec and were from middle- to high-income families. Among the four, one resident was older than the others and had past professional life experience in an area other than medicine. Two social science researchers—a sociologist and an anthropologist —were also part of the photovoice group, for a total of eight participants.
Design and procedure:
We conducted this study in an academic primary care unit of the Faculty of Medicine at Sherbrooke University, Quebec, Canada. The university’s research ethics committee approved the project, and participants signed a consent form. Our study, conducted in 2010–2011, consisted of six three-hour meetings over seven months. All meetings were recorded and transcribed for analysis. After each meeting, we took field notes and researchers discussed their interpretations to deepen the understanding of the experiential learning.
In the first meeting, the researchers provided training on the photovoice methodology, related concepts, and goals. In line with the research objectives, the medical residents formulated a research question: What are the barriers between medical residents and socioeconomically disadvantaged persons? The second author, a visual anthropologist, prepared the team to use photography as a means of responding to the research question and to begin a reflexive process. All eight participants were invited to take photos, over a four-week period, that responded in some way to the research question, to foster a reflexive approach in all the participants. Participants were free to take as many photos as they wanted to, but were asked to bear in mind that they would need to choose five photos to share with the group at the next meeting. Participants could take photos anywhere, as long as they preserved the security and the anonymity of any persons they photographed on the street. A majority of the photos were taken outside, in the city where the participants were living. The photographs were very diverse: an empty fridge, a pharmacy shelf showing drug price labels, a wrecked car, a homeless shelter, etc.
The second meeting took place four weeks later, to allow participants time to reflect on the research question and take photos. At this meeting, each participant presented five photos, explaining the reasons for taking them and for sharing them with the group. After participants had shared their interpretations of their own photos, the others were invited to comment.
In the four remaining meetings, the medical residents analyzed the data using qualitative research methodology. During this analysis phase, two of them also participated on a voluntary basis in a one-day activity with an anti-poverty community organization, ATD Fourth World, which involved selecting photos that reflect happiness. They listened as socioeconomically disadvantaged persons explained why they selected certain photos and what happiness was, from their perspective. Following this activity, the participants shared critical reflections with the other photovoice participants.
The researchers trained the residents in qualitative analysis. Assisted by the researchers, the residents themselves created a coding scheme and coded the transcripts of the second meeting (at which the photos were exhibited). The analysis progressed between meetings through emails and telephone calls among researchers and residents. With the researchers’ support, the residents analyzed data, did peer debriefing, created a table synthesizing the data, and discussed interpretations with researchers.
At the end, the residents gave an oral presentation of the results to medical residents, supervisors, and managers at the Annual Research Day of the Faculty of Medicine. They received two significant awards in recognition of their work.
Data analysis:
All meetings were audio-recorded and transcribed. Data analysis consisted of data coding, peer debriefing, and validation of interpretation. The medical residents coded and analyzed the second meeting, and a visual anthropologist coded and analyzed the transcripts of all the meetings in collaboration with two researchers. The researchers validated the coding and were involved in data interpretation through reading transcripts and independently coding the data and by attending regular meetings. An interpretive content analysis framework [24] was used to interpret the data. Our aim was to identify verbal exchanges that indicated reflexivity and critical consciousness directly sparked by the photovoice project. Data analysis involved three interrelated steps: 1) repeated reading of each discussion transcript by the medical residents and the researchers to “get a sense of the whole” [25]; 2) extraction from the transcripts of storylines discussed; and 3) establishing links between storylines, which became the core of the analysis section of this article. In this study, data reliability was maintained by data triangulation, participant validation, supervision, and peer review.
Results:
The medical residents acknowledged at the outset that they had, in their words, “few tools” to respond to the situations of socioeconomically disadvantaged patients. Their involvement in the photovoice project with supervisors and researchers who study poverty improved their knowledge and raised their awareness of issues of poverty and primary care. Residents felt encouraged to consider adopting a different care approach and trying to overcome barriers between themselves and their socioeconomically disadvantaged patients. In the photovoice project, medical residents underwent a three-phase learning process that could be summarized as: (1) engaging reflexively; (2) break(ing) through; and (3) taking action. The characteristics of each phase are described below.
Phase1: engaging reflexively with poverty The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.
Defining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
The discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.
The photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.
The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.
Defining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
The discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.
The photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.
Phase 2: break(ing)through Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.
It was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.
The first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
The professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.
The changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.
It was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.
The first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
The professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.
The changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
Phase 3: taking action Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.
Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.
Phase1: engaging reflexively with poverty:
The process of reflexivity started in the first meeting. The residents expressed discomfort with scrutinizing their experience through a photovoice project (thereby becoming what they called “photovoice subjects”). They learned that photovoice projects are often conducted among vulnerable populations, and did not see themselves in that category. However, after long and intense discussion with supervisors and researchers, they admitted their lack of knowledge of how to interact with socioeconomically disadvantaged persons. They reported feelings of incompetence and powerlessness that prevented them from addressing certain issues with socioeconomically disadvantaged patients. They realized that, in that sense, they were somewhat in a position of professional vulnerability. This moment of collective awareness prompted them to commit to the photovoice process. The project also encouraged them to explore what poverty meant to each of them and how they could represent it visually.
Defining poverty from the participants’ perspectives was a key part of the process throughout, but reached a turning point in the second meeting when one participant presented her final photograph, that of a 50-year old man who had given up his medical practice:This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
This is a nice picture because this gentleman … decided to quit [his job] to live in a state of voluntary simplicity. … He was tired of being super rich and having 56,000 things to do. So, I just wanted to introduce the notion that in a very small percentage of cases, it’s possible that some people choose to live in poverty. … Poverty is just that: it is the absence of choice, the absence of means, that puts you in a particular situation. … But when you have the option to be able to get out of it, then it’s different.
The discussion around this picture and the notion of “real poverty” had a major impact on the resident’s level of sensitivity concerning what it is to experience poverty. The process of defining poverty also touched upon their own feelings and personal stories. One striking case emerged when a medical resident revealed, through her photos, that one of her close relatives with children was living in poverty. In fact, the perception of what poverty means became both a personal and a professional issue for all participants. Residents spoke of the tension between “doing something” and “being powerless” when confronted with certain aspects of poverty. They expressed their desire to provide good-quality care to socioeconomically disadvantaged patients but said they lacked tools and resources to respond to needs related to patients’ precarious economic situation.
The photovoice project allowed residents to reflect on their own prejudices and fears concerning poverty. Admitting publicly that one has prejudices is not easy, but the openness of participants who shared similar thoughts provided a safe environment for such confidences. Several forms of prejudices were uncovered during the discussions. They included the ideas that “poor people should not have leisure activities that cost money because they can’t afford it”, that socioeconomically disadvantaged patients abuse the healthcare system, that they look down on rich people and are envious of them, and that poor and rich people live in two different worlds.
Phase 2: break(ing)through:
Zigon [26] argues that moral breakdowns are significant ethical moments to consider when looking at issues of morality. As a methodological approach encouraging reflexivity, photovoice invites people to step back and take positions on certain issues and problems. As such, it has the potential to provoke moral breakdowns, or moments of reflexive questioning. Break(ing)through, or making breakthroughs, refers to moments of self-critical awareness during which one acknowledges one’s biases, prejudices, and social position in relation to others. It is destabilizing, in that it fosters introspection based on ethical principles. An ethical imperative to solve the breakdown—break(ing)through— is placed on the person or group experiencing this moment, after which, according to Zigon [26], they are able to return to an “unreflective everydayness”, a much less troublesome state of being.
It was during residents’ discussions of their photos that most of the breakthroughs generated by moral breakdowns occurred. These breakthroughs were facilitated by interactive feedback from the professionals involved in the project, which fostered two interrelated results: 1) the residents accepted that they could be photovoice subjects; and 2) they acknowledged having emotions that could be discussed openly by naming the barriers and prejudices that hindered their effectiveness with socioeconomically disadvantaged patients.
The first breakthrough occurred during the first meeting. Residents argued vehemently that they could not be the subjects of a study:… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
… It’s rare that we, doctors, are included in that category [of vulnerable people], which makes us uncomfortable with this topic. However, it is somewhat embarrassing to say that we are uncomfortable, and have grey zones and areas where we feel powerless. So … I think we become a photovoice subject, if we’re forced to face our own prejudices.
The professional team’s mentoring of the residents was a crucial part of the photovoice project. Constant probing from the supervisors brought the residents to accept that they could become the subjects of their own questioning and of a qualitative project that would not necessarily give them yes/no answers. With guidance, they were afforded time and space in which to identify and name, through photos and guided conversations, the barriers, obstacles, prejudices, and stereotypes they each had related to poverty and socioeconomically disadvantaged persons.
The changes that occurred during the process were enabled by open-ended discussions that encouraged the residents to express themselves—an experience that runs counter to their training, which teaches them to make decisions based on objective facts and rational observations. Despite initial resistance, many “breakthroughs” occurred in the residents’ conversations, mainly facilitated by the supervisors’ mentoring. They encouraged the residents to express themselves openly and to pursue their reasoning further. For instance, at the end of the project the residents all acknowledged the social distance that prevails between them and persons who live in poverty. They realized how hard it could be for persons living in poverty to deal with the biomedical language of doctors if they are not familiar with it. Also, residents recognized they may be predisposed to making negative initial judgments concerning people in poverty:There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
There is always a small voice of prejudice in the back of my head saying that a person in a situation of poverty […], I have the feeling that these are people who tend to use drugs, drink a lot of alcohol, and smoke.
Phase 3: taking action:
Being reflexive in relation to a situation was empowering for all the participants, but particularly for the medical residents. One resident reported that discussing issues of poverty with the other participants empowered her to ask one of her patients personal questions she would not have to ask before, which opened up the possibility of exploring what resources in the community were available for her patient. She directly attributed her new professional behavior to the reflexive process fostered by the photovoice project. Instead of ignoring the problem, she addressed it directly, without shouldering all the responsibility. Residents expressed feeling better equipped to serve patients with health problems caused in part by poverty. Also, after medical residents presented their data to the Faculty members, a “social competence” component was incorporated into the spectrum of competencies that medical residents should acquire during their training and a course on poverty was developed and given to medical residents.
Discussion:
This study explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons. It also examined how residents’ involvement in a photovoice project helped them uncover their prejudices and assumptions concerning poverty. In this project, residents underwent a three-phase learning process of: (1) engaging reflexively; (2) break(ing)through; and (3) taking action. Sharing experiences involved lengthy conversations in which participants responded to photos presented by others, made connections with other participants’ comments, and highlighted differences and similarities. In fact, most “individual” photos became “group” photos as they were appropriated by each of the participants, who were stimulated by the photos to reflect on their own concerns.
As in other photovoice projects, this research generated a reflexive process that led to sustained critical consciousness and constituted “transformative learning” [27,28]. As noted by Sandars [29], the medical residents reported undergoing transformations in their perceptions of themselves and of the world triggered by a process of introspection during which feelings of powerlessness, sadness, shame, and anger emerged.
We believe the medical residents’ experience with the photovoice project encouraged them to take poverty into serious consideration in their practice. The entire photovoice project, encompassing three phases, was a learning process for all participants. Residents’ concrete actions in direct response to the project could be seen as evidence that it was an effective teaching tool. As reported by Carlson, Engebretson, and Chamberlain [17], photovoice can be a means for expanding social consciousness and triggering social change, because its use fosters both reflection and action.
We acknowledge that the research was limited by the small number of participants. Small sample size in photovoice research initiatives is not uncommon because of the time requirements and the nature of the participants’ involvement. For example, in their oft-cited article on African-American mothers, Killion & Wang [30] had five participants. Another limitation of our study relates to the fact that the medical supervisors fulfilled multiple roles with the medical residents. A posteriori we considered it to be an asset for our data collection, because the supervisors contributed valuable input to the photovoice meetings. However, we observed that power relations could arise among the participants because the supervisors were involved in the residents’ evaluations.
By involving both medical residents and supervisors in this photovoice study, we made one of the first attempts in a medical academic milieu to involve healthcare professionals in identifying ways to improve physicians’ competencies in delivering care to socioeconomically disadvantaged patients. The study and its outcomes also generated momentum for changes to the family medicine curriculum at a teaching university. Thus, to improve the knowledge and skills of future primary care doctors and incorporate a “social competence” component into residency programs, we recommend developing an experiential learning approach by giving residents opportunities to be involved in underserved community activities and in reflexive discussion with medical supervisors.
In conclusion, photovoice appears to be a promising and innovative teaching approach in medical education, especially for medical residents. More specifically, our participatory research project helped future physicians become aware of their prejudices and motivated them to acquire skills for delivering healthcare to socioeconomically disadvantaged patients. Photovoice can be an effective tool for raising health professionals’ awareness of socioeconomic realities of their patients.
Conclusions:
This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients.
| Background:
Clinicians face challenges in delivering care to socioeconomically disadvantaged patients. While both the public and academic sectors recognize the importance of addressing social inequities in healthcare, there is room for improvement in the training of family physicians, who report being ill-equipped to provide care that is responsive to the living conditions of these patients. This study explored: (i) residents' perceptions and experience in relation to providing care for socioeconomically disadvantaged patients, and (ii) how participating in a photovoice study helped them uncover and examine some of their prejudices and assumptions about poverty.
Methods:
We conducted a participatory photovoice study. Participants were four family medicine residents, two medical supervisors, and two researchers. Residents attended six photovoice meetings at which they discussed photos they had taken. In collaboration with the researchers, the participants defined the research questions, took photos, and participated in data analysis and results dissemination. Meetings were recorded and transcribed for analysis, which consisted of coding, peer debriefing, thematic analysis, and interpretation.
Results:
The medical residents uncovered and examined their own prejudices and misconceptions about poverty. They reported feeling unprepared to provide care to socioeconomically disadvantaged patients. Supported by medical supervisors and researchers, the residents underwent a three-phase reflexive process of: (1) engaging reflexively, (2) break(ing) through, and (3) taking action. The results indicated that medical residents subsequently felt encouraged to adopt a care approach that helped them overcome the social distance between themselves and their socioeconomically disadvantaged patients.
Conclusions:
This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients. | Background:
Poverty and inequities of access to primary care present serious threats to health. Socioeconomically disadvantaged persons are the least well-served in terms of healthcare services [1] and are least likely to have a family physician [2]. They report more unmet health needs than do people with higher incomes [3]. They experience negative healthcare interactions and sometimes feel judged by physicians [3-6].
Clinicians may face challenges in delivering care to socioeconomically disadvantaged persons and are not well prepared to take into account the social context to create therapeutic alliances with them. Health professionals, particularly physicians, have little understanding of these patients’ social situation. Their lack of knowledge and mistaken perceptions of poverty affect the quality of clinical interactions [7,8].
Physicians tend to be more directive with socioeconomically disadvantaged patients, spend less time with them, and provide less information concerning treatment options [9]. In a study of residents in medicine, 25% thought poverty was a consequence of laziness, 50% thought the poor were more likely to abuse the healthcare system, and 50% thought the poor were less attentive to their health than the rest of the population [10]. Primary care physicians, being close to patients’ personal and day-to-day experiences, occupy an important position with major impact on people’s lives [11].
Hence, there is a need to strengthen family medicine residents’ training and better prepare them to consider the impact of poverty on health and healthcare. Unfortunately, few medical residency programs offer satisfactory and well-resourced training programs that would prepare future family physicians to cope with poverty issues in the healthcare process. There are some innovative and promising training programs, such as Oregon Health and Science University’s (OHSU) social medicine program for homeless and addicted patients that provides seminars and experiential learning in the community [12]. While medical schools’ efforts to foster humanism and/or cultural competence are diverse and not much evaluated, three elements seem to be essential in training related to poverty: high involvement of a mentor as role model, experiential learning with supportive supervision, and time for critical reflection and discussion about the learning experience [13-15].
Photovoice is a participatory action research method that uses photography to enable participants to share experiences and develop critical consciousness of a variety of topics [16,17]. There have been very few studies using photovoice with medical residents, supervisors, and researchers in a family medicine teaching context. A brief note by Wang, Anderson, and Stern [18] explaining how they used photovoice with final-year medical students is one rare case. This elective course explored professional values and health policy issues. Students photographed aspects of healthcare delivery that could be changed through policies. Three participants presented their work to policy-makers and university representatives. Leipert and Anderson [19] used photovoice with 38 nursing students to promote and recognize the value of nurses’ care delivery in Canadian rural areas. More recently, also in nursing, Garner [20] encouraged the use of photovoice for teaching and learning, arguing specifically that photovoice facilitated students’ cultural awareness and competence in geriatric nursing. Except for such rare cases, there seems to be a gap in medical education concerning the use of photovoice as a tool to foster medical residents’ awareness of discrimination of certain patient groups, such as immigrants or socioeconomically disadvantaged persons. Photovoice may help raise medical residents’ awareness and increase their skills for delivering care to patients from these groups [21].
This participatory study conducted in a primary care teaching unit in Quebec, Canada, explored residents’ perceptions and experience in relation to providing care for socioeconomically disadvantaged persons and examined how their participation in a photovoice study, including in a one-day activity for some of them with an anti-poverty community organization, helped them uncover their prejudices and assumptions about poverty.
Conclusions:
This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients. | Background:
Clinicians face challenges in delivering care to socioeconomically disadvantaged patients. While both the public and academic sectors recognize the importance of addressing social inequities in healthcare, there is room for improvement in the training of family physicians, who report being ill-equipped to provide care that is responsive to the living conditions of these patients. This study explored: (i) residents' perceptions and experience in relation to providing care for socioeconomically disadvantaged patients, and (ii) how participating in a photovoice study helped them uncover and examine some of their prejudices and assumptions about poverty.
Methods:
We conducted a participatory photovoice study. Participants were four family medicine residents, two medical supervisors, and two researchers. Residents attended six photovoice meetings at which they discussed photos they had taken. In collaboration with the researchers, the participants defined the research questions, took photos, and participated in data analysis and results dissemination. Meetings were recorded and transcribed for analysis, which consisted of coding, peer debriefing, thematic analysis, and interpretation.
Results:
The medical residents uncovered and examined their own prejudices and misconceptions about poverty. They reported feeling unprepared to provide care to socioeconomically disadvantaged patients. Supported by medical supervisors and researchers, the residents underwent a three-phase reflexive process of: (1) engaging reflexively, (2) break(ing) through, and (3) taking action. The results indicated that medical residents subsequently felt encouraged to adopt a care approach that helped them overcome the social distance between themselves and their socioeconomically disadvantaged patients.
Conclusions:
This study highlights the importance of providing medical training on issues related to poverty and increasing awareness about social inequalities in medical education to counteract prejudices toward socioeconomically disadvantaged patients. Future studies should examine which elective courses and training could provide suitable tools to clinicians to improve their competence in delivering care to socioeconomically disadvantaged patients. | 10,155 | 355 | [
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|||||
TH1/TH2 Cytokine profile in relapsing-remitting multiple sclerosis patients treated with Glatiramer acetate or Natalizumab. | 22989378 | The balance between T helper cells Th2- and Th1-related cytokines plays a key role in multiple sclerosis (MS). A shift from a Th1 towards a Th2 cytokine profile could have a beneficial effect on the clinical course of the disease. The objective of this study was to assess Th2/Th1 cytokine profile in relapsing-remitting MS (RRMS) patients receiving an immunosuppressive treatment with natalizumab (NAT), or an immunomodulatory treatment with glatiramer acetate (GA) after one year of treatment. | BACKGROUND | This was an observational cross-sectional study. All consecutive patients diagnosed with RRMS who had received GA or NAT for 12 months were included in the study. We determined serum levels of Th1 and Th2 cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) by flow cytometry. Th2/Th1 bias was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2 cytokines and proinflammatory INF-γ or TNF-α Th1 cytokines. | METHODS | Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated. RRMS patients treated with NAT showed significantly higher levels of IL-6 (p < 0.05), MCP-1 (p < 0.01), and GM-CSF (p < 0.05) compared to GA patients after one year of treatment. A trend for increasing of IL-12p70, IL-1b, TNF- α and IFN- γ levels was also found in patients receiving NAT compared to GA patients. IL-4/IFN-γ, IFN-γ/TNF-α and IL-10/IFN-γ ratios as markers of Th2/Th1 ratio were significantly elevated in GA patients compared to those receiving NAT (p < 0.05). | RESULTS | In conclusion, our findings suggest that GA promotes a superior Th2-biased anti-inflammatory response as compared with NAT in the systemic circulation of RRMS patients. Future studies with larger cohorts will determine whether this immune Th2 shift in GA patients is associated with a beneficial effect on disease outcome. | CONCLUSION | [
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] | 3517482 | Background | Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.
Treatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].
In the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment. | Methods | The present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.
Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.
All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.
Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.
Serum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.
Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.
Serum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.
Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.
Th2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.
Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.
Th2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented. | Results | Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.
The study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.
Mean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.
A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.
The study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.
Mean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.
Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).
Serum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.
IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).
Th2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.
Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).
Serum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.
IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).
Th2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab. | Conclusion | In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA. | [
"Background",
"Patients",
"Cytokine analysis",
"Statistical analysis",
"Patient characteristics",
"Th1/Th2 cytokine profile",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.",
"All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.",
"Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.",
"Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.",
"A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.",
"Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.",
"E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.",
"All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Patients",
"Cytokine analysis",
"Statistical analysis",
"Results",
"Patient characteristics",
"Th1/Th2 cytokine profile",
"Discussion",
"Conclusion",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.\nTreatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].\nIn the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.",
"The present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.\n Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\nAll consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.\n Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\nBriefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.\n Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.\nMedian serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.",
"All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.",
"Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.\nSerum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.",
"Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.\nTh2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.",
" Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\nA total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.\n Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.\nOverall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.",
"A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.\nThe study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.\nMean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.",
"Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).\n\nSerum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.\nIL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).\n\nTh2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.",
"The results of the present study suggest a Th2/Th1 balance shift in favour of a Th2 cytokine profile on GA-treated patients, while NAT causes a predominant Th1-biased response. This superior anti-inflammatory shift of GA seems to be mainly mediated by raising IL-4 and IL-10 levels which could lead to a down-regulation of Th1 cytokine secretion. Accordingly, the enhancement of circulating IL-4 and IL-10 and the subsequent detrimental effect on IFN-γ and TNF-α seen in GA-treated RRMS patients may play a protective role from inflammatory response that could affect the clinical course of disease in these patients. In this regard, a stabilization of disability score was found in both NAT and GA patients after one year of treatment. The potential clinical implications of immune response in GA-treated patients have been previously assessed [30-32]. Indeed, Valenzuela et al. [31] reported an increased IL-4/IFN-γ ratio to be associated with a favourable clinical outcome in a study of 36 RRMS patients treated with GA. However, the available findings suggesting the potential association between specific cytokine patterns and clinical response to GA were controversial primarily due to the short follow-up period. A recent study with a longer follow-up period of 3 years has demonstrated that IL-2 + IFN-γ/IL-10 + IL-4 ratio was significantly elevated in those patients with RRMS that suffered from relapses and progressing brain atrophy, suggesting that a specific pattern of Th2/Th1 cytokines may predict clinical response to GA therapy [33]. Moreover, this study suggests that the quotient IL-4 + IL-10/IL-2 + IFN-γ could be a promising parameter to identify patients associated with a highly beneficial response to GA therapy. However, although follow-up data over 3 years were available in this study, the sample size was relatively small to draw firm conclusions. Consequently, further studies including larger cohorts of patients will be required to validate that clinical and immune response correlate in patients treated with GA. Additionally, the mechanisms underlying the relation between cytokine response and clinical outcome in GA treated patients remain as a matter of debate.\nThe results of the present study show that patients treated with NAT exhibit higher levels of circulating proinflammatory cytokines and chemokines than those treated with GA. These findings are in agreement with previous studies where NAT treatment has been associated with an increased expression of proinflammatory cytokines in peripheral blood mononuclear cells [34,35]. Accordingly, an increase in activated leukocytes producing proinflammatory cytokines has been found in peripheral blood of NAT-treated patients [34,36]. Although it is not clear, these findings could probably be due to the inhibition of transmigration of lymphocytes into CNS, resulting in sequestration of activated T cells in the peripheral circulation [36]. The prolonged T-cell activation could result in decreased local immunosurveillance, reactivation of latent viral infections or opportunistic CNS infections, as evidenced by the rare but severe occurrence of progressive multifocal leukoencephalopathy caused by JC virus in NAT-treated patients [37]. Recent evidence has suggested that NAT seem to exert its beneficial effect without affecting regulatory T cell function [28]. Interestingly, NAT therapy has been associated with an increase in some pro-inflammatory and anti-inflammatory cytokines within the first 2 months of therapy, whereas relevant cytokines for MS such as IL-2, IL-7, or IL-1β have been found to be increased after one year of treatment, suggesting different immunological mechanisms [28]. The changes in the Th1/Th2 paradigm do not appear to be applicable to explain the beneficial effect of NAT. The increase of circulating Th1 cytokines could be related to a “rebound” effect that led to the development of new and enlarging T2 lesions previously seen in cohorts of patients discontinuing NAT due to safety issues related to this therapy, particularly regarding PML [38]. This finding has led to the concern that cessation of NAT might promote a worsening of MS disease by increasing inflammatory activity. The most likely explanation is that short exposure to NAT (e.g., 2 infusions) results in blockade of migration and accumulation of activated lymphocytes in the periphery that retain their capacity to cause CNS disease [39].\nThe main limitations of this study include the small sample size and a one-point measurement of cytokine patterns. However, despite these limitations, our findings are potentially interesting given that to our knowledge, this is the first study to compare the Th1/Th2 bias between GA and NAT treated RRMS patients.",
"In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA.",
"E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.",
"All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2377/12/95/prepub\n"
] | [
null,
"methods",
null,
null,
null,
"results",
null,
null,
"discussion",
"conclusions",
null,
null,
null
] | [
"Multiple sclerosis",
"Th2",
"Th1",
"Cytokines",
"Glatiramer acetate",
"Natalizumab"
] | Background:
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.
Treatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].
In the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.
Methods:
The present study was a cross-sectional, observational study conducted at a University Hospital. Written informed consent was obtained from all patients before they were included in the study. The study was approved by the Ethics Committee of the University Hospital La Paz, Madrid.
Patients All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.
All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.
Cytokine analysis Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.
Serum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.
Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.
Serum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.
Statistical analysis Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.
Th2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.
Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.
Th2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.
Patients:
All consecutive patients diagnosed with RRMS who had received GA (Copaxone®) or NAT (Tysabri®) for 12 months were included in the study. All patients fulfilled the McDonald criteria for RRMS [29]. Patients underwent a complete neurological examination to quantify patients’ disability by the Kutzke's Expanded Disability Status Scale (EDSS) every 6 months. All patients treated with NAT had been previously treated with immunomodulatory treatments (GA or β-interferon; minimum wash-out period of 1 month). The GA-treated patients were naïve for previous treatments.
Cytokine analysis:
Briefly, blood samples were collected from patients after 12 months of treatment and serum was obtained by centrifugation and stored at −80°C until cytokine determination. All samples were collected before each drug administration.
Serum levels of Th1- and Th2-related, MS-relevant cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) were determined by flow cytometry (FacsCalibur, BD Biosciense, CA, USA) using CBA Flex Set kit (BD Bioscience, Bedford, MA, USA) following manufacturer’s instructions. CBA Flex Set analysis was performed using FCAP array v1.0 software (Soft Flow Inc., USA). Protein values were converted to NIBSC/WHO protein standards for further comparisons.
Statistical analysis:
Median serum cytokines levels between GA-treated patients and those receiving NAT were compared using Student´s t-test.
Th2/Th1 ratio was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2-related cytokines and proinflammatory INF-γ or TNF-α Th1-related cytokines. The median Th2/Th1 ratio was calculated for each group. The non-parametric Mann-Whitney U test was performed in order to compare both groups using GraphPad Prism 5.0 software (San Diego, CA, USA), and t-test under log transformation of Th2/Th1 ratio is presented.
Results:
Patient characteristics A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.
The study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.
Mean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.
A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.
The study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.
Mean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.
Th1/Th2 cytokine profile Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).
Serum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.
IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).
Th2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.
Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).
Serum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.
IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).
Th2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.
Patient characteristics:
A total of 23 RRMM patients treated with GA or NAT for 12 months were included in the study. Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated.
The study population was comprised by 9 females in each group (82% and 75% in NAT and GA group, respectively) and 2 and 3 males (18% and 25%) in NAT and GA group, respectively. The mean age was 37.73 ± 7.24 and 37.67 ± 10.58 years old for NAT and GA-treated patients correspondingly.
Mean disease duration for patients treated with NAT and GA was 7.90 ± 3.42 and 4.25 ± 2.70 years, respectively. The mean EDSS score at the start of treatment ranged from 0 to 6.50 for all patients, with a mean of 4.05 ± 1.67 in NAT-treated patients and 1.67 ± 1.77 in those receiving GA (p = 0.005). After 12 months of treatment, mean EDSS decreased by 0.2 and 0.1 in the NAT and GA groups, respectively.
Th1/Th2 cytokine profile:
Overall, patients receiving NAT showed significantly higher levels of proinflammatory cytokines IL-6 (p < 0.05), MCP-1 (p < 0.01) and GM-CSF (p < 0.05) compared to GA-treated patients after one year of treatment. Median levels of IL-6, MCP-1 and GM-CSF in NAT patients were 5.21 pg/L (3.80-6.36), 121.23 pg/L (95.86-157.68) and 4.05 pg/L (1.38-12.19), respectively, whereas GA patients showed median levels of 2.88 pg/L (1.05-5.75), 65.41 pg/L (41.70-75.89) and 1.56 pg/L (0.68-3.23) for IL-6, MCP-1 and GM-CSF, respectively. Moreover, serum levels of Th1-related cytokines IL-12p70, IL-1b, TNF-α, and IFN-γ showed a clear tendency to be higher in patients receiving NAT as compared to GA patients (Figure 1).
Serum levels of cytokines after one year of treatment with GA or NAT in RRMS patients. Cytokines are grouped as Th1-related (A) or Th2-related (B). Cytokines were determined by Flow cytometry. All concentrations are given in pg/mL and were converted to international WHO/MIBBSC standards. Data are shown as median and interquartile ranges. NAT-treated patients showed significantly higher levels of proinflammatory cytokines MCP-1, and GM-CSF compared to those patients treated with GA. IL-6 Th2-related cytokine was significantly higher in NAT group. *p < 0.05; **p < 0.01. GA = glatiramer acetate; NAT = natalizumab.
IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio were significantly higher for GA-treated patients as compared to those receiving NAT after one year of treatment (p < 0.05) (Figure 2).
Th2/Th1 ratio in GA and NAT patients. Comparative Th2/Th1 ratios considering anti-inflammatory IL-4, IL-5, IL-6, IL-10 cytokines and pro-inflammatory cytokine INF-γ (A) or TNF-α (B). Box and whiskers plots showing median and interquartile ranges are presented. IL-4/IFN-γ, IL-4/TNF-α and IL-10/IFN-γ ratio as markers of Th2/Th1 ratio are significantly higher for GA patients compared to patients receiving NAT. *p < 0.05. GA = glatiramer acetate; NAT = natalizumab.
Discussion:
The results of the present study suggest a Th2/Th1 balance shift in favour of a Th2 cytokine profile on GA-treated patients, while NAT causes a predominant Th1-biased response. This superior anti-inflammatory shift of GA seems to be mainly mediated by raising IL-4 and IL-10 levels which could lead to a down-regulation of Th1 cytokine secretion. Accordingly, the enhancement of circulating IL-4 and IL-10 and the subsequent detrimental effect on IFN-γ and TNF-α seen in GA-treated RRMS patients may play a protective role from inflammatory response that could affect the clinical course of disease in these patients. In this regard, a stabilization of disability score was found in both NAT and GA patients after one year of treatment. The potential clinical implications of immune response in GA-treated patients have been previously assessed [30-32]. Indeed, Valenzuela et al. [31] reported an increased IL-4/IFN-γ ratio to be associated with a favourable clinical outcome in a study of 36 RRMS patients treated with GA. However, the available findings suggesting the potential association between specific cytokine patterns and clinical response to GA were controversial primarily due to the short follow-up period. A recent study with a longer follow-up period of 3 years has demonstrated that IL-2 + IFN-γ/IL-10 + IL-4 ratio was significantly elevated in those patients with RRMS that suffered from relapses and progressing brain atrophy, suggesting that a specific pattern of Th2/Th1 cytokines may predict clinical response to GA therapy [33]. Moreover, this study suggests that the quotient IL-4 + IL-10/IL-2 + IFN-γ could be a promising parameter to identify patients associated with a highly beneficial response to GA therapy. However, although follow-up data over 3 years were available in this study, the sample size was relatively small to draw firm conclusions. Consequently, further studies including larger cohorts of patients will be required to validate that clinical and immune response correlate in patients treated with GA. Additionally, the mechanisms underlying the relation between cytokine response and clinical outcome in GA treated patients remain as a matter of debate.
The results of the present study show that patients treated with NAT exhibit higher levels of circulating proinflammatory cytokines and chemokines than those treated with GA. These findings are in agreement with previous studies where NAT treatment has been associated with an increased expression of proinflammatory cytokines in peripheral blood mononuclear cells [34,35]. Accordingly, an increase in activated leukocytes producing proinflammatory cytokines has been found in peripheral blood of NAT-treated patients [34,36]. Although it is not clear, these findings could probably be due to the inhibition of transmigration of lymphocytes into CNS, resulting in sequestration of activated T cells in the peripheral circulation [36]. The prolonged T-cell activation could result in decreased local immunosurveillance, reactivation of latent viral infections or opportunistic CNS infections, as evidenced by the rare but severe occurrence of progressive multifocal leukoencephalopathy caused by JC virus in NAT-treated patients [37]. Recent evidence has suggested that NAT seem to exert its beneficial effect without affecting regulatory T cell function [28]. Interestingly, NAT therapy has been associated with an increase in some pro-inflammatory and anti-inflammatory cytokines within the first 2 months of therapy, whereas relevant cytokines for MS such as IL-2, IL-7, or IL-1β have been found to be increased after one year of treatment, suggesting different immunological mechanisms [28]. The changes in the Th1/Th2 paradigm do not appear to be applicable to explain the beneficial effect of NAT. The increase of circulating Th1 cytokines could be related to a “rebound” effect that led to the development of new and enlarging T2 lesions previously seen in cohorts of patients discontinuing NAT due to safety issues related to this therapy, particularly regarding PML [38]. This finding has led to the concern that cessation of NAT might promote a worsening of MS disease by increasing inflammatory activity. The most likely explanation is that short exposure to NAT (e.g., 2 infusions) results in blockade of migration and accumulation of activated lymphocytes in the periphery that retain their capacity to cause CNS disease [39].
The main limitations of this study include the small sample size and a one-point measurement of cytokine patterns. However, despite these limitations, our findings are potentially interesting given that to our knowledge, this is the first study to compare the Th1/Th2 bias between GA and NAT treated RRMS patients.
Conclusion:
In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA.
Competing interests:
E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials and as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb,Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, EVER Neuro Pharma. C. Oreja- Guevara has collaborated as speaker and in clinical trials with Biogen Idec, Merck Serono, Teva, Sanofi, Bayer-Schering and Novartis.
Authors’ contributions:
All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: COG. Acquisition of data: COG, BC. Analysis and interpretation of data: COG, JRC, LSA. Drafting of the manuscript: COG, JRC, EDT. Statistical analysis: COG, JRC. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2377/12/95/prepub
| Background:
The balance between T helper cells Th2- and Th1-related cytokines plays a key role in multiple sclerosis (MS). A shift from a Th1 towards a Th2 cytokine profile could have a beneficial effect on the clinical course of the disease. The objective of this study was to assess Th2/Th1 cytokine profile in relapsing-remitting MS (RRMS) patients receiving an immunosuppressive treatment with natalizumab (NAT), or an immunomodulatory treatment with glatiramer acetate (GA) after one year of treatment.
Methods:
This was an observational cross-sectional study. All consecutive patients diagnosed with RRMS who had received GA or NAT for 12 months were included in the study. We determined serum levels of Th1 and Th2 cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) by flow cytometry. Th2/Th1 bias was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2 cytokines and proinflammatory INF-γ or TNF-α Th1 cytokines.
Results:
Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated. RRMS patients treated with NAT showed significantly higher levels of IL-6 (p < 0.05), MCP-1 (p < 0.01), and GM-CSF (p < 0.05) compared to GA patients after one year of treatment. A trend for increasing of IL-12p70, IL-1b, TNF- α and IFN- γ levels was also found in patients receiving NAT compared to GA patients. IL-4/IFN-γ, IFN-γ/TNF-α and IL-10/IFN-γ ratios as markers of Th2/Th1 ratio were significantly elevated in GA patients compared to those receiving NAT (p < 0.05).
Conclusions:
In conclusion, our findings suggest that GA promotes a superior Th2-biased anti-inflammatory response as compared with NAT in the systemic circulation of RRMS patients. Future studies with larger cohorts will determine whether this immune Th2 shift in GA patients is associated with a beneficial effect on disease outcome. | Background:
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). T helper (Th) cells appear to play a pivotal role in the autoreactive immune response of the CNS in MS, primarily characterized by inflammation and demyielination [1,2]. Cytokines are key factors in the regulation of inflammatory responses and may reflect the disease process in MS [3,4]. Th cells can be divided into subtypes based on the characteristic cytokine secretion patterns and their effector functions. While Th2-related cytokines such as interleukin IL-4, IL-10, or IL-5 have been associated with inflammation reduction and improvement of symptoms in MS patients, Th1 cytokines such as interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) have been shown to increase inflammation, therefore leading to disease progression and worsening of symptoms [5-10]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines. Induction of Th1 cytokines toward Th2 could achieve the suppression of undesirable autoimmunity and have a beneficial effect on the clinical course of the disease [11]. In this scenario, effective treatments should induce a shift toward Th2 cytokine anti-inflammatory response.
Treatment of relapsing-remitting MS (RRMS) with glatiramer acetate (GA) (Copaxone®) reduces frequency of relapses and the appearance of new lesions in gadolinium enhanced magnetic resonance imaging (MRI) [12,13]. As other immunomodulatory treatments, GA seems to interfere with the Th1/Th2 balance by promoting a shift from the Th1 to the Th2 anti-inflammatory cytokine pathway [14-22]. Natalizumab (NAT) (Tysabri®), a humanized monoclonal antibody against very late activation antigen (VLA)-4 on leukocytes, has demonstrated to reduce the relapse rate by about 70% in RRMS patients [23,24]. The widely proposed mode of action of NAT is based on the reduction of transmigration of leukocytes into the CNS [25,26]. However, other immunological effects may also be operative accounting for changes of some Th1/Th2 cytokines levels in plasma of RRMS patients treated with NAT [27,28].
In the present study, we aimed to evaluate the patterns of Th2/Th1 cytokines in RRMS patients receiving an immunosuppressive treatment with NAT, or an immunomodulatory treatment with GA after one year of treatment.
Conclusion:
In summary, GA seems to modulate Th1/Th2 balance in the systemic circulation with a shift toward the anti-inflammatory Th2 profile response in RRMS patients. This effect may have a beneficial effect on disease activity in these patients. Further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune Th2 shift in GA patients correlates with a favourable clinical response. NAT seems to exert is beneficial effect through different mechanisms than immunomodulators such as GA. | Background:
The balance between T helper cells Th2- and Th1-related cytokines plays a key role in multiple sclerosis (MS). A shift from a Th1 towards a Th2 cytokine profile could have a beneficial effect on the clinical course of the disease. The objective of this study was to assess Th2/Th1 cytokine profile in relapsing-remitting MS (RRMS) patients receiving an immunosuppressive treatment with natalizumab (NAT), or an immunomodulatory treatment with glatiramer acetate (GA) after one year of treatment.
Methods:
This was an observational cross-sectional study. All consecutive patients diagnosed with RRMS who had received GA or NAT for 12 months were included in the study. We determined serum levels of Th1 and Th2 cytokines (interleukin [IL]-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, monocyte chemotactic protein [MCP]-1, tumor-necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte macrophage colony stimulating factor [GM-CSF]) by flow cytometry. Th2/Th1 bias was defined based on the ratio of IL-4, IL-5, IL-6 or IL-10 Th2 cytokines and proinflammatory INF-γ or TNF-α Th1 cytokines.
Results:
Eleven patients under treatment with NAT and 12 patients treated with GA were evaluated. RRMS patients treated with NAT showed significantly higher levels of IL-6 (p < 0.05), MCP-1 (p < 0.01), and GM-CSF (p < 0.05) compared to GA patients after one year of treatment. A trend for increasing of IL-12p70, IL-1b, TNF- α and IFN- γ levels was also found in patients receiving NAT compared to GA patients. IL-4/IFN-γ, IFN-γ/TNF-α and IL-10/IFN-γ ratios as markers of Th2/Th1 ratio were significantly elevated in GA patients compared to those receiving NAT (p < 0.05).
Conclusions:
In conclusion, our findings suggest that GA promotes a superior Th2-biased anti-inflammatory response as compared with NAT in the systemic circulation of RRMS patients. Future studies with larger cohorts will determine whether this immune Th2 shift in GA patients is associated with a beneficial effect on disease outcome. | 5,026 | 427 | [
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] | [CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY] | [CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY] | [CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY] | [CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY] | [CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY] | [CONTENT] Multiple sclerosis | Th2 | Th1 | Cytokines | Glatiramer acetate | Natalizumab [SUMMARY] | [CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY] | [CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY] | [CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY] | [CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY] | [CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY] | [CONTENT] Adjuvants, Immunologic | Adult | Antibodies, Monoclonal, Humanized | Cytokines | Glatiramer Acetate | Humans | Male | Multiple Sclerosis | Natalizumab | Peptides | Secondary Prevention | Th1-Th2 Balance | Treatment Outcome [SUMMARY] | [CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY] | [CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY] | [CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY] | [CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY] | [CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY] | [CONTENT] th2 related cytokines | cytokines ms | relevant cytokines ms | th1 cytokines interferon | inflammation demyielination cytokines [SUMMARY] | [CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] patients | il | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] cytokines | th2 | th1 cytokines | ms | th1 | disease | inflammation | cns | inflammatory | treatment [SUMMARY] | [CONTENT] il | il il | patients | il il il | usa | protein | test | ratio | il il il il | th1 [SUMMARY] | [CONTENT] nat | il | patients | ga | 05 | pg | higher | respectively | treated | mean [SUMMARY] | [CONTENT] effect | larger | patients | beneficial effect | shift | beneficial | response | th2 | ga | systemic [SUMMARY] | [CONTENT] il | patients | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] il | patients | ga | nat | th2 | treated | th1 | cytokines | il il | ratio [SUMMARY] | [CONTENT] ||| ||| RRMS | NAT | one year [SUMMARY] | [CONTENT] ||| RRMS | NAT | 12 months ||| IL-1b | IL-5 | IL-6 | IL-10 | IL-12p70 | monocyte ||| ||| GM-CSF ||| Th2 | IL-5 | IL-6 | IL-10 | INF | TNF [SUMMARY] | [CONTENT] NAT | 12 ||| RRMS | NAT | IL-6 | 0.05 | MCP-1 | 0.01 | GM-CSF | 0.05 | one year ||| IL-12p70 | IL-1b | NAT ||| IFN | IFN | IL-10 | IFN | GA | NAT | 0.05 [SUMMARY] | [CONTENT] NAT | RRMS ||| GA [SUMMARY] | [CONTENT] ||| ||| RRMS | NAT | one year ||| ||| RRMS | NAT | 12 months ||| IL-1b | IL-5 | IL-6 | IL-10 | IL-12p70 | monocyte ||| ||| GM-CSF ||| Th2 | IL-5 | IL-6 | IL-10 | INF | TNF ||| ||| NAT | 12 ||| RRMS | NAT | IL-6 | 0.05 | MCP-1 | 0.01 | GM-CSF | 0.05 | one year ||| IL-12p70 | IL-1b | NAT ||| IFN | IFN | IL-10 | IFN | GA | NAT | 0.05 ||| NAT | RRMS ||| GA [SUMMARY] | [CONTENT] ||| ||| RRMS | NAT | one year ||| ||| RRMS | NAT | 12 months ||| IL-1b | IL-5 | IL-6 | IL-10 | IL-12p70 | monocyte ||| ||| GM-CSF ||| Th2 | IL-5 | IL-6 | IL-10 | INF | TNF ||| ||| NAT | 12 ||| RRMS | NAT | IL-6 | 0.05 | MCP-1 | 0.01 | GM-CSF | 0.05 | one year ||| IL-12p70 | IL-1b | NAT ||| IFN | IFN | IL-10 | IFN | GA | NAT | 0.05 ||| NAT | RRMS ||| GA [SUMMARY] |
||||||
PURIFICATION AND FRACTIONAL ANALYSIS OF METHANOLIC EXTRACT OF | 28480428 | Wedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity. | BACKGROUND | The methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components. | MATERIALS AND METHODS | Out of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC. | RESULTS | The single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis. | CONCLUSION | [
"Antineoplastic Agents, Phytogenic",
"Apoptosis",
"Cell Line, Tumor",
"Chromatography, Thin Layer",
"Humans",
"Megakaryocytes",
"Methanol",
"Methylene Chloride",
"Plant Extracts",
"Wedelia"
] | 5412222 | Introduction | Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.
Leukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.
Asteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.
A brief explanation of the entire process done in the study | null | null | Results | Thin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.
Thin layer chromatogram showing the separation of methanolic extract into two bands under UV light.
Thymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.
The reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.
From the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.
DNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.
lane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.
Silica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.
Thymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.
The graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.
Nuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.
Nuclear staining of MEG01 cell with negative, positive control and fraction 3.
Flow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.
Detection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).
HPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.
HPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes. | Conclusion | The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis. | [
"Collection of plant and obtaining crude extracts of Wedelia trilobata",
"Thin layer chromatography (TLC)",
"Activity analysis of the fractions obtained from TLC",
"Activity analysis of the fractions obtained from Silica gel chromatography"
] | [
"The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).",
"This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.",
"Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.",
"Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016)."
] | [
null,
null,
null,
null
] | [
"Introduction",
"Materials and methods",
"Collection of plant and obtaining crude extracts of Wedelia trilobata",
"Thin layer chromatography (TLC)",
"Activity analysis of the fractions obtained from TLC",
"Activity analysis of the fractions obtained from Silica gel chromatography",
"Results",
"Discussion",
"Conclusion"
] | [
"Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.\nLeukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.\nAsteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.\nA brief explanation of the entire process done in the study",
" Collection of plant and obtaining crude extracts of Wedelia trilobata The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\nThe characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).\n Thin layer chromatography (TLC) This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\nThis is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.\n Activity analysis of the fractions obtained from TLC Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\nCell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.\n Activity analysis of the fractions obtained from Silica gel chromatography Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).\nThymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).",
"The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).",
"This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.\nTLC done with the mentioned solvent combinations at different ratios with the results as noted.",
"Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).\nDNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).\nSilica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.",
"Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).\nNuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).\nFlow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.\nThe cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.\nHPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).",
"Thin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.\nThin layer chromatogram showing the separation of methanolic extract into two bands under UV light.\nThymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.\nThe reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.\nFrom the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.\nDNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.\nlane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.\nSilica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.\nThymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.\nThe graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.\nNuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.\nNuclear staining of MEG01 cell with negative, positive control and fraction 3.\nFlow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.\nDetection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).\nHPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.\nHPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes.",
"TLC: The initial separation of methanolic extract through TLC (Sasidharan et al. 2011) showed two prominent bands with methanol and dichloromethane solvent mixture taken at the ratio 6:4 whereas, the separation was improper with other solvent mixtures and ratios tried. Among the two bands obtained, the second band still retains certain color mixture which may indicate the presence of different compositions. Before going to the further purification, the characteristic analysis of both the bands was assessed for anti-proliferation and apoptosis activity.\nThymidine uptake assay: The thymidine uptake assay (Giridharan et al. 2002; Belakavadi et al. 2005) to check antiproliferation activity in MEG01 cells was performed twice, first was done on the bands obtained from TLC and analyzed that the second band showed the reduction in proliferation of MEG01 cells in comparison to the first band indicating that band 2 posses more active compounds in it. Second on the fraction obtained through silica gel column chromatography and in result the fractions 2, 3 and 4 showed the anti-proliferation activity among which the fraction 3 was more prominent. This gave the further lead to concentrate on fraction 3 for analysis.\nDNA fragmentation assay: The apoptosis activity of TLC separated bands when checked with the DNA degradation assay (Park et al. 2013) showed that, the band 2 possess the prominent bioactive compound having both antiproliferation as well as the apoptosis activity. This made the band 2 a clear selection for further purification.\nNuclear staining assay: The apoptosis activity was analyzed for fraction 3 in comparison with the activity of 2-Amino-N-quinolin-8-yl-benzenesulfonamide (Sigma Aldrich), taken as positive control. Both the positive control and fraction 3 produced orange fluorescent with green fluorescent in background due to apoptotic activity indicating that, the ruptured nucleus released apoptotic bodies containing DNA binds to EtBr and produced orange fluorescent (Srinivas et al, 2003).\nFlow cytometry analysis: Further analysis through flow cytometry using Ca2+ dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide; the fraction 3 was confirmed that it has more apoptotic activity. In the figure 7 the presence of cells in lower left square indicates the apoptotic cells and this clearly indicates the apoptotic activity by fraction 3 (Donal et al, 2009).\nHPLC: The HPLC analysis of fraction 3 produced a single peak at a retention time 3.65 min. The single peak and the early elution indicate the presence of pure and polar compound (Panawan et al, 2015; Ji-Yeon et al, 2016).",
"The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis."
] | [
"intro",
"materials|methods",
null,
null,
null,
null,
"results",
"discussion:",
"conclusion"
] | [
"\nWedelia trilobata\n",
"MEG-01",
"Antiproliferation",
"Apoptosis"
] | Introduction:
Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.
Leukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.
Asteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.
A brief explanation of the entire process done in the study
Materials and methods:
Collection of plant and obtaining crude extracts of Wedelia trilobata The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).
The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).
Thin layer chromatography (TLC) This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.
TLC done with the mentioned solvent combinations at different ratios with the results as noted.
This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.
TLC done with the mentioned solvent combinations at different ratios with the results as noted.
Activity analysis of the fractions obtained from TLC Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).
DNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).
Silica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.
Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).
DNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).
Silica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.
Activity analysis of the fractions obtained from Silica gel chromatography Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).
Nuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).
Flow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.
The cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.
HPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).
Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).
Nuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).
Flow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.
The cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.
HPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).
Collection of plant and obtaining crude extracts of Wedelia trilobata:
The characteristic features for identification of plant and its habitat and also procedure for the preparation of plant extracts of Wedelia trilobata has been followed as explained earlier (Uday et al. 2016).
Thin layer chromatography (TLC):
This is the rapid and inexpensive method for the identification of number of compounds present in a crude sample and for the separation of plant extracts into different fractions (Sasidharan et al. 2011). The TLC plates of size 15cm in length and 8cm in width along with 0.5mm thickness (E. Merck, Germany) were used. The extract was spotted on the plate and solvent front was made to run up to 10 cm. Then the plates were dried and visualized under UV light. The two combination systems of solvents with different concentration ratio were used to separate the compositional mixtures in methanolic extract of the plant as mentioned below. The bands obtained were scraped from the plate and dissolved in methanol. The mixture was then centrifuged to separate from silica and the obtained supernatant was further used for antiproliferation and apoptotic assays.
TLC done with the mentioned solvent combinations at different ratios with the results as noted.
Activity analysis of the fractions obtained from TLC:
Cell line culture and Thymidine uptake assay: 5 × 104 MEG-01 human Megakaryoblastic cells (Sigma Aldrich, India) were seeded per well and cultured in RPMI 1640 (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) and 5% CO2 at 37°C for 24h. When the cells becomes 80% confluent, 1 μl (μg/mL) of [3H] Thymidine was added and followed by the addition of TLC separated samples in a quantity of 2μl, 4μl and 8μl respectively. After 48h of incubation, the media was removed and cells were processed to measure the radioactivity using liquid scintillation counter. The proliferation rate was correlated through graphical method (Giridharan et al. 2002; Belakavadi et al. 2005).
DNA fragmentation assay: The MEG01 cells treated with TLC fractions and the untreated cells were subjected to DNA isolation by incubation with lysis buffer for 1h at room temperature. The lysate was centrifuged to separate the contaminants, then the DNA was precipitated using phenol: chloroform: isoamyl alcohol mixture. The precipitated DNA sample was pooled out and dried through vacuum evaporator. The obtained DNA samples were assessed electrophoretically in 1% agarose gel (Park et al. 2013).
Silica gel chromatography (SGC): The bulk quantity of the positive fraction was obtained through preparative TLC method and subjected to further purification using silica gel (Merk) column chromatography (Fatma et al. 2016). The minimum quantity of sample was dissolved in the solvent mixture of methanol and dichloromethane at 6:4 ratio and loaded to the silica packed column. The five different fractions separated (confirmed through TLC) were collected and used for the study of activity.
Activity analysis of the fractions obtained from Silica gel chromatography:
Thymidine uptake assay: The five fractions obtained from column chromatography were analyzed for the anti-proliferation activity through thymidine uptake assay and the procedure was followed as explained in earlier methods (Giridharan et al. 2002; Belakavadi et al. 2005).
Nuclear staining: The apoptotic activity of the five different column chromatography fractions was analyzed through nuclear staining by (EtBr) Ethidium Bromide and Acridine orange solutions. The MEG-01 cells treated with the SGC obtained fractions and the untreated cells were washed in ice-cold Phosphate buffer saline (PBS) and incubated with 3.7% paraformaldehyde for 10 min at room temperature for fixation of cells. The cells were washed again with PBS and 4’,6-diamidino-2-phenylindole (DAPI) solution was added to stain for 10 min at room temperature and cells were rewashed with PBS and the fluorescent cell nuclei were analyzed through microscopy as reported earlier (Srinivas et al, 2003).
Flow cytometry analysis: The induction of apoptosis in MEG-01 cells by the sample (column purified third fraction) was analyzed using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience). The principle behind the study was to analyze the translocated phosphatidylserine from inside to outer plasma membrane of the apoptotic cell which is not present in the normal cells. Fluorochrome tagged Annexin V a Ca2+-dependent phospholipid-binding protein has high affinity towards phosphatidylserine and serves as a probe in flow cytometry assay (Donai et al, 2009). Propidium iodide a dye being impermeable into viable cells can easily penetrate inside dead cells due to the permeability of dead cell membrane. Considering these features, the Annexin V and Propidium iodide were conjugated to use for the early and late apoptotic detection.
The cultured treated and untreated MEG-01 cells were suspended and centrifuged for 5 min at room temperature. The cell pellet was used according to BD Annexin V: FITC Apoptosis Detection Kit protocol and analyzed the samples using flow cytometry.
HPLC: Compositional analysis of positive fraction obtained after column chromatography was done using a HPLC system. The fraction was subjected to a reversed-phase HPLC on a semi preparative C-18 column, at 25 °C with the DAD detector at wavelength 210 nm. Two solvent mixtures (solvent A is 100% methanol and solvent B is 100% dichloromethane) were used for the elution. The column was equilibrated with solvent mixture in the ratio 1:1. After injecting the sample of 20μl the elution was carried out at a flow rate of 1 ml/min for about 60 min (Panawan et al, 2015; Ji-Yeon et al, 2016).
Results:
Thin layer chromatography: TLC was used to separate the crude plant extract into different fractions present if any in the methanolic extract.
Thin layer chromatogram showing the separation of methanolic extract into two bands under UV light.
Thymidine uptake assay: Antiproliferation activity for TLC separated fractions were assayed on MEG01 (human Megakaryoblastic) cell line through thymidine uptake assay.
The reduction in cell proliferation of band 1 and band 2 along with increase in quantity has been indicated in the graph. Among two bands, the band 2 showed prominent reduction in cell viability of MEG01.
From the figure 3 it is confirmed that, the band 2 was having more anti-proliferation activity when compare to the band 1.
DNA fragmentation assay: Antiproliferation assay was also in correlation with the DNA degradation assay carried out in our study for the two fractions separated by TLC. The figure 4 indicates that, the lane B2 shows clear fragmentation of the sample indicating the apoptotic activity possessed by band 2 compounds.
lane B1 indicates the DNA sample treated with band 1, lane B2 indicates the DNA sample treated with band 2 and lane PC indicates the DNA sample treated with positive control.
Silica gel Column Chromatography: Further dilution and elution of band 2 (obtained from TLC separation) through column chromatography has resulted in five fractions. Approximately eight to ten ml of elution for each fraction were collected (confirmed through TLC) and used for the study of activity.
Thymidine uptake assay: The fractions from column chromatography were analyzed for antiproliferation activity on MEG01 cells through thymidine uptake assay.
The graph indicates the reduction of MEG01 cell proliferation rate by the column chromatography fractions accordingly with increase in quantity.
Nuclear staining assay: Nuclear staining assay was processed to confirm the apoptotic activity of fraction 3. Nuclear staining of MEG01 cell in negative control shows the viable cells that are permeable only to Acridine orange causing green fluorescence where as in positive control and fraction 3 treated cells shows orange fluorescence due to binding of EtBr to nucleic acids and thereby indicating the apoptotic activity.
Nuclear staining of MEG01 cell with negative, positive control and fraction 3.
Flow cytometry analysis: Flow cytometry analysis for Fraction 3 was also done using BD Annexin V: FITC Apoptosis Detection Kit I (BD bioscience) to estimate the apoptosis activity. The MEG01 cells either untreated (Ctrl) or treated with fraction 3 sample were subsequently stained with Annexin V and Propidium Iodide (PI). The logarithmic amplification of green and red fluorescent signals was measured by flow cytometry. Viable cells (LL) are both Annexin V and PI negative. Early apoptotic cells (LR) bind to Annexin V but not to PI. Late apoptotic (UR) are both Annexin V and PI positive. Some very late apoptotic cells (UL) stain with PI only.
Detection of apoptotic cells through flow cytometry was done by Ca2+-dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide (PI).
HPLC: The composition analysis of fraction 3 was conducted through reversed-phase HPLC on a semi preparative C18 column.
HPLC chromatogram of 3rd fraction, showed a single peak at retention time 3.65 minutes.
Discussion:
TLC: The initial separation of methanolic extract through TLC (Sasidharan et al. 2011) showed two prominent bands with methanol and dichloromethane solvent mixture taken at the ratio 6:4 whereas, the separation was improper with other solvent mixtures and ratios tried. Among the two bands obtained, the second band still retains certain color mixture which may indicate the presence of different compositions. Before going to the further purification, the characteristic analysis of both the bands was assessed for anti-proliferation and apoptosis activity.
Thymidine uptake assay: The thymidine uptake assay (Giridharan et al. 2002; Belakavadi et al. 2005) to check antiproliferation activity in MEG01 cells was performed twice, first was done on the bands obtained from TLC and analyzed that the second band showed the reduction in proliferation of MEG01 cells in comparison to the first band indicating that band 2 posses more active compounds in it. Second on the fraction obtained through silica gel column chromatography and in result the fractions 2, 3 and 4 showed the anti-proliferation activity among which the fraction 3 was more prominent. This gave the further lead to concentrate on fraction 3 for analysis.
DNA fragmentation assay: The apoptosis activity of TLC separated bands when checked with the DNA degradation assay (Park et al. 2013) showed that, the band 2 possess the prominent bioactive compound having both antiproliferation as well as the apoptosis activity. This made the band 2 a clear selection for further purification.
Nuclear staining assay: The apoptosis activity was analyzed for fraction 3 in comparison with the activity of 2-Amino-N-quinolin-8-yl-benzenesulfonamide (Sigma Aldrich), taken as positive control. Both the positive control and fraction 3 produced orange fluorescent with green fluorescent in background due to apoptotic activity indicating that, the ruptured nucleus released apoptotic bodies containing DNA binds to EtBr and produced orange fluorescent (Srinivas et al, 2003).
Flow cytometry analysis: Further analysis through flow cytometry using Ca2+ dependent phospholipid-binding fluorochrome tagged Annexin V along with Propidium Iodide; the fraction 3 was confirmed that it has more apoptotic activity. In the figure 7 the presence of cells in lower left square indicates the apoptotic cells and this clearly indicates the apoptotic activity by fraction 3 (Donal et al, 2009).
HPLC: The HPLC analysis of fraction 3 produced a single peak at a retention time 3.65 min. The single peak and the early elution indicate the presence of pure and polar compound (Panawan et al, 2015; Ji-Yeon et al, 2016).
Conclusion:
The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis.
| Background:
Wedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity.
Methods:
The methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components.
Results:
Out of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC.
Conclusions:
The single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis. | Introduction:
Approach towards natural products, like plant extracts, for healthcare needs is increasing consistently due to the side-effects of chemically synthesized drugs and the decrease in the potency of these drugs for effective treatment is due to the development of resistance by various pathogens. As per WHO, more than 80% of the world’s population depends on traditional medicine. Though traditional medicine being less potent in its action compared to chemically synthesized drugs, it has many other advantages, such as: minimal side-effects and maintaining being effective for the disease. Bioactive compounds from plant extracts provide unmatched chemically diverse compositions possessing the biological activity such as anti-microbial, anti-oxidants, anti-cancer, analgesic etc., which can be used to treat infectious as well as chronic diseases (Veeramuthu et al. 2006). Certain plant extracts like diterpenes possessing basic biological activity are also used as structural building block to synthesize bioactive compounds (Barrero et al. 2012; Barrero et al. 2006). Relying on the plant extracts for drug discovery and designing is a neve rending process which also gives maximum possibility for combinatorial treatment with other drugs to increase the effectiveness.
Leukemia, one of the major diseases in the world, has a statistical record of 1,450 deaths in United States during 2015 and overall survival rate of adults with ALC is very less (American Cancer Society. 2015). Though advanced treatment strategies were being developed against leukemia in parallel drug designing through traditional method via plant extracts place an important role to create wide range of cost effective treatment.
Asteraceae family plants are well known for its biological activities like antimicrobial, antifungal, antioxidant (Casiglia et al. 2016; Na An et al. 2016) and anticancer activity. Methanolic extract of Wedelia trilobata (Asteraceae family) has shown anti-leukemia activity (Uday et al. 2016), fortified by the previous results, here we have purified and screened further for the anti-leukemic activity. Chromatographic techniques like TLC and HPLC was employed for the stepwise screening of positive fractions and studied for their anti-leukemia and apoptotic assays using MEG01 cell lines.
A brief explanation of the entire process done in the study
Conclusion:
The phytomedicines can be developed as an alternative and are relatively inexpensive than modern drugs (Huang et al, 2006; Neelam et al, 2014). In our current study emphasis is laid on the apoptotic and anti-leukemic activity of the phytoconstituent where in purification and analytical study of methanolic extract of WT revealed a single peak in HPLC indicating the presence of pure compound with apoptotic and anti-leukemia activities and encouraging for further study on structural analysis. | Background:
Wedelia trilobata (L.) Hitch (WT), commonly known as yellow dots or creeping daisy, is a shrub possessing potent biological activities, and is traditionally used a medicinal plant in Ayurveda, Siddha and Unani systems of medicines, and it has also been tried against leukemia cell line MEG- 01. In the present study, purification and screening of the plant was done for bioactive compounds in methanolic extract of WT for apoptotic and anti-leukemia activity.
Methods:
The methanolic extract of WT was initially purified through thin layer chromatography (TLC) and screened for the apoptotic and anti-leukemia activities. The positive band of TLC was subjected to silica gel column chromatography for further purification and the fractions obtained from it were screened again for anti-leukemia activity through thymidine uptake assay and apoptotic activity by DNA fragmentation, nuclear staining and flow cytometry assays. The fraction with positive result was subjected to HPLC for analysis of bioactive components.
Results:
Out of many combinations of solvents, the methanol and dichloromethane combination in the ratio 6:4 has revealed two bands in TLC, among which the second band showed positive results for apoptotic and anti-leukemic activities. Further purification of second band through silica gel chromatography gave five fractions in which the 3rd fraction gave positive results and it shows single peak during compositional analysis through HPLC.
Conclusions:
The single peak revealed through HPLC indicates the presence of pure compound with apoptotic and anti-leukemia activities encouraging for further structural analysis. | 4,738 | 284 | [
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Wedelia trilobata
| MEG-01 | Antiproliferation | Apoptosis [SUMMARY] | null | [CONTENT]
Wedelia trilobata
| MEG-01 | Antiproliferation | Apoptosis [SUMMARY] | [CONTENT]
Wedelia trilobata
| MEG-01 | Antiproliferation | Apoptosis [SUMMARY] | [CONTENT]
Wedelia trilobata
| MEG-01 | Antiproliferation | Apoptosis [SUMMARY] | [CONTENT]
Wedelia trilobata
| MEG-01 | Antiproliferation | Apoptosis [SUMMARY] | [CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia [SUMMARY] | null | [CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia [SUMMARY] | [CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia [SUMMARY] | [CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia [SUMMARY] | [CONTENT] Antineoplastic Agents, Phytogenic | Apoptosis | Cell Line, Tumor | Chromatography, Thin Layer | Humans | Megakaryocytes | Methanol | Methylene Chloride | Plant Extracts | Wedelia [SUMMARY] | [CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY] | null | [CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY] | [CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY] | [CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY] | [CONTENT] relying plant extracts | plant extracts healthcare | bioactive compounds plant | extracts like diterpenes | disease bioactive compounds [SUMMARY] | [CONTENT] cells | activity | tlc | column | fractions | assay | obtained | fraction | apoptotic | chromatography [SUMMARY] | null | [CONTENT] cells | activity | tlc | column | fractions | assay | obtained | fraction | apoptotic | chromatography [SUMMARY] | [CONTENT] cells | activity | tlc | column | fractions | assay | obtained | fraction | apoptotic | chromatography [SUMMARY] | [CONTENT] cells | activity | tlc | column | fractions | assay | obtained | fraction | apoptotic | chromatography [SUMMARY] | [CONTENT] cells | activity | tlc | column | fractions | assay | obtained | fraction | apoptotic | chromatography [SUMMARY] | [CONTENT] treatment | like | leukemia | drugs | plant extracts | extracts | anti | traditional | chemically | biological [SUMMARY] | null | [CONTENT] band | pi | assay | fraction | cells | indicates | meg01 | lane | annexin | activity [SUMMARY] | [CONTENT] apoptotic anti | study | anti | revealed | single peak hplc | study emphasis | phytoconstituent purification | phytoconstituent purification analytical | phytoconstituent purification analytical study | extract wt [SUMMARY] | [CONTENT] cells | activity | plant | fraction | assay | tlc | column | apoptotic | dna | band [SUMMARY] | [CONTENT] cells | activity | plant | fraction | assay | tlc | column | apoptotic | dna | band [SUMMARY] | [CONTENT] Wedelia | Ayurveda | Siddha | MEG- 01 ||| WT [SUMMARY] | null | [CONTENT] 6:4 | two | TLC | second ||| second | five | 3rd | HPLC [SUMMARY] | [CONTENT] HPLC [SUMMARY] | [CONTENT] Wedelia | Ayurveda | Siddha | MEG- 01 ||| WT ||| WT ||| TLC ||| HPLC ||| 6:4 | two | TLC | second ||| second | five | 3rd | HPLC ||| HPLC [SUMMARY] | [CONTENT] Wedelia | Ayurveda | Siddha | MEG- 01 ||| WT ||| WT ||| TLC ||| HPLC ||| 6:4 | two | TLC | second ||| second | five | 3rd | HPLC ||| HPLC [SUMMARY] |
|||||
Melatonin mitigates the adverse effect of hypoxia during myocardial differentiation in mouse embryonic stem cells. | 34313039 | Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. | BACKGROUND | Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. | METHODS | Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. | RESULTS | This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway. | CONCLUSIONS | [
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] | 8318788 | INTRODUCTION | Abnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.
Oxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].
Embryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].
The pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].
Previous studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation. | null | null | RESULTS | Hypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.
*p < 0.05 versus Normoxia (Control).
Hypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.
HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control).
To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.
*p < 0.05 versus Normoxia (Control).
Hypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.
HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control).
Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.
mESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Protein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.
mESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.
mESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Protein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.
mESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.
ERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.
Melatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.
Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
To investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.
Con, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.
ERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.
Melatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.
Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
To investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.
Con, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. | null | null | [
"Mouse embryonic stem cell (mESC) culture",
"Differentiation into cardiomyocytes",
"Hypoxia induction and melatonin and/or luzindole treatment",
"Assessment of beating ratio in mESCs-derived cardiomyocytes",
"Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR)",
"Western blotting analysis",
"Statistical analyses",
"Hypoxia-induced inhibition on myocardial differentiation",
"Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia",
"Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation"
] | [
"mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].",
"The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.",
"Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.",
"Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.",
"Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.",
"Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].",
"Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.",
"To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).",
"The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.",
"Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Mouse embryonic stem cell (mESC) culture",
"Differentiation into cardiomyocytes",
"Hypoxia induction and melatonin and/or luzindole treatment",
"Assessment of beating ratio in mESCs-derived cardiomyocytes",
"Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR)",
"Western blotting analysis",
"Statistical analyses",
"RESULTS",
"Hypoxia-induced inhibition on myocardial differentiation",
"Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia",
"Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation",
"DISCUSSION"
] | [
"Abnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.\nOxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].\nEmbryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].\nThe pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].\nPrevious studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation.",
"Mouse embryonic stem cell (mESC) culture mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\nmESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].\nDifferentiation into cardiomyocytes The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\nThe mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.\nHypoxia induction and melatonin and/or luzindole treatment Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\nDifferentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.\nAssessment of beating ratio in mESCs-derived cardiomyocytes Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\nContraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.\nTotal RNA extraction and real-time quantitative polymerase chain reaction (qPCR) Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\nTotal RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.\nWestern blotting analysis Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\nProteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].\nStatistical analyses Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.\nSignificant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.",
"mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].",
"The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.",
"Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.\nmEB, mouse embryo body; mESC, mouse embryonic stem cell.",
"Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.",
"Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.\nqPCR, quantitative polymerase chain reaction; F, forward; R, reverse.",
"Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].",
"Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.",
"Hypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nTo determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).\nEffect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nThe effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nEffect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nMelatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.",
"To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.\n*p < 0.05 versus Normoxia (Control).\nHypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.\nHIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control).",
"The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.\nmESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.\nProtein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.\nmESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\n*p < 0.05 versus Normoxia (Control). #\np < 0.05 versus Hypoxia.",
"Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.\nERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.\nMelatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.\nCon, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.\nTo investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.\nCon, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.",
"In the present study, it was observed that differentiation into cardiomyocytes is impaired under hypoxic conditions. In addition, it was observed that the differentiation of hypoxia-treated cardiomyocytes was recovered by treatment with melatonin. Our results suggest that supraphysiological melatonin treatment can counteract the adverse effect of hypoxia during the myocardial differentiation of mESCs.\nmRNA expression of Mtnr1a was decreased under hypoxic conditions. It was determined that the adverse effect of hypoxia was altered by melatonin pretreatment, which affected the mRNA expressions of melatonin receptors during myocardial differentiation of mESCs.\nMelatonin is reported to have a neuroprotective effect by relieving brain edema and improving nerve function [2526]. Albeit physiological concentration of melatonin has been known as between 30 to 400 pM in normal plasma; however, supraphysiological dose of melatonin treatment has been reported to hold anti-tumor activity in head and neck squamous cell carcinoma cell lines [2728]. In addition, Kudová et al. [29] reported that 100 mM promoted myocardial differentiation., while physiological dose of melatonin did not show a protective role against hypoxia. In the present study, three different concentrations (50–100 mM) of melatonin were set. We performed MTT assay and confirmed that melatonin per se did not affect cell viability in every concentration. In hypoxia-treated mESCs, treatment with melatonin 500 μM stabilized the cells' beating ratio. Beating ratio of stem cell-derived cardiomyocyte has been known to be sensitive to environmental stress during differentiation [30]. Our results suggest that 500 µM of melatonin treatment hold a protective effect against hypoxic stress during differentiation. Melatonin significantly reduced protein expression of HIF-1α; the mRNA expression of the melatonin receptors was restored in a concentration-dependent way. These results indicated that melatonin alleviates hypoxia-induced inhibition on cardiomyocytes differentiation.\nThe mechanism associated with the protective action of melatonin on hypoxia-induced effects in mESCs-derived cardiomyocytes was investigated. Previous studies have shown that activation of the ERK/AKT pathway is involved with melatonin-mediated anti-proliferative effects [3132]. In the present study, hypoxia increased the expression of p-ERK in cardiomyocytes during differentiation, while melatonin suppressed the expression of p-ERK in hypoxia-treated cardiomyocytes in a concentration-dependent manner. These results provide that melatonin can inhibit phosphorylation of ERK, which is consistent with the findings in previous studies; PI3K and AKT regulate various cellular events and are involved in cellular survival and apoptosis [3334]. In the present study, hypoxia reduced the protein expression of p-AKT and PI3K, while melatonin increased those expressions in hypoxia-treated cardiomyocytes in a concentration-dependent manner, in contrast to the results presented in other studies. Alteration of expression of Bcl-2 family members associated with apoptosis was also identified. Bcl-2 expression was decreased in hypoxia-treated cardiomyocytes during differentiation but was increased by melatonin pretreatment. In contrast, the expression of Bax increased in hypoxia-treated cardiomyocytes but decreased with melatonin pretreatment. Taken together, the results show that melatonin, acting via the AKT and PI3K pathway, can mitigate hypoxia-related effects on the myocardial differentiation of mESCs.\nROS are generated as a byproduct during cellular metabolism and involve in cellular signalling and homeostasis. When ROS significant increase in the cytoplasm, it leads to oxidative stress [3536]. Melatonin reduces oxidative stress by directly promoting the ROS neutralizers and by increasing the expression of antioxidant enzymes [3738]. The expression level of Cu/Zn-SOD, Mn-SOD, and catalase in differentiating cardiomyocytes was decreased under hypoxic conditions, but they were increased by pretreatment of melatonin before the induction of hypoxia.\nThis study demonstrates that melatonin can partially mitigate the adverse effect of hypoxia in hypoxia-treated cardiomyocytes during differentiation by regulating apoptosis and by reducing and oxidative stress of the cells via the actions of the AKT and PI3K pathway. Previous evidence suggests that melatonin hold protective roles in vitro and clinically. Kudová et al. [29] reported that melatonin promoted myocardial differentiation in HIF-1α deficient mESCs by stabilizing HIF-2α. Albeit Kudová et al. [29] did not implement a physical hypoxia environment, their observation and our result both concluded that melatonin exerted a protect effect during myocardial differentiation. In addition, in ischemic heart disease, melatonin can prevent cardiac malfunctions related to ischemic heart disease and myocardial cell death through its antioxidant and anti-inflammatory properties [39]. Overexpression of melatonin receptor 2 can attenuate the hypoxia derived cell injury via the Notch1/HES1/RORa signalling [40]. Furthermore, Guerra-Librero et al. [28] reported that high-dose of melatonin treatment exerted anticancer activity in neck and head squamous cell carcinoma cell line, suggesting the potential possibility for melatonin as clinical use. Our results can support that melatonin hold a regulatory function against hypoxia also in differentiation stage. For a reason, findings from the present study may provide helpful information for understanding the mechanism of melatonin as a mitigator of hypoxic damage."
] | [
"intro",
"materials|methods",
null,
null,
null,
null,
null,
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null,
"results",
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] | [
"Melatonin",
"hypoxia",
"cardiomyocytes",
"mESCs",
"Apoptosis"
] | INTRODUCTION:
Abnormal heart development and abnormal cardiovascular processes in the fetus can lead to congenital heart disease [12]. Studies have shown that chronic hypoxia in fetal stage can cause heart failure in the fetus and increase cardiovascular disease [345]. Therefore, early cardiac development of the fetus and the process of cardiovascular formation are significant.
Oxygen is an essential component of cardiac viability and function (heart contraction) [6]. Lack of oxygen can cause cardiac dysfunction (heart failure) or death, so a proper oxygen supply level is necessary. In preeclampsia patient, which is the most common consequence of complex pregnancy, as blood supply to the placenta reduces, the oxygen supplement to the fetus is affected, leading to abnormal growth [7]. Hypoxia is known to associate with myocardial ischemia and cause oxidative damage on cells by changing the redox balance. Hypoxia alters the cytochrome chain activity responsible for mitochondrial oxidative phosphorylation, increasing reactive oxygen species (ROS) production and resulting in improper oxidation of cytoplasm and nucleus, leading to apoptosis, necrosis [89].
Embryonic stem cells (ESCs) have pluripotency and differentiate into specific cell types; mouse ESCs can be differentiated into cardiomyocytes in vitro [1011]. The growth of the heart occurs due to the division of cardiomyocytes during the fetal stage. Stem cells have been used in myocardial differentiation studies [1012].
The pineal gland produces melatonin, a powerful antioxidant that directly removes radicals of cells [131415]. Melatonin has been known to have a protective effect in the growth and fetal cardiovascular function of the fetus, but it is unclear whether the antioxidant action of melatonin directly protects the heart and circulation functions of the fetus [1617]. With its antioxidant activity, melatonin can be used to treat cardiovascular diseases [18]. Moreover, melatonin is efficient in limiting the loss of vital cardiac tissue due to abnormal cardiac physiology or ischemia/reperfusion injury; it helps correct heart failure by reducing cardiac hypertrophy [19].
Previous studies have shown that both melatonin and hypoxia are associated with remodeling of the heart [20]. However, during the early stage of myocardial differentiation, the effects of melatonin have not been fully described. Thus, the present study aimed to investigate: i) the effects of hypoxia on the early stages of cardiomyocytes differentiation and ii) the effects of melatonin pretreatment on the hypoxia-induced effects on cardiomyocytes differentiation.
MATERIALS AND METHODS:
Mouse embryonic stem cell (mESC) culture mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].
mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].
Differentiation into cardiomyocytes The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.
The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.
Hypoxia induction and melatonin and/or luzindole treatment Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.
mEB, mouse embryo body; mESC, mouse embryonic stem cell.
Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.
mEB, mouse embryo body; mESC, mouse embryonic stem cell.
Assessment of beating ratio in mESCs-derived cardiomyocytes Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.
Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.
Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR) Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.
qPCR, quantitative polymerase chain reaction; F, forward; R, reverse.
Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.
qPCR, quantitative polymerase chain reaction; F, forward; R, reverse.
Western blotting analysis Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].
Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].
Statistical analyses Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.
Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.
Mouse embryonic stem cell (mESC) culture:
mESCs (ES-E14TG2a) were purchased from the American Type Culture Collection (USA). mESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts (mEFs) at humidified culture incubator (37°C with 5% CO2). The growth medium was prepared as in previously described [10].
Differentiation into cardiomyocytes:
The mESCs were suspended without mouse leukemia inhibitory factor (mLIF) in the differentiation medium containing 15% fetal bovine serum (FBS). Aside from mLIF and 15% FBS, other ingredients were the same as the growth medium. The mouse embryo bodies (mEBs) were formed (25 μL, 800 cells/drop, 84 drops per plate) on the lid of a Petri dish (SPL Inc., Korea). PBS was added on the bottom of the Petri dish to avoid evaporation of differentiation medium; then the top plate hanging the EBs was inverted and then cultured. After 3 days, mEBs formed on the lid were transferred to an uncoated Petri dish containing 6 mL of differentiation medium. After one day of suspension, the EBs (6–7 EBs per well) were transplanted into a 6-well plate containing 2 mL of differentiation medium.
Hypoxia induction and melatonin and/or luzindole treatment:
Differentiation into cardiomyocytes was induced under normoxic (95% O2, 5% CO2) and hypoxic conditions (94% N2, 1% O2, 5% CO2) as described in Fig. 1. To confirm the effects of melatonin (Sigma-Aldrich, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich), it was treated before inducing hypoxia as shown in Fig. 1B.
mEB, mouse embryo body; mESC, mouse embryonic stem cell.
Assessment of beating ratio in mESCs-derived cardiomyocytes:
Contraction of mESCs that had differentiated into cardiomyocytes was observed manually via phase-contrast microscopy. The beating ratio was measured by the number of contracted cells relative to the total number of attached embryoid bodies, expressed as a percentage.
Total RNA extraction and real-time quantitative polymerase chain reaction (qPCR):
Total RNA was collected from the cells using TRI reagent, then 1 μg of total RNA was transcribed to produce first-strand cDNA. The real-time qPCR was performed and the fluorescence intensity was measured. The material information and processes for PCR were already described in our previous study [10]. The primer sequences used are shown in Table 1. The expression level of each gene is normalized by that of Gapdh.
qPCR, quantitative polymerase chain reaction; F, forward; R, reverse.
Western blotting analysis:
Proteins were extracted using RIPA buffer. Then, protein concentration was measured at 562 nm using a BCA assay. Fifty microgram of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with primary antibodies for 1 h at room temperature and then overnight at 4°C: hypoxia-inducible factor (HIF)-1α (#14179, 1:1,000 dilution, Cell Signaling Technology, USA), HIF-2α (#7096, 1:1,000 dilution, Cell Signaling Technology), phospho-extracellular signal-related kinase (p-ERK; sc-7383, 1:500 dilution, Santa Cruz Biotechnology), ERK(sc-93, 1:500 dilution, Santa Cruz Biotechnology), phospho-protein kinase B (p-AKT; #4051, 1:1,000 dilution, Cell Signaling Technology), AKT (sc-8321, 1:500 dilution, Santa Cruz Biotechnology), B-cell lymphoma 2 (Bcl-2; sc-7382, 1:500, Santa Cruz Biotechnology), Bcl-2-associated X proteins (Bax; sc-7480, 1:500, Santa Cruz Biotechnology), HSP70 (1:1,000, Cell Signaling Technology), Cu/Zn-SOD (1:1,000, Cell Signaling Technology), Mn-SOD (1:1,000, Cell Signaling Technology), Catalase (1:1,000, Cell Signaling Technology), and GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778, 1:500 dilution, Santa Cruz Biotechnology). Membranes were incubated with anti-rabbit IgG (#7074, 1:1,000, Cell Signaling Technology) or anti-mouse IgG (#7076, 1:1,000, Cell Signaling Technology)-conjugated horseradish peroxidase secondary antibodies. Other materials and processes were already described in our previous study [10].
Statistical analyses:
Significant differences were detected by using analysis of variance. The analyses were performed with the Graph Pad Prism (v.5.0, Graph Pad Software, USA). Each value is expressed as means ± SD of three separate experiments at least, and a representative result is depicted in the figures. The p values < 0.05 were considered statistically significant.
RESULTS:
Hypoxia-induced inhibition on myocardial differentiation To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.
*p < 0.05 versus Normoxia (Control).
Hypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.
HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control).
To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.
*p < 0.05 versus Normoxia (Control).
Hypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.
HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control).
Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.
mESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Protein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.
mESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.
mESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Protein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.
mESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.
ERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.
Melatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.
Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
To investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.
Con, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.
ERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.
Melatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.
Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
To investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.
Con, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Hypoxia-induced inhibition on myocardial differentiation:
To determine the effect of hypoxia on mESCs, hypoxic states were induced during various differentiation periods. The mRNA expression of mesodermal markers, including Brachyury and cardiac-specific markers Tbx20 and cTn1, were investigated with real-time qPCR (Fig. 2). When hypoxia was induced during differentiation at 6–10 days and 2–10 days, Brachyury mRNA expression was reduced (Fig. 2A). Tbx20 mRNA expression was decreased when hypoxia was induced in differentiation at 6-10 days (Fig. 2B). cTn1 mRNA expression was reduced in all groups, causing hypoxia (Fig. 2C). These results show that hypoxia can inhibit cardiogenesis of mESCs.
*p < 0.05 versus Normoxia (Control).
Hypoxic environment was induced during myocardial differentiation of mESCs; the expression level of HIF-1α, a hypoxia marker, in mECSs was shifted corresponding with the induction of hypoxia. In Fig. 3A, the upregulated protein expression of HIF-1α indicated that the mESCs were properly exposed to hypoxia during each differential stages. To identify the effect of melatonin during hypoxic induction, melatonin receptor Mtnr1a was investigated in transcriptional level. Under normal oxygen conditions, the expression of Mtnr1a mRNA increased during the differentiation of mESCs into cardiomyocytes (Supplementary Fig. 1). However, at 6–10 days and 2–10 days of mESC differentiation under hypoxic conditions, Mtnr1a mRNA expression was reduced (Fig. 3B). These results indicate that hypoxic conditions can influence Mtnr1a expression in mESCs during their differentiation into cardiomyocytes.
HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; EB, embryo body; Diff, differentiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control).
Effect of melatonin pretreatment on cardiomyocytes suppressed by hypoxia:
The effects of melatonin on hypoxia-induced cardiomyocytes were examined (Fig. 4). Melatonin (50, 100, and 500 μM) was added to the differentiation medium two days before hypoxia induction and cells were continuously maintained in the melatonin-treated media except for the control group. We preemptively performed cell counting kit-8 (CCK-8) assay to screen the potential hazard of our experimental design for the melatonin treatment; from 50 to 500 µM, melatonin per se did not exert inhibition on cell viability in mESCs (Supplementary Fig. 2). In all hypoxia-induced groups, the beating ratio of differentiated cardiomyocytes was reduced but recovered when 500 μM of melatonin had been treated before hypoxia induction (Fig. 4A). The expression of cardiac progenitor marker Tbx20 was decreased in all hypoxia induction groups, and there was no significant change in Tbx20 when exposed to melatonin (Fig. 4B). By contrast, cardiac-specific marker cTn1 mRNA expression was significantly reduced in hypoxia but was recovered by the 500 μM melatonin administration (Fig. 4C). These results indicate that 500 μM of melatonin can alter the hypoxia-induced changes during cardiomyocytes differentiation.
mESCs, mouse embryonic stem cells; qPCR, quantitative polymerase chain reaction.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Protein levels of HIF-1α as a hypoxia marker and HIF-2α as a control marker were examined at day 4–10 of differentiation (Fig. 5). As shown in Fig. 5A, melatonin reduced the expression of HIF-1α protein in a concentration-dependent manner, but the HIF-2α level did not change. Also, under these conditions, the expression of melatonin receptor Mtnr1a increased in a concentration-dependent way (Fig. 5B). These findings support that melatonin can influence hypoxia during cardiomyocytes differentiation by reducing the HIF-1α protein level and increasing the expression of melatonin receptor Mtnr1a mRNA.
mESC, mouse embryonic stem cell; HIF, hypoxia-inducible factor; qPCR, quantitative polymerase chain reaction; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
*p < 0.05 versus Normoxia (Control). #
p < 0.05 versus Hypoxia.
Effect of melatonin on ERK/AKT pathway in hypoxia-induced cardiomyocytes differentiation:
Melatonin has been known to inhibit ROS and regulate apoptosis [2122]. To determine that the ERK/AKT pathway is involved by melatonin in hypoxic conditions during myocardial cell differentiation, it was evaluated using Western blot analysis. As depicted in Fig. 6A, the induction of hypoxia increased the protein expression of p-ERK, and melatonin suppressed the level of p-ERK in hypoxia-induced cardiomyocytes according to an increase of melatonin concentration. On the other hand, the levels of p-AKT and phosphatidylinositol 3-kinase (PI3K) were reduced during hypoxia induction but increased under melatonin-plus-hypoxia treatment. Apoptosis is a complicated cell signalling process which many genes involved, including Bcl-2, Bax. Bcl-2 protein has an anti-apoptotic and anti-autophagic action, and its expression during cardiomyocytes differentiation decreased while under hypoxic conditions but increased with melatonin pretreatment (Fig. 6B). However, the level of the pro-apoptotic protein Bax increased during cardiomyocytes differentiation under hypoxic conditions but decreased with melatonin pretreatment. Overall, the results indicate that melatonin can regulate the inhibition of p-ERK and activation of p-AKT during the differentiation of mESCs into cardiomyocytes.
ERK, extracellular signal-related kinase; AKT, protein kinase B; PI3K, phosphatidylinositol 3-kinase; Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, cl-2-associated X proteins.
Melatonin efficiently neutralizes free-radical and indirectly protects against oxidative stress by stimulating the production of antioxidant enzymes [23]. Melatonin stimulates the expression of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [24]. As shown in Fig. 7, protein expression of Cu/Zn-SOD, Mn-SOD, and catalase were reduced in the cardiomyocytes under hypoxic conditions; but the expression of those enzymes were rescued with melatonin pretreatment. These results indicate that melatonin inhibits hypoxia-induced effects on cardiomyocytes differentiation by stimulating the production of Cu/Zn-SOD, Mn-SOD, and catalase enzymes.
Con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
To investigate the involvement of melatonin receptors, the melatonin receptor antagonist luzindole was treated simultaneously with melatonin. Luzindole abolished the melatonin-induced effects in the expression of p-AKT, p85 (PI3K), Cu/Zn-SOD, Mn-SOD, and catalase proteins (Fig. 8). The results suggest that melatonin pretreatment can mitigate hypoxia effects via the p-Akt and PI3K pathway in the myocardial differentiation of mESCs.
Con, control; Mel, melatonin; Luz, luzindole; p-AKT, phospho-protein kinase B; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
DISCUSSION:
In the present study, it was observed that differentiation into cardiomyocytes is impaired under hypoxic conditions. In addition, it was observed that the differentiation of hypoxia-treated cardiomyocytes was recovered by treatment with melatonin. Our results suggest that supraphysiological melatonin treatment can counteract the adverse effect of hypoxia during the myocardial differentiation of mESCs.
mRNA expression of Mtnr1a was decreased under hypoxic conditions. It was determined that the adverse effect of hypoxia was altered by melatonin pretreatment, which affected the mRNA expressions of melatonin receptors during myocardial differentiation of mESCs.
Melatonin is reported to have a neuroprotective effect by relieving brain edema and improving nerve function [2526]. Albeit physiological concentration of melatonin has been known as between 30 to 400 pM in normal plasma; however, supraphysiological dose of melatonin treatment has been reported to hold anti-tumor activity in head and neck squamous cell carcinoma cell lines [2728]. In addition, Kudová et al. [29] reported that 100 mM promoted myocardial differentiation., while physiological dose of melatonin did not show a protective role against hypoxia. In the present study, three different concentrations (50–100 mM) of melatonin were set. We performed MTT assay and confirmed that melatonin per se did not affect cell viability in every concentration. In hypoxia-treated mESCs, treatment with melatonin 500 μM stabilized the cells' beating ratio. Beating ratio of stem cell-derived cardiomyocyte has been known to be sensitive to environmental stress during differentiation [30]. Our results suggest that 500 µM of melatonin treatment hold a protective effect against hypoxic stress during differentiation. Melatonin significantly reduced protein expression of HIF-1α; the mRNA expression of the melatonin receptors was restored in a concentration-dependent way. These results indicated that melatonin alleviates hypoxia-induced inhibition on cardiomyocytes differentiation.
The mechanism associated with the protective action of melatonin on hypoxia-induced effects in mESCs-derived cardiomyocytes was investigated. Previous studies have shown that activation of the ERK/AKT pathway is involved with melatonin-mediated anti-proliferative effects [3132]. In the present study, hypoxia increased the expression of p-ERK in cardiomyocytes during differentiation, while melatonin suppressed the expression of p-ERK in hypoxia-treated cardiomyocytes in a concentration-dependent manner. These results provide that melatonin can inhibit phosphorylation of ERK, which is consistent with the findings in previous studies; PI3K and AKT regulate various cellular events and are involved in cellular survival and apoptosis [3334]. In the present study, hypoxia reduced the protein expression of p-AKT and PI3K, while melatonin increased those expressions in hypoxia-treated cardiomyocytes in a concentration-dependent manner, in contrast to the results presented in other studies. Alteration of expression of Bcl-2 family members associated with apoptosis was also identified. Bcl-2 expression was decreased in hypoxia-treated cardiomyocytes during differentiation but was increased by melatonin pretreatment. In contrast, the expression of Bax increased in hypoxia-treated cardiomyocytes but decreased with melatonin pretreatment. Taken together, the results show that melatonin, acting via the AKT and PI3K pathway, can mitigate hypoxia-related effects on the myocardial differentiation of mESCs.
ROS are generated as a byproduct during cellular metabolism and involve in cellular signalling and homeostasis. When ROS significant increase in the cytoplasm, it leads to oxidative stress [3536]. Melatonin reduces oxidative stress by directly promoting the ROS neutralizers and by increasing the expression of antioxidant enzymes [3738]. The expression level of Cu/Zn-SOD, Mn-SOD, and catalase in differentiating cardiomyocytes was decreased under hypoxic conditions, but they were increased by pretreatment of melatonin before the induction of hypoxia.
This study demonstrates that melatonin can partially mitigate the adverse effect of hypoxia in hypoxia-treated cardiomyocytes during differentiation by regulating apoptosis and by reducing and oxidative stress of the cells via the actions of the AKT and PI3K pathway. Previous evidence suggests that melatonin hold protective roles in vitro and clinically. Kudová et al. [29] reported that melatonin promoted myocardial differentiation in HIF-1α deficient mESCs by stabilizing HIF-2α. Albeit Kudová et al. [29] did not implement a physical hypoxia environment, their observation and our result both concluded that melatonin exerted a protect effect during myocardial differentiation. In addition, in ischemic heart disease, melatonin can prevent cardiac malfunctions related to ischemic heart disease and myocardial cell death through its antioxidant and anti-inflammatory properties [39]. Overexpression of melatonin receptor 2 can attenuate the hypoxia derived cell injury via the Notch1/HES1/RORa signalling [40]. Furthermore, Guerra-Librero et al. [28] reported that high-dose of melatonin treatment exerted anticancer activity in neck and head squamous cell carcinoma cell line, suggesting the potential possibility for melatonin as clinical use. Our results can support that melatonin hold a regulatory function against hypoxia also in differentiation stage. For a reason, findings from the present study may provide helpful information for understanding the mechanism of melatonin as a mitigator of hypoxic damage.
| Background:
Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase.
Methods:
Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated.
Results:
Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects.
Conclusions:
This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway. | null | null | 7,977 | 318 | [
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] | null | null | null | [CONTENT] Melatonin | hypoxia | cardiomyocytes | mESCs | Apoptosis [SUMMARY] | null | [CONTENT] Melatonin | hypoxia | cardiomyocytes | mESCs | Apoptosis [SUMMARY] | null | [CONTENT] Melatonin | hypoxia | cardiomyocytes | mESCs | Apoptosis [SUMMARY] | null | [CONTENT] Animals | Biomarkers | Cell Differentiation | Embryonic Stem Cells | Gene Expression Regulation | Heart | Hypoxia | Melatonin | Myocytes, Cardiac | Oxygen [SUMMARY] | null | [CONTENT] Animals | Biomarkers | Cell Differentiation | Embryonic Stem Cells | Gene Expression Regulation | Heart | Hypoxia | Melatonin | Myocytes, Cardiac | Oxygen [SUMMARY] | null | [CONTENT] Animals | Biomarkers | Cell Differentiation | Embryonic Stem Cells | Gene Expression Regulation | Heart | Hypoxia | Melatonin | Myocytes, Cardiac | Oxygen [SUMMARY] | null | [CONTENT] fetus unclear antioxidant | hypoxia fetal stage | cardiac development fetus | heart failure fetus | hypoxia inhibit cardiogenesis [SUMMARY] | null | [CONTENT] fetus unclear antioxidant | hypoxia fetal stage | cardiac development fetus | heart failure fetus | hypoxia inhibit cardiogenesis [SUMMARY] | null | [CONTENT] fetus unclear antioxidant | hypoxia fetal stage | cardiac development fetus | heart failure fetus | hypoxia inhibit cardiogenesis [SUMMARY] | null | [CONTENT] melatonin | hypoxia | differentiation | expression | cardiomyocytes | fig | cell | mescs | protein | induced [SUMMARY] | null | [CONTENT] melatonin | hypoxia | differentiation | expression | cardiomyocytes | fig | cell | mescs | protein | induced [SUMMARY] | null | [CONTENT] melatonin | hypoxia | differentiation | expression | cardiomyocytes | fig | cell | mescs | protein | induced [SUMMARY] | null | [CONTENT] heart | fetus | cardiovascular | melatonin | abnormal | oxygen | cardiac | heart failure | failure | cause [SUMMARY] | null | [CONTENT] melatonin | hypoxia | fig | expression | differentiation | control | cardiomyocytes | mrna | induced | protein [SUMMARY] | null | [CONTENT] melatonin | hypoxia | differentiation | expression | fig | cardiomyocytes | cell | mescs | control | hypoxic [SUMMARY] | null | [CONTENT] ||| Melatonin [SUMMARY] | null | [CONTENT] Tbx20 ||| mESCs ||| ||| p-ERK | Bcl-2 | Bax | 3 | 2 | Bcl-2 | Cu/Zn-SOD | Mn-SOD ||| [SUMMARY] | null | [CONTENT] ||| Melatonin ||| mESCs ||| ||| ||| ||| Tbx20 ||| mESCs ||| ||| p-ERK | Bcl-2 | Bax | 3 | 2 | Bcl-2 | Cu/Zn-SOD | Mn-SOD ||| ||| the p-AKT | PI3K [SUMMARY] | null |
|||
Multi-contrast atherosclerosis characterization (MATCH) of carotid plaque with a single 5-min scan: technical development and clinical feasibility. | 25184808 | Multi-contrast weighted imaging is a commonly used cardiovascular magnetic resonance (CMR) protocol for characterization of carotid plaque composition. However, this approach is limited in several aspects including low slice resolution, long scan time, image mis-registration, and complex image interpretation. In this work, a 3D CMR technique, named Multi-contrast Atherosclerosis Characterization (MATCH), was developed to mitigate the above limitations. | BACKGROUND | MATCH employs a 3D spoiled segmented fast low angle shot readout to acquire data with three different contrast weightings in an interleaved fashion. The inherently co-registered image sets, hyper T1-weighting, gray blood, and T2-weighting, are used to detect intra-plaque hemorrhage (IPH), calcification (CA), lipid-rich necrotic core (LRNC), and loose-matrix (LM). The MATCH sequence was optimized by computer simulations and testing on four healthy volunteers and then evaluated in a pilot study of six patients with carotid plaque, using the conventional multi-contrast protocol as a reference. | METHODS | On MATCH images, the major plaque components were easy to identify. Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation. Based on Cohen's kappa tests, moderate to excellent agreement in the image-based or artery-based component detection between the two protocols was obtained for LRNC, IPH, CA, and LM, respectively. Compared with the conventional multi-contrast protocol, the MATCH protocol yield significantly higher signal contrast ratio for IPH (3.1±1.3 vs. 0.4±0.3, p<0.001) and CA (1.6±1.5 vs. 0.7±0.6, p=0.012) with respect to the vessel wall. | RESULTS | To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for characterization of carotid plaque composition. Our pilot clinical study suggests that the MATCH-based protocol may outperform the conventional multi-contrast protocol in several respects. With further technical improvements and large-scale clinical validation, MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup. | CONCLUSIONS | [
"Aged",
"Algorithms",
"Carotid Arteries",
"Carotid Stenosis",
"Computer Simulation",
"Contrast Media",
"Feasibility Studies",
"Fibrosis",
"Hemorrhage",
"Humans",
"Image Interpretation, Computer-Assisted",
"Imaging, Three-Dimensional",
"Magnetic Resonance Angiography",
"Male",
"Middle Aged",
"Models, Cardiovascular",
"Necrosis",
"Pilot Projects",
"Plaque, Atherosclerotic",
"Predictive Value of Tests",
"Reproducibility of Results",
"Signal-To-Noise Ratio",
"Vascular Calcification"
] | 4222690 | Background | Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].
To date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].
A highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].
Second, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].
Third, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].
In this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference. | Methods | Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).
Schematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.
At the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.
Criteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle
IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.
The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).
Schematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.
At the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.
Criteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle
IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.
Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].
A low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.
A TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).
Relevant imaging parameters for the sequences used
*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.
To verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.
A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].
A low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.
A TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).
Relevant imaging parameters for the sequences used
*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.
To verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.
Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.
Artery-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.
Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.
Artery-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.
Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.
In healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.
For patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.
Blinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.
The design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.
All the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.
All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.
In healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.
For patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.
Blinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.
The design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.
All the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations. | Results | Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.
Computer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).
Good image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).
Healthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.
For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.
Computer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).
Good image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).
Healthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.
Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.
The remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.
(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.
(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.
(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.
Slice-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.
Note: the plaque with IPH detected was also counted as a plaque with LRNC.
The MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).
MATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.
The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.
The remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.
(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.
(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.
(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.
Slice-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.
Note: the plaque with IPH detected was also counted as a plaque with LRNC.
The MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).
MATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol. | Conclusions | A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup. | [
"Background",
"Sequence design",
"Optimization",
"Patient studies",
"Image analysis",
"Computer simulations and in vivo verification",
"Patient studies",
"Competing interests",
"Authors’ contributions"
] | [
"Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.",
"The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.",
"A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.",
"Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.",
"All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.",
"For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.",
"The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.",
"The authors declare that they have no competing interests.",
"ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Sequence design",
"Optimization",
"Patient studies",
"Image analysis",
"Results",
"Computer simulations and in vivo verification",
"Patient studies",
"Discussion",
"Conclusions",
"Competing interests",
"Authors’ contributions"
] | [
"Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].\nTo date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].\nA highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].\nSecond, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].\nThird, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].\nIn this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.",
" Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\nThe proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.\n Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\nA hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.\n Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\nSix male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.\n Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.\nAll image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.",
"The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).\nSchematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.\nAt the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.\nCriteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle\nIPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.",
"A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].\nA low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.\nA TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).\nRelevant imaging parameters for the sequences used\n*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.\nTo verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.",
"Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.\nArtery-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.",
"All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.\nIn healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.\nFor patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.\nBlinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.\nThe design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.\nAll the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.",
" Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\nFor the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.\n Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.\nThe two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.",
"For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.\nComputer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).\nGood image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).\nHealthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.",
"The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.\nThe remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.\n(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.\n(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.\n(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.\nSlice-based composition analyses: MATCH vs. Conventional protocol\nLRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.\nNote: the plaque with IPH detected was also counted as a plaque with LRNC.\nThe MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).\nMATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.",
"To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for adequate characterization of carotid plaques. Compared with the conventional multi-contrast protocol, the presented MATCH CMR protocol features substantially shortened scan time and simplified image interpretation. The pilot study on patients demonstrated that the MATCH protocol is in good agreement with the conventional protocol in the detection of IPH, CA, LM, and LRNC as well as differentiation of IPH age.\nTo enable a short scan time and expedited identification of these components, the MATCH protocol included a minimum of three contrast weightings. The first contrast weighting, hyper T1-w, adopted a nonselective inversion preparation that has been employed in the MPRAGE method and proven to outperform several other T1-w sequences for the detection of IPH [29]. Such an approach, however, may not adequately suppress the blood signals while achieving good contrast between the IPH and vessel wall. Although a phase-sensitive (PS) reconstruction method could be used to address the issue [25],[39], an FSD black-blood preparation module was used instead to eliminate the need of post-processing and avoid phase manipulation-related errors. With such a combination of magnetization preparations, computer simulations revealed an IPH-wall signal contrast of 0.173 that is substantially higher than that theoretically available from regular MPRAGE imaging [39]. In our pilot study, 7 images with IPH-like signal characteristics were observed by the MATCH protocol only, suggesting its higher sensitivity to IPH than the conventional multi-contrast protocol that included T1-w TSE instead of MPRAGE and thus could be of limited performance according to Ota et al. [29].\nThe second contrast weighting, gray blood, was designed specifically for the detection of CA, especially the superficial calcified nodules. Due to hypointense signals and juxtaluminal location, superficial calcified nodules are often poorly visualized on the black-blood images and likely mistaken as the lumen or wall surface ulceration. On the other hand, the shadowing effect from the bright lumen on the TOF images may limit the discrimination of this constituent from others. In contrast, gray blood contrast makes the lumen slightly brighter than calcification while maintaining an adequate contrast between the lumen and the vessel wall. In contrast to the original implementation by Koktzoglou I. [28], gray blood contrast in this work was acquired following relatively long blood T1-recovery, resulting in a slightly brighter lumen. Nevertheless, the advantages associated with the gray blood contrast were preserved and have been clearly demonstrated in the patient studies.\nThe third contrast weighting, T2-w, was chosen to provide overall plaque morphology, detect LM and LRNC, and, when combined with the hyper T1-w contrast, differentiate acute and recent hemorrhage. With the optimized parameters, the T2-w MATCH images were nearly identical to the conventional T2-w TSE images with respect to overall image contrast. Therefore, concordant composition analyses were obtained with both characterization protocols in our study.\nAn interleaved acquisition of the three contrast weightings is one of noteworthy features in MATCH. This may significantly reduce the likelihood of mis-registration between multiple contrasts and potentially avoid substantial data exclusion in patient studies [11],[14]. Easier identification of co-existent components and better appreciation of their spatial relations were perceived by the reviewer when using MATCH. As an important ingredient for the interleaved acquisition strategy, a low-flip-angle segmented FLASH readout employed in this work allowed for a large amount of k-space lines to be acquired and three-phase signal sampling per TR with a very minimal interruption of T1-recovery.\nLong scan times commonly associated with 3D imaging for plaque characterization is a limitation because some patients, particularly the elderly in whom carotid atherosclerosis is common, may not tolerate the long procedure. In a recent multi-center trial using the conventional plaque characterization protocol, patient movement-induced motion artifact accounted for 46% of failed cases (15% of all) [40]. Thus, the proposed MATCH protocol was developed so that imaging could be completed within a clinically acceptable scan time, i.e. 5 minutes. The use of fast FLASH readout, interleaved image acquisition, and parallel imaging has contributed to the achievement of this goal. As a trade-off, however, high slice resolution (e.g. 1 mm) or even isotropic spatial resolution was not attempted in this work. Therefore, the reduced partial volume effect by 3D imaging was essentially not demonstrated herein. Nevertheless, the MATCH sequence has the potential to yield higher spatial resolution images if advanced fast imaging methods such as compressed sensing can be integrated into it.\nDue to the interleaved acquisition manner and relatively long scan times, MATCH could be more prone to motion than the conventional sequences. Random motion events, such as bulk motion or swallowing, would corrupt all three MATCH data sets and necessitate a repeat scan. In contrast, the conventional protocol may still have part of scans providing valid information. Although no evident motion artifacts were observed in MATCH with the limited patient size in this work, this shortcoming merits a systematic investigation and further technical improvement such as motion self-gating [41].\nIt should be acknowledged that MATCH is not capable of identifying all important features in carotid atherosclerotic plaques. Thinning/rupture of the fibrous cap has been shown to have the highest hazard ratio compared with IPH and LRNC and therefore serves as one of important imaging marker for plaque vulnerability [42]. However, the current non-contrast MRI techniques have been shown to be poor in reproducibility of identifying fibrous cap [43]. Hence, MATCH, as a non-contrast technique, was not designed in this first development work to characterize the fibrous status. Further innovation is desirable to incorporate the ability into MATCH. Alternatively, contrast-enhanced MRI that has proven good reproducibility of fibrous cap status assessment [44] may be combined with MATCH.\nThere are several limitations in this study. First, plaque histology, the gold standard for plaque characterization, was not available in the pilot study. The capability of MATCH in detecting each of histological components was evaluated through a comparison with the conventional multi-contrast protocol. Despite good agreement in image-based composition analysis, there was some discrepancy between the two protocols. Histological verification is needed to determine the accuracy of composition analysis. Second, the patient sample size is rather small. This preliminary work presents technical aspects and the feasibility of the new sequence. While the pilot data have been encouraging, a large-scale investigation is warranted to further elucidate its clinical utility. Third, the conventional multi-contrast protocol that served as a reference could be further refined. For example, the TOF sequence should have used the same slice thickness and in-plane resolution as in other scans; MPRAGE [29] and contrast-enhanced T1-w TSE [45]-[47] might be included for an improved detection of IPH and lipid core, respectively, although this could be unsuitable for patients who cannot tolerate longer imaging protocol or have renal insufficiency. Last, some potential technical advantages of the MATCH sequence over the conventional protocol await more stringent validation. For example, higher slice resolution was not demonstrated due to the consideration of scan time and patient tolerance; more simplified image interpretation was only subjectively reported by the image reader but not quantitatively validated by a comparison of interpretation times. Further investigations with focus on these aspects are highly necessary.",
"A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup.",
"The authors declare that they have no competing interests.",
"ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript."
] | [
null,
"methods",
null,
null,
null,
null,
"results",
null,
null,
"discussion",
"conclusions",
null,
null
] | [
"Carotid plaque",
"Atherosclerosis",
"Multi-contrast",
"Composition characterization",
"Magnetic resonance"
] | Background:
Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].
To date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].
A highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].
Second, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].
Third, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].
In this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.
Methods:
Sequence design The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).
Schematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.
At the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.
Criteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle
IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.
The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).
Schematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.
At the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.
Criteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle
IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.
Optimization A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].
A low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.
A TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).
Relevant imaging parameters for the sequences used
*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.
To verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.
A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].
A low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.
A TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).
Relevant imaging parameters for the sequences used
*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.
To verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.
Patient studies Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.
Artery-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.
Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.
Artery-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.
Image analysis All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.
In healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.
For patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.
Blinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.
The design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.
All the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.
All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.
In healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.
For patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.
Blinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.
The design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.
All the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.
Sequence design:
The proposed technique employs a 3D spoiled segmented fast low angle shot (FLASH) readout to acquire data with three different contrast weightings following a nonselective inversion pulse and various inversion-recovery times (TIs) (Figure 1a).
Schematic diagram (a) and signal simulations (b) of the MATCH sequence. a. Three contrast weightings following a nonselective inversion (Nonsel Inv) pulse and various inversion-recovery times (TIs) are acquired during each TR (4800 ms). At the first TI, TI1, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals. At the second TI (TI1 + 1200 ms), blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI (TI1 + 3600 ms), all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. The acquisition of each contrast weighting is based on a low-flip-angle FLASH readout that is immediately after a chemically selective fat saturation module (Fat Sat). The two FSD preparative modules consist of 2 and 4 composite refocusing pulses, respectively, to minimize (17-ms-long) or strengthen (40-ms-long) T2-decay effects while maintaining homogenous B1 field. b. With the TI1 optimized, steady-state signal evolutions for the normal vessel wall, IPH, and arterial blood during a TR period were simulated. The arterial blood signal involution shown herein is based on the assumption that inflow fresh blood is ignored.
At the first TI, hyper T1 weighting is created to suppress the signals from all non-hemorrhagic vessel wall tissues while highlighting IPH because of its shorter T1. Flow-sensitive dephasing (FSD) preparation is applied before data acquisition to suppress luminal blood signals [31]-[33]. At the second TI, blood and non-hemorrhagic vessel wall tissues will both recover moderately to create neutral weighting or gray-blood images to highlight the dark CA. At the third TI, all tissues have largely recovered and black-blood T2-w images are acquired with a combined T2 and FSD preparation. T2-weighted images are used to characterize LM and LRNC and facilitate differentiation between acute and recent IPH. The criteria for resolving plaque components based on the three sets of images acquired using the MATCH sequence are summarized in Table 1.
Criteria for determining plaque components from the MATCH imaging protocol based on components’ signal intensity relative to adjacent sternocleidomastoid muscle
IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix; LRNC: lipid-rich necrotic core. +: hyper-intense; =: iso-intense; -: hypo-intense.
Optimization:
A hyperbolic secant adiabatic inversion pulse was used for uniform inversion at 3 T. The FSD module in the hyper T1-w acquisition had two composite (90°x-180°y-90°x) refocusing pulses weighted in an MLEV pattern to ensure second-order corrections to B1-field inhomogeneity [34]. A cumulative first-order gradient moment (m1) of 990 mT∙ms2/m, which has been suggested to be adequate for carotid flow suppression given the voxel dimension used in this work [32], was chosen to allow a relatively short preparation time of 17 ms to alleviate the T2 weighting. The second FSD module was 40 ms long to generate T2 weighting. To further improve the insensitivity to B1-field inhomogeneity, four composite refocusing pulses were used [34].
A low flip angle of 8° was used in the FLASH readout for minimal interruption of the T1-recovery of vessel wall magnetizations and reducing the incidence of image artifact due to a strong k-space signal modulation. Chemically selective fat saturation was applied immediately before each readout train to improve outer wall boundary definition. To preserve the effects of the FSD preparation and fat saturation, centric phase-encoding was employed in readouts.
A TR of 4800 ms was chosen as a trade-off between the total scan time and the need of near full T1 recovery prior to the T2-w acquisition. In this context, the first TI, denoted as TI1, was optimized through computer simulations based on the Bloch equations in MATLAB (R2009b, Mathworks, Natick, MA); TI1 should approximately null the signals from all non-hemorrhagic vessel wall tissue and IPH appears as a “hot spot” in images. The second and third TIs were shifted by 1200 ms (for moderate T1 recovery) and 3600 ms (for near full T1 recovery), respectively, relative to TI1. The simulated tissues included arterial wall media (T1/T2 = 1115/50 ms), arterial blood (1550/275 ms), IPH (500/25 ms) [26],[35]. Fibrotic tissue (T1 = 1000 ms) [28], a major intra-plaque occupant, was also considered in the optimization of TI1. The sequence parameters used in simulations were the same as in in-vivo scans (Table 2).
Relevant imaging parameters for the sequences used
*The # of segments varied with oversampling in the phase-encoding direction which ranged from 20-30%.
To verify the simulation results and characterize general image contrast, the sequence was tested on four healthy subjects. A 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and a 4-channel carotid coil (Machnet BV, Roden, The Netherlands) were used for data acquisitions.
Patient studies:
Six male patients (aged 56-77, mean age = 67) with ultrasonography-documented carotid artery stenosis (Table 3) were recruited in a feasibility study using a 3 T whole-body system (MAGNETOM Verio; Siemens AG, Erlangen, Germany) and an 8-channel carotid coil (Shanghai Chenguang Medical Technologies, Shanghai, China). After obtaining informed consent from each patient, MATCH imaging was conducted during their scheduled clinical CMR examination that included the conventional multi-contrast (black-blood multi-slices 2D T1-w and T2-w TSE with saturation bands, multi-slab 3D TOF) imaging protocol [11]. All these scans were performed axially with the same imaging volume centered at the bilateral bifurcations. The slice thickness (2 mm) and in-plane spatial resolution of MATCH matched those of TSE. TOF had different slice thickness and in-plane spatial resolution as a part of the clinical protocol.
Artery-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. Conv.: Conventional protocol. +: detected; -: undetected. *The symbol in the parenthesis denotes the results when only IPH-free slices were read.
Image analysis:
All image data sets were processed on a workstation (Leonardo; Siemens AG, Erlangen, Germany). T2-w MATCH images served as a reference of arterial wall anatomy for determining plaque composition using the other two sets of images of MATCH.
In healthy subjects, for each of MATCH contrast weightings, the center 5 slices of each artery were chosen for signal measurement. On every slice, two regions-of-interest (ROI) were manually drawn to respectively outline the arterial lumen and wall for measuring their signal intensity Sl and Sw. Noise level (σn) was measured as the standard deviation of signals from an ROI (~100 mm2) manually drawn in an artifact-free air region that was near the artery. The signal-to-noise ratio (SNR) of the wall and lumen (calculated as Sl (w)/σn) as well as the wall-lumen contrast-to-noise ratio (CNR) (calculated as |Sw-Sl|/σn) were calculated for each slice and averaged over all 5 slices. The above ROIs were first prescribed on the T2-w images and then copied to the other two contrast weightings to ensure consistency of measurement locations. Due to the use of parallel imaging, absolute SNR and CNR were difficult to quantify. Instead, the values calculated herein were counted as apparent SNR and apparent CNR, respectively. They were aimed to help appreciate the relative image contrasts of different MATCH image sets and reveal whether they were in accord with the theoretical sequence design.
For patient studies, the images from each artery underwent the location matching (including image reformation in 3D TOF) process to account for the inconsistency in slice number and thickness between the two protocols and inter-scan motion. The images that had all four spatially registered scans were further screened for diagnostic quality (i.e. overall image quality, vessel wall clarity). Diagnostic images were finally included in subsequent analyses.
Blinded image review for composition identification was performed by a radiologist (with 9-year experience in carotid plaque MR characterization) with the two imaging protocols separated by two weeks. The presence of IPH, CA, LRNC, and LM were determined using the criteria summarized in Table 1 for the MATCH protocol and those in a recent review article for the conventional protocol [36]. In addition, for both protocols, the age of each identified IPH, i.e. acute or recent, was recorded according to its signal intensity relative to adjacent sternocleidomastoid muscle on T2-w images: iso-intensity or hypo-intensity for the acute type and hyper-intensity for the recent type [37]. Artery-based and image-based agreements in the detection of individual components by the two protocols were respectively determined using a Cohen’s kappa test. According to Landis and Koch [38], the agreement was rated as follows: kappa 0 to 0.2 indicated slight agreement, 0.21 to 0.4 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.8 substantial agreement, and 0.81 upward excellent agreement. Note that the plaque with detected IPH was counted as a plaque with LRNC during review, and LRNCs underwent agreement analysis for both scenarios – IPH present and IPH absent.
The design of hyper T1-w and gray-blood contrasts is relatively unique in MATCH, aiming for better discerning IPH and CA, respectively. To unravel such, the contrast ratio (CR) between the component and the regular vessel wall was calculated as [S1-S2]/S2 and compared between the two protocols using a paired Student’s t test. More specifically, the signal was measured in all images where IPH or CA was identified and clearly demarcated in both protocols; for simplicity, IPH (S1) and the vessel wall (S2) was analyzed on T1-w TSE and hyper T1-w MATCH, whereas CA (S2) and the vessel wall (S1) on TOF and gray-blood MATCH since these contrast weightings were the most relevant for the discrimination of IPH or superficial calcification. Window level adjustment was performed on each of images to ensure optimal contrast between the vessel wall and lumen. The component of interest was then manually outlined along its boundary that was visually determined by the reviewer (with 7-year carotid wall MRI) based on signal contrast (hypo- or hyper-intense). SNR or CNR was not measured herein because parallel imaging was used in the MATCH protocol.
All the above statistical tests were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL). Statistical significance was defined at the p < 0.05 level. Data are presented as means ± standard deviations.
Results:
Computer simulations and in vivo verification For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.
Computer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).
Good image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).
Healthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.
For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.
Computer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).
Good image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).
Healthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.
Patient studies The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.
The remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.
(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.
(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.
(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.
Slice-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.
Note: the plaque with IPH detected was also counted as a plaque with LRNC.
The MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).
MATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.
The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.
The remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.
(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.
(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.
(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.
Slice-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.
Note: the plaque with IPH detected was also counted as a plaque with LRNC.
The MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).
MATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.
Computer simulations and in vivo verification:
For the interrogated range of 400 to 600 ms, an optimal TI (490 ms) existed that could approximately null the signals of the “normal” arterial wall and provided a maximal difference of 0.185 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2a, b). However, the largely distributed fibrotic tissue required a shorter TI, 460 ms. As a trade-off, 480 ms was selected as the optimal TI1, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (Figure 2b cross hair). With the choice, steady-state signal evolutions (after 10 iterations) for the normal vessel wall, IPH, and arterial blood during a TR period were simulated and shown in Figure 1b.
Computer simulations for the optimization of the first inversion-recovery time (TI). A TI of 490 ms can approximately null the signals of the “normal” arterial wall, but the fibrotic tissue required a shorter TI time, i.e. 460 ms (a). As a trade-off, 480 ms was selected as the optimal delay time, providing a difference of 0.173 in normalized signals between the wall and IPH on the hyper T1-w contrast (b cross hair).
Good image quality was observed in MATCH images of all 4 healthy subjects (Figure 3a). The three image sets were intrinsically co-registered in spatial location. The normal vessel wall and lumen were both substantially attenuated on hyper T1-w images using the optimized delay time. They were of moderate signal intensity on gray blood images with the lumen appearing a little brighter, presumably due to the inflow effect. On T2-w images, the carotid wall was well depicted with the lumen and epivascular fat sufficiently attenuated. The above image contrast was also corroborated by the signal measurements from the 8 healthy carotid arteries (Figure 3b).
Healthy subject example images (a. 6 contiguous slices around the left carotid bifurcation) and signal measurements from 8 carotid arteries (b) from the MATCH imaging protocol. All three contrast weightings are inherently registered. Note that blood attenuation is sufficient in both hyper T1-w and T2-w images. A.U.: arbitrary unit. The error bars in b denotes a standard deviation of the specific quantity.
Patient studies:
The two protocols were successfully performed in all 6 patients. The scan time was 12-15 min for the conventional protocol (including sequence set up and B0 field shimming) and 5 min for the MATCH protocol. During the studies, patients were instructed to remain still throughout the imaging session. No evident motion artifacts were observed within MATCH and each of the conventional sequences. All three MATCH contrasts were spatially co-registered. However, slight mis-registration was observed between T1-w TSE and T2-w TSE in one subject or between TOF and TSE in two subjects. We stipulate that the patient could remain still well in a scan due to the prior instruction and reminder right before the scan, but more likely he/she moved the head during the gap between scans. For a total of 192 artery images (6 patients × 16 slices × 2 arteries), 32 arterial images (from two arteries) were excluded from analyses because of the presence of stenting and surgical removal of intima, respectively, 7 excluded due to spatial mismatch between the two protocols, and 17 excluded due to poor diagnostic quality (11, incomplete wall structure due to low signals on both MATCH and TSE/TOF images; 4, incomplete wall structure due to low signals on MATCH; 2, flow artifacts on MATCH). The remaining 136 images were subjected to analyses.
The remaining 10 arteries all have at least one of 4 major components. On MATCH images, the major components were easy to identify by the reader. IPH (Figures 4 and 5, arrows) appeared hyper-intense on hyper T1-w MATCH images, but hyper-intense (recent IPH) or iso-intense (acute IPH) on T2-w MATCH images. CA (Figure 5, dashed arrow) appeared as focal signal voids on gray-blood MTCH images. LM (Figure 5, arrowheads) appeared hyper-intense on T2-w MATCH but not on hyper T1-w MATCH images. LRNC was depicted as a hypo-intense region on T2-w MATCH images when there was no IPH in it (Figure 6). Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation, as exemplified by the case shown in Figure 5. For the artery-based component detection, excellent agreement was obtained for LRNC (regardless of the presence of IPH) (κ = 1.0) and IPH (κ = 1.0), good agreement obtained for LM (κ = 0.62), and moderate agreement obtained for CA (κ = 0.41) (Table 3). For the image-based component detection between the two protocols, excellent agreement was obtained for LRNC (including images with IPH detected) (κ = 0.89) and IPH (κ = 0.83), and substantial agreement obtained for CA (κ = 0.70) and LM (κ = 0.78) (Table 4). Furthermore, IPH-free LRNCs received slice-based good agreement (κ = 0.86; MATCH: detected in 20 out of 107 images; conventional protocol: detected in 23 out of 107 images; 19 images in common) between the two protocols as well. In the 22 images with IPH detected by both protocols, a complete consensus in the classification of IPH (22 images with IPH in common) into the acute (6 images) and recent (16 images) types was achieved.
(Patient 3) For the recent intra-plaque hemorrhage, hyper-intense contrast is its characteristic appearance on hyper T1-w and T2-w MATCH. The component is also verified on conventional T1-w (hyper-intense), T2-w (hyper-intense) TSE and TOF (hyper-intense). Notably, the contrast of the hemorrhage in this case is much sharper on hyper T1-w MATCH than on T1-w TSE.
(Patient 4) A slice with co-existent plaque components including acute intra-plaque hemorrhage (solid arrows), calcification (dashed arrows), and loose matrix (arrowheads). With the MATCH protocol, the unique contrast weightings and spatial coregistration facilitate easier identification of co-existent components and better appreciation of their spatial relations. The loose matrix is also hyper-intense on T1-w TSE, mimicking hemorrhage. However, it is not as hyper-intense as hemorrhage on TOF.
(Patient 2) A lipid-rich necrotic core without hemorrhage appears hypo-intense on both T2-w TSE and T2-w MATCH. Notice that there is signal drop in the lumen on the gray blood image due to flow signal dephasing associated with fast flow at the high degree stenosis.
Slice-based composition analyses: MATCH vs. Conventional protocol
LRNC: lipid-rich necrotic core; IPH: intra-plaque hemorrhage; CA: calcification; LM: loose matrix. +: detected; -: undetected.
Note: the plaque with IPH detected was also counted as a plaque with LRNC.
The MATCH protocol yielded more incidence of IPH (Figure 7a) and CA (Figure 7b) compared with the conventional protocol. All IPH-like and all but one CA-like locations as detected by the conventional protocol were also identified on MATCH images; however, the MATCH protocol indicated 7 additional “IPH” (categorized as the recent type) and 15 additional “CA” (Table 3). Five locations appeared hyper-intense on both hyper T1-w and T2-w MATCH images, but only on T2-w TSE, and therefore were counted as LM by the conventional protocol (Figure 7 a, arrows). Both of the two components had significantly higher signal contrast (based on 22 images for IPH: CR = 3.1 ± 1.3 vs. 0.4 ± 0.3, p < 0.001; 23 images for CA: CR = 1.6 ± 1.5 vs. 0.7 ± 0.6, p = 0.012) with respect to the vessel wall on MATCH images, making their detection remarkably straightforward (Figure 7b).
MATCH sequence yielded more detection of hemorrhage-like and calcification-like locations. a. (Patient 3) The recent hemorrhage (arrows) indicated by MATCH is hyper-intense on T2-w TSE but not on T1-w TSE; thus it is counted as loose matrix by the conventional protocol. b. (Patient 7) A superficial calcified nodule is clearly depicted on gray blood MATCH. Such a component, when protruding into the arterial lumen, is usually not well observable with the conventional multi-contrast protocol. c. The contrast ratios between the hemorrhage/calcification and the regular arterial wall are significantly higher (p < 0.001 and p = 0.012, respectively) with the MATCH protocol.
Discussion:
To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for adequate characterization of carotid plaques. Compared with the conventional multi-contrast protocol, the presented MATCH CMR protocol features substantially shortened scan time and simplified image interpretation. The pilot study on patients demonstrated that the MATCH protocol is in good agreement with the conventional protocol in the detection of IPH, CA, LM, and LRNC as well as differentiation of IPH age.
To enable a short scan time and expedited identification of these components, the MATCH protocol included a minimum of three contrast weightings. The first contrast weighting, hyper T1-w, adopted a nonselective inversion preparation that has been employed in the MPRAGE method and proven to outperform several other T1-w sequences for the detection of IPH [29]. Such an approach, however, may not adequately suppress the blood signals while achieving good contrast between the IPH and vessel wall. Although a phase-sensitive (PS) reconstruction method could be used to address the issue [25],[39], an FSD black-blood preparation module was used instead to eliminate the need of post-processing and avoid phase manipulation-related errors. With such a combination of magnetization preparations, computer simulations revealed an IPH-wall signal contrast of 0.173 that is substantially higher than that theoretically available from regular MPRAGE imaging [39]. In our pilot study, 7 images with IPH-like signal characteristics were observed by the MATCH protocol only, suggesting its higher sensitivity to IPH than the conventional multi-contrast protocol that included T1-w TSE instead of MPRAGE and thus could be of limited performance according to Ota et al. [29].
The second contrast weighting, gray blood, was designed specifically for the detection of CA, especially the superficial calcified nodules. Due to hypointense signals and juxtaluminal location, superficial calcified nodules are often poorly visualized on the black-blood images and likely mistaken as the lumen or wall surface ulceration. On the other hand, the shadowing effect from the bright lumen on the TOF images may limit the discrimination of this constituent from others. In contrast, gray blood contrast makes the lumen slightly brighter than calcification while maintaining an adequate contrast between the lumen and the vessel wall. In contrast to the original implementation by Koktzoglou I. [28], gray blood contrast in this work was acquired following relatively long blood T1-recovery, resulting in a slightly brighter lumen. Nevertheless, the advantages associated with the gray blood contrast were preserved and have been clearly demonstrated in the patient studies.
The third contrast weighting, T2-w, was chosen to provide overall plaque morphology, detect LM and LRNC, and, when combined with the hyper T1-w contrast, differentiate acute and recent hemorrhage. With the optimized parameters, the T2-w MATCH images were nearly identical to the conventional T2-w TSE images with respect to overall image contrast. Therefore, concordant composition analyses were obtained with both characterization protocols in our study.
An interleaved acquisition of the three contrast weightings is one of noteworthy features in MATCH. This may significantly reduce the likelihood of mis-registration between multiple contrasts and potentially avoid substantial data exclusion in patient studies [11],[14]. Easier identification of co-existent components and better appreciation of their spatial relations were perceived by the reviewer when using MATCH. As an important ingredient for the interleaved acquisition strategy, a low-flip-angle segmented FLASH readout employed in this work allowed for a large amount of k-space lines to be acquired and three-phase signal sampling per TR with a very minimal interruption of T1-recovery.
Long scan times commonly associated with 3D imaging for plaque characterization is a limitation because some patients, particularly the elderly in whom carotid atherosclerosis is common, may not tolerate the long procedure. In a recent multi-center trial using the conventional plaque characterization protocol, patient movement-induced motion artifact accounted for 46% of failed cases (15% of all) [40]. Thus, the proposed MATCH protocol was developed so that imaging could be completed within a clinically acceptable scan time, i.e. 5 minutes. The use of fast FLASH readout, interleaved image acquisition, and parallel imaging has contributed to the achievement of this goal. As a trade-off, however, high slice resolution (e.g. 1 mm) or even isotropic spatial resolution was not attempted in this work. Therefore, the reduced partial volume effect by 3D imaging was essentially not demonstrated herein. Nevertheless, the MATCH sequence has the potential to yield higher spatial resolution images if advanced fast imaging methods such as compressed sensing can be integrated into it.
Due to the interleaved acquisition manner and relatively long scan times, MATCH could be more prone to motion than the conventional sequences. Random motion events, such as bulk motion or swallowing, would corrupt all three MATCH data sets and necessitate a repeat scan. In contrast, the conventional protocol may still have part of scans providing valid information. Although no evident motion artifacts were observed in MATCH with the limited patient size in this work, this shortcoming merits a systematic investigation and further technical improvement such as motion self-gating [41].
It should be acknowledged that MATCH is not capable of identifying all important features in carotid atherosclerotic plaques. Thinning/rupture of the fibrous cap has been shown to have the highest hazard ratio compared with IPH and LRNC and therefore serves as one of important imaging marker for plaque vulnerability [42]. However, the current non-contrast MRI techniques have been shown to be poor in reproducibility of identifying fibrous cap [43]. Hence, MATCH, as a non-contrast technique, was not designed in this first development work to characterize the fibrous status. Further innovation is desirable to incorporate the ability into MATCH. Alternatively, contrast-enhanced MRI that has proven good reproducibility of fibrous cap status assessment [44] may be combined with MATCH.
There are several limitations in this study. First, plaque histology, the gold standard for plaque characterization, was not available in the pilot study. The capability of MATCH in detecting each of histological components was evaluated through a comparison with the conventional multi-contrast protocol. Despite good agreement in image-based composition analysis, there was some discrepancy between the two protocols. Histological verification is needed to determine the accuracy of composition analysis. Second, the patient sample size is rather small. This preliminary work presents technical aspects and the feasibility of the new sequence. While the pilot data have been encouraging, a large-scale investigation is warranted to further elucidate its clinical utility. Third, the conventional multi-contrast protocol that served as a reference could be further refined. For example, the TOF sequence should have used the same slice thickness and in-plane resolution as in other scans; MPRAGE [29] and contrast-enhanced T1-w TSE [45]-[47] might be included for an improved detection of IPH and lipid core, respectively, although this could be unsuitable for patients who cannot tolerate longer imaging protocol or have renal insufficiency. Last, some potential technical advantages of the MATCH sequence over the conventional protocol await more stringent validation. For example, higher slice resolution was not demonstrated due to the consideration of scan time and patient tolerance; more simplified image interpretation was only subjectively reported by the image reader but not quantitatively validated by a comparison of interpretation times. Further investigations with focus on these aspects are highly necessary.
Conclusions:
A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contributions:
ZF designed and implemented the sequence, performed numerical simulations and in-vivo validation on healthy volunteers, and analyzed all data and prepared manuscript. WY was in charge of recruiting clinical patients, performing clinical studies, and helped analyzing data. YX participated in the sequence implementation and data acquisition on healthy volunteers. LD and LY participated in the coordination, data acquisition, and data analysis for clinical studies. ZW participated in the data acquisition of clinical studies. AHC participated in design and coordination of healthy volunteer studies. XB and GL helped with the sequence design and implementation. JA and TZ helped with coordination of clinical studies. PKS and ZZ participated in the design of the clinical studies. DL conceived of the sequence design and provided supervision of the whole project and critical review of the manuscript. All authors read and approved the final manuscript.
| Background:
Multi-contrast weighted imaging is a commonly used cardiovascular magnetic resonance (CMR) protocol for characterization of carotid plaque composition. However, this approach is limited in several aspects including low slice resolution, long scan time, image mis-registration, and complex image interpretation. In this work, a 3D CMR technique, named Multi-contrast Atherosclerosis Characterization (MATCH), was developed to mitigate the above limitations.
Methods:
MATCH employs a 3D spoiled segmented fast low angle shot readout to acquire data with three different contrast weightings in an interleaved fashion. The inherently co-registered image sets, hyper T1-weighting, gray blood, and T2-weighting, are used to detect intra-plaque hemorrhage (IPH), calcification (CA), lipid-rich necrotic core (LRNC), and loose-matrix (LM). The MATCH sequence was optimized by computer simulations and testing on four healthy volunteers and then evaluated in a pilot study of six patients with carotid plaque, using the conventional multi-contrast protocol as a reference.
Results:
On MATCH images, the major plaque components were easy to identify. Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation. Based on Cohen's kappa tests, moderate to excellent agreement in the image-based or artery-based component detection between the two protocols was obtained for LRNC, IPH, CA, and LM, respectively. Compared with the conventional multi-contrast protocol, the MATCH protocol yield significantly higher signal contrast ratio for IPH (3.1±1.3 vs. 0.4±0.3, p<0.001) and CA (1.6±1.5 vs. 0.7±0.6, p=0.012) with respect to the vessel wall.
Conclusions:
To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for characterization of carotid plaque composition. Our pilot clinical study suggests that the MATCH-based protocol may outperform the conventional multi-contrast protocol in several respects. With further technical improvements and large-scale clinical validation, MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup. | Background:
Disrupted carotid atherosclerotic plaques can lead to transient ischemic attack and cerebral thrombo-embolic stroke, significantly contributing to mortality and morbidity worldwide [1]-[3]. Accurate assessment of plaque stability and identification of the lesion at high risk for disruption is of vital importance for improving disease management as well as reducing public health burden. The vulnerability of a plaque is primarily related to its compositional characteristics [4]-[6]. High-resolution cardiovascular magnetic resonance (CMR) is a viable tool for non-invasive characterization of plaque composition [7],[8].
To date, the most commonly used CMR technique for plaque characterization is multi-contrast weighted imaging that involves a series of scans (e.g. T1- and T2-weighted [T1-w, T2-w] black-blood turbo spin-echo [TSE], and bright-blood time-of-flight [TOF]) to differentiate major plaque components, including lipid-rich necrotic core (LRNC), intra-plaque hemorrhage (IPH), calcification (CA), loose matrix (LM), and fibrous tissue [9]-[14]. Despite numerous successes in previous studies, the multi-contrast approach has four major limitations: (a) limited slice resolution associated with two-dimensional (2D) TSE imaging and potential repositioning error when used in serial studies; (b) long acquisition time (approximately 20-30 min); (c) image mis-registration due to inter-scan subject motion; (d) complex image interpretation for differentiating various components based on their signal patterns on multi-contrast weightings. While continued technical improvements and research efforts have been made during the past decade, routine application of CMR for plaque characterization in clinical work-up has not become standard practice [15].
A highly desired CMR technique would be capable of detecting multiple plaque components while mitigating all of the above issues. First, the technique should be based on a three-dimensional (3D) acquisition to reduce partial volume effects and relax the requirements for positioning, both of which are relevant to the accuracy of plaque assessment in tortuous carotid arteries [16],[17]. In fact, 3D imaging has been attempted for assessing carotid wall morphology [18]-[23] or detecting a specific plaque constituent such as IPH [24]-[26], LRNC [27], and CA [28].
Second, the technique should ideally require one scan only that provides multiple image contrasts in an interleaved fashion. This may help shorten the examination time and avoid the mis-registration issue. Multi-echo TSE is perhaps one of the earliest techniques adopting this idea to generate both T2- and proton density-weighted contrasts in carotid plaque imaging. Recent developments on multi-contrast imaging were still limited to offering only two contrasts in a single scan, one for general wall or lumen geometry and the other for one specific plaque component, such as IPH [25] and CA [28].
Third, the multiple image contrasts provided by the technique should be optimized and, if possible, respectively be tailored to one of major components to allow a simplified analysis of composition. Several component-specific contrast weightings have previously been proposed. Heavily T1-weighted contrast created by a nonselective inversion preparation (i.e. MPRAGE), for example, has been shown to be highly sensitive to IPH [24],[26],[29],[30]. Another example is the gray-blood contrast that better discriminates superficial calcific nodules from the carotid wall and lumen irrespective of its hypo-intense signals and juxtaluminal location [28].
In this work, a 3D CMR technique, named Multi-contrast ATherosclerosis CHaracterization (MATCH), was developed to meet the aforementioned needs. The technique features an interleaved acquisition of three image sets with different contrast weightings in a 5-min scan. The first two contrasts, black-blood hyper T1-w and gray-blood, are used to identify IPH and CA, respectively. A third T2-w contrast, in addition to providing overall plaque morphology, can detect LM and LRNC and differentiate acute and recent hemorrhage when combined with the hyper T1-w contrast. The MATCH sequence was optimized for 3.0-Tesla (3 T) based on computer simulations and testing on healthy volunteers and then evaluated in a pilot study of patients with carotid plaque, using the conventional multi-contrast protocol as a reference.
Conclusions:
A 3D CMR technique, MATCH, is presented that requires one scan only to concurrently collect three spatially co-registered multi-contrast image sets for comprehensive characterization of carotid plaque composition. Our pilot patient study demonstrates that the MATCH-based protocol has a comparable ability to detect carotid plaque components such as LRNC, IPH, CA, and LM in less time with no problems of mis-registration compared with the conventional multi-contrast protocol. Further technical improvements to reduce scan time and increase slice resolution (better than the current implementation of 2-mm thick slice) are needed to make the technique more useful in the assessment of carotid plaques. Large scale clinical validation, in particular with histology as reference, is warranted to elucidate whether MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup. | Background:
Multi-contrast weighted imaging is a commonly used cardiovascular magnetic resonance (CMR) protocol for characterization of carotid plaque composition. However, this approach is limited in several aspects including low slice resolution, long scan time, image mis-registration, and complex image interpretation. In this work, a 3D CMR technique, named Multi-contrast Atherosclerosis Characterization (MATCH), was developed to mitigate the above limitations.
Methods:
MATCH employs a 3D spoiled segmented fast low angle shot readout to acquire data with three different contrast weightings in an interleaved fashion. The inherently co-registered image sets, hyper T1-weighting, gray blood, and T2-weighting, are used to detect intra-plaque hemorrhage (IPH), calcification (CA), lipid-rich necrotic core (LRNC), and loose-matrix (LM). The MATCH sequence was optimized by computer simulations and testing on four healthy volunteers and then evaluated in a pilot study of six patients with carotid plaque, using the conventional multi-contrast protocol as a reference.
Results:
On MATCH images, the major plaque components were easy to identify. Spatial co-registration between the three image sets with MATCH was particularly helpful for the reviewer to discern co-existent components in an image and appreciate their spatial relation. Based on Cohen's kappa tests, moderate to excellent agreement in the image-based or artery-based component detection between the two protocols was obtained for LRNC, IPH, CA, and LM, respectively. Compared with the conventional multi-contrast protocol, the MATCH protocol yield significantly higher signal contrast ratio for IPH (3.1±1.3 vs. 0.4±0.3, p<0.001) and CA (1.6±1.5 vs. 0.7±0.6, p=0.012) with respect to the vessel wall.
Conclusions:
To the best of our knowledge, the proposed MATCH sequence is the first 3D CMR technique that acquires spatially co-registered multi-contrast image sets in a single scan for characterization of carotid plaque composition. Our pilot clinical study suggests that the MATCH-based protocol may outperform the conventional multi-contrast protocol in several respects. With further technical improvements and large-scale clinical validation, MATCH has the potential to become a CMR method for assessing the risk of plaque disruption in a clinical workup. | 14,891 | 433 | [
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] | [CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY] | [CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY] | [CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY] | [CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY] | [CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY] | [CONTENT] Carotid plaque | Atherosclerosis | Multi-contrast | Composition characterization | Magnetic resonance [SUMMARY] | [CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY] | [CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY] | [CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY] | [CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY] | [CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY] | [CONTENT] Aged | Algorithms | Carotid Arteries | Carotid Stenosis | Computer Simulation | Contrast Media | Feasibility Studies | Fibrosis | Hemorrhage | Humans | Image Interpretation, Computer-Assisted | Imaging, Three-Dimensional | Magnetic Resonance Angiography | Male | Middle Aged | Models, Cardiovascular | Necrosis | Pilot Projects | Plaque, Atherosclerotic | Predictive Value of Tests | Reproducibility of Results | Signal-To-Noise Ratio | Vascular Calcification [SUMMARY] | [CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY] | [CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY] | [CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY] | [CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY] | [CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY] | [CONTENT] assessment plaque stability | carotid plaque imaging | plaque mr characterization | application cmr plaque | cmr plaque characterization [SUMMARY] | [CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY] | [CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY] | [CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY] | [CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY] | [CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY] | [CONTENT] match | images | iph | contrast | t1 | wall | hyper | t2 | protocol | blood [SUMMARY] | [CONTENT] plaque | contrast | technique | multi | multi contrast | cmr | contrasts | weighted | carotid | multiple [SUMMARY] | [CONTENT] ms | wall | images | iph | match | t1 | blood | t2 | vessel | vessel wall [SUMMARY] | [CONTENT] images | match | hyper | iph | figure | intense | hyper intense | t1 | protocol | tse [SUMMARY] | [CONTENT] carotid plaque | carotid | plaque | match | technique | cmr | clinical | multi contrast | multi | validation particular [SUMMARY] | [CONTENT] match | images | iph | contrast | wall | t1 | ms | protocol | hyper | t2 [SUMMARY] | [CONTENT] match | images | iph | contrast | wall | t1 | ms | protocol | hyper | t2 [SUMMARY] | [CONTENT] CMR ||| ||| CMR | Multi [SUMMARY] | [CONTENT] three ||| T1 | T2 | IPH | CA | LRNC ||| four | six [SUMMARY] | [CONTENT] ||| three ||| Cohen | two | LRNC | IPH | CA ||| IPH | 3.1±1.3 | 0.4±0.3 | CA | 1.6±1.5 [SUMMARY] | [CONTENT] first | CMR ||| ||| CMR [SUMMARY] | [CONTENT] CMR ||| ||| CMR | Multi ||| three ||| T1 | T2 | IPH | CA | LRNC ||| four | six ||| ||| three ||| Cohen | two | LRNC | IPH | CA ||| IPH | 3.1±1.3 | 0.4±0.3 | CA | 1.6±1.5 ||| first | CMR ||| ||| CMR [SUMMARY] | [CONTENT] CMR ||| ||| CMR | Multi ||| three ||| T1 | T2 | IPH | CA | LRNC ||| four | six ||| ||| three ||| Cohen | two | LRNC | IPH | CA ||| IPH | 3.1±1.3 | 0.4±0.3 | CA | 1.6±1.5 ||| first | CMR ||| ||| CMR [SUMMARY] |
||||||
Factors Associated with Health Literacy, Self-Efficacy, Social Support, and Oral Health Care Behaviors Among Elderly in Northern Border Community Thailand. | 34326634 | Oral health problems among elderly people are an important public health issues worldwide. Oral healthcare is essential to the health and well-being of elders and is one of the key indicators determining their quality of life. This research aimed to study oral health literacy, self-efficacy, social support, and demographic characteristic factors associated with the oral health care behaviors of elderly people living in the rural areas of northern Thailand. | BACKGROUND | This research was a cross-sectional study that recruited 406 elderly participants using convenience and snowball samplings. Participants' names were obtained from the registration list of the Java Health Center Information System (JHCIS) program, where they received a health service between 2018 and 2020. Data were obtained through face-to-face interviews with participants, while they were waiting to receive a health service or through a phone interview. Linear regression was analyzed to determine the factors associated with oral healthcare behaviors. | METHODS | The majority of participants (85%) had inadequate functional health literacy, 52% had moderate self-efficacy toward oral health behaviors, 91.9% had moderate social support, and 53% admitted to moderate oral health behaviors. The results from the model show that self-efficacy, social support, and oral health literacy are positively associated with oral health care behaviors among the elderly (p-value < 0.05). The multiple regression model can account for 47.2% of the variance in oral health care behaviors. | RESULTS | Improving oral health care behaviors among elderly people should be considered by health care providers and those who provide social support. Self-esteem, communication skills among service providers and service receivers, and self-management of oral healthcare should receive special attention. Moreover, social support and relevant agencies can help promote oral healthcare by collaborating with other healthcare providers for better oral health outcomes among elderly people. | CONCLUSION | [
"Aged",
"Aged, 80 and over",
"Cross-Sectional Studies",
"Delivery of Health Care",
"Female",
"Health Literacy",
"Humans",
"Male",
"Middle Aged",
"Quality of Life",
"Self Efficacy",
"Social Support",
"Thailand"
] | 8314679 | Introduction | Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3
A Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15
Health literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice. | null | null | null | null | Conclusion | In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information. | [
"Methodology",
"Statistical Analysis",
"Results",
"Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly",
"Discussion",
"Conclusion"
] | [
"This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.",
"Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.",
"According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\n\nDemographic Characteristics of Elderly (N=406)\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\n\nOral Health Characteristics of Elderly (N=406)\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\n\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).",
"Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).",
"The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.",
"In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information."
] | [
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"Introduction",
"Methodology",
"Statistical Analysis",
"Results",
"Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly",
"Discussion",
"Conclusion"
] | [
"Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3\nA Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15\nHealth literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.",
"This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.\nThe study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.\nThe questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33\n3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.\nStatistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.\nStatistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.",
"Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.",
"According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7\n\nDemographic Characteristics of Elderly (N=406)\nIn terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7\n\nOral Health Characteristics of Elderly (N=406)\nTable 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)\n\nOral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)\nOral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).\nLinear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).",
"Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).\n\nFactors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)\nNote: *Significant at the 0.05 level (2-tailed).",
"The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nOral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.\nIn terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45\nIn the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.\nIn terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40\nSocial support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44\nIn terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.\nWhen analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.\nThis study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.",
"In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information."
] | [
"intro",
null,
null,
null,
null,
null,
null
] | [
"health literacy",
"self-efficacy",
"social support",
"oral health",
"elderly"
] | Introduction:
Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3
A Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15
Health literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.
Methodology:
This research was a cross-sectional study conducted in the rural Mae Rim district, Chiang Mai province, in northern Thailand between March – and May 2021. Chiang Mai was selected because it has the third highest number of elderly people living in Thailand according to the Department of Older Persons Thailand.11 The number of elderly people living in the province is expected to increase continuously.4,11 The mountainous area in which the study was conducted is located 30 kilometers from Chiang Mai city. Simple random sampling was employed using the drawing technique to select sub-districts out of 14 sub-districts. San Pong sub-district, Rim Tai sub-district, and Mae Rim sub-district were selected. The research was carried out at primary health service centers run by the Ministry of Public Health, where dentists can provide oral health examinations and services in the study area’s Health Promoting Hospital. Eligible participants were selected through convenience and snowball samplings. The inclusion criteria were: 1) Both males and females aged 60 years and older; 2) Living in the area of Mae Rim district for at least two years and having their names in the civil registry; 3) Registered as a patient in the Java Health Center Information System (JHCIS) 2018–2020 and received health services from the primary health center; 4) Ever received oral health service at the study area’s Health Promoting Hospitals; 5) No severe periodontitis, such as loose teeth, oral lesions, and swelling of the face or jaw; 6) The absence of any cognitive disorders; 7) Able to communicate in the local language; and 8) Willing to participate in the study. The sample size was calculated using the correlation coefficient formula31 with a 95% confidence level, 90% test power, and a correlation coefficient (r) = 0.163.32 The sample size was increased by five percent to address potential dropouts from the study by participants. The total number of participants in the study was 410 based on the calculation.
The study procedures started by recruiting 10 research assistants who were able to communicate in the local northern language. These assistants were public health personnel comprising three dental nurses, four public health scholars, and three nurses. A meeting was organized by the researcher on a single day from 9:00 am-12:00 pm to clarify the study’s objectives, data collection techniques, and the procedure for answering the questionnaires, schedule appointments, and to outline the rights and privacy of the participants. The researcher ensured that everyone obtained a common understanding regarding the research process. The researcher translated research materials into the local northern language so the research assistants could better understand the research context. In the process of data collection, the researcher contacted and cooperated with the director of the District Health Office, Health Promoting Hospital, and key persons in the community before data collection. Once the researcher obtained the written informed consent form, the research team started performing data collection in the study area. In the process of data collection, the elderly were interviewed face-to-face while waiting to receive health services at a health center. A telephone interview was performed in some cases when elderly participants could not come to a health center due to the COVID-19 pandemic situation. The process of data collection was conducted during the hours of 9:00 am and 16:00 pm; and the duration of each interview was approximately 20–30 minutes.
The questionnaires were used as an instrument to collect research information. The questionnaires were checked and validated by three experts in the field of dental and oral health, health behavioral science, and aging. The questionnaires comprised five parts including: 1) Part 1: Demographic characteristics such as gender, age, marital status, occupation, education level, chronic health conditions, current medication, smoking, alcohol consumption, income, and health and treatment benefits. 2) Part 2: An oral health literacy questionnaire adapted from related literature reviews to be suitable for the context of elderly people living in a rural community.25 The questionnaire comprised 37 items, and six components including: a) Assessing skills toward health information and services, which consisted of six questions; b) Cognitive skills, perceived knowledge and understanding of health information, which consisted of 10 questions; c) Communication skills, which consisted of four questions; d) Self-management skills, which consisted of seven questions;, e) Media literacy skills, which consisted of six questions; and f) Decision-making skills, which consisted of four questions. There were three choices for selecting an answer: Yes, “No”, and “Do not know.” The elderly people were allowed to choose only one answer. The scoring criteria were one point for a correct answer and 0 points for a wrong or “do not know” answer. The criteria for scoring were divided into three levels: scores ranging from 80–100% are considered as good; 60–69% is considered moderate; and 0–59% is considered poor. Therefore, scores greater than, or equal to, 30 indicates Adequate Health Literacy); scores of 22–29 indicate Marginal Functional Health Literacy; and scores less than, or equal to, 21 indicates Inadequacy Functional Health Literacy.33
3) Part 3: The Self-efficacy questionnaire on oral health behaviors was modified to be suitable for the context of elderly people living in a rural community.34 The questionnaire focused on the ability to regularly maintain oral healthcare behaviors, such as eating healthy food, cleaning or brushing teeth, and regular oral health check-ups. The questionnaire comprised 10 items. The questionnaire is a rating scale with three levels. The elderly participants could only select one answer from “Agree,” “Uncertain,” and “Disagree”. Scores between 24 and 30 indicates a good level, 18–23 indicates a moderate level, and 0–17 indicates a poor level.34,35 4) Part 4: A social support questionnaire toward periodontal status among elderly people which was adapted from related research and literature reviews to be suitable for the context of the elderly living in a rural community.36,37 Examples of social support questions were: 1. Have you ever received dental health information from dentists?; 2. Have you ever been supported, encouraged or reinforced by public health workers, family members or caregivers toward teeth brushing; and 3.Have you ever communicated with family caregivers regarding oral healthcare. The questionnaire comprised of 10 items using a Likert scale. Participants could choose one of the following answers: “Regularly,” “Sometimes,” and “Never”. Social support questionnaire was divided into three levels: Low (scores ≤ 17), Moderate (scores of 18–23), and High (scores ≥24). 5) Part 5: The oral healthcare behaviors questionnaire was modified from previous studies34,38 and included cleaning or brushing teeth, using dental floss, oral healthcare check-up, and healthy food consumption. The questionnaire comprises 12 items, and elderly participants could choose only one answer for activities they did during the previous week. The questionnaire is characterized by three scale levels: “Always Practice,” (5–7 times/week), “Sometimes Practice,” (2–4 times/week), and “Never Practice.” The scoring criteria are classified as follows: 28–36 indicates good oral healthcare behaviors; 21–27 indicates moderate oral health care behaviors; 0–20 indicates poor oral healthcare behaviors that need to be improved. The overall questionnaires were tested in a pilot study on 30 elderly people who had similar characteristics and lived in a rural community. A reliability test was performed on parts 2–5 of the questionnaires, with Cronbach’s alpha coefficient at 0.82, 0.87, 0.84, and 0.85, respectively.
Statistical Analysis Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.
Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.
Statistical Analysis:
Statistical analysis was performed using the SPSS software version 17, licensed from Chiang Mai University (SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to describe the demographic characteristics and general information among older adults. Linear regression was analyzed to determine the factors associated with oral health care behaviors. Initially, each independent variable was investigated using univariate analysis, with a p-value of 0.20. The significant variables were then included in a multivariate analysis to examine factors that were still linked to oral health care behaviors. The predictors, which were statistically significant at the 0.05 level, were put into the final regression model.
Results:
According to Table 1, the results represent the demographic characteristics of the total of 406 elderly participants in the study. The average age of the participants was 66.97 years (SD = 4.34), and more than half of them were female (52.5%). Just over half (50.7%) of the participants had obtained primary education; 73.6% were married, and 56.2% were employed. More than half of the participants (50.7%) had insufficient income. In terms of health behaviors and health status, just over a third (34.2%) of participants smoked cigarettes and drank alcohol (46.6%). Half of the participants had a medical history, involving one or more of the following: hypertension (HT), stress, diabetes mellitus (DM), chronic kidney disease (CKD), hyperlipidemia, stroke, coronary heart disease (CHD), and tuberculosis (TB).Table 1Demographic Characteristics of Elderly (N=406)Characteristicsn%Gender Male19347.5 Female21352.5Age 60–70 years31577.6 71–80 years9122.4 Min = 60, Max = 77, mean (SD) = 66.97 (4.336)Education No20049.3 Yes20650.7Marital status Single/widowed/divorced/separated10726.4 Married29973.6Working status Not working17443.8 Currently working22856.2Financial status Insufficient20650.7 Sufficient20049.3Smoking No26765.8 Yes13934.2Alcohol drinking No21753.4 Yes18946.6Incurrent disease No20350.0 Yes20350.0 Hypertension (HT) (yes)11528.3 Stress (yes)7618.7 Diabetes mellitus (DM) (yes)6917.0 Chronic kidney disease (CKD) (yes)276.7 Hyperlipidemia (yes)204.9 Stroke (yes)153.7 Coronary heart disease (CHD) (yes)61.5 Tuberculosis51.2Receiving information about dental health No21553.0 Yes19147.0 By health volunteer (yes)17543.1 By public health officer (yes)16741.1 By family (yes)245.9 By online media (yes)235.7
Demographic Characteristics of Elderly (N=406)
In terms of perceived health information, nearly half (47%) of the participants had ever received oral health information. The sources of health information came from village health volunteers, public health officers, family members, and online media. The oral health characteristics of the elderly are shown in Table 2. For health check-ups and health services, it was reported that over a third (37.4%) of participants had ever received an oral health examination; and the last check-up was over a year previously. The elderly had received services that included tooth extraction, denture and tooth replacement, cleaning and polishing teeth, fillings, addressing toothache, and other problems related to oral health.Table 2Oral Health Characteristics of Elderly (N=406)Characteristicsn%Have had dental services No25462.6 Yes15237.4When did you last visit the dentist? (N=152) ≤ 6 month1610.5 6–12 month4529.7 ≥ 1 year9159.8Have you had any problems? (N=152) Pull a tooth7046.0 Put false tooth3120.4 Clean and polish2214.4 Fillings1912.6 Dental pain106.6Why did you go to the dentist? (N=152) Medical Checkup4328.3 Illnesses symptom10971.7
Oral Health Characteristics of Elderly (N=406)
Table 3 illustrates the percentages and scores of the studied variables. Participants had inadequate functional health literacy scores (85%), moderate functional health literacy scores (13.3%), and good functional health literacy scores (1.7%), respectively; and the mean score of functional health literacy was 17.63 (SD = 3.92). In terms of self-efficacy toward oral health behaviors, we found that more than half of the participants obtained scores at a moderate level (52.5%), poor level (45.8%), and high level (1.7%), respectively, with a mean score of 17.75 (SD = 2.28). In terms of social support, most of the participants obtained scores at the moderate level (91.9%), followed by poor and high levels (6.4% and 1.7%), respectively; with a mean score of 20.11 (SD = 1.58). In terms of oral health behaviors, more than half of participants (53%) obtained scores at the moderate level, (43%) obtained scores at the poor level, and 3.9% received a high level. The mean score was 21.16 (SD = 3.21).Table 3Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)Variablesn%Oral health Literacy Inadequate Functional Health literacy (scores ≤ 21)34585.0 Marginal Functional Health literacy (scores of 22–29)5413.3 Adequate Functional Health literacy (scores ≥ 30)71.7 Min = 11, Max = 31, mean (SD) = 17.63 (3.918)Self-efficacy about oral health Low level (scores ≤ 17)18645.8 Moderate level (scores of 18–23)21352.5 High level (scores ≥24)71.7 Min = 13, Max = 24, mean (SD) = 17.75 (2.285)Social support about oral health Low level (scores ≤ 17)266.4 Moderate level (scores of 18–23)37391.9 High level (scores ≥24)71.7 Min = 14, Max = 25, mean (SD) = 20.11 (1.580)Oral health care behaviors Low level (scores ≤ 20)17543.1 Moderate level (scores of 21–27)21553.0 High level (scores ≥28)163.9 Min = 14, Max = 29, mean (SD) = 21.16 (3.207)
Oral Health Literacy, Self-Efficacy, Social Support and Oral Health Care Behaviors of Elderly (N=406)
Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).
Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)
Note: *Significant at the 0.05 level (2-tailed).
Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).
Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)
Note: *Significant at the 0.05 level (2-tailed).
Oral Health Literacy, Self-Efficacy and Social Support Associated with Oral Health Care Behaviors Among Elderly:
Linear regression analysis was used to investigate factors associated with oral health care behaviors among older adults (Table 4). The results of the univariate analysis revealed that significant factors at a p-value=0.20 included gender, financial status, current disease, smoking, drinking alcohol, receiving information about oral health, oral health literacy, self-efficacy, and social support about oral health. These variables were entered into the multivariate analysis. The results from the last model showed that self-efficacy, social support, and oral health literacy, were positively associated with oral health care behaviors among the elderly (p-value<0.05). The single factor of oral health literacy can explain 40.7% of the variance in oral health care behaviors, whereas the multiple regression models can account for 47.2%.Table 4Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)VariableUnivariable AnalysisMultivariable AnalysisBBetaSig.BBetaSig.Female−0.470−0.0730.140Age (years)−0.002−0.0020.960Married−0.350−0.0480.333Getting an education−0.074−0.0120.817Currently working−0.081−0.0120.802Sufficient income0.8620.1350.007*Incurrent disease−1.842−0.288<0.001*Smoking−0.915−0.1350.006*Alcohol drinking−0.918−0.1430.004*Receiving information0.7510.1170.018*Oral health literacy (scores)0.5230.639<0.001*0.4320.528<0.001*Self-efficacy (scores)0.5490.391<0.001*0.1220.0870.035*Social support (scores)0.8820.434<0.001*0.4890.241<0.001*Note: *Significant at the 0.05 level (2-tailed).
Factors Associated with Oral Health Care Behaviors Among Elderly by Linear Regression (N=406)
Note: *Significant at the 0.05 level (2-tailed).
Discussion:
The study’s findings show that there was a statistically significant association between the primary variables and oral healthcare behaviors among elderly people living in a rural area of Thailand. The majority of the elderly had scores that revealed inadequate and moderate functional health literacy, which suggests the majority of these people obtained education at a primary level. Moreover, the way they manage their health is aligned with their perceived traditional way of life. These issues limit their ability to understand and seek health information, as well as to decide on their own oral healthcare behaviors. This is consistent with a previous study, which found that elderly participants had poor oral health literacy scores because they had limited knowledge of oral health information.39 Even though health providers communicated with the elderly in their local language, their limited health literacy prevented them from understanding health information.39 One study similar to ours also found that one third of elderly people had poor health literacy regarding oral healthcare behaviors.22 It was also found that elderly people with an insufficient level of health literacy could have a decreased ability to learn and understand knowledge related to oral healthcare behaviors. As a result, they would practice self-care behaviors toward oral healthcare incorrectly; for instance, they would brush their teeth incorrectly, not use dental floss, and consume sugary food, which leads to cavities and tooth decay.39,40 Poor health literacy is one of the barriers that prevents oral health hygiene and appropriate oral healthcare practices among the elderly.40
Oral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.
Oral health literacy was found to be a highly significant positive predictor of oral healthcare behaviors. The results are consistent with the concept of cognitive behaviors.28 Cognitive skills have positive effects on the practice of self-healthcare behaviors,28 which is consistent with the principle of functional health literacy that demonstrates that cognitive and social skills could defines a person’s motivation and ability to understand health information.41 As a previous study reported, a positive correlation between oral health knowledge and health behaviors resulted in people implementing correct practices, such as teeth brushing skills and maintaining personal oral hygiene.39,42 Therefore, to increase cognitive skills, specific communication methods to improve the elderly’s oral hygiene should be appropriate in their context and should be easy to understand and comprehend.
In terms of self-efficacy toward oral healthcare behaviors, the findings of the study show that most of the elderly participants obtained moderate to poor scores on self-efficacy. Most of them had a poor perception of self-managing their healthcare, which leads to a decrease in decision making regarding their own healthcare. Self-efficacy determines a person’s behavioral expression and the trend that drives their health outcomes.43 In this study, the elderly participants had poor and moderate self-efficacy in preventing oral health problems, which resulted in inappropriate oral healthcare behaviors and an increase in oral health problems, such as tooth decay, and cavities.44 One study showed that low self-efficacy indicates poor self-assessment toward oral health and is associated with a high risk of caries and periodontal disease.44,45
In the analysis of factors, the study showed that self-efficacy was statistically significant and positively associated with oral healthcare behaviors. Consistent with previous studies, it is believed self-efficacy is a predictor of teeth brushing behaviors of the elderly. Self-efficacious elders are confident in their ability to brush their teeth regularly and believe in the results, which are called outcome-expectancy; moreover, they are able to brush their teeth successfully on their own and observe oral health hygiene. One study highlighted that people’s improved ability to manage their own oral healthcare is associated with better outcomes; for instance, improved brushing time, associated with having less plaque and bleeding, reduces the risk of long-term periodontal treatment.46,47 Therefore, encouraging elderly people to recognize the importance of self-management is one effective way of helping the individuals develop good healthcare behaviors.43 This is consistent with the concept of self-efficacy, which allows a person to achieve expected behaviors through cognitive motivation, the use of persuasive speech and emotional stimulation, and the decision-making process.28,43 This enhances the elderly’s ability to decide on or determine any behaviors that are appropriate for managing care by themselves.
In terms of social support, this study found that the majority of the elderly obtained a moderate score level. The study shows that they need help from people around them to take care of themselves. In addition, there is limited access to dental care in rural areas, and dental health information is not yet comprehensive.40 As one study mentioned, if elderly people were encouraged by social support, including receiving health information from healthcare providers and getting help from family members and information from media, it would positively impacts the elderly’s self-management of oral healthcare.44 This is consistent with a study which found that the role of social support influenced the elderly’s attitude toward oral healthcare and self-management skills.40
Social support is one of the important variables we found to be significantly positively associated with oral health behaviors.30,37 Social support consists of groups of people in society; in which individuals interact with family members, neighbors, and key persons in the community, such as health personnel and, health volunteers. Such interactions have a positive impact on individuals’ health and well-being.37,48 One study found that social support toward elders oral health behaviors is significantly associated with good behaviors on dental healthcare, wearing dentures, and other health-related behaviors among the elderly.49 This finding is consistent with the social support concept, which states that social support has a direct impact on one’s health. Social support networks enhance individuals’ ability to manage their own health by boosting confidence and increasing motivation.36,37,44
In terms of oral healthcare behaviors, we found that the majority of the elderly obtained moderate and poor scores. This group generally did not go to a health center for an annual check-up or receive an oral examination. Generally, they only visit health centers when illness occurs or they need to have a tooth extracted, false teeth made, or cleaning and polishing teeth and fillings. Their self-efficacy scores regarding oral healthcare behaviors were at a moderate level and need to be improved. Elders may experience an increase in medical expenses, which could be a barrier to visiting a health center for oral health check-ups.50 Similar to a study on oral health among the elderly, we found that oral healthcare behaviors among older people may become inappropriate due to them having developed low self-confidence and low self-efficacy in performing self-healthcare behaviors, resulting in performing inappropriate or, irregular behaviors.32,36,50 Therefore, supporting, promoting, and providing educations for the elderly on how to implement self-care behaviors on a continuous basis is important; and oral hygiene, primary oral care, and oral health knowledge among the aging population should receive attention.
When analyzing the demographic characteristics of the elderly participants, this study found that sufficient income and perceived health information had statistically significant relationships with oral healthcare behaviors. This is consistent with a previous study, which mentioned that the elderly with high incomes demonstrated better healthcare behaviors than those with low incomes; and insufficient economic status was associated with elders’ healthcare behaviors.38,50 In terms of perceived health information, it is similar to a previous study which mentioned that elders who were advised by public health workers had significantly higher oral healthcare behaviors than those who were not advised by public health workers.38,50 Our study also found that chronic health conditions, smoking, and drinking alcohol, had statistically significant relationships with oral healthcare behaviors among the elderly. This is consistent with previous studies that have shown chronic health conditions may lead to poorer oral healthcare behaviors and decreased quality of life.22,50 Moreover, smoking was significantly associated with periodontal health conditions. Smoking and drinking alcohol had negative relationships with healthcare behaviors, resulting in increased oral health problems.22,50,51 Oral healthcare should be promoted among elderly people with underlying disease so that they can manage their care properly.
This study has strengths and limitations. Many variables are being studied in this paper, including oral health literacy, self-efficacy, social support, and other demographic characteristics associated with oral healthcare behaviors of the elderly. This is the strength of this research. On the other hand, there are several limitations, which include 1) The data obtained from non-probability samplings may not be proven by the registered list in the public health center and primary care system (Java Health Center Information System: JHCIS). Therefore, the results are representative only of elderly people living in the Mae Rim District, Chiang Mai Province, Thailand. However, this study has provided critical information that can be extended to large-scale multistage studies to identify key trends for promoting policy and intervention activities. Secondly, other important factors such as health insurance rights, psychosocial factors, and social capital are not included in the data set used in the study. Thirdly, the outcome variables reported in the study are specific to oral healthcare behaviors; future studies could switch this focus to other factors, such as the (DMFT index, the number of natural teeth, or occluding pairs of teeth). Future research could consider focusing on brushing teeth skills that influence oral health status; for instance, forced brushing, the frequency of toothbrush replacement, and self-efficacy in the proper use of brushing techniques among the elderly. Other aspect of oral healthcare, such as flossing, could be studied in greater depth.
Conclusion:
In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information.
| Background:
Oral health problems among elderly people are an important public health issues worldwide. Oral healthcare is essential to the health and well-being of elders and is one of the key indicators determining their quality of life. This research aimed to study oral health literacy, self-efficacy, social support, and demographic characteristic factors associated with the oral health care behaviors of elderly people living in the rural areas of northern Thailand.
Methods:
This research was a cross-sectional study that recruited 406 elderly participants using convenience and snowball samplings. Participants' names were obtained from the registration list of the Java Health Center Information System (JHCIS) program, where they received a health service between 2018 and 2020. Data were obtained through face-to-face interviews with participants, while they were waiting to receive a health service or through a phone interview. Linear regression was analyzed to determine the factors associated with oral healthcare behaviors.
Results:
The majority of participants (85%) had inadequate functional health literacy, 52% had moderate self-efficacy toward oral health behaviors, 91.9% had moderate social support, and 53% admitted to moderate oral health behaviors. The results from the model show that self-efficacy, social support, and oral health literacy are positively associated with oral health care behaviors among the elderly (p-value < 0.05). The multiple regression model can account for 47.2% of the variance in oral health care behaviors.
Conclusions:
Improving oral health care behaviors among elderly people should be considered by health care providers and those who provide social support. Self-esteem, communication skills among service providers and service receivers, and self-management of oral healthcare should receive special attention. Moreover, social support and relevant agencies can help promote oral healthcare by collaborating with other healthcare providers for better oral health outcomes among elderly people. | Introduction:
Oral health problems among elderly people are an important public health issues in Thailand and worldwide.1 Poor oral health care among the elderly is seen in the high level of tooth loss, dental caries, and the prevalence of periodontal diseases that have a major bearing on the quality of life, and lead to increasing dental and medical expenses.2,3
A Thailand report released 2017,4 found that of the elderly aged between 60–74 years (56.1%) have at least 20 active permanent teeth and (40.2%) have four pairs of posterior occlusion.4 Poor occlusion performance and periodontitis with tissue and root canal damage, increases the risk of inflammation, pain, swelling, infection, and loss of teeth among the elderly.4 The report also found untreated tooth decay and, root canal decay associated with receding gingival recession in older age is prevalent in Thailand.4,5 Furthermore, a previous report found an association between oral health and health conditions such as heart disease, stroke, and diabetes,; which become more common with increased age.6,7 Some studies have shown that poor oral healthcare among the elderly may lead to tooth loss and poor oral health function,8,9 such as reduced occlusion performance, causing digestive problems and other health complications.2 According to a study the emotional effects of tooth loss in edentulous people the pain of having oral health problems may affect the mental state of the elderly.10,11 This in turn makes them avoid socializing with people because they are worried about their image.1 Consequently, the elderly with dental problems tend to be more isolated from social gatherings than other age groups.1,10 Chiang Mai province, the largest province in northern Thailand, has the third highest aged population in the country.11 In 2018, 2019, and -2020, periodontal disease was reported to be 24.8%, 55.2%, and 57.3%, respectively, among the elderly aged 65–80 years.11,12 Periodontal disease, affects occlusion efficiency and the quality of life.11,12 Elderly people in Chiang Mai are experiencing oral health problems, due mainly to poor oral healthcare behaviors, such as wrong brushing and not cleaning their teeth regularly.4,12 The elderly also have restricted access to clinical oral examinations and services as well as communication problems with dentists.1,12 This is similar to the study which was found that dental health care problem is associated with a lack of oral health literacy.1,13 The elderly with high oral health literacy tend to have more permanent teeth than those who have low oral health literacy.13 Almost half of the elderly recognized that tooth loss can be replaced by a denture, but they ignore the importance of tooth replacement.14 Some studies have found that less than 50% of elderly people have mild oral hygiene habits such as brushing twice a day and seeing the dentist only if they experience oral health problems.15
Health literacy is the ability to obtain, process, and understand the basics of health information and, the services needed to make appropriate health decisions.16 Health literacy also facilitates the effective communication of health- related information and the importance of maintaining good health.17 An individual’s health literacy is the ability to perceive health depending upon education and knowledge adequacy, attributes that are affected by the culture, language, way of life, and health related practices of people in diverse environments.18,19 Health literacy is a strong predictor of an individual’s health, health behavior, and health outcomes.18 Limited knowledge toward health literacy is associated with poor self-ratings on health, poor adherence to medical instructions, poor self-management skills, increased mortality risks, poor health outcomes, and higher healthcare costs.19 Oral health literacy has been popular in the dental literature over the past decade.17,20 A person with limited oral health literacy has been reported to be at higher risk of oral diseases and problems associated with them.20 One study found that people with poor oral health literacy were more likely to miss dental health appointments.21 Non-adherence to dental recommendations has been reported to cause higher caries experiences22 and poor periodontal status.23 Oral health problems and poor oral health behaviors among the elderly should be an important focus in public health.24,25 Communication and preventive and therapeutic behaviors once the disease occurs are important for enhancing oral health among elderly people.15,24–26 This study applies the concept of health literacy as a person’s ability to access, understand, assess, use and communicate health information on demand to promote and maintain a healthy lifelong state.27 Moreover, in promoting oral healthcare behaviors it is important to advocate self-efficacy and social support, which affect the outcomes of self-care behaviors.28–30 This study is part of an on-going intervention research that explores oral health literacy, self-efficacy, social support, and demographic factors associated with oral health care behaviors among elderly living in a northern border community in Thailand. Importantly, this study aims to contribute to the development of oral health literacy and expectations of outcomes of their own proper oral health practice.
Conclusion:
In conclusion, oral healthcare behavior among elderly people is an important issue within public health. Many aspects must be considered when assessing the elderly’s health, including health behaviors, health knowledge, and other related factors. In addition, oral health literacy, self-efficacy, and social support have a significant impact on the elderly’s oral healthcare behaviors. As age progresses, oral health problems among older people increase so comprehensive oral healthcare services should be made available to the elderly. Dental clinics can use health information to organize training and create key strategies for appropriate oral healthcare behaviors among elderly people living in rural areas. Therefore, accessibility to comprehensive services and policy plans should cover the areas of program activities, health campaigns, and media and public relations. Related information should be simple, understandable, and suitable for the context of elderly people living in the rural community. Moreover, family members, relatives, or other people from relevant agencies should provide social support to promote oral healthcare among elderly people and offer integrated programs, such as organizing health activities through clubs for the elderly in conjunction with family care and home visits. Also, appropriate healthcare behaviors should be included in community activities to enhance the skills of the elderly regarding self-health care management, decision-making, following medical advices from health personnel, and accessing health information. | Background:
Oral health problems among elderly people are an important public health issues worldwide. Oral healthcare is essential to the health and well-being of elders and is one of the key indicators determining their quality of life. This research aimed to study oral health literacy, self-efficacy, social support, and demographic characteristic factors associated with the oral health care behaviors of elderly people living in the rural areas of northern Thailand.
Methods:
This research was a cross-sectional study that recruited 406 elderly participants using convenience and snowball samplings. Participants' names were obtained from the registration list of the Java Health Center Information System (JHCIS) program, where they received a health service between 2018 and 2020. Data were obtained through face-to-face interviews with participants, while they were waiting to receive a health service or through a phone interview. Linear regression was analyzed to determine the factors associated with oral healthcare behaviors.
Results:
The majority of participants (85%) had inadequate functional health literacy, 52% had moderate self-efficacy toward oral health behaviors, 91.9% had moderate social support, and 53% admitted to moderate oral health behaviors. The results from the model show that self-efficacy, social support, and oral health literacy are positively associated with oral health care behaviors among the elderly (p-value < 0.05). The multiple regression model can account for 47.2% of the variance in oral health care behaviors.
Conclusions:
Improving oral health care behaviors among elderly people should be considered by health care providers and those who provide social support. Self-esteem, communication skills among service providers and service receivers, and self-management of oral healthcare should receive special attention. Moreover, social support and relevant agencies can help promote oral healthcare by collaborating with other healthcare providers for better oral health outcomes among elderly people. | 6,606 | 359 | [
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||||
Comprehension and Practice Patterns of Korean Urologists for Retractile and Gliding Testes. | 35347906 | It is quite difficult to distinguish retractile testis from gliding testis, which requires different treatment planning in the clinic setting. We evaluated practice patterns of urologists in Korea regarding the diagnosis and management of retractile and gliding testes. | BACKGROUND | We mailed or e-mailed self-completion questionnaires consisting of 20 items to 106 urologists practicing in Korean hospitals concerning the diagnosis and treatment of cryptorchidism. We collected and analyzed the responses statistically. | METHODS | Responses were received from 62 urologists. The response rate was 58.5%. Thirty-seven urologists (59.7%) actually felt they had difficulty in distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than for pediatric urologists (40.0%) (P = 0.006). In cases of infant retractile testis, only five urologists (8.1%) said that they would perform orchiopexy immediately, with 54 (87.1%) urologists saying they would do follow-up. In cases of preschool-age children with retractile testis, 17 urologists (27.4%) said that they would perform orchiopexy immediately with 41 (66.1%) urologists saying they would do follow-up. In cases of infant gliding testis, 37 urologists (59.7%) said that they would perform orchiopexy immediately with 24 (38.7%) urologists saying they would do a follow-up. | RESULTS | More than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing a practical guideline. | CONCLUSION | [
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] | 8960942 | INTRODUCTION | Recently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.
The normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4
In prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9
Retractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey. | METHODS | We conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.
The questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.
We received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.
Thirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.
Statistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.
We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.
Ethics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.
The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled. | RESULTS |
Fig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.
Thirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).
GT = gliding testis, RT = retractile testis.
Most of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).
Case scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.
Only five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).
Values are presented as number (%).
Respondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).
Values are presented as number (%).
Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.
Only five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).
Values are presented as number (%).
Respondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).
Values are presented as number (%).
Case scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.
Seventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).
Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.
Seventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).
Case scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.
Thirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).
Responses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).
Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.
Thirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).
Responses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).
Summary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.
None chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.
Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.
None chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied. | null | null | [
"Statistical analysis",
"Ethics statement",
"Case scenario of an infant with retractile testis",
"Case scenario of a 6-year-old child with retractile testis",
"Case scenario of an infant with a gliding testis",
"Summary"
] | [
"We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.",
"The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.",
"Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).",
"Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).",
"Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).",
"Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied."
] | [
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"METHODS",
"Statistical analysis",
"Ethics statement",
"RESULTS",
"Case scenario of an infant with retractile testis",
"Case scenario of a 6-year-old child with retractile testis",
"Case scenario of an infant with a gliding testis",
"Summary",
"DISCUSSION"
] | [
"Recently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.\nThe normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4\nIn prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9\nRetractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey.",
"We conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.\nThe questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.\nWe received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.\nThirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.\nStatistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nWe performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.\nEthics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.\nThe present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.",
"We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.",
"The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.",
"\nFig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.\nThirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).\nGT = gliding testis, RT = retractile testis.\nMost of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).\nCase scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nOf the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).\nCase scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nOf the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).\nCase scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nOf the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).\nSummary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.\nScrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.",
"Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.\nOnly five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).\nValues are presented as number (%).\nRespondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).\nValues are presented as number (%).",
"Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.\nSeventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).",
"Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.\nThirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).\nResponses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).",
"Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.\nNone chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.",
"Although cryptorchidism is one of the most common diseases in pediatric urology, there have been no systematic investigations into the recognition and treatment of unstable high scrotal testis, such retractile or gliding testes.\nGliding testis should be clearly distinguished from retractile testis. There are many tips on how to distinguish gliding from retractile testis. Van Essen10 described that having a patient take a squatting position on physical examination is essential in distinguishing gliding from retractile testis. Wyllie11 stated that retractile testis could be brought into a low, stable scrotal position and that traction on the cord structures is not painful. In contrast, gliding testis can only be brought into a high unstable scrotal position, and further traction on cord structures is painful.11 Ferro et al.12 defines retractile testis as a phenomenon in which a testis can manipulated into the scrotum and remain in position without tension. A gliding testis is defined as when it can be manipulated into the upper scrotum, but then it retracts when released.12 However, both are two different and distinct non-scrotal entities, and each definition is so clear, but there are many cases where it is often difficult to apply it in practice.\nIn the beginning, both conditions were thought to be identical, and a gliding testis was therefore also called a “pathological retractile testis”.1314 Many studies about retractile testis could have some bias because they include some cases of gliding testis in their studies. Often it was concluded that the course of retracted testis was similar to that of cryptorchidism. It should be realized that this can create vague fears about retractile testis. However, most urologists believe that a retractile testis is a physiological variant of a fully descended testis, and therefore active treatment and long-term follow-up are usually not indicated. In contrast to a retractile testis, a gliding testis belongs to the spectrum of undescended testis. Therefore, it should be noted that retractile testis do not need surgery but indicates careful physical examinations and periodic follow-ups. However, in this study, 59.7% of urologists felt they had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than pediatric urologists (40.0%).\nThe treatment for retractile testis remains controversial, whereas treatment methods for undescended testis have been well established through many studies.151617 The most typical indication for orchidopexy is when a retractile testis has any decrease in testicular volume and when it ascends because of increased spermatic cord tension. This makes it impossible to reposition the testis into its correct anatomic location. In this study, treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. They recommended follow-up or orchiopexy at a similar rate.\nA retractile testis is attributed to an overactive cremasteric reflex or to alterations within the contractile properties of the cremasteric muscle.18 It is especially common in boys around the school starting age (5–6 years).19 Some authors have suggested that retractile testis may not be as innocuous as has been widely believed.20 In addition, recent evidence suggests that a retractile testis may evolve into an acquired-undescended testis.8\nIt has been reported that retractile testis is accompanied by histological changes. Abnormalities on semen analysis have been found during follow-up when patients with retractile testis had become adults.2122\nIn this study, most of the urologists (98.4%) agreed that cryptorchidism could affect infertility and testicular function in the future. Fifty urologists (80.6%) agreed that these adverse effects were also true with gliding testis, and selected surgical treatment. The distribution of treatment decisions shows no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108). Meanwhile, 13 urologists (21.0%) agreed that retractile testis lead to adverse effects, including 31.2% of non-pediatric urologists and 10% of pediatric urologists. Regarding infant with retractile testis, treatment distribution varied among groups (P = 0.030). These differences show the result of a more conservative treatment trend among pediatric urologists than in non-pediatric urologists. To overcome such discrepancies in clinical practice, it is necessary to establish an appropriate guideline for the proper management of retractile testis.\nLa Scala and Ein7 reported that 150 boys with retractile testis needed periodic follow-up. In their series, only 22.7% of the patients required orchidopexy. They recommend that patients with retractile testis be attentively followed annually. They suggested that most patients with retractile testis can expect a spontaneously favorable resolution without surgical treatment, even after 14 years of age. In this study, the follow-up period in retractile and gliding testes is not clear. Half of the urologists wanted to follow-up before school age or until 2 years of age. Some urologists (12.5–24.1%) wanted to follow-up after puberty. Therefore, we need further evaluation about what is the proper follow-up period for retractile and gliding testes.\nIn this study, there are some limitations. This is a survey study of urologists. As a result of raising questions about the treatment trends of these urologists, we do not know if this occurs in the real clinical setting. However, we tried to elicit answers that are as realistic as possible, with scenarios for patients seen in the actual clinical field. Second, the number of respondents is small. We think it is likely that the urologists who seldom treat pediatric patients did not respond. However, we tried to include all pediatric urologists in the Korean Society of Pediatric Urology, and most members of our society participated in this survey study.\nMoreover, we found some differences in practice patterns between pediatric urologists and non-pediatric urologists. Overall, non-pediatric urologists found it challenging to distinguish retractile testis from gliding testis. Compared to pediatric urologists, they had a higher tendency to agree with the statement that retractile testis could cause infertility and testicular dysfunction in the future and chose to perform orchiopexy as early as the time of the first detection. To overcome these differences, adequate education about retractile and gliding testes is essential. The results of this study are expected to be utilized as basic data for establishing a practical guideline for the diagnosis and treatment of retractile testis in Korea.\nIn conclusion, more than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. Among urologists, non-pediatric urologists had more difficulty than pediatric urologists, and this disparity was more pronounced in children compared to infants. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing consistent management plans. The results of this study are expected to be used as basic data for making a practical guideline for the diagnosis and treatment of retractile testis in Korea."
] | [
"intro",
"methods",
null,
null,
"results",
null,
null,
null,
null,
"discussion"
] | [
"Cryptorchidism",
"Child",
"Testis",
"Practice Patterns, Physicians",
"Surveys and Questionnaires",
"Practice Guideline"
] | INTRODUCTION:
Recently, parents have been increasingly concerned and anxious about their children’s testicles. Cryptorchidism, or undescended testis, is a common genital anomaly that is extensively studied yet incompletely understood. Orchiopexy, a treatment option for cryptorchidism, is a common operation in pediatric urology.
The normal scrotal position of the testis has been defined as the positioning of the testis at the midpoint, or at or below mid scrotum.1 Although “high scrotal testis” is not routinely considered undescended by most clinicians, this has been included in the definition of undescended testis in some epidemiologic studies.2 This is likely a heterogeneous group that includes stable descended “retractile” testis and undescended “gliding” testis.3 Retractile testis is scrotal testis that retract easily out of the scrotum but can be manually replaced in a stable scrotal position and remain there at least temporarily until there is a recurrent stimulation. On the other hand, gliding testis is not stable and include low undescended testicles.4
In prepubertal boys, what seems like an undescended testis is often a retractile testis that results from a brisk cremasteric reflex and does not require a surgical intervention. A retractile testis must be differentiated from an ascending testis that may require an orchidopexy. However, the management of retractile testis is still controversial. Recent articles have recommended that only observation in cases of retractile testis is needed.56 In more than 70% of patients with a retractile testis, the condition evolves favorably without the need for surgery.7 However, there is a 25% risk of these testis ascending and becoming acquired cryptorchidism in adolescence.8 In a Korean study, about 16.3% of patients with retractile testis required surgical correction during long-term follow-up.9
Retractile testis, in some cases, may be difficult to distinguish from the gliding testis that present as high scrotal testes in the clinic setting. In this case, some non-specialists often choose unnecessary surgery for defensive and preventive treatment. Therefore, it will be meaningful to understand the actual practice patterns of all urologists for retractile testis in order to develop proper clinical guidelines. Therefore, we evaluated the actual practice patterns of Korean urologists regarding the diagnosis and management of retractile and gliding testes and the difference between pediatric urologists and general urologists via a questionnaire survey.
METHODS:
We conducted a questionnaire survey of 106 urologists who worked in a Korean training hospital from March to July 2018. We distributed a self-administered questionnaire consisting of 20 items on the diagnosis and treatment of retractile and gliding testes (Appendix 1) via email or via mail with letters.
The questionnaires dealt with cases of retractile and gliding testes in infants and cases of retractile testis in childhood. If errors were found in the retrieved questionnaire, they were corrected or supplemented by telephone or face-to-face interviews. Prior to the questionnaires, we checked the period of urologic practice and the rate of pediatric patient treatment at their hospital.
We received responses from 62 urologists. The response rate was 58.5%. We divided the urologists into two groups, according to the number of pediatric patients who had visited each hospital. If the urologist treated pediatric patients accounting for more than 25 percent of total patients, or if the individual was a member of the Korean Society of Pediatric Urology, this urologist was regarded as a pediatric urologist. Non-pediatric urologist was defined as treating pediatric patients less than 25 percent of total patients.
Thirty (48.4%) were pediatric urologists, and 32 (51.6%) were non-pediatric urologists. We compared and statistically analyzed the two groups’ distribution of responses.
Statistical analysis We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.
We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.
Ethics statement The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.
The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.
Statistical analysis:
We performed statistical analysis by using the IBM SPSS statistics software, version 22.0 (IBM Co., Armonk, NY, USA), and the two groups were compared using the Fisher's exact test and the chi-squared test. A P value < 0.05 was considered to be statistically significant.
Ethics statement:
The present study protocol was reviewed and approved by the Institutional Review Board of Pusan National University Yangsan Hospital (approval No. 05-2018-073). Informed consent was submitted by all subjects when they were enrolled.
RESULTS:
Fig. 1 shows the distribution of practice duration according to the duration of the urologists’ qualifications.
Thirty-seven urologists (59.7%) had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher in non-pediatric urologists (78.1%) than in pediatric urologists (40.0%) (P = 0.006) (Fig. 2). The treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. Each one-third of urologists preferred surgical correction (38.7%) or follow-up (33.9%) for when it was difficult to distinguish either a retractile or a gliding testis. The treatment planning showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.083) (Fig. 3).
GT = gliding testis, RT = retractile testis.
Most of the urologists (98.4%) agreed that cryptorchidism could cause infertility and testicular dysfunction in the future. Although 50 of these urologists (80.6%) agreed on the adverse effects of gliding testis, 13 of them (21.0%) agreed this was the case with retractile testis. The rate difference was especially notable among the urologists, with 31.2% of non-pediatric urologists (10 of 32) compared to 10.0% of pediatric urologists (3 of 30) (P > 0.05).
Case scenario of an infant with retractile testis Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.
Only five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).
Values are presented as number (%).
Respondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).
Values are presented as number (%).
Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.
Only five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).
Values are presented as number (%).
Respondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).
Values are presented as number (%).
Case scenario of a 6-year-old child with retractile testis Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.
Seventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).
Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.
Seventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).
Case scenario of an infant with a gliding testis Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.
Thirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).
Responses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).
Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.
Thirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).
Responses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).
Summary Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.
None chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.
Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.
None chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.
Case scenario of an infant with retractile testis:
Of the 62 respondents, 54 urologists (87.1%) agreed with the diagnosis of retractile testis in this case scenario, and 22 urologists (35.5%) wanted to check an ultrasound study for further evaluation.
Only five urologists (8.1%) said that they would have performed orchiopexy immediately, but 54 (87.1%) urologists responded that they would do follow-up. None wanted to use hormone therapy. Three (4.8%) urologists said that they would no longer need to follow-up. This distribution of treatment decisions showed a statistically significant difference between the pediatric and non-pediatric urologists (P = 0.030) (Table 1 and Fig. 4A).
Values are presented as number (%).
Respondents had various opinions on the follow-up period. However, there was no statistical significance between pediatric and non-pediatric urologists (P = 0.164) (Table 2 and Fig. 4B).
Values are presented as number (%).
Case scenario of a 6-year-old child with retractile testis:
Of the total 62 respondents, 53 urologists (85.5%) agreed with the diagnosis of retractile testis in this case scenario, and 29 urologists (46.8%) wanted an ultrasound study for further evaluation.
Seventeen urologists (27.4%) said that they would have performed orchiopexy immediately, and 41 (66.1%) urologists said that they would do a follow-up. None wanted to use hormone therapy, and three (4.8%) urologists said follow-up was no longer needed. This distribution of treatment decisions showed a statistically significant difference between pediatric and non-pediatric urologists (P = 0.010) (Table 1 and Fig. 5).
Case scenario of an infant with a gliding testis:
Of the 62 respondents, 49 (79.0%) agreed with the diagnosis of gliding testis in this case scenario, and 36 urologists (58.1%) wanted to do an ultrasound study for further evaluation.
Thirty-seven urologists (59.7%) responded that they would have performed orchiopexy immediately. Twenty-four urologists (38.7%) said they would do a follow-up. None wanted to use hormone therapy or to stop following up. This distribution of treatment decisions showed no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108) (Table 1 and Fig. 6A).
Responses about the follow-up period showed various but statistically insignificant differences between pediatric and non-pediatric urologists (P = 0.109) (Table 2 and Fig. 6B).
Summary:
Scrotal ultrasound was chosen by 35.5% to 58.1% of respondents to establish a treatment plan. Immediate orchiopexy was selected by 8.1% to 59.7% of respondents. This imaging modality was frequently utilized in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Immediate orchiopexy was frequently selected in the order of infants with gliding testis, children with retractile testis, and infants with retractile testis. Especially half of the non-pediatric urologists responded that they would have performed orchiopexy immediately even in the case of a child with retractile testis that generally does not require surgical treatment. In the case of an infant with retractile testis, 15.6% of non-pediatric urologists responded that they would have performed orchiopexy immediately.
None chose to use hormone therapy for either retractile or gliding testes. Opinions on the follow-up period for both retractile and gliding testes were inconsistent, and their responses varied.
DISCUSSION:
Although cryptorchidism is one of the most common diseases in pediatric urology, there have been no systematic investigations into the recognition and treatment of unstable high scrotal testis, such retractile or gliding testes.
Gliding testis should be clearly distinguished from retractile testis. There are many tips on how to distinguish gliding from retractile testis. Van Essen10 described that having a patient take a squatting position on physical examination is essential in distinguishing gliding from retractile testis. Wyllie11 stated that retractile testis could be brought into a low, stable scrotal position and that traction on the cord structures is not painful. In contrast, gliding testis can only be brought into a high unstable scrotal position, and further traction on cord structures is painful.11 Ferro et al.12 defines retractile testis as a phenomenon in which a testis can manipulated into the scrotum and remain in position without tension. A gliding testis is defined as when it can be manipulated into the upper scrotum, but then it retracts when released.12 However, both are two different and distinct non-scrotal entities, and each definition is so clear, but there are many cases where it is often difficult to apply it in practice.
In the beginning, both conditions were thought to be identical, and a gliding testis was therefore also called a “pathological retractile testis”.1314 Many studies about retractile testis could have some bias because they include some cases of gliding testis in their studies. Often it was concluded that the course of retracted testis was similar to that of cryptorchidism. It should be realized that this can create vague fears about retractile testis. However, most urologists believe that a retractile testis is a physiological variant of a fully descended testis, and therefore active treatment and long-term follow-up are usually not indicated. In contrast to a retractile testis, a gliding testis belongs to the spectrum of undescended testis. Therefore, it should be noted that retractile testis do not need surgery but indicates careful physical examinations and periodic follow-ups. However, in this study, 59.7% of urologists felt they had difficulty distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than pediatric urologists (40.0%).
The treatment for retractile testis remains controversial, whereas treatment methods for undescended testis have been well established through many studies.151617 The most typical indication for orchidopexy is when a retractile testis has any decrease in testicular volume and when it ascends because of increased spermatic cord tension. This makes it impossible to reposition the testis into its correct anatomic location. In this study, treatment planning varied among the respondents when they had difficulty distinguishing between retractile and gliding testes in the clinic setting. They recommended follow-up or orchiopexy at a similar rate.
A retractile testis is attributed to an overactive cremasteric reflex or to alterations within the contractile properties of the cremasteric muscle.18 It is especially common in boys around the school starting age (5–6 years).19 Some authors have suggested that retractile testis may not be as innocuous as has been widely believed.20 In addition, recent evidence suggests that a retractile testis may evolve into an acquired-undescended testis.8
It has been reported that retractile testis is accompanied by histological changes. Abnormalities on semen analysis have been found during follow-up when patients with retractile testis had become adults.2122
In this study, most of the urologists (98.4%) agreed that cryptorchidism could affect infertility and testicular function in the future. Fifty urologists (80.6%) agreed that these adverse effects were also true with gliding testis, and selected surgical treatment. The distribution of treatment decisions shows no statistically significant differences between pediatric and non-pediatric urologists (P = 0.108). Meanwhile, 13 urologists (21.0%) agreed that retractile testis lead to adverse effects, including 31.2% of non-pediatric urologists and 10% of pediatric urologists. Regarding infant with retractile testis, treatment distribution varied among groups (P = 0.030). These differences show the result of a more conservative treatment trend among pediatric urologists than in non-pediatric urologists. To overcome such discrepancies in clinical practice, it is necessary to establish an appropriate guideline for the proper management of retractile testis.
La Scala and Ein7 reported that 150 boys with retractile testis needed periodic follow-up. In their series, only 22.7% of the patients required orchidopexy. They recommend that patients with retractile testis be attentively followed annually. They suggested that most patients with retractile testis can expect a spontaneously favorable resolution without surgical treatment, even after 14 years of age. In this study, the follow-up period in retractile and gliding testes is not clear. Half of the urologists wanted to follow-up before school age or until 2 years of age. Some urologists (12.5–24.1%) wanted to follow-up after puberty. Therefore, we need further evaluation about what is the proper follow-up period for retractile and gliding testes.
In this study, there are some limitations. This is a survey study of urologists. As a result of raising questions about the treatment trends of these urologists, we do not know if this occurs in the real clinical setting. However, we tried to elicit answers that are as realistic as possible, with scenarios for patients seen in the actual clinical field. Second, the number of respondents is small. We think it is likely that the urologists who seldom treat pediatric patients did not respond. However, we tried to include all pediatric urologists in the Korean Society of Pediatric Urology, and most members of our society participated in this survey study.
Moreover, we found some differences in practice patterns between pediatric urologists and non-pediatric urologists. Overall, non-pediatric urologists found it challenging to distinguish retractile testis from gliding testis. Compared to pediatric urologists, they had a higher tendency to agree with the statement that retractile testis could cause infertility and testicular dysfunction in the future and chose to perform orchiopexy as early as the time of the first detection. To overcome these differences, adequate education about retractile and gliding testes is essential. The results of this study are expected to be utilized as basic data for establishing a practical guideline for the diagnosis and treatment of retractile testis in Korea.
In conclusion, more than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. Among urologists, non-pediatric urologists had more difficulty than pediatric urologists, and this disparity was more pronounced in children compared to infants. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing consistent management plans. The results of this study are expected to be used as basic data for making a practical guideline for the diagnosis and treatment of retractile testis in Korea.
| Background:
It is quite difficult to distinguish retractile testis from gliding testis, which requires different treatment planning in the clinic setting. We evaluated practice patterns of urologists in Korea regarding the diagnosis and management of retractile and gliding testes.
Methods:
We mailed or e-mailed self-completion questionnaires consisting of 20 items to 106 urologists practicing in Korean hospitals concerning the diagnosis and treatment of cryptorchidism. We collected and analyzed the responses statistically.
Results:
Responses were received from 62 urologists. The response rate was 58.5%. Thirty-seven urologists (59.7%) actually felt they had difficulty in distinguishing retractile testis from gliding testis in the clinic setting. This rate was higher for non-pediatric urologists (78.1%) than for pediatric urologists (40.0%) (P = 0.006). In cases of infant retractile testis, only five urologists (8.1%) said that they would perform orchiopexy immediately, with 54 (87.1%) urologists saying they would do follow-up. In cases of preschool-age children with retractile testis, 17 urologists (27.4%) said that they would perform orchiopexy immediately with 41 (66.1%) urologists saying they would do follow-up. In cases of infant gliding testis, 37 urologists (59.7%) said that they would perform orchiopexy immediately with 24 (38.7%) urologists saying they would do a follow-up.
Conclusions:
More than half (59.7%) of Korean urologists revealed it challenging to distinguish retractile testis and gliding testis in the clinical setting. The more it was difficult to diagnose retractile testis with certainty, the more frequent surgical correction was chosen for treatment. Therefore, it is essential to prevent unnecessary surgical treatment by establishing a practical guideline. | null | null | 4,576 | 334 | [
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"acquired undescended testis",
"testicles cryptorchidism undescended",
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] | null | null | [CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY] | [CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY] | [CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY] | null | [CONTENT] Cryptorchidism | Child | Testis | Practice Patterns, Physicians | Surveys and Questionnaires | Practice Guideline [SUMMARY] | null | [CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY] | [CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY] | [CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY] | null | [CONTENT] Asian People | Child | Child, Preschool | Comprehension | Cryptorchidism | Humans | Infant | Male | Urologists [SUMMARY] | null | [CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY] | [CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY] | [CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY] | null | [CONTENT] acquired undescended testis | testicles cryptorchidism undescended | high scrotal testis | scrotal testis retract | retractile testis scrotal [SUMMARY] | null | [CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY] | [CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY] | [CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY] | null | [CONTENT] testis | urologists | retractile | retractile testis | pediatric | gliding | pediatric urologists | treatment | non | follow [SUMMARY] | null | [CONTENT] testis | retractile | undescended | retractile testis | scrotal | stable | undescended testis | cryptorchidism | gliding | testis retractile testis [SUMMARY] | [CONTENT] hospital | urologist | pediatric | patients | test | ibm | groups | 05 | pediatric patients | questionnaire [SUMMARY] | [CONTENT] urologists | testis | pediatric | retractile | retractile testis | fig | gliding | pediatric urologists | non pediatric urologists | non pediatric [SUMMARY] | null | [CONTENT] urologists | testis | retractile | pediatric | retractile testis | gliding | follow | pediatric urologists | non pediatric | said [SUMMARY] | null | [CONTENT] ||| Korea [SUMMARY] | [CONTENT] 20 | 106 | Korean ||| [SUMMARY] | [CONTENT] 62 ||| 58.5% ||| Thirty-seven | 59.7% ||| 78.1% | 40.0% ||| 0.006 ||| only five | 8.1% | 54 | 87.1% ||| 17 | 27.4% | 41 | 66.1% ||| 37 | 59.7% | 24 | 38.7% [SUMMARY] | null | [CONTENT] ||| Korea ||| 20 | 106 | Korean ||| ||| ||| 62 ||| 58.5% ||| Thirty-seven | 59.7% ||| 78.1% | 40.0% ||| 0.006 ||| only five | 8.1% | 54 | 87.1% ||| 17 | 27.4% | 41 | 66.1% ||| 37 | 59.7% | 24 | 38.7% ||| More than half | 59.7% | Korean ||| ||| [SUMMARY] | null |
||||
Comparison of perceived quality amongst migrant and local patients using primary health care delivered by community health centres in Shenzhen, China. | 24779564 | Providing good quality primary health care to all inhabitants is one of the Chinese Government's health care objectives. However, information is scarce regarding the difference in quality of primary health care delivered to migrants and local residents respectively. This study aimed to compare patients' perceptions of quality of primary health care between migrants and local patients, and their willingness to use and recommend primary health care to others. | BACKGROUND | A cross-sectional survey was conducted. 787 patients in total were chosen from four randomly drawn Community Health Centers (CHCs) for interviews. | METHODS | Local residents scored higher than migrants in terms of their satisfaction with types of drugs available (3.62 vs. 3.45, p=0.035), attitude of health workers (4.41 vs. 4.14, p=0.042) and waiting time (4.30 vs. 3.86, p<0.001). Even though there was no significant difference in overall satisfaction between local residents and migrants (4.16 vs. 3.91, p=0.159), migrants were more likely to utilize primary health care as the first choice for their usual health problems (94.1% vs. 87.1%, p=0.032), while local residents were more inclined to recommend Traditional Chinese Medicine to others (65.6% vs. 56.6%, p=0.026). | RESULTS | Quality of primary health care given to migrants is less satisfactory than to local residents in terms of attitude of health workers and waiting time. Our study suggests quality of care could be improved through extending opening hours of CHCs and strengthening professional ethics education. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals' recommendations for traditional Chinese medicine were possibly because of cultural differences. | CONCLUSIONS | [
"Attitude of Health Personnel",
"China",
"Community Health Centers",
"Cross-Sectional Studies",
"Female",
"Health Services Accessibility",
"Humans",
"Interviews as Topic",
"Male",
"Primary Health Care",
"Quality of Health Care",
"Transients and Migrants",
"Waiting Lists"
] | 4012177 | Background | China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.
China has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.
Migrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.
Patient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others. | Methods | Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.
This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.
Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.
Questions to measure patient satisfaction
Moreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).
Questions on patients’ willingness to use and recommend primary health care to others
Socio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.
In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.
Questions to measure patient satisfaction
Moreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).
Questions on patients’ willingness to use and recommend primary health care to others
Socio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.
Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).
We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).
Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).
Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441). | Results | In total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).
Socio-economic and demographic characteristics of respondents
The mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).
The mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status
†Adjusted for demographic and socio-economic characteristics of the respondents.
Compared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).
The willingness to use by the respondents, and to recommend to others by registration type
†Adjusted for demographic and socio-economic characteristics of the respondents. | Conclusions | In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences. | [
"Background",
"Study design, settings and data collection process",
"Key measures",
"Statistical analysis",
"Ethical approval",
"Major findings",
"Strengths and limitations",
"Comparisons with existing literature",
"Implications for research and practice",
"Competing interests",
"Authors’ contribution",
"Pre-publication history"
] | [
"China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.",
"This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.",
"In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.",
"We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).",
"Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).",
"The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.",
"A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.",
"Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].",
"Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.",
"The authors declare that they have no competing interests.",
"XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\n"
] | [
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] | [
"Background",
"Methods",
"Study design, settings and data collection process",
"Key measures",
"Statistical analysis",
"Ethical approval",
"Results",
"Discussion",
"Major findings",
"Strengths and limitations",
"Comparisons with existing literature",
"Implications for research and practice",
"Conclusions",
"Competing interests",
"Authors’ contribution",
"Pre-publication history"
] | [
"China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.\nChina has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.\nMigrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.\nPatient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.",
" Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\nThis cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.\n Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\nIn this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.\n Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\nWe compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).\n Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).\nEthical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).",
"This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.",
"In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.\nQuestions to measure patient satisfaction\nMoreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).\nQuestions on patients’ willingness to use and recommend primary health care to others\nSocio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.",
"We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).",
"Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).",
"In total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).\nSocio-economic and demographic characteristics of respondents\nThe mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).\nThe mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status\n†Adjusted for demographic and socio-economic characteristics of the respondents.\nCompared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).\nThe willingness to use by the respondents, and to recommend to others by registration type\n†Adjusted for demographic and socio-economic characteristics of the respondents.",
" Major findings The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\nThe findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.\n Strengths and limitations A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\nA particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.\n Comparisons with existing literature Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\nUnlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].\n Implications for research and practice Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.\nLocal residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.",
"The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.",
"A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.\nThere were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.",
"Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.\nThe mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.\nThe mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.\nMigrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].",
"Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.\nThe findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.",
"In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences.",
"The authors declare that they have no competing interests.",
"XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2296/15/76/prepub\n"
] | [
null,
"methods",
null,
null,
null,
null,
"results",
"discussion",
null,
null,
null,
null,
"conclusions",
null,
null,
null
] | [
"Patient satisfaction",
"Quality of care",
"Primary health care",
"Migration"
] | Background:
China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.
China has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.
Migrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.
Patient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.
Methods:
Study design, settings and data collection process This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.
This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.
Key measures In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.
Questions to measure patient satisfaction
Moreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).
Questions on patients’ willingness to use and recommend primary health care to others
Socio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.
In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.
Questions to measure patient satisfaction
Moreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).
Questions on patients’ willingness to use and recommend primary health care to others
Socio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.
Statistical analysis We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).
We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).
Ethical approval Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).
Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).
Study design, settings and data collection process:
This cross-sectional study was conducted in Shenzhen in June, 2012. We recruited a site-based (i.e., CHC based) sample aged 18 years and older using a multistage random sampling process. In the first stage, 10 districts in Shenzhen were stratified into four geographical areas within or outside Shenzhen Economic Zone (SEZ), and in the eastern or western part of the city. We then randomly selected one district in each category to arrive at four districts in total, namely Luohu, Futian, Bao’an and Longgang Districts. In the second stage, we randomly selected one CHC from each randomly drawn district, so that four CHCs were chosen as study settings in total. In the third stage, we selected primary health care users through a systematic sampling method. We planned to invite 800 patients to participate in the study, 200 from each CHC. The interval for sampling was calculated by dividing the estimated total number of consultations of each CHC by the expected number of respondents to be invited in each day. The inclusion criteria for respondents were: i) aged 18 and above, ii) CHC visit at least once before the survey, iii) ability to communicate and give informed consent. Interviewers were trained extensively by the research team. Three interviewers were allocated to each CHC for five days to conduct face-to-face interviews. Patients were informed orally of the study’s purpose and then asked for their written informed consent before the interview.
Key measures:
In this study, patient satisfaction was measured by a questionnaire with 13 questions, including one question measuring overall satisfaction (Table 1). The questionnaire was based on a model by Ware et al. [14]. Factor analysis with varimax rotation was conducted to test the reliability and internal consistency of the 13 items. The Cronbach’s alpha of the scale was 0.82, indicating good reliability of the items. For consistency in response and scoring, all items were represented by a 5-point Likert-type scale (1 = very dissatisfied; 2 = dissatisfied; 3 = not sure; 4 = satisfied; 5 = very satisfied). Accordingly, a score of “1” was assigned to the lowest satisfaction rating, and “5” to the highest. Patient satisfaction score for each item was calculated by dividing the score of each item by the number of respondents for the corresponding item.
Questions to measure patient satisfaction
Moreover, two questions concerning willingness to choose primary health care for usual body checks and treatment of new health problems were asked (Table 2). The willingness to recommend services, including any consultations, preventive care and Traditional Chinese Medicine, to others was estimated by three questions. The answers to these five questions were classified into two groups–“yes” (i.e., “definitely” and “probably”) and “no” (i.e., “probably not” and “definitely not”).
Questions on patients’ willingness to use and recommend primary health care to others
Socio-demographic characteristics of respondents were also collected. We grouped employment status into two groups–the respondents who had a job (including employed and self-employed) and the respondents who did not have a job (including students, housewives, the retired, and the unemployed). Information on respondents’ self-reported chronic diseases/conditions was collected. These conditions included hypertension, diabetes, cardiovascular disease, chronic respiratory or pulmonary disease, liver disease, thyroid disease, skeleton-muscular disease, gastrointestinal disorders, mental illness and disability. The respondents classified as with health insurance were those covered by any type of social health insurance scheme including MISM and CHIS. The respondents were classified into three economic groups depending on monthly household poverty line and mean monthly household income level in 2011 [15,16], i.e., below RMB 3,000/US$484 as having low income, between RMB 3,000/US$484 and RMB10,000/US$1282 as having middle income, and above RMB10,000/US$1282 as having high income. As for marital status, the respondents were classified into two groups–the singles (including never married, widowed and divorced) and the currently married.
Statistical analysis:
We compared the respondents’ socio-demographic characteristics between migrants and local residents using chi-square tests. The differences in individual and overall satisfaction mean scores between migrants and local residents were examined by both two sample t-test and multiple linear regressions. The differences in the willingness to use and to recommend primary health care between the migrants and local residents were tested by both chi-square tests and multiple logistic regression analyses. Confounding variables, including all characteristics of the respondents, i.e., gender, age, marital status, household income, education, occupation, health insurance status, presence of chronic diseases, health status and length of time with CHC, were adjusted in regression models. In multiple linear regression models, dummy variables were created for age, education and household income. Model fittings were conducted using backward elimination with a threshold of 0.10 for variable inclusion in the model. For all tests conducted in the study, a p-value less than 0.05 was considered to be statistically significant. All analyses were conducted using SPSS19.0 (IBM, USA).
Ethical approval:
Ethical approval was obtained for the study from the ethics committee of the Chinese University of Hong Kong and New Territories East Cluster Clinical Research (Ref. No. CRE-2012.441).
Results:
In total, 787 eligible respondents, of whom 140 were locals and 647 were migrants, completed the face-to-face interviews. Locals tended to be older and to have higher educational status than migrants (p < 0.001). 36.8% of locals did not have a job, while the figure was 23.2% for migrants (p = 0.003). Compared to locals, migrants were more likely to have lower income, but less likely to have any chronic existing physical, mental or psychological problems (p < 0.001). The majority of locals had health insurance (94.7%), which was higher than that of migrants (72.2%, p < 0.001). The insured migrant respondents were all covered by MISM, while the local respondents with insurance were all insured by CHIS. However, not all local residents were insured. Compared with locals, migrants had been utilizing services provided by the CHCs for a shorter period (p < 0.001). Differences found between locals and migrants in gender, marital status and health status were statistically not significant (Table 3).
Socio-economic and demographic characteristics of respondents
The mean scores reported by migrants were lower than those by locals for all dimensions of satisfaction except for the environment of CHCs. Using independent two sample t-test, the significant differences were identified in satisfaction with types of drugs available (p ~ 0.036), effectiveness of primary health care (p < 0.001), attitude of health workers (p < 0.001), waiting time (p < 0.001), convenience (p ~ 0.013), patience of health workers (p ~ 0.001) and involvement of patients in therapeutic decision-making (p ~ 0.003). The mean score of overall satisfaction was also found to be higher for locals than for migrants (4.16 vs. 3.91, p < 0.001). However, after adjusting for the characteristics of the respondents, only the differences in the satisfaction with types of drugs available to them, attitude of health workers, and waiting time remained statistically significant (Table 4).
The mean (SD) scores of different dimensions of satisfaction reported by the respondents by migrant status
†Adjusted for demographic and socio-economic characteristics of the respondents.
Compared to locals, the migrants were more likely to consider primary health care as their first choice for their usual body check (50.7% vs. 61.0%, p ~ 0.029) and usual treatment of health problems (87.1% vs. 94.1%, p ~ 0.006). The difference remained statistically significant for the latter after controlling for the characteristics of the respondents (p ~ 0.032). Locals were more inclined to recommend Traditional Chinese Medicine to others than migrants (65.6% vs. 56.6%, 0.026), after adjusting for the characteristics of the respondents (Table 5).
The willingness to use by the respondents, and to recommend to others by registration type
†Adjusted for demographic and socio-economic characteristics of the respondents.
Discussion:
Major findings The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.
The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.
Strengths and limitations A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.
There were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.
A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.
There were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.
Comparisons with existing literature Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.
The mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.
The mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.
Migrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].
Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.
The mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.
The mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.
Migrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].
Implications for research and practice Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.
The findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.
Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.
The findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.
Major findings:
The findings of the study showed that the mean scores in satisfaction with types of drugs available, attitude of health workers and waiting time were higher for the locals than for migrants. Even though there was no statistically significant difference in the overall satisfaction between local residents and migrants, the latter were more likely to utilize primary health care as the first choice for their usual health problems, while the former were more inclined to recommend Traditional Chinese Medicine to their friends and relatives.
Strengths and limitations:
A particular strength of the study was that quality of primary health care was assessed from the patients’ perspective, which allowed patients to provide feedback to health care providers for quality of care improvement. The comprehensive coverage of information on health status, chronic diseases, health care measures, and socio-demographic characteristics of the respondents was another strength of this study.
There were some limitations. Firstly, the extent to which the sample was representative of the general population was limited. Only four out of more than six hundred CHCs in Shenzhen were selected for study; on the other hand, the sampling approach was CHC-based but not community-based. Even though we employed random sampling method to choose CHCs to improve the representativeness, the conclusions of the study could not be extended to the population in general. Moreover, the study was conducted in Shenzhen and the conclusion could not be generalized to other cities. Secondly, given that the scores were patient-rated, our estimates might be subject to recall bias which could not have been accounted for by statistical adjustments. However, there was no reason why the recall bias would occur systematically. Thirdly, there are other factors which could influence the quality of care provided such as language (dialect), culture and religion. These factors have not been controlled for in our regression models.
Comparisons with existing literature:
Unlike previous studies, the difference in overall satisfaction between migrants and local residents was not found in the present study after patient characteristics were controlled for. Previous studies showed a general trend toward a positive relationship between migration and poorer patient satisfaction. The study by Else et al. in Norway showed that non-western immigrants were less satisfied with visits to general practitioners than Norwegians (40.6% vs. 62.8%; p < 0.05). The study conducted in the USA by Taira et al. [17] showed that Asian-Americans rated overall satisfaction significantly lower than whites did after adjusting for potential confounders (65% vs. 72%; p < 0.01). A qualitative study in Germany which was performed on two focus groups of black immigrants from the Democratic Republic of Congo underlined that German medical staff tended to be unfriendly and to show a diffuse lack of respect towards the migrants [18]. The findings of our study differed from previous studies in that no apparent disparity was found between migrants and local residents in quality of primary health care as measured by overall patient satisfaction at multivariate analysis stage, although univariate analysis showed differences in overall satisfaction scores. The impacts that insurance and chronic diseases/conditions had on patients’ satisfaction with primary health care warrant further investigations in the future.
The mean rating score in satisfaction with the types of drugs available was higher for the locals than the migrants. In Shenzhen, two major health insurance schemes that cover out-patient services are MISM which is for migrant employees, and CHIS which mainly covers local residents. Locals who are registered with CHIS can choose health care providers without any constraint or referral from the primary health care facilities (i.e., CHCs) if secondary or tertiary care facilities were their preference for the first contact, while migrants, who are mainly covered by MISM, have to visit primary care providers and to obtain referrals from their first-contact, i.e., CHCs, to visit health facilities at higher levels (i.e., secondary and tertiary hospitals) due to health insurance reimbursement regulations. For locals, the availability at CHCs of drugs that meet their health needs, and the lower co-payment ratio at CHCs as compared with that in higher level health care facilities, may be two major reasons that motivate them also to seek health care from CHCs, even though they are given options to choose hospital out-patient services. However, since the National Essential Medicine Scheme, which aims to reduce unreasonable drug prescription and consumption, has been implemented in CHCs, the disparity found concerning drug availability was not substantial between the two groups.
The mean scores reported by the migrants for both the attitude of health workers and the waiting time were lower than those of the locals. Higher satisfaction with the attitude of health workers as perceived by the locals may reflect discrimination against migrants by the receiving societies because of their relatively lower socio-economic status. In China, the hukou system plays an important role in the allocation of economic resources, educational opportunities and other welfare benefits. Till now, how the hukou system shapes individuals’ attitudes towards disadvantaged migrants is not known. Nonetheless, Lei and Li [19] recommended that the abolition of the hukou system may reduce discrimination. Migrants reported to have waited longer than they expected to obtain health care services, possibly because they were mostly employed and their employment feature did not allow them paid sick leave [20]. In these circumstances they could visit the CHCs only during non-working hours, when most of their sick peers also came to visit the CHCs.
Migrants were more inclined to consider CHCs as their first point of contact, while local residents were more likely to recommend Traditional Chinese Medicine to their friends and relatives. Studies by Tung et al. [21] and Platonova et al. [22] demonstrated that patients’ satisfaction was a very strong and significant predicator of patients’ intention to choose the doctor and to recommend the primary care physician to others. Although no statistically significant difference in overall satisfaction between locals and migrants was identified in our study, significant differences in willingness to choose and to recommend primary health care were found. The choice of using CHCs as the first point of contact for migrants may be due to the structure of their health insurance scheme, which is often interrelated with their employers and working contracts, but may not necessarily be related to the quality of the services per se [9,23]. Traditional Chinese Medicine services, which were provided by CHCs as signature services, might be another reason prompting locals who were older as a group to seek health services from CHCs and to recommend them to others. This might reflect cultural belief differences between the old and the young [24].
Implications for research and practice:
Local residents and migrants in Shenzhen appeared to be equally satisfied with the quality of primary health care as measured by the overall patient satisfaction score. While this might reflect the true situation, we need to be aware that the respondents in this study were restricted to CHC users and caution should be used when interpreting the findings. Future studies using other indicators measuring primary health care quality, such as primary health care experiences as well as studies amongst non-CHC users, should be conducted to ensure diversity in the subjects.
The findings showed that migrants were reliant on the primary health care system, especially western medical services delivered by the CHCs, more so than the locals. In case disparity in actual waiting time (as opposed to perceived waiting time) really existed between migrants and locals, it would be a greater concern to the migrants who solely relied on the services under insurance constraint but were treated differently at CHCs, as compared to the locals, who would have more options in seeking care. Extending opening hours of CHCs may be one possible solution from the health care providers’ side to reduce migrants’ visits during peak hours, and thus shorten waiting times In addition, policies to encourage employers to provide paid sick leave to employees for health care should be considered. Furthermore, although the abolition of the hukou system may be an alternative to reduce discrimination (e.g., attitude) by health workers against migrants in the future, how to strengthen professional ethics education for health workers in CHCs should be considered by policy makers.
Conclusions:
In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences.
Competing interests:
The authors declare that they have no competing interests.
Authors’ contribution:
XLW, SG, SW and MW conceived of the study, and took part in its design. DZ and YJZ participated in the data collection and helped to draft the manuscript. HTL, RC and XLW were responsible for data analysis and interpretation. HTL, RC and XLW drafted the manuscript. XLW, SW, MW, JM and SG revised the draft for intellectual content. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2296/15/76/prepub
| Background:
Providing good quality primary health care to all inhabitants is one of the Chinese Government's health care objectives. However, information is scarce regarding the difference in quality of primary health care delivered to migrants and local residents respectively. This study aimed to compare patients' perceptions of quality of primary health care between migrants and local patients, and their willingness to use and recommend primary health care to others.
Methods:
A cross-sectional survey was conducted. 787 patients in total were chosen from four randomly drawn Community Health Centers (CHCs) for interviews.
Results:
Local residents scored higher than migrants in terms of their satisfaction with types of drugs available (3.62 vs. 3.45, p=0.035), attitude of health workers (4.41 vs. 4.14, p=0.042) and waiting time (4.30 vs. 3.86, p<0.001). Even though there was no significant difference in overall satisfaction between local residents and migrants (4.16 vs. 3.91, p=0.159), migrants were more likely to utilize primary health care as the first choice for their usual health problems (94.1% vs. 87.1%, p=0.032), while local residents were more inclined to recommend Traditional Chinese Medicine to others (65.6% vs. 56.6%, p=0.026).
Conclusions:
Quality of primary health care given to migrants is less satisfactory than to local residents in terms of attitude of health workers and waiting time. Our study suggests quality of care could be improved through extending opening hours of CHCs and strengthening professional ethics education. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals' recommendations for traditional Chinese medicine were possibly because of cultural differences. | Background:
China’s health challenges are similar to many other transitioning countries, where rural–urban migrants are at higher risk for many health problems than their local counterparts [1]. According to the World Health Organization (WHO), the right to the highest attainable health is a fundamental human right [2]. High quality primary health care is essential in protecting and maintaining the population’s health. China is implementing a primary health care led system through Community Health Centers (CHCs) as a strategy to create a more equitable and efficient health system. Therefore, to achieve the overall goal of better health for all, China needs to provide quality primary health care to all inhabitants including migrants. However, information on the differences in quality of primary health care delivered by CHCs to migrants and local residents, information essential for better future health care planning, is lacking.
China has a large number of internal migrants (nearly 230 million by the end of 2011) [3]. They are normally from rural areas and seeking better paid jobs in cities [4]. The term internal migrants in China usually refers to those who do not change their official hukou registration to the new location which they move to (i.e., floating or non-permanent residents) [5]. Hukou refers to a household registration status officially issued, often on a family basis, to identify a person’s official place of residence. Hukou defines a person’s access to employment, housing, social welfare, educational opportunities and medical and other services. Shenzhen is one of the most populous metropolitan areas located in the Pearl River Delta in Southern China, attracting millions of rural laborers to work as non-permanent migrants [4]. By the end of 2012, the number of internal migrants in Shenzhen reached nearly 12.6 million, which was about 80% of the total population of Shenzhen [6]. To facilitate migrant population management and reduce gaps between migrants and local hukou holders in accessing social welfare, including government-sponsored housing, education, employment and medical insurance, Shenzhen initiated its resident card system in August 2008 to replace the former, discriminatory, temporary residence regime. This new system is open to all non-hukou non-student migrants above 16 years old and has covered more than 10 million people since 2009. Registration into the system is convenient and free. A Shenzhen resident card ensures, under certain additional conditions, legitimacy for migrants to join the city’s Comprehensive Health Insurance Scheme (CHIS) as an individual (instead of as an employee within a working unit). However, due to the higher premium fee, migrants tend not to join CHIS. Whilst all employed migrants are entitled to join the Medical Insurance System for Migrant Employees (MISM), which strictly defines workers’ first contact to be with the city’s 611 primary care providers, i.e., Community Health Centers (CHCs). A referral system from CHCs exists to make sure that care in the community leads the following triage into any necessary secondary or tertiary care. Whilst CHIS participants can choose between hospital outpatient services and CHC clinics as the first contact, they generally pay higher premiums than MISM insurees.
Migrants are generally less skilled and minimally educated, and hence tend to have lower incomes than their local counterparts, leading to poorer access to health care [7]. Their lack of health insurance makes the situation even worse, resulting in unsatisfactory health outcomes [8]. Research suggests that migrants are more likely than local residents to seek health care from CHCs, the major primary health care providers in China, due to the lower costs as compared to the costs of using secondary and tertiary care facilities [9]. Given the important role played by primary health care in protecting migrants’ health, the quality of primary health care is of great concern for quality improvement for services and health improvement among migrants.
Patient satisfaction is one of the most widely used outcome indicators to measure quality of care from patients’ perspective [10]. The assessment of patients’ perception can be seen as a direct measure of quality of care received [11]. Literature indicates that quality of care, specifically patient satisfaction, is an important area because it helps physicians and health care organizations better understand patients’ points of view, and use this feedback to increase accountability and improve the services provided [12,13]. In this paper, we compare patient satisfaction with primary health care provided by the CHCs in Shenzhen between non-permanent migrants and local hukou residents, as well as assessing their willingness to use and recommend primary health care to others.
Conclusions:
In summary, quality of primary health care given to migrants is less satisfactory than to local residents in terms of the attitude of health workers and waiting time. Our study suggests that extending opening hours of CHCs and providing paid sick leave for health care are possible approaches to shorten waiting time for migrants, while abolishing the hukou system and strengthening professional ethics education should be considered to reduce discrimination. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals’ recommendations for TCM were possibly because of cultural differences. | Background:
Providing good quality primary health care to all inhabitants is one of the Chinese Government's health care objectives. However, information is scarce regarding the difference in quality of primary health care delivered to migrants and local residents respectively. This study aimed to compare patients' perceptions of quality of primary health care between migrants and local patients, and their willingness to use and recommend primary health care to others.
Methods:
A cross-sectional survey was conducted. 787 patients in total were chosen from four randomly drawn Community Health Centers (CHCs) for interviews.
Results:
Local residents scored higher than migrants in terms of their satisfaction with types of drugs available (3.62 vs. 3.45, p=0.035), attitude of health workers (4.41 vs. 4.14, p=0.042) and waiting time (4.30 vs. 3.86, p<0.001). Even though there was no significant difference in overall satisfaction between local residents and migrants (4.16 vs. 3.91, p=0.159), migrants were more likely to utilize primary health care as the first choice for their usual health problems (94.1% vs. 87.1%, p=0.032), while local residents were more inclined to recommend Traditional Chinese Medicine to others (65.6% vs. 56.6%, p=0.026).
Conclusions:
Quality of primary health care given to migrants is less satisfactory than to local residents in terms of attitude of health workers and waiting time. Our study suggests quality of care could be improved through extending opening hours of CHCs and strengthening professional ethics education. Considering CHCs as the first choice by migrants might be due to their health insurance scheme, while locals' recommendations for traditional Chinese medicine were possibly because of cultural differences. | 9,543 | 316 | [
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] | [CONTENT] Patient satisfaction | Quality of care | Primary health care | Migration [SUMMARY] | [CONTENT] Patient satisfaction | Quality of care | Primary health care | Migration [SUMMARY] | [CONTENT] Patient satisfaction | Quality of care | Primary health care | Migration [SUMMARY] | [CONTENT] Patient satisfaction | Quality of care | Primary health care | Migration [SUMMARY] | [CONTENT] Patient satisfaction | Quality of care | Primary health care | Migration [SUMMARY] | [CONTENT] Patient satisfaction | Quality of care | Primary health care | Migration [SUMMARY] | [CONTENT] Attitude of Health Personnel | China | Community Health Centers | Cross-Sectional Studies | Female | Health Services Accessibility | Humans | Interviews as Topic | Male | Primary Health Care | Quality of Health Care | Transients and Migrants | Waiting Lists [SUMMARY] | [CONTENT] Attitude of Health Personnel | China | Community Health Centers | Cross-Sectional Studies | Female | Health Services Accessibility | Humans | Interviews as Topic | Male | Primary Health Care | Quality of Health Care | Transients and Migrants | Waiting Lists [SUMMARY] | [CONTENT] Attitude of Health Personnel | China | Community Health Centers | Cross-Sectional Studies | Female | Health Services Accessibility | Humans | Interviews as Topic | Male | Primary Health Care | Quality of Health Care | Transients and Migrants | Waiting Lists [SUMMARY] | [CONTENT] Attitude of Health Personnel | China | Community Health Centers | Cross-Sectional Studies | Female | Health Services Accessibility | Humans | Interviews as Topic | Male | Primary Health Care | Quality of Health Care | Transients and Migrants | Waiting Lists [SUMMARY] | [CONTENT] Attitude of Health Personnel | China | Community Health Centers | Cross-Sectional Studies | Female | Health Services Accessibility | Humans | Interviews as Topic | Male | Primary Health Care | Quality of Health Care | Transients and Migrants | Waiting Lists [SUMMARY] | [CONTENT] Attitude of Health Personnel | China | Community Health Centers | Cross-Sectional Studies | Female | Health Services Accessibility | Humans | Interviews as Topic | Male | Primary Health Care | Quality of Health Care | Transients and Migrants | Waiting Lists [SUMMARY] | [CONTENT] better health china | migrants china | health china implementing | internal migrants china | health care migrants [SUMMARY] | [CONTENT] better health china | migrants china | health china implementing | internal migrants china | health care migrants [SUMMARY] | [CONTENT] better health china | migrants china | health china implementing | internal migrants china | health care migrants [SUMMARY] | [CONTENT] better health china | migrants china | health china implementing | internal migrants china | health care migrants [SUMMARY] | [CONTENT] better health china | migrants china | health china implementing | internal migrants china | health care migrants [SUMMARY] | [CONTENT] better health china | migrants china | health china implementing | internal migrants china | health care migrants [SUMMARY] | [CONTENT] health | care | migrants | health care | chcs | satisfaction | study | primary | primary health care | primary health [SUMMARY] | [CONTENT] health | care | migrants | health care | chcs | satisfaction | study | primary | primary health care | primary health [SUMMARY] | [CONTENT] health | care | migrants | health care | chcs | satisfaction | study | primary | primary health care | primary health [SUMMARY] | [CONTENT] health | care | migrants | health care | chcs | satisfaction | study | primary | primary health care | primary health [SUMMARY] | [CONTENT] health | care | migrants | health care | chcs | satisfaction | study | primary | primary health care | primary health [SUMMARY] | [CONTENT] health | care | migrants | health care | chcs | satisfaction | study | primary | primary health care | primary health [SUMMARY] | [CONTENT] health | migrants | care | china | health care | quality | system | primary | better | hukou [SUMMARY] | [CONTENT] respondents | questions | including | disease | income | chc | health | 000 | classified | study [SUMMARY] | [CONTENT] 001 | locals | respondents | migrants | characteristics respondents | locals migrants | characteristics | health | vs | compared locals migrants [SUMMARY] | [CONTENT] health | migrants | waiting time | waiting | time | education considered reduce discrimination | providing paid sick leave | providing paid sick | providing paid | providing [SUMMARY] | [CONTENT] health | migrants | care | health care | chcs | satisfaction | respondents | study | locals | primary [SUMMARY] | [CONTENT] health | migrants | care | health care | chcs | satisfaction | respondents | study | locals | primary [SUMMARY] | [CONTENT] the Chinese Government's ||| ||| [SUMMARY] | [CONTENT] ||| 787 | four | Community Health Centers [SUMMARY] | [CONTENT] 3.62 | 3.45 | 4.41 | 4.14 | 4.30 | 3.86 ||| 4.16 | 3.91 | first | 94.1% | 87.1% | Traditional Chinese Medicine | 65.6% | 56.6% [SUMMARY] | [CONTENT] ||| opening hours ||| first | Chinese [SUMMARY] | [CONTENT] the Chinese Government's ||| ||| ||| ||| 787 | four | Community Health Centers ||| 3.62 | 3.45 | 4.41 | 4.14 | 4.30 | 3.86 ||| 4.16 | 3.91 | first | 94.1% | 87.1% | Traditional Chinese Medicine | 65.6% | 56.6% ||| ||| opening hours ||| first | Chinese [SUMMARY] | [CONTENT] the Chinese Government's ||| ||| ||| ||| 787 | four | Community Health Centers ||| 3.62 | 3.45 | 4.41 | 4.14 | 4.30 | 3.86 ||| 4.16 | 3.91 | first | 94.1% | 87.1% | Traditional Chinese Medicine | 65.6% | 56.6% ||| ||| opening hours ||| first | Chinese [SUMMARY] |
||||||
Simultaneous resection of coexisting pulmonary and mediastinal lesions by video-assisted thoracic surgery: a case-series study. | 35725438 | With the growing number of patients with coexisting pulmonary and mediastinal lesions detected, reports about simultaneous video-assisted thoracic surgery (VATS) for these concurrent diseases are still rare. To further explore the safety and effectiveness of simultaneous resection of pulmonary and mediastinal lesions by uniportal or biportal VATS, we retrospectively analyzed the clinical data of the largest series of cases to date. | BACKGROUND | From July 2018 to July 2021, all patients whose pulmonary lesions and mediastinal tumors were resected simultaneously in our institution were retrospectively reviewed. Their demographic and clinical data were collected and analyzed. | METHODS | A total of 54 patients were enrolled, of whom 44 underwent unilateral uniportal VATS, 3 underwent bilateral uniportal VATS and 7 underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. For the remaining 47 patients with various demographic and clinical characteristics, most of the operations were completed within 3 h (n = 33, 70.2%) with blood loss of no more than 100 mL (n = 43, 91.5%). The duration of chest tube drainage was 5.66 ± 3.34 days, and the average daily volume was 196.90 ± 122.31 mL. Four cases of postoperative complications occurred during hospitalization. The length of postoperative hospital stay was 8.60 ± 3.63 days. No severe complications or deaths were observed during follow-up. | RESULTS | Uniportal and biportal VATS are safe and effective for simultaneous resection of selected coexisting pulmonary and mediastinal lesions, but the indications and operational details need more evaluation. | CONCLUSIONS | [
"Humans",
"Lung Neoplasms",
"Pneumonectomy",
"Retrospective Studies",
"Thoracic Surgery, Video-Assisted",
"Thoracotomy"
] | 9208703 | Background | Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.
Video-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety. | null | null | Results | The general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.
Table 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019
General information of the patients
ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019
For the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.
Table 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.
Lung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1
Pulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Locations and pathological diagnosis of isolated pulmonary lesions
AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Table 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone
Locations and pathological diagnosis of isolated mediastinal lesions
ACTH adrenocorticotropic hormone
Nineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.
Table 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Pathological diagnosis of lesions suspected to invade surrounding tissues
LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Surgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.
Table 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3
For the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18
Operative details and characteristics
After thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.
Table 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1
General postoperative conditions of the 47 cases of non-converted VATS | Conclusions | We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy. | [
"Background",
"Methods",
"Study design and patient collection",
"Preoperative assessment",
"Surgical technique",
"Data collection and analysis"
] | [
"Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.",
"Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.",
"Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.",
"All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy",
"General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.",
"Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out."
] | [
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Study design and patient collection",
"Preoperative assessment",
"Surgical technique",
"Data collection and analysis",
"Results",
"Discussion",
"Conclusions"
] | [
"Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.\nVideo-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.",
"Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPatients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.\nPreoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nAll patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nSurgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nGeneral anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.\nData collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.\nData including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.",
"Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.",
"All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.\n\nFig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy\nChest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy",
"General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.\n\nFig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nIntraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach\nResection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).\nIf two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.\nFor the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.",
"Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.",
"The general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.\n\nTable 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nGeneral information of the patients\nASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019\nFor the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.\n\nTable 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.\nLung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1\nPulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nLocations and pathological diagnosis of isolated pulmonary lesions\nAIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\n\nTable 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone\nLocations and pathological diagnosis of isolated mediastinal lesions\nACTH adrenocorticotropic hormone\nNineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.\n\nTable 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nPathological diagnosis of lesions suspected to invade surrounding tissues\nLUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe\nSurgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.\n\nTable 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3\nFor the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18\nOperative details and characteristics\nAfter thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.\n\nTable 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1\nGeneral postoperative conditions of the 47 cases of non-converted VATS",
"Yoon et al. indicated that the expected prevalence of anterior mediastinal nodular lesions is approximately 1% in populations aged 55–74 at high risk for lung cancer during low-dose chest CT screening [11]. Similarly, combined surgery for coexisting pulmonary and mediastinal lesions accounted for approximately 0.93% of the pulmonary operations between July 2018 and July 2021 in our hospital and 6.64% of the mediastinal operations. Although the mechanism has not been clarified, thymoma is associated with an increased risk of a secondary neoplasm, such as primary lung cancer [12, 13]. With the help of rapidly developed medical imaging technology and regular examinations before thoracic surgery, coexisting pulmonary and mediastinal lesions are being increasingly detected clinically, but their management remains a challenge due to the lack of surgical guidelines or reports of mass cases.\nThe adjacent anatomical locations of the lung and mediastinum allow the possibility of combined resection, and some surgeons have already tried this approach and shared their valuable experience [1–7]. As a minimally invasive surgery, VATS has been applied widely in the diagnosis and treatment of thoracic diseases. Since first introduced by Migliore in 2001, uniportal VATS has been increasingly adopted by virtue of its advantages, such as a smaller incision, less intraoperative bleeding, rapider recovery, and a shorter duration of surgery, drainage, and hospitalization than multiportal VATS [14–17]. Taken together, VATS, especially uniportal VATS, seems to be an optimal choice for coexisting pulmonary and mediastinal lesions, but the potential risks from complex techniques and prolonged surgery make it necessary for more assessment of its feasibility and safety.\nFor the 54 cases enrolled in this study, simultaneous VATS was successfully performed in 47. Most of the non-converted VATS were completed within 3 h (n = 33, 70.2%) and with bleeding no more than 100 mL (n = 43, 91.5%), which is comparable to the data shown in other studies [4–6]. Specifically, 12 cases (25.5%) of combined surgery were completed within 2 h, 29 cases (61.7%) were between 2 and 4 h, and 6 cases (12.8%) took more than 4 h. The pleural adhesions, large sizes of lesions, and dissection of lymph nodes and invaded tissues were the main causes of prolonged surgical duration. Besides severe pleural adhesions, another factor that converted VATS into thoracotomy was the large size (54.67 ± 14.99 mm vs. 32.87 ± 17.95 mm) or extensive invasion of the mediastinal lesions, especially when the number of local metastases was more than two. In addition to the surgeons’ habits, biportal VATS was usually chosen instead of uniportal VATS when both coexisting lesions were relatively large but had no severe local invasion.\nTo ensure that all lesions are resected, median sternotomy is generally chosen for invasive thymoma [18]. We gathered the data of combined thoracotomy as well, from which it could be found that the average size of the mediastinal lesions was 79.91 ± 26.99 mm, and almost all of them (84.8%) had invaded the surrounding lung tissues (Fig. 1G, H). As shown in Fig. 3, a diameter of 50 mm for mediastinal lesions can be chosen as a turning point by comparing the data between successful VATS and open surgery (including the operations converted to thoracotomy). Therefore, for mediastinal lesions no larger than 50 mm coexisting with pulmonary lesions, if there is no severe local invasion on imaging, it will be feasible to perform uniportal VATS exploration first and decide whether to continue thoracoscopic resection or convert to thoracotomy.\n\nFig. 3Sizes of mediastinal lesions between VATS and thoracotomy\nSizes of mediastinal lesions between VATS and thoracotomy\nFor the resection of pulmonary lesions, the incision should be located in the midaxillary line, slightly anterior for segmental resection near the anterior hilum and slightly posterior for segmental resection near the posterior hilum. For combined VATS, because the location of the single port needed to take both the pulmonary and mediastinal lesions into account, the operation was more difficult. It is necessary to choose the location of the surgical incision with flexibly, and typically a single incision is made in the midaxillary line of the 5th ICS near the posterior axillary line. For the right thoracic incision, if the incision is slightly anterior, the internal thoracic vein will block the visual field and affect the exposure of the left innominate vein. In this case, the internal thoracic vein can be cut (Fig. 2C). For left thoracic incision, surgical instruments are susceptible to interference from the heart if the incision is too far posterior, and resection of segments or lobes at certain sites may not be easy. None of our patients who underwent uniportal VATS had a thoracotomy or auxiliary port added because of poor placement of the incision.\nIt seems that our drainage duration (5.66 ± 3.34 days) was longer than that in other studies, but the difference in standards for the removal of chest tubes should not be ignored. In our department, the last 24-h drainage volume should be less than 200 mL. Most cases of massive drainage occurred after systematic lymph node dissection, and the other case of pleural effusion was mostly due to the large inner wound surface left by a 78 mm mediastinal lesion. All of them were resolved by conservative treatment. Besides, the standards for discharge also delayed some patients’ leaving. The chest imaging (X-ray photograph or CT) and routine blood test (especially the WBC count and neutrophil proportion) were required to become normal before discharge, otherwise the patients should stay to receive further observation and treatment.\nTwo cases of severe postoperative complications occurred during hospitalization. One case of ectopic ACTH syndrome broke out acutely 3 days after uniportal VATS. Fortunately, the condition gradually became controlled, and the patient left the hospital 15 days after surgery. The other case showed difficultly weaning from mechanical ventilation. Although MG had not been diagnosed before surgery, the cause was discovered to be postoperative myasthenic crisis (PMC). The patient recovered and left the hospital 23 days after surgery. In sum, these severe complications were mainly caused by the primary diseases of the patients rather than the operations.\nTelephone follow-up was performed for all 47 non-converted VATS cases, and 2 were lost to follow-up. One case of new-onset bronchospasm and associated paroxysmal hypoxemia was reported. According to the Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score, this case of long-duration (290 min) intrathoracic surgery can be leveled as high risk for PPCs [8, 9]. For combined surgery, relative extension of operation time and trauma is inevitable, but the risk remains controllable. Other complaints reported during follow-up included pain, cough, and small amounts of pleural effusion, which were gradually improved over time.\nConsistent with normal thoracoscopic operations, combined uniportal or biportal VATS does not have many absolute contraindications. Besides enough physical toleration and relatively early clinical stages required by most thoracic tumor operations, coexisting lesions that are difficult to be resected through the same surgical incision might be a relative contraindication, although the clinical judgment lacks quantitative criteria and needs abundant operational experience. The history of thoracic surgery should also be taken into account in view of the fact that severe pleural adhesion is one of the main factors that turned VATS into thoracotomy.\nIt is worth discussing whether pulmonary lesions or mediastinal tumors should be removed first. Normally, surgeons tend to first resect the pulmonary lesions [4–6], which is the same in our study (38:9). Technically, the resection of pulmonary lesions can provide more space for the exposure of mediastinal lesions. When the mediastinal lesions are large but have not invaded surrounding tissues, mediastinum-first resection is preferred. However, if mediastinal tumor invades or adheres closely to the surrounding tissues, the invaded tissues should be removed first. In general, lung-first VATS helps to maintain a clear field of vision for both pulmonary and mediastinal surgery. If mediastinal lesions are treated first before pulmonary lesions, the exudation of mediastinal surgical wounds will flow to the lung, thus affecting the clear field of pulmonary surgery.\nAlthough the patients enrolled in this study were various, its reliability is limited by the retrospective design and relatively small sample size. Items such as the incision length were not analyzed due to the missing of records, and it failed to establish a case-control study since there lacked enough comparable cases of multiportal VATS or staged operations for coexisting pulmonary and mediastinal lesions in our hospital. We tried to assess the indications by comparing the characteristics of cases between VATS and thoracotomy, but this was inevitably influenced by the subjective bias and habits of surgeons.\nIn brief, this is thus far the largest case series analysis about combined resection of pulmonary and mediastinal lesions through uniportal VATS. We analyzed the clinical pathological characteristics of these patients and the efficacy and safety of the operations. The surgical indications and selection of the appropriate incision site were also explored. The reliability of this study is limited due to the small number of cases and non-comparative design. Further researches with larger sample sizes are needed.",
"We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy."
] | [
null,
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusion"
] | [
"Lung neoplasms",
"Mediastinal neoplasms",
"Simultaneous operation",
"Thoracic surgery",
"Video-assisted thoracoscopic surgery"
] | Background:
Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.
Video-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.
Methods:
Study design and patient collection Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.
Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.
Preoperative assessment All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.
Fig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy
Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy
All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.
Fig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy
Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy
Surgical technique General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.
Fig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach
Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach
Resection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).
If two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.
For the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.
General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.
Fig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach
Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach
Resection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).
If two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.
For the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.
Data collection and analysis Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.
Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.
Study design and patient collection:
Patients who underwent simultaneous operation for pulmonary and mediastinal lesions at the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between July 2018 and July 2021 were reviewed. A total of 87 patients were identified, of whom 33 underwent thoracotomy and 54 underwent VATS. These 54 patients were analyzed in this study. (The other 33 cases were also reviewed for comparison and discussion, but their data are not listed directly.) Forty-four patients underwent unilateral uniportal VATS, 3 patients underwent bilateral uniportal VATS, and 7 patients underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. Therefore, there were 47 cases of VATS performed successfully. Follow-up data were obtained through telephone interviews. All patients were followed up for 3–36 months (mean 12.5 months), with the exception of 2.
Preoperative assessment:
All patients underwent chest contrast-enhanced computed tomography (CT), from which the coexistence of pulmonary and mediastinal lesions was found and carefully evaluated (Fig. 1). Mediastinal lymph node enlargement was examined by enhanced chest CT as well, and some patients underwent mediastinal magnetic resonance imaging (MRI) to determine whether the mediastinal lesions were cystic or solid. Tumor markers of lung cancer and T-Spot test were used for differential diagnosis. To determine the presence of distant metastasis, head MRI, whole-body osteonuclide scans, and liver and adrenal gland ultrasonography were routinely performed, while positron emission tomography (PET)-CT was also used in a few cases. For patients suspected of myasthenia gravis (MG) with mediastinal tumors, electromyography (EMG) was performed routinely, and autoantibodies including Acetylcholine receptor (AChR) antibody, Muscle-specific kinase (MuSK) antibody and other antibodies were also detected for some patients. In addition to the typical clinical features of MG, patients with immunological, pharmacological, and/or neuroelectrophysiological manifestations can be clinically diagnosed as MG. Meanwhile, the patients received ultrasonography to check the conditions of their cardiovascular system and major organs, including the spleen, pancreas and kidney. Preoperative assessment also included an electrocardiogram, routine blood test, routine urine test, renal function test, blood gas analysis, and pulmonary function test to comprehensively evaluate the patients’ physiological status and tolerance for surgical treatment. Ground glass nodules (GGNs) were observed for at least three months and removed only when the nodules persisted or increased on subsequent imaging examination. Pulmonary nodules that may be inflammatory according to imaging were re-examined after anti-inflammatory treatment and removed when there was no reduction. Preoperative neoadjuvant therapy was performed for lung cancer that was clinically assessed as stage IIIA or above.
Fig. 1Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy
Chest CT images of representative patients. The arrows denote lesions. A, B A 10 mm left upper lobe GGO (microinvasive adenocarcinoma) associated with a 38 mm anterior mediastinal mass (hemangioma). C, D A 35 mm left upper lobe mass (invasive adenocarcinoma) associated with a 13 mm anterior mediastinal mass (bronchogenic cyst). E, F A 50 mm anterior mediastinal lesion (thymoma of type B2) invading surrounding lung tissues. G, H A 70 mm mediastinal mass (thyroid follicular neoplasm) and its coexisting 15 mm left upper lobe lesion (microinvasive adenocarcinoma), which were difficult for VATS and resected by thoracotomy
Surgical technique:
General anesthesia was applied with double-lumen endotracheal intubation. For patients with coexisting lesions on the same side, unilateral uniportal VATS was performed while the patient was placed in the lateral decubitus position. After disinfection, a single incision of approximately 3 cm was made in the 5th intercostal space (ICS). The location of the incision was posterior to the midaxillary line (Fig. 2A, B). For biportal VATS, incisions were made in the 5th and 7th ICSs. Exploration was carried out for the locations, sizes and invasion of lesions, the quality of lungs, and the conditions of lung fissures. Meanwhile, pleural adhesions were separated carefully when found. If thoracoscopic separation of pleural adhesions was difficult, thoracoscopic surgery was converted to thoracotomy.
Fig. 2Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach
Intraoperative photos of uniportal VATS. A The surgical incision of uniportal VATS for the resection of the posterior segment of the right upper lobe and an anterior mediastinal mass. B The surgical incision of uniportal VATS for the resection of the posterior segment of the left upper lobe and an anterior mediastinal mass. C The ligation of the internal thoracic vein on the right-sided approach
Resection of pulmonary lesions was usually performed first. For visible peripheral pulmonary nodules, wedge resection was performed for intraoperative frozen section examination. The surgical method including the type of pulmonary resection and whether lymph node dissection or sampling was necessary, was selected according to the results of intraoperative frozen section examination and preoperative examination. For patients requiring segmentectomy or lobectomy, anatomic resection was performed. After complete hemostasis, we examined air leakage of the lung. In some cases, the mediastinal lesions were excised first due to their large sizes or the surgeons’ personal preference. For the resection of mediastinal tumors, tissues in the area surrounded by the left innominate vein, the contralateral pleura, and the phrenic nerve on the surgical side were excised in one piece. For the right thoracic incision, the internal thoracic vein would be cut when it blocked the visual field and affected the exposure of the left innominate vein (Fig. 2C).
If two coexisting lesions were located on different sides, bilateral uniportal VATS was selected. The selection of surgical incision and surgical procedures were similar to uniportal VATS. For patients with MG, the mediastinal tumors were resected, and bilateral mediastinal fat clearance was performed by bilateral uniportal VATS.
For the lesions suspected to invade surrounding tissues (it was most common that mediastinal lesions invaded lung tissues), according to the tumor-free principle, the adjoined tissues invaded or attached tightly by the primary lesions were resected first. After all the surrounding tissues were separated, the primary lesion was finally removed.
Data collection and analysis:
Data including the demographic information, clinical manifestations, operative details, and perioperative outcomes were collected. The ASA category was established by the American Society of Anesthesiologists to classify patients undergoing anesthesia according to their physical conditions and the risk of surgery, which includes six levels from the best (1st) to the worst (6th). Data processing was performed using SPSS (ver. 20; SPSS, Inc., Chicago, IL, USA) for Windows. A descriptive analysis of the variables collected in this research was carried out.
Results:
The general characteristics of all 54 patients are summarized in Table 1. One patient had a history of coronavirus disease 2019 (COVID-19), and her operation and postoperative course were uneventful. The most common symptoms were cough (n = 7, 13.0%), chest tightness (n = 7, 13.0%) and chest pain (n = 6, 11.1%). Three patients had muscle weakness, two of whom were diagnosed with MG, while the remaining patient had ectopic adrenocorticotropic hormone (ACTH) syndrome. In sum, 31 patients had no associated symptoms and found thoracic lesions incidentally during routine examinations or diagnosis for diseases of other systems.
Table 1General information of the patientsVariableMean ± SD (range) or no.Gender (n) Men26 Women28Age at diagnosis (year)53.87 ± 11.58Medical history (n) Hypertension17 Diabetes6 Pulmonary emphysema3 Chronic bronchitis3 COPD1 Asthma2 Coronary heart disease5 Arrhythmia4 Cerebral infarction3 Thyroid cancer2 Chest wall tuberculosis1 COVID-191 History of surgery25 Thoracic surgery0Smoking history (n)16ASA category (n) 1th5 2th31 3th18Symptoms (n) Cough7 Chest tightness7 Chest pain6 Expectoration6 Dyspnea4 Muscle weakness3 Myasthenia gravis2 Limb pain2 Hemoptysis1 Fever1 Edema of lower limbs1 Asymptomatic or no related symptoms31ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019
General information of the patients
ASA American Society of Anesthesiologists, COPD chronic obstructive pulmonary disease, COVID-19 Coronavirus Disease 2019
For the 35 patients whose pulmonary and mediastinal lesions were anatomically isolated and pathologically irrelevant to each other, Table 2 showed the pathological features of their pulmonary lesions, which included ground glass opacity (GGO), solid nodules, and masses associated with mediastinal lesions of varied sizes on imaging (Fig. 1A–D). Their anatomical locations included all five lung lobes, and the TNM stage of lung cancer ranged from 0 to IIIA. The only case of stage IIIA was a squamous cell carcinoma treated with radiotherapy and chemotherapy before surgery. Various pathological types were found in pulmonary benign lesions, with 3 cases of benign nodules unclassified pathologically. Table 3 shows the pathological features of the isolated mediastinal lesions. Most of them were located in the anterior mediastinum (n = 27, 77.1%). Benign cysts (n = 15, 42.9%) were the most common types among them, which included bronchogenic cysts, thymic cysts, and pericardial cysts. Almost half of these lesions (n = 16, 45.7%) were related to the thymus.
Table 2Locations and pathological diagnosis of isolated pulmonary lesionsVariableMean ± SD (range) or no.
Lung cancer21 Size (mm)19.52 ± 12.32 Anatomical site (n) LUL6 LLL4 RUL7 RLL2 RML + RLL2 Pathological type (n) Adenocarcinoma18 AIS3 MIA4 ICA11 Squamous cell carcinoma1 Adenosquamous carcinoma1 Lymphoepithelioma-like carcinoma1 Stage (n) 03 IA15 IA24 IA33 IB4 IIB1 IIIA1
Pulmonary benign diseases14 Anatomical site (n) LUL2 LLL2 RUL3 RML2 RLL4 LUL + LLL1 Pathological type (n) Chronic inflammation4 Reactive lymph node hyperplasia2 Tuberculosis2 Sclerosing lung cell tumor1 Lung cyst1 Infectious pneumonia1 Benign nodules3AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Locations and pathological diagnosis of isolated pulmonary lesions
AIS adenocarcinoma in situ, ICA invasive adenocarcinoma, LUL left upper lobe, LLL left lower lobe, MIA minimally invasive adenocarcinoma, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Table 3Locations and pathological diagnosis of isolated mediastinal lesionsVariableMean ± SD (range) or no.Size (mm)27.71 ± 16.39Anatomical site (n) Upper1 Anterior27 Middle1 Posterior2 Upper anterior4Pathological type and stage (n) Thymoma7 AB3 B1 + B21 B23 Bronchogenic cyst7 Thymic cyst6 Pericardial cyst2 Thymic carcinoma2 Thymic hyperplasia1 Lipoma1 Hemangioma1 Neuroendocrine tumor (ACTH)1 Unclassified benign lesions7ACTH adrenocorticotropic hormone
Locations and pathological diagnosis of isolated mediastinal lesions
ACTH adrenocorticotropic hormone
Nineteen patients underwent simultaneous resection because the mediastinal tumors were suspected to invade the lung (n = 17, 89.5%), or vice versa (n = 2, 10.5%) (Fig. 1E, F). As shown in Table 4, the invasive lesions were much larger than the isolated ones mentioned above (51.29 ± 12.06 mm vs. 27.71 ± 16.39 mm for mediastinal lesions as example). It was no surprise that thymoma and thymic carcinoma constituted the majority of cases (n = 11, 57.9%), but there were also some lesions, such as bronchogenic cysts and pulmonary abscesses, found to be noninvasive after surgery, for which the surrounding tissues were resected due to the tight adhesion between them. Two cases of invasive lesions were located at both the anterior and posterior mediastinum. One of them was an anterior mediastinal thymic cyst and posterior mediastinal ganglioneuroma, and the other one was thymoma (type B2) shown as two lesions. Finally, 11 cases of invasion were confirmed by postoperative pathological examination.
Table 4Pathological diagnosis of lesions suspected to invade surrounding tissuesVariableMean ± SD (range) or no.Sizes of primary lesions (mm)50.95 ± 12.75Anatomical sites of primary lesions (n) Mediastinum17 Anterior12 Posterior1 Upper anterior2 Anterior + Posterior2 Lung2 LUL1 LLL1Pathological type (n) Thymoma8 B12 B1 + B21 B24 B31 Thymic carcinoma3 Hodgkin lymphoma2 Thymic Cyst + Ganglioneuroma1 Tuberculosis1 Bronchogenic cyst1 Sclerosing lung cell tumor1 Pulmonary abscess1 Mature teratoma1Invaded site (n) Mediastinum2 Anterior1 Posterior1 Lung17 LUL6 LLL1 RUL6 RLL3 LLL + LUL1Invasion confirmed by biopsy (n)11LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Pathological diagnosis of lesions suspected to invade surrounding tissues
LUL left upper lobe, LLL left lower lobe, RUL right upper lobe, RML right middle lobe, RLL right lower lobe
Surgical details and intraoperative characteristics are stated in Table 5. Seven cases of VATS were converted to thoracotomy. Two cases of conversion were because of the severe pleural adhesions, and 2 cases were respectively due to the massive invasion on lung or pericardium. The other 3 operations were converted on account of the large sizes and severe local invasion of mediastinal lesions. These cases were converted to thoracotomy just after exploration by VATS. For the remaining 47 non-converted VATS, 38 patients had pulmonary lesions resected first. Coincidentally, all 3 cases of bilateral uniportal VATS (including the one converted to thoracotomy) had resection of the mediastinal lesions first. The only frequent intraoperative unplanned events were pleural adhesions (n = 18, 38.3%), which could usually be resolved without much difficulty. Most of the operations were completed within 3 h (n = 33, 70.2%). No heavy bleeding occurred, and most cases (n = 43, 91.5%) had blood loss of no more than 100 mL during surgery. A few cases of large mediastinal tumors (50 mm, 70 and 78 mm) resulted in more bleeding (300 mL, 300 mL, and 800 mL, respectively) due to their abundant blood supply.
Table 5Operative details and characteristicsVariableMean ± SD (range) or no.Surgical approach (n) Unilateral uniportal VATS44 Converted to thoracotomy5 Left side16 Right side23 Bilateral uniportal VATS3 Converted to thoracotomy1 Unilateral biportal VATS7 Converted to thoracotomy1 Left side3 Right side3
For the 47 cases of non-converted VATS Operational sequence (n) Lung first38 Mediastinum first9 Type of pulmonary surgery (n) Lobectomy12 Double lobectomy2 Segmentectomy8 Wedge resection27 Lymph node dissection (n)11 Number of lymph nodes excised20.00 ± 5.74 Groups of mediastinal lymph nodes removed (n) Left side 4L1 53 63 74 81 94 Right side 2R7 4R7 77 95 Operation time (min)167.83 ± 69.22 Intraoperative blood loss (mL)57.55 ± 128.33 Range (n) ≤ 10043 > 1004 Intraoperative unplanned events (n) Pleural adhesions18
Operative details and characteristics
After thoracoscopic surgery (Table 6), patients with MG or major surgery were taken back to the intensive care unit (ICU) with tracheal intubation for ventilator-assisted breathing. One patient was repeatedly put on ventilators, with the overall duration of 285 h, which was caused by a myasthenic crisis. There were 3 other cases of postoperative complications. One of them presented acutely with convulsions, ventricular fibrillation, hypertension, and hypoxemia, which was related to the patient’s primary disease-induced ectopic ACTH syndrome and was eventually controlled. The other two patients had massive pleural effusion cured by thoracentesis. The 47 patients left the hospital 8.60 ± 3.63 days (range 5–23 days) after surgery with no perioperative mortality. One patient reported bronchospasm after discharge, which is one of the common postoperative pulmonary complications (PPCs) [8–10]. No recurrence or death occurred during follow-up.
Table 6General postoperative conditions of the 47 cases of non-converted VATSVariableMean ± SD (range) or no.Postoperative mechanical ventilation (n)7 ≤ 24 h5 > 24 h2Duration of chest tube drainage (d)5.66 ± 3.34Average daily volume of drainage (mL/d)196.90 ± 122.31Postoperative hospital stay (d)8.60 ± 3.63Postoperative complications (n)4 Cardiovascular dysfunction + convulsions1 Myasthenic crisis1 Massive pleural effusion2Perioperative mortality (n)0Postoperative adjuvant therapy (n) Chemotherapy7 Radiotherapy3 Chemotherapy + Radiotherapy2 Targeted therapy1 Immunotherapy1Complications reported during follow-up (n) Bronchospasm1
General postoperative conditions of the 47 cases of non-converted VATS
Discussion:
Yoon et al. indicated that the expected prevalence of anterior mediastinal nodular lesions is approximately 1% in populations aged 55–74 at high risk for lung cancer during low-dose chest CT screening [11]. Similarly, combined surgery for coexisting pulmonary and mediastinal lesions accounted for approximately 0.93% of the pulmonary operations between July 2018 and July 2021 in our hospital and 6.64% of the mediastinal operations. Although the mechanism has not been clarified, thymoma is associated with an increased risk of a secondary neoplasm, such as primary lung cancer [12, 13]. With the help of rapidly developed medical imaging technology and regular examinations before thoracic surgery, coexisting pulmonary and mediastinal lesions are being increasingly detected clinically, but their management remains a challenge due to the lack of surgical guidelines or reports of mass cases.
The adjacent anatomical locations of the lung and mediastinum allow the possibility of combined resection, and some surgeons have already tried this approach and shared their valuable experience [1–7]. As a minimally invasive surgery, VATS has been applied widely in the diagnosis and treatment of thoracic diseases. Since first introduced by Migliore in 2001, uniportal VATS has been increasingly adopted by virtue of its advantages, such as a smaller incision, less intraoperative bleeding, rapider recovery, and a shorter duration of surgery, drainage, and hospitalization than multiportal VATS [14–17]. Taken together, VATS, especially uniportal VATS, seems to be an optimal choice for coexisting pulmonary and mediastinal lesions, but the potential risks from complex techniques and prolonged surgery make it necessary for more assessment of its feasibility and safety.
For the 54 cases enrolled in this study, simultaneous VATS was successfully performed in 47. Most of the non-converted VATS were completed within 3 h (n = 33, 70.2%) and with bleeding no more than 100 mL (n = 43, 91.5%), which is comparable to the data shown in other studies [4–6]. Specifically, 12 cases (25.5%) of combined surgery were completed within 2 h, 29 cases (61.7%) were between 2 and 4 h, and 6 cases (12.8%) took more than 4 h. The pleural adhesions, large sizes of lesions, and dissection of lymph nodes and invaded tissues were the main causes of prolonged surgical duration. Besides severe pleural adhesions, another factor that converted VATS into thoracotomy was the large size (54.67 ± 14.99 mm vs. 32.87 ± 17.95 mm) or extensive invasion of the mediastinal lesions, especially when the number of local metastases was more than two. In addition to the surgeons’ habits, biportal VATS was usually chosen instead of uniportal VATS when both coexisting lesions were relatively large but had no severe local invasion.
To ensure that all lesions are resected, median sternotomy is generally chosen for invasive thymoma [18]. We gathered the data of combined thoracotomy as well, from which it could be found that the average size of the mediastinal lesions was 79.91 ± 26.99 mm, and almost all of them (84.8%) had invaded the surrounding lung tissues (Fig. 1G, H). As shown in Fig. 3, a diameter of 50 mm for mediastinal lesions can be chosen as a turning point by comparing the data between successful VATS and open surgery (including the operations converted to thoracotomy). Therefore, for mediastinal lesions no larger than 50 mm coexisting with pulmonary lesions, if there is no severe local invasion on imaging, it will be feasible to perform uniportal VATS exploration first and decide whether to continue thoracoscopic resection or convert to thoracotomy.
Fig. 3Sizes of mediastinal lesions between VATS and thoracotomy
Sizes of mediastinal lesions between VATS and thoracotomy
For the resection of pulmonary lesions, the incision should be located in the midaxillary line, slightly anterior for segmental resection near the anterior hilum and slightly posterior for segmental resection near the posterior hilum. For combined VATS, because the location of the single port needed to take both the pulmonary and mediastinal lesions into account, the operation was more difficult. It is necessary to choose the location of the surgical incision with flexibly, and typically a single incision is made in the midaxillary line of the 5th ICS near the posterior axillary line. For the right thoracic incision, if the incision is slightly anterior, the internal thoracic vein will block the visual field and affect the exposure of the left innominate vein. In this case, the internal thoracic vein can be cut (Fig. 2C). For left thoracic incision, surgical instruments are susceptible to interference from the heart if the incision is too far posterior, and resection of segments or lobes at certain sites may not be easy. None of our patients who underwent uniportal VATS had a thoracotomy or auxiliary port added because of poor placement of the incision.
It seems that our drainage duration (5.66 ± 3.34 days) was longer than that in other studies, but the difference in standards for the removal of chest tubes should not be ignored. In our department, the last 24-h drainage volume should be less than 200 mL. Most cases of massive drainage occurred after systematic lymph node dissection, and the other case of pleural effusion was mostly due to the large inner wound surface left by a 78 mm mediastinal lesion. All of them were resolved by conservative treatment. Besides, the standards for discharge also delayed some patients’ leaving. The chest imaging (X-ray photograph or CT) and routine blood test (especially the WBC count and neutrophil proportion) were required to become normal before discharge, otherwise the patients should stay to receive further observation and treatment.
Two cases of severe postoperative complications occurred during hospitalization. One case of ectopic ACTH syndrome broke out acutely 3 days after uniportal VATS. Fortunately, the condition gradually became controlled, and the patient left the hospital 15 days after surgery. The other case showed difficultly weaning from mechanical ventilation. Although MG had not been diagnosed before surgery, the cause was discovered to be postoperative myasthenic crisis (PMC). The patient recovered and left the hospital 23 days after surgery. In sum, these severe complications were mainly caused by the primary diseases of the patients rather than the operations.
Telephone follow-up was performed for all 47 non-converted VATS cases, and 2 were lost to follow-up. One case of new-onset bronchospasm and associated paroxysmal hypoxemia was reported. According to the Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score, this case of long-duration (290 min) intrathoracic surgery can be leveled as high risk for PPCs [8, 9]. For combined surgery, relative extension of operation time and trauma is inevitable, but the risk remains controllable. Other complaints reported during follow-up included pain, cough, and small amounts of pleural effusion, which were gradually improved over time.
Consistent with normal thoracoscopic operations, combined uniportal or biportal VATS does not have many absolute contraindications. Besides enough physical toleration and relatively early clinical stages required by most thoracic tumor operations, coexisting lesions that are difficult to be resected through the same surgical incision might be a relative contraindication, although the clinical judgment lacks quantitative criteria and needs abundant operational experience. The history of thoracic surgery should also be taken into account in view of the fact that severe pleural adhesion is one of the main factors that turned VATS into thoracotomy.
It is worth discussing whether pulmonary lesions or mediastinal tumors should be removed first. Normally, surgeons tend to first resect the pulmonary lesions [4–6], which is the same in our study (38:9). Technically, the resection of pulmonary lesions can provide more space for the exposure of mediastinal lesions. When the mediastinal lesions are large but have not invaded surrounding tissues, mediastinum-first resection is preferred. However, if mediastinal tumor invades or adheres closely to the surrounding tissues, the invaded tissues should be removed first. In general, lung-first VATS helps to maintain a clear field of vision for both pulmonary and mediastinal surgery. If mediastinal lesions are treated first before pulmonary lesions, the exudation of mediastinal surgical wounds will flow to the lung, thus affecting the clear field of pulmonary surgery.
Although the patients enrolled in this study were various, its reliability is limited by the retrospective design and relatively small sample size. Items such as the incision length were not analyzed due to the missing of records, and it failed to establish a case-control study since there lacked enough comparable cases of multiportal VATS or staged operations for coexisting pulmonary and mediastinal lesions in our hospital. We tried to assess the indications by comparing the characteristics of cases between VATS and thoracotomy, but this was inevitably influenced by the subjective bias and habits of surgeons.
In brief, this is thus far the largest case series analysis about combined resection of pulmonary and mediastinal lesions through uniportal VATS. We analyzed the clinical pathological characteristics of these patients and the efficacy and safety of the operations. The surgical indications and selection of the appropriate incision site were also explored. The reliability of this study is limited due to the small number of cases and non-comparative design. Further researches with larger sample sizes are needed.
Conclusions:
We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy.
| Background:
With the growing number of patients with coexisting pulmonary and mediastinal lesions detected, reports about simultaneous video-assisted thoracic surgery (VATS) for these concurrent diseases are still rare. To further explore the safety and effectiveness of simultaneous resection of pulmonary and mediastinal lesions by uniportal or biportal VATS, we retrospectively analyzed the clinical data of the largest series of cases to date.
Methods:
From July 2018 to July 2021, all patients whose pulmonary lesions and mediastinal tumors were resected simultaneously in our institution were retrospectively reviewed. Their demographic and clinical data were collected and analyzed.
Results:
A total of 54 patients were enrolled, of whom 44 underwent unilateral uniportal VATS, 3 underwent bilateral uniportal VATS and 7 underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. For the remaining 47 patients with various demographic and clinical characteristics, most of the operations were completed within 3 h (n = 33, 70.2%) with blood loss of no more than 100 mL (n = 43, 91.5%). The duration of chest tube drainage was 5.66 ± 3.34 days, and the average daily volume was 196.90 ± 122.31 mL. Four cases of postoperative complications occurred during hospitalization. The length of postoperative hospital stay was 8.60 ± 3.63 days. No severe complications or deaths were observed during follow-up.
Conclusions:
Uniportal and biportal VATS are safe and effective for simultaneous resection of selected coexisting pulmonary and mediastinal lesions, but the indications and operational details need more evaluation. | Background:
Lung cancer is a major health problem worldwide. Because of their anatomical proximity, the coexistence of pulmonary and mediastinal lesions has been detected more frequently with the development and popularization of medical imaging technology [1–7]. Correspondingly, more attention has been concentrated on the optimal measures to deal with these concurrent diseases and invasive lesions. Staged operations are available for some cases, but theoretically, combined resection could better reduce the financial burden on patients and avoid delays in treatment. For invasive lesions, open surgery is still preferred, but minimally invasive treatment should also be utilized as much as possible.
Video-assisted thoracic surgery (VATS) has been applied widely in the clinical diagnosis and treatment of thoracic diseases. Simultaneous thoracoscopic resection seems to be an ideal treatment option for coexisting pulmonary and mediastinal lesions, but convincing evidence is required and most of the studies thus far are small-sample and the types of patients are limited [4–6]. Our institution is one of the largest thoracic surgery centers in China with numerous operations performed every year, and has abundant surgical experience in uniportal VATS. Here, the aim of this study is to present the largest sample of uniportal VATS for simultaneous resection of pulmonary and mediastinal lesions and retrospectively analyze these data to help evaluate their feasibility and safety.
Conclusions:
We found that simultaneous resection of pulmonary and mediastinal lesions by an experienced thoracic surgeon using uniportal or biportal VATS is safe and feasible. This surgical method has certain indications: in general, mediastinal lesions that are less than 50 mm without serious local invasion, and pulmonary lesions that are in the early or middle stages. In addition, the importance of appropriate surgical procedures is noteworthy. | Background:
With the growing number of patients with coexisting pulmonary and mediastinal lesions detected, reports about simultaneous video-assisted thoracic surgery (VATS) for these concurrent diseases are still rare. To further explore the safety and effectiveness of simultaneous resection of pulmonary and mediastinal lesions by uniportal or biportal VATS, we retrospectively analyzed the clinical data of the largest series of cases to date.
Methods:
From July 2018 to July 2021, all patients whose pulmonary lesions and mediastinal tumors were resected simultaneously in our institution were retrospectively reviewed. Their demographic and clinical data were collected and analyzed.
Results:
A total of 54 patients were enrolled, of whom 44 underwent unilateral uniportal VATS, 3 underwent bilateral uniportal VATS and 7 underwent unilateral biportal VATS. Seven cases were converted to thoracotomy during surgery. For the remaining 47 patients with various demographic and clinical characteristics, most of the operations were completed within 3 h (n = 33, 70.2%) with blood loss of no more than 100 mL (n = 43, 91.5%). The duration of chest tube drainage was 5.66 ± 3.34 days, and the average daily volume was 196.90 ± 122.31 mL. Four cases of postoperative complications occurred during hospitalization. The length of postoperative hospital stay was 8.60 ± 3.63 days. No severe complications or deaths were observed during follow-up.
Conclusions:
Uniportal and biportal VATS are safe and effective for simultaneous resection of selected coexisting pulmonary and mediastinal lesions, but the indications and operational details need more evaluation. | 8,547 | 298 | [
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] | null | [CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY] | null | [CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY] | [CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY] | [CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY] | [CONTENT] Lung neoplasms | Mediastinal neoplasms | Simultaneous operation | Thoracic surgery | Video-assisted thoracoscopic surgery [SUMMARY] | [CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY] | null | [CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY] | [CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY] | [CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY] | [CONTENT] Humans | Lung Neoplasms | Pneumonectomy | Retrospective Studies | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY] | [CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY] | null | [CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY] | [CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY] | [CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY] | [CONTENT] resection pulmonary mediastinal | pulmonary mediastinal surgery | video assisted thoracic | simultaneous thoracoscopic resection | thoracic surgery vats [SUMMARY] | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY] | null | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY] | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY] | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY] | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | lobe | uniportal | left | uniportal vats [SUMMARY] | [CONTENT] treatment | lesions | invasive | invasive lesions | largest | sample | resection | operations | diseases | pulmonary mediastinal lesions [SUMMARY] | null | [CONTENT] lobe | table | right | pathological | lower | lesions | range | lower lobe | thymic | cases [SUMMARY] | [CONTENT] lesions | mm local invasion pulmonary | vats safe | local invasion pulmonary lesions | method certain | surgical procedures noteworthy | method certain indications | method certain indications general | vats safe feasible surgical | vats safe feasible [SUMMARY] | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | uniportal | lobe | uniportal vats | resection [SUMMARY] | [CONTENT] mediastinal | lesions | vats | patients | mm | pulmonary | uniportal | lobe | uniportal vats | resection [SUMMARY] | [CONTENT] VATS ||| [SUMMARY] | null | [CONTENT] 54 | 44 | VATS | 3 | VATS | 7 | VATS ||| Seven ||| 47 | 3 | 33 | 70.2% | no more than 100 mL (n = | 43 | 91.5% ||| 5.66 | 3.34 days | daily | 196.90 | 122.31 mL. Four ||| 8.60 | 3.63 days ||| [SUMMARY] | [CONTENT] [SUMMARY] | [CONTENT] VATS ||| ||| July 2018 to July 2021 ||| ||| 54 | 44 | VATS | 3 | VATS | 7 | VATS ||| Seven ||| 47 | 3 | 33 | 70.2% | no more than 100 mL (n = | 43 | 91.5% ||| 5.66 | 3.34 days | daily | 196.90 | 122.31 mL. Four ||| 8.60 | 3.63 days ||| ||| [SUMMARY] | [CONTENT] VATS ||| ||| July 2018 to July 2021 ||| ||| 54 | 44 | VATS | 3 | VATS | 7 | VATS ||| Seven ||| 47 | 3 | 33 | 70.2% | no more than 100 mL (n = | 43 | 91.5% ||| 5.66 | 3.34 days | daily | 196.90 | 122.31 mL. Four ||| 8.60 | 3.63 days ||| ||| [SUMMARY] |
|||||
Histologic features and genomic alterations of primary colorectal adenocarcinoma predict growth patterns of liver metastasis. | 31341365 | Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established. | BACKGROUND | A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis. | METHODS | In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5). | RESULTS | The HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis. | CONCLUSION | [
"Adenocarcinoma",
"Adult",
"Aged",
"Aged, 80 and over",
"B7-H1 Antigen",
"Class I Phosphatidylinositol 3-Kinases",
"Colon",
"Colorectal Neoplasms",
"DNA Mismatch Repair",
"Female",
"Humans",
"Kaplan-Meier Estimate",
"Liver",
"Liver Neoplasms",
"Male",
"Middle Aged",
"Mutation",
"Predictive Value of Tests",
"Prognosis",
"Proto-Oncogene Proteins B-raf",
"Rectum",
"Retrospective Studies",
"Exome Sequencing"
] | 6639555 | INTRODUCTION | Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].
According to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.
It is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis. | MATERIALS AND METHODS | Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.
With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.
Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.
The HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).
Representative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).
For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.
The HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).
Representative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).
Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].
The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].
Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.
Automated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.
The IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.
All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.
Automated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.
The IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.
Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.
Whole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].
Work flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.
Sequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).
Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.
Whole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].
Work flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.
Sequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).
Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.
Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis. | null | null | Research conclusion | We thank Dr. Logan Walsh at Department of Human Genetics, Rosalind and Morris Goodman Cancer Research Center, McGill University for his assistance in analyzing the whole exome sequencing data, and associate professor Jianfeng Luo at Department of Biostatistics, School of Public Health, Fudan University for the help of biostatistical analysis. | [
"INTRODUCTION",
"Patients",
"Evaluation of HGPs of primary and metastatic CRC",
"Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC",
"Immunohistochemistry",
"Whole exome sequencing",
"Statistical analysis",
"RESULTS",
"Clinical parameters",
"Pathological features",
"Value of selected pathological features in predicting the HGPs of CRCLMs",
"Whole-exome sequencing",
"DISCUSSION",
"ARTICLE HIGHLIGHTS",
"Research background",
"Research motivation",
"Research objective",
"Research methods",
"Research results",
"Research conclusion"
] | [
"Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.",
"With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.",
"For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).",
"The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].",
"All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.",
"Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).",
"Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.",
" Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.",
"The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).",
"Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.",
"EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.",
"Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.",
"Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.",
" Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.",
"Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.",
"Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis",
"To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.",
"A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.",
"In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).",
"The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern."
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"INTRODUCTION",
"MATERIALS AND METHODS",
"Patients",
"Evaluation of HGPs of primary and metastatic CRC",
"Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC",
"Immunohistochemistry",
"Whole exome sequencing",
"Statistical analysis",
"RESULTS",
"Clinical parameters",
"Pathological features",
"Value of selected pathological features in predicting the HGPs of CRCLMs",
"Whole-exome sequencing",
"DISCUSSION",
"ARTICLE HIGHLIGHTS",
"Research background",
"Research motivation",
"Research objective",
"Research methods",
"Research results",
"Research conclusion"
] | [
"Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].\nAccording to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.\nIt is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.",
" Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\nWith the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.\n Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\nFor each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).\n Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\nThe tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].\n Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\nAll 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.\n Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\nFormalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).\n Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.\nStatistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.",
"With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.",
"For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.\nThe HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).\nRepresentative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).",
"The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].",
"All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.\nAutomated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.\nThe IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.",
"Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.\nWhole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].\nWork flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.\nSequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).",
"Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.",
" Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\nThe clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).\n Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\nAmong 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.\n Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\nEGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.\n Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.\nHierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.",
"The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.\nMain clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study\nDGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.\nAssociation between clinicopathological characteristics and growth patterns\nSD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.\nThe mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).\nKaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).",
"Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)\nTBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).\nInterestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nSubtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.\nAs shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.\nRepresentative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.",
"EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.\nValue of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis\nHGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.\nIGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.",
"Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.\nWhole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.\nComparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.",
"Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.\nOur results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.\nDuring our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.\nUsing a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.\nThe prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.\nIt is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.\nAlthough the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.\nIn summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.",
" Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\nDifferent histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.\n Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\nThrough studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis\n Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\nTo understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.\n Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\nA total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.\n Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\nIn the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).\n Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\nThe primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.\n Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.\nMulticenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.",
"Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.",
"Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis",
"To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.",
"A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.",
"In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).",
"The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern."
] | [
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] | [
"Colorectal carcinoma",
"Liver metastasis",
"Histologic growth pattern",
"Clinicopathological characteristics",
"Whole exome sequencing"
] | INTRODUCTION:
Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].
According to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.
It is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.
MATERIALS AND METHODS:
Patients With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.
With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.
Evaluation of HGPs of primary and metastatic CRC For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.
The HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).
Representative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).
For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.
The HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).
Representative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).
Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].
The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].
Immunohistochemistry All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.
Automated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.
The IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.
All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.
Automated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.
The IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.
Whole exome sequencing Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.
Whole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].
Work flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.
Sequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).
Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.
Whole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].
Work flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.
Sequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).
Statistical analysis Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.
Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.
Patients:
With the approval of McGill University Health Center Research Ethics Board (No. 11-196-HGP), formalin-fixed, paraffin-embedded tissue blocks of matched primary CRC and liver metastases from 29 patients were obtained from the archives (2012-2017) of the Department of Pathology, McGill University Health Center. The primary cases were divided into two groups according to HGPs of matched CRCLMs: A (15 cases with matched CRCLMs showing desmoplastic growth pattern [DGP]) and B (14 cases with matched CRCLMs showing replacement growth pattern [RGP]). Tumor tissues of primary CRCs from five randomly selected cases in each group and their matched normal large intestinal mucosa were collected for whole exome sequencing. Clinical variables including age, gender, serum carcinoembryonic antigen (CEA) level, and survival data were obtained from patients’ health records. Pathological characteristics of the primary colorectal tumor including localization, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor budding score (TBS), tumor deposit, lymphovascular invasion, and perineural invasion were retrieved from patients’ pathology reports.
Evaluation of HGPs of primary and metastatic CRC:
For each case, two pathologists (Gao ZH and Wu JB ) reviewed at least three slides stained with hematoxylin and eosin (H&E) under a light microscope (BX45, Olympus, Tokyo, Japan). Discrepancies of HGPs were resolved by reevaluation and discussion in a multi-head microscope setting.
The HGPs of liver metastasis were evaluated according to the international consensus on the HGPs of liver metastasis[4]. In the DGP, the metastatic cancer cells are separated from the liver tissue by a band of desmoplastic tissue. In the RGP, cancer cells form cell plates that are continuous with the hepatocyte plates. The invasive front of the primary cancers were classified as either expanding growth pattern (EGP) or infiltrating growth pattern (IGP) based on the predominant morphology, as defined by Jass et al[10], where the expanding type had been described as the pushing growth type of adenocarcinoma, whereas the infiltrating type had been described as the wide spread streaming form of adenocarcinoma (Figure 1A-D).
Representative histological images of the growth pattern of primary colorectal carcinoma and liver metastasis. A: Primary colorectal carcinoma (CRC) with an expanding growth pattern where the tumor gland pushes the surrounding stroma with a sharper dividing interface (arrows show expanding growth pattern); B: Liver metastasis with a desmoplastic growth pattern showing a stroma band separating the tumor with the surrounding liver parenchyma (arrows show desmoplastic growth pattern), the same case as A; C: Primary CRC with an infiltrating growth pattern where streams of tumor cells permeate into the stroma with an irregular interface (arrows show infiltrating growth pattern); D: Liver metastasis with a replacement growth pattern showing tumors cells in direct contact with hepatocytes and grow into the liver cell plate (arrows show replacement growth pattern), the same case as C; E: Negative inflammatory cell band (ICB) negative. There is no lymphocyte infiltration at the tumor-normal tissue interface, as arrows show; F: Low grade ICB. Lymphocyte infiltration is present in less than 50% tumor-normal tissue interface, as arrows show; G: High grade ICB. Lymphocyte infiltration is present in more than 50% tumor-normal tissue interface, as arrows show; H: Positive Crohn’s disease-like response (CDR) showing lymphoid aggregates at the deeper aspects of the bowl wall (arrows show aggregated lymphoid tissue); I: Positive CDR showing lymphoid aggregates at the mucosal-submucosal junction (arrows show aggregated lymphoid tissue).
Evaluation of peri-tumoral inflammatory cell band (ICB) and Crohn’s disease-like response (CDR) in primary CRC:
The tumor-normal tissue interface was assessed for the degree of ICB and the presence or absence of CDR in H&E stained serial sections (Figure 1E-I). If ICB is present in less than 10% area of the tumor-normal tissue interface, the case was categorized as negative ICB; if between 10% and 50%, the case was categorized with low grade ICB; if ICB was observed in 50% or more of the tumor-normal tissue interface, the case was categorized with high grade ICB. CDR was defined as dense lymphoid aggregates, with or without germinal centers present at the mucosal-submucosal junction, or in the deeper aspects of the bowl wall, including the subserosal adipose tissue (“Crohn’s rosary”)[11].
Immunohistochemistry:
All 29 formalin-fixed, paraffin-embedded colorectal tumor tissues and one normal intestinal tissue were used for tissue microarray construction, as previously described by Zhang et al[12]. Three 1.5 mm representative punches were included from each tumor tissue.
Automated immunohistochemistry (IHC) was performed on 3-μm-thick TMA slides using a Ventana benchmark machine according to the manufacturer’s instructions (Ventana Medical Systems, Inc., Tucson, AZ, United States). The following commercially available antibodies were used in the IHC: MLH1 [clone ES05, 1:100, Dako (Glostrup, Denmark)], MSH2 (clone FE11, 1:100, Dako), MSH6 (clone EP49, 1:100, Dako), PMS2 (clone EP51, 1:100, Dako), programmed death receptor ligand-1 (PDL1) [clone ES05, SP263, VENTANA (Roche, United States)], and BRAFV600E [clone VE1, 1:100, Abcam (Cambridge, United Kingdom)]. On-slide positive and negative controls were used and the expected reaction was confirmed.
The IHC slides were evaluated independently by two gastrointestinal pathologists blindly using images of full slides obtained with a digital slide scanner (Aperio ScanScope AT Turbo, Leica microsystem, Concord, ON, Canada) and analyzed using the Aperio Image Analyzer. Deletion of MLH1, MSH2, MSH6, or PMS2 in cancerous tissues was defined as the absence of detectable nuclear staining of tumor cells. BRAF V600E staining was considered positive if the cytoplasmic staining was similar to the positive control in each batch. It was determined that any isolated nuclear staining was negative. In line with Zoroquiain et al[13], we defined PDL1 positivity by a threshold of 5% of tumor cells with strong cytoplasmic expression and membrane-accentuation or single membrane pattern. The expression of negative or equivocal protein expression detected in TMA was validated by IHC staining on the corresponding large tumor sections of the resection specimen using the same protocol.
Whole exome sequencing:
Formalin-fixed, paraffin-embedded tumor tissue blocks and matched normal blocks were microdissected to remove residual normal tissue and enhance neoplastic cellularity. The DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Netherlands) according to the manufacturer’s protocol. The DNA concentration was determined using a Qubit 2.0 Fluorimeter (Invitrogen, Carlsbad, CA, United States). After DNA extraction, we obtained 282 to 6480 ng of DNA per lesion from 10 CRCs and matched normal tissues.
Whole exome sequencing was performed at McGill University and Genome Quebec Innovation Center. The gDNA library was prepared using a TruSeq DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions, followed by sequencing on an Illumina HiSeq, as previously described[14]. As shown in Figure 2, the raw DNA sequences were aligned, trimmed, and duplicates flagged to the NCBI human genome build 38 version 93, using Isaac aligner (Version: Isaac-04.18.01.19)[15]. Structural variant (SV) analysis calls were generated using Manta (version manta-1.4.0)[16]. Small variants [single nucleotide variant (SNV) and small indels] in germline and somatic variations in tumor/normal sample pairs were achieved using Strelka (Version: strelka-2.9.0)[17]. Copy number variant (CNV) calls were generated using Canvas (Version: Canvas/1.11.0/) with the tumor-normal-enrichment workflow[18]. The callers share output, and therefore there is no double counting of calls. Manta and Strelka were run with Python (Version: 2.7.14). Annotation of the resulting calls was done with the Ensembl Variant Effect Predictor (VEP) using version 93.3 in a Perl (Version 5.22.4) environment[19]. Fastp (version 0.19.4) was used to collect QC metrics of the raw reads[20].
Work flow of whole exome sequencing bioinformatics analysis. SV: Structural variant; CNV: Copy number variant.
Sequencing result analyses and figures were prepared in the R statistical software suite (version 3.5.1) using standard R functions in custom code[21]. Circlize (version 0.4.4) was used to generate the Circos plots[22]. GGplot2 (version 3.0.0) was used to generate the heat maps. Custom R code was developed and run in RStudio (Version: 1.1.453).
Statistical analysis:
Statistical analyses were performed using Prism 7.00 statistical program (GraphPad, 2015, San Diego, CA, United States); P < 0.05 was considered statistically significant. The statistical review of the study was performed by a biomedical statistician. Comparison of clinicopathological features and immunohistochemical staining results between groups was performed using the t-test, Wilcoxon Signed rank test, and Fisher’s exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the prediction capacity of histologic features and genomic alternations of primary CRC for the growth patterns of liver metastasis.
RESULTS:
Clinical parameters The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.
Main clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study
DGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.
Association between clinicopathological characteristics and growth patterns
SD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.
The mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).
Kaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).
The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.
Main clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study
DGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.
Association between clinicopathological characteristics and growth patterns
SD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.
The mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).
Kaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).
Pathological features Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)
TBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).
Interestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.
Subtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.
As shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.
Representative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.
Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)
TBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).
Interestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.
Subtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.
As shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.
Representative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.
Value of selected pathological features in predicting the HGPs of CRCLMs EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.
Value of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis
HGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.
IGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.
EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.
Value of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis
HGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.
IGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.
Whole-exome sequencing Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.
Whole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.
Comparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.
Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.
Whole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.
Comparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.
Clinical parameters:
The clinical characteristics of the study population are shown in Table 1. The statistical comparison of the clinicopathological characteristics between Group A and Group B is shown in Table 2. Cases 1 to 15 belong to Group A, and cases 16 to 29 belong to Group B. With respect to age and gender, no significant differences were identified between Group A and Group B. The mean serum CEA level in Group B (46.9 μg/L) was noticeably higher than that in Group A (15.5 μg/L), although not statistically significant.
Main clinical demographics and histological growth patterns in patients with colorectal carcinoma and liver metastasis in the study
DGP: Desmoplastic growth pattern; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern; RGP: Replacement growth pattern; L: Low; M: Intermediate; H: High; NA: Not available.
Association between clinicopathological characteristics and growth patterns
SD: Standard deviation; CEA: Carcinoembryonic antigen; NA: Not available; EGP: Expanding growth pattern; IGP: Infiltrating growth pattern.
The mean length of follow-up for all patients was 36.8 mo (95%CI: 30.81-42.85 mo). Seventy-nine percent (23/29) patients were alive on completion of the study. There was no death in group A. Five cases were alive with liver, bone, or lung metastasis, the remaining 10 cases were alive without evidence of tumor recurrence or metastasis. In group B, however, 6 cases died of liver, lung, or brain metastasis and the remaining 8 cases had no evidence for tumor recurrence or metastasis. The length time for liver metastasis was calculated from the date of diagnosis of the primary CRC to the date of diagnosis of liver metastasis. The disease-free survival (DFS) was defined as the time from CRCLM resection to recurrence, or to the date of censoring in October, 2018, when the patient had no signs or symptoms of tumor recurrence or metastasis. Overall survival (OS) was calculated from the date of diagnosis of the primary carcinoma to the date of death, or to the date of censoring of live patients in October, 2018. With respect to the length time of liver metastasis and the DFS, there were no significant differences between Group A and Group B (Figure 3A and B, P > 0.05). However, OS was significantly longer in Group A than Group B (Figure 3C, P = 0.0337).
Kaplan-Meier curves for colorectal cancer liver metastasis between two groups. A: Comparison of length time of liver metastasis, showing no significant difference; B: Comparison of disease-free survival, showing no significant difference; C: Comparison of overall survival, showing a statistically significant difference (P = 0.0337).
Pathological features:
Among 29 cases, 37.9% (11/29) of the primary cancers were EGP and 62.1% (18/19) were IGP. Primary CRC with an EGP were more likely to have desmoplastic phenotype liver metastasis (81.8% vs 33.3%, P < 0.05). On the other hand, primary cancers with an IGP were significantly more likely to form replacement pattern liver metastasis (66.7% vs 18.2 %, P < 0.05)
TBS and tumor deposit were evaluated according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (Eight Edition). Most of TBS was low (66.7%, 10/15) in Group A, and intermediate or high in group B (71.4%, 10/14). CDR was identified in 46.7% (7/15) of cases in Group A, and only in 7.1% (1/14) in Group B. There was no significant difference between Group A and Group B regarding tumor location, size, gross configuration, histologic grade, tumor depth, lymph node metastasis, tumor deposit, lymphovascular invasion, perineural invasion, or peri-tumoral ICB. With respect to TBS and CDR, there was significant higher TBS in Group B than in Group A (P < 0.05) and significantly more CDR in Group A than in Group B (P < 0.05).
Interestingly, as shown in Figure 4, we observed that the EGP of primary cancer could be divided into four subtypes: (1) Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; (2) Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; (3) Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure, and there is a clear dividing line between the tumor and the surrounding stroma; and (4) Micropapillary expanding, where the tumor cells at the invasive front form micropapillary architecture, and there is a clear dividing line between the tumor and the surrounding stroma. On the other hand, infiltrating growth pattern could be divided into two subtypes: (1) Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; and (2) Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.
Subtypes of histologic growth pattern of primary colorectal carcinoma. A: Ordinary expanding, where the tumor gland pushes the stroma and forms a sharp dividing line at the interface; B: Mucinous expanding, where pools of tumor epithelium containing mucin pushes the stroma and forms a sharp dividing line at the interface; C: Cribriform expanding, where the tumor gland at the invasive front forms a round cribriform structure and there is a clear dividing line between the tumor and the surrounding stroma; D: Micropapillary expanding, where the tumor cells at the invasive front forms micropapillary architecture and there is a clear dividing line between the tumor and the surrounding stroma; E: Ordinary infiltrating, where tumor cells infiltrate to the stroma and connect to the original tumor mass; F: Skip infiltrating, where there is a gap (>0.5 cm) of non-neoplastic tissue between the main tumor bulk and the invasive front.
As shown in Figure 5, there was no significant differences between the two groups in the tumor cell expression of MLH1, MSH2, MSH6, PMS2, BRAFV600E, or PDL1. Interestingly, the three MSI deficiency cases were all MSH2 protein deficient.
Representative immunohistochemical images. A: Positive staining for MLH1; B: Positive staining for MSH2; C: Positive staining for MSH6; D: Positive staining for PMS2; E: Positive staining for MLH2; F: Negative staining for MSH2; G: Positive staining for MSH6; H: Positive staining for PMS2; I: Positive staining for BRAF V600E; J: Negative staining for BRAF V600E; K: Positive staining for PD-L1; L: Negative staining for PD-L1. A-D is the same case; E-H is the same case.
Value of selected pathological features in predicting the HGPs of CRCLMs:
EGP, low TBS score, and positive CDR in the primary tumor were shown to have predictive value for the DGPs of CRCLMs. As shown in Table 3, HGP + TBS + CDR is the most sensitive and has the highest negative predictive value, whereas CDR alone is the most specific and has the highest positive predictive value.
Value of selected pathological characteristics in predicting the histological growth patterns of colorectal carcinoma liver metastasis
HGP: Histological growth pattern; 95%CI: 95% confidence interval; SE: Sensitivity; SF: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; TBS: Tumor budding score; CDA: Crohn’s disease-like appearance.
IGP, high or intermediate TBS score, and negative CDR were shown to have predictive value for the RGP of CRCLMs. As shown in Table 3, CDR alone or combined with HGP and TBS are the most sensitive; TBS alone is the most specific; HGP or TBS has the highest PPV, whereas CDR has the highest NPV. When combining HGP, TBS, and CDR, although the sensitivity is 92.9%, the specificity is only 20.0%.
Whole-exome sequencing:
Hierarchical clustering of Groups A and B based on SNV and indels is shown in Figure 6A, and frequency of SNV or indels in Group A and Group B is shown in Figure 6B. There was no statistical difference between the two groups in SNV or indels. However, our whole-exome sequencing revealed 14 major gene mutations, as shown in Table 1 and Figure 7. The most prevalent major mutation occurred in the APC gene, which occurred in 80% (4/5) of Group A cases and 60% (3/5) cases of Group B, followed by TP53 and KRAS mutations. Both Group A and Group B had the same frequency of TP53 and DNAH5 mutations. Interestingly, PIK3CA mutations were showed in 40% (2/5) cases of Group A but no one case in Group B, similarly, the mutations of BRCA1, BRCA2, and BRAF were only observed in Group A, and the mutations of SMAD, ERBB2, ERBB3, LRP2, and SDK1 were identified only in Group B, each in one patient.
Whole exome sequencing analysis. A: Hierarchical clustering of Groups A and B based on single nucleotide variant (SNV) and indels, showing no significant difference between the two groups. B: Frequency of SNV or indels in Group A and Group B, showing no significant difference between the two groups. SNV: Single nucleotide variant.
Comparison of the major mutations between Group A and Group B. Fourteen major gene mutations were identified. PIK3CA, BRCA1, BRCA2, and BRAF mutations were only observed in Group A, and SMAD, ERBB2, ERBB3, LRP2, and SDK1 mutations were identified only in Group B.
DISCUSSION:
Colorectal adenocarcinoma liver metastasis has been categorized into three growth patterns: desmoplastic, pushing, and replacement, each of which has characteristic morphological features[4]. Using this classification, Van den Eynden et al[6] reported that the pushing pattern was an independent predictor of poor survival. In contrast, Nielsen et al showed that patients with an RGP had a death risk 2 to 2.5 times higher than patients with a pushing growth pattern or mixed growth pattern, and almost 4 times more than patients with a DGP[5]. According to Eefsen et al[3], similar findings in chemonaive patients and patients receiving neoadjuvant therapy were identified. Histologically differentiating DGP and invasive growth pattern were more clearly cut with almost no interobserver discrepancies and the clinical relevance was more obvious[4]. For these reasons, only those CRCLMs with well-defined desmoplastic or replacement growth patterns were enrolled in this study.
Our results demonstrated that expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating CRCs tended to develop replacement liver metastasis, which is consistent with previous findings of Rajaganeshan et al[8]. Our study also confirmed that patients with expanding primary cancers had a significant longer OS than those with infiltrating primary tumors, which is in line with the previously reported findings of Cianchi et al[23], Nystrom et al[24], and Pinheiro et al[25]. Therefore, the growth pattern of primary CRC could, to a certain extent, predict the growth pattern of CRCLMs and patients’ prognosis.
During our study, we identified four subtypes of primary expanding CRC and two subtypes of primary infiltrating cancers. Awareness of these morphological variations is critical in order to appropriately subclassify the primary CRCs. Zoning on the epithelial-stromal interface, we found that the infiltrating CRC had higher TBS, whereas the expanding cancer was more prone to form CDR. Our observation is in keeping with the previous report on the prognostic value of TBS and CDR of primary CRC[26-29]. To the best of our knowledge, this study is the first comprehensive morphological sub-classification of the growth patterns of primary CRCs in association with clinical follow-up data and corresponding HGPs of CRCLMs.
Using a standard formula, we analyzed the sensitivity, specificity, PPV, and NPV of selected pathological characteristics (HGP, TBS, and CDR) in predicting the HGPs of CRCLMs. Our results indicated that combined EGP, low TBS, and positive CDR of primary cancer could be used to predict the DGP of CRCLMs, whereas IGP alone of primary cancer could be the best to predict the RGP of CRCLMs. Once validated by larger set of cases, these parameters have the potential for clinical application.
The prognostic value of immunohistochemical markers such as microsatellite instability (MSI), BRAF V600E mutation, and PD-L1 expression has been reported[30-33]. However, there were no studies on the association between the expression of these IHC markers and the HGPs of CRC. Our study did not reveal any significant differences in the tumor cell expression of these IHC markers between the two groups.
It is reasonable to expect that the genomic makeup of the primary CRC plays an important role in determining the HGP of CRCLMs. However, to the best of our knowledge, there has been no report on the specific genomic drivers of the primary tumor that determine the specific growth pattern of liver metastasis. If the growth patterns of liver metastasis could be predicted based on the molecular biomarkers present in the primary CRC and each of the growth patterns could be associated with a different underlying biology, this could have important implications in the stratification of patients for the oncological treatment[34]. We compared the mutation rate of genes related to metastasis (WNA5A, TIMP1, MMP-1, MMP-2, COX-2, and HIF-1α), angiogenesis (VEGF, TGF, EGF, and TNF), epithelial-mesenchymal transition (E-cadherin, FGF, P63, and FOXC2), oncogenes (C-myc, K-ras, and Bcl-2), tumor suppressor genes (p53, APC, and NGX6), and other genes, such as Survivin and CIAPIN1, between the two groups[35-39]. The only gene that stands out is pho-sphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), which was present in 40% of tumors with desmoplastic group pattern in the CRCLMs. PIK3CA is an essential element of the signaling pathway of phosphatidylinositol-3 kinase (PIK3) downstream of EGFR. The PIK3CA mutation activates the PIK3 signaling pathway, improves cell proliferation, and ultimately leads to carcinogenesis[40]. It is associated with angiogenesis as it is essential in endothelial cell migration during vascular development through vascular endothelial growth factor-A (VEGFA) signaling, possibly by regulating RhoA activity[41]. The significantly higher expression of PIK13CA in primary CRC with desmoplastic liver metastasis indicates that metastasis can become vascularized through sprouting angiogenesis, in a process stimulated by VEGFA. In addition to PIK3CA, we also found that BRCA-1, BRCA-2, and BRAF genes were present only in primary CRC with desmoplastic liver metastasis, and SMAD, ERBB-2, ERBB-3, LRP2, and SDK1 genes were present only in CRCs with invasive liver metastasis. Once these findings are validated with a larger set of cases, these growth pattern-related genomic abnormalities could become new targets for precision therapy.
Although the existence of two invasive phenotypes (expanding or infiltrating) in CRC is now well recognized and their prognostic implications proven, the biological mechanisms for their existence remain unexplained. It is unclear why some tumors cause a desmoplastic and angiogenic response while others adopt the replacement growth pattern and acquire nutrition through vessel co-option. In addition to the biology of primary tumor cells, the surrounding liver microenvironment may also play a role in determining the growth pattern or dependence of angiogenesis. One possible explanation for the desmoplastic or replacement liver metastasis is that these HGPs summarize the different responses of the liver to injury[4]. Fibrosis in the desmoplastic liver metastasis may be mediated by the same biological mechanisms which drive liver fibrosis in response to an injury[42]. In addition, replacement HGP is similar to liver regeneration because cancer cells replace liver cells in the same way that new liver cells replace old liver cells during liver regeneration[43]. Another explanation is that the different tumor growth patterns are related to differential gene expression, which may be a driving factor for HGP. Sakariassen et al[44] investigated that vessel co-opting glioblastomas (GBMs) upregulated gene expression associated with fetal development and cell motility, whereas angiogenic GBMs had higher angiogenic regulatory factors, such as VEGF and angiopoetin-2 expression. In the present study, mutation of the PIK3CA gene was present only in the primary CRC with desmoplastic CRCLMs, which further validated its role as a marker for sprouting angiogenesis and a potential target for anti-angiogenic gene therapy.
In summary, primary CRCs with an EGP have a better OS than those with an IGP. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an EGP showed PIK3CA gene mutations in contrast to 0% of primary CRCs with an IGP. These genomic differences, if validated in a larger cohort of cases, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.
ARTICLE HIGHLIGHTS:
Research background Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.
Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.
Research motivation Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis
Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis
Research objective To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.
To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.
Research methods A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.
A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.
Research results In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).
In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).
Research conclusion The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.
The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.
Research perspectives Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.
Multicenter collaborative studies with a larger number of patients and prospective studies to assess the predictive value of the clinicopathological features of primary CRC on the HGPs of its liver metastasis could help to further validate our results. These genomic differences between the two groups of primary CRC, if validated in a larger cohort of case, have the potential to become not only clinically applicable diagnostic and prognostic biomarkers but also therapeutic targets of genomic engineering.
Research background:
Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy.
Research motivation:
Through studying the relationship between the different HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer, we aimed to evaluate whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis
Research objective:
To understand the biology of the primary CRCs in association with different HGPs of liver metastasis, and to identify histological and biology markers in the primary tumor that could predict the HGPs of liver metastasis.
Research methods:
A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases from each group were randomly selected for WES analysis.
Research results:
In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).
Research conclusion:
The primary CRCs with an expanding growth pattern have a better overall survival that those with an infiltrating growth pattern. Expanding CRCs tend to develop desmoplastic liver metastasis, whereas infiltrating cancers tend to develop replacement liver metastasis. Combined HGP, TBS, and CDR of primary CRC could be used to predict the HGPs of liver metastasis. Up to 40% of primary CRCs with an expanding growth pattern show PIK3CA gene mutations in contrast to 0% of primary CRCs with an invasive growth pattern.
| Background:
Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established.
Methods:
A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis.
Results:
In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).
Conclusions:
The HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis. | INTRODUCTION:
Colorectal carcinoma (CRC) is the third and second most commonly diagnosed cancer in men and women, respectively, with an estimated global incidence of 1.4 million new cases, and 693900 deaths in the world every year[1]. The prognosis after curative resection depends entirely on the development of metastasis, especially liver metastasis. At the time of diagnosis, 25% of patients with CRC had liver metastases (CRCLMs) and another 50% of patients without initial liver metastases had liver metastasis during follow-up[2]. Metastatic CRC is one of the leading causes of cancer-related deaths worldwide[3].
According to the international consensus guidelines for scoring the histological growth patterns (HGPs) of liver metastasis[4], CRCLMs present with distinct HGPs, including desmoplastic, pushing, replacement, and two rarer HGPs (sinusoidal and portal HGPs). The HGPs are defined based on the distinct interfaces between cancer cells and adjacent normal liver parenchyma. Importantly, the HGPs of liver metastases were shown to have prognostic and predictive value by some retrospective studies. Several studies showed that patients with desmoplastic liver metastasis had a longer recurrence free survival than patients with non-desmoplastic liver me-tastasis[5,6]. They attributed the poorer survival in patients with non-desmoplastic growth pattern to the higher recurrence rate. Frentzas et al[7] confirmed that CRC with replacement liver metastasis did not respond well to bevacizumab treatment, due to the fact that these tumors utilize vessel co-option instead of angiogenesis. On the other hand, desmoplastic liver metastasis, which relies on sprouting angiogenesis for its blood supply, showed a better response to bevacizumab. They proposed that the HGPs of CRCLMs could be used as a predictive biomarker for anti-angiogenic therapy.
It is natural to expect that the HGP and other clinicopathological characteristics of primary cancer may have biological, predictive, and important prognostic information. However, the relationship between HGPs of liver metastasis and clinicopathological characteristics of primary cancer have not been well established. In the study of Rajaganeshan et al[8], 69.2% of primary expanding CRCs developed capsulated liver metastases, whereas only 17.2% of infiltrating primary CRCs developed a capsulated phenotype (P < 0.001). Although almost all liver metastases from breast cancer adopt a replacement growth pattern, CRCLMs may present different HGPs[7,9]. Furthermore, the underlying genetic abnormalities and molecular pathways that drive the distinct HGPs remain unknown. In this study, we first analyzed the clinicopathological features of CRC patients with two distinct pattern of liver metastases. We then studied the genomic differences of primary CRCs between these two groups using whole exome sequencing analysis. Our study will provide new insights into the primary tumor in terms of their value in predicting the growth pattern of liver metastasis, response to antiangiogenic therapy, and patients’ prognosis.
Research conclusion:
We thank Dr. Logan Walsh at Department of Human Genetics, Rosalind and Morris Goodman Cancer Research Center, McGill University for his assistance in analyzing the whole exome sequencing data, and associate professor Jianfeng Luo at Department of Biostatistics, School of Public Health, Fudan University for the help of biostatistical analysis. | Background:
Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established.
Methods:
A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis.
Results:
In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5).
Conclusions:
The HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis. | 14,357 | 335 | [
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"tumor",
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] | [
"cancer liver metastasis",
"liver metastasis statistical",
"liver metastases crclms",
"colorectal adenocarcinoma liver",
"colorectal carcinoma crc"
] | null | [CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY] | [CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY] | null | [CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY] | [CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY] | [CONTENT] Colorectal carcinoma | Liver metastasis | Histologic growth pattern | Clinicopathological characteristics | Whole exome sequencing [SUMMARY] | [CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY] | [CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY] | null | [CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY] | [CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY] | [CONTENT] Adenocarcinoma | Adult | Aged | Aged, 80 and over | B7-H1 Antigen | Class I Phosphatidylinositol 3-Kinases | Colon | Colorectal Neoplasms | DNA Mismatch Repair | Female | Humans | Kaplan-Meier Estimate | Liver | Liver Neoplasms | Male | Middle Aged | Mutation | Predictive Value of Tests | Prognosis | Proto-Oncogene Proteins B-raf | Rectum | Retrospective Studies | Exome Sequencing [SUMMARY] | [CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY] | [CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY] | null | [CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY] | [CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY] | [CONTENT] cancer liver metastasis | liver metastasis statistical | liver metastases crclms | colorectal adenocarcinoma liver | colorectal carcinoma crc [SUMMARY] | [CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY] | [CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY] | null | [CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY] | [CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY] | [CONTENT] tumor | liver | metastasis | group | primary | growth | liver metastasis | pattern | growth pattern | cases [SUMMARY] | [CONTENT] liver | liver metastases | metastases | hgps | distinct | patients | metastasis | cancer | liver metastasis | crclms [SUMMARY] | [CONTENT] version | tissue | tumor | arrows | normal | icb | dna | growth | pattern | tumor normal [SUMMARY] | null | [CONTENT] crcs | growth pattern | primary crcs | crcs expanding growth pattern | primary crcs expanding growth | crcs expanding growth | crcs expanding | primary crcs expanding | pattern | growth [SUMMARY] | [CONTENT] liver | metastasis | liver metastasis | primary | tumor | group | growth | pattern | growth pattern | hgps [SUMMARY] | [CONTENT] liver | metastasis | liver metastasis | primary | tumor | group | growth | pattern | growth pattern | hgps [SUMMARY] | [CONTENT] CRC ||| [SUMMARY] | [CONTENT] 29 | CRC | two | 15 | 14 ||| ||| ||| Five | WES [SUMMARY] | null | [CONTENT] CDR | CRC [SUMMARY] | [CONTENT] CRC ||| ||| 29 | CRC | two | 15 | 14 ||| ||| ||| Five | WES ||| ||| Crohn | CDR ||| WES | APC | 4/5 | 3/5 | KRAS | PIK3CA | 2/5 | BRCA2 | BRAF | DNAH5 (1/5 | APC | 3/5 | KRAS | DNH5 | SMAD | ERBB2 | ERBB3 | 1/5 ||| CDR | CRC [SUMMARY] | [CONTENT] CRC ||| ||| 29 | CRC | two | 15 | 14 ||| ||| ||| Five | WES ||| ||| Crohn | CDR ||| WES | APC | 4/5 | 3/5 | KRAS | PIK3CA | 2/5 | BRCA2 | BRAF | DNAH5 (1/5 | APC | 3/5 | KRAS | DNH5 | SMAD | ERBB2 | ERBB3 | 1/5 ||| CDR | CRC [SUMMARY] |
|||||
Eradication rate and safety of a "simplified rescue therapy": 14-day vonoprazan and amoxicillin dual regimen as rescue therapy on treatment of Helicobacter pylori infection previously failed in eradication: A real-world, retrospective clinical study in China. | 35877765 | The currently recommended quadruple regimens as rescue therapy on Helicobacter pylori infection were not as effective as being supposed, especially in those who had failed two or more times. Dual regimen composed of vonoprazan (a potassium-competitive acid blocker) and amoxicillin might be an option since it's effective in eradication therapy as first-line treatment. | BACKGROUND | From May 2020 to June 2021, the clinical data of patients who had failed in Helicobacter pylori infection treatment were collected in GI department of Peking University First Hospital, Beijing, China. Patients were given vonoprazan 20 mg or 40 mg per day and amoxicillin 3000 mg per day (VA dual therapy) for 14 days as rescue treatment. Helicobacter pylori status was evaluated by 13 C-urease breath test 6 weeks after treatment. All adverse effects during treatment were recorded. | METHODS | A total of 186 patients were enrolled, including 67 males and 119 females. All of them had failed for 1 ~ 7 times in their previous treatment. Successful eradication was achieved in 172 patients (92.5%, 172/186). The adverse effects (referring to skin rash, abdominal pain, diarrhea, and headache), mainly mild and did not cause quit of treatment, occurred in 14 patients (7.5%, 14/186) and all symptoms relieved spontaneously. | RESULTS | Dual regimen composed of vonoprazan and amoxicillin for 14 days was effective and safe as rescue therapy in Helicobacter pylori infection treatment. It could be chosen as a "simplified rescue therapy" with relatively high eradication rate no matter how many times the patients had failed and what regimens they had used previously. | CONCLUSIONS | [
"Amoxicillin",
"Anti-Bacterial Agents",
"Clarithromycin",
"Drug Therapy, Combination",
"Female",
"Helicobacter Infections",
"Helicobacter pylori",
"Humans",
"Male",
"Potassium",
"Proton Pump Inhibitors",
"Pyrroles",
"Retrospective Studies",
"Sulfonamides",
"Treatment Outcome",
"Urease"
] | 9542484 | INTRODUCTION |
Helicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.
1
,
2
The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.
Different from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.
3
,
4
In recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.
5
,
6
,
7
It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,
8
which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment. | null | null | RESULTS | Patients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.
The demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.
A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.
The demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.
Eradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).
In 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).
According to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).
In 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4
).
All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).
In 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).
According to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).
In 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4
).
MIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).
According to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.
Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).
According to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.
Compliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.
Totally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.
Adverse events happened
Note: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.
Abbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).
Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.
Totally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.
Adverse events happened
Note: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.
Abbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg). | CONCLUSION | The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before. | [
"INTRODUCTION",
"Study design and participants",
"Diagnosis of H. pylori infection and treatment regimen",
"Antibiotic susceptibility test",
"Statistical analysis",
"Patients enrolled and baseline characteristics",
"Eradication of H. pylori infection",
"\nMIC to antibiotics of isolated H. pylori strains",
"Compliance and adverse events"
] | [
"\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.",
"A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.",
"\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.",
"Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n",
"Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.",
"A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.",
"All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).",
"Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.",
"Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg)."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Study design and participants",
"Diagnosis of H. pylori infection and treatment regimen",
"Antibiotic susceptibility test",
"Statistical analysis",
"RESULTS",
"Patients enrolled and baseline characteristics",
"Eradication of H. pylori infection",
"\nMIC to antibiotics of isolated H. pylori strains",
"Compliance and adverse events",
"DISCUSSION",
"CONCLUSION",
"CONFLICT OF INTEREST"
] | [
"\nHelicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.\n1\n, \n2\n The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.\nDifferent from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.\n3\n, \n4\nIn recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.\n5\n, \n6\n, \n7\n It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,\n8\n which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.",
"Study design and participants A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\nA real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.\nDiagnosis of H. pylori infection and treatment regimen \nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\n\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.\nAntibiotic susceptibility test Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\nSome of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n\nStatistical analysis Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.\nData collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.",
"A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.\nDemographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy\n\nNote: Data are n (%), or mean (SD, standard deviation).\nAbbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.\n*Compliance, taken >80% of tablets.",
"\nH. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.\nVonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.",
"Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.\nResistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.\n9\n, \n10\n\n",
"Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.",
"Patients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nA total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.\nEradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\nAll the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).\n\nMIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nTwenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.\nCompliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).\nOf all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).",
"A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.\nThe demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.",
"All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).\nIn 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).\nAccording to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).\nIn 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4\n).",
"Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).\nAccording to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.",
"Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.\nTotally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.\nAdverse events happened\nNote: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.\nAbbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).",
"In our study, VA dual regimen was designed as rescue treatment used in patients who failed one or more times before, no matter what regimen they had used, including those who had used PPI + amoxicillin dual therapy. The overall eradication rate was 92.5% (95% CI 87.4%–95.7%) with minimal side effects (7.5%, 95% CI 4.3%–12.6%).\nCauses of treatment failure of anti‐H. pylori therapy include antibiotics resistance, poor compliance of patients, low gastric pH, and high bacterial load.\n11\n, \n12\n The prevalence of multidrug‐resistant H. pylori strains is increasing, especially in cases with multiple eradication failure, which makes rescue treatment difficult.\n13\n, \n14\n However, since the resistance rate to amoxicillin is low even after the failure of eradication, amoxicillin can be a candidate of antimicrobial agent for the rescue therapy.\n8\n, \n15\n, \n16\n, \n17\n\n\nBeyond the traditional quadruple therapy, dual therapy, which was composed of PPI + amoxicillin, was testified to be an effective regimen used in treatment of H. pylori infection in recent years.\n5\n, \n16\n, \n18\nAfter the first report in 1989, the efficacy of AMX‐contained dual therapy was unstable and being abandoned for many years.\n19\n, \n20\nWhile in recent 10 years, there were more and more studies showing that it could be pretty effective.\n21\n It was believed that there were two critical variables/factors that affected the efficacy of the treatment.\n21\n One was to achieve and maintain a relatively high intragastric pH value, in which the antibactericidal effect of amoxicillin would be stable to get a better bioavailability in gastric cavity. The second was the concentration of amoxicillin in stomach.\n21\n Amoxicillin is a time‐dependent antibiotic, which is rapidly absorbed into plasma and then to be excreted in 6 ~ 8 h after administration. Comparing with 1000 mg twice daily, a dosage of 500 ~ 750 mg per 6 h might be more likely to maintain a higher plasma concentration.\n6\n, \n22\n\n\nAccording to the choice of acid inhibitors, since the intragastric pH value might vary according to the potency of different PPIs and ethnic difference in PPI metabolism (cytochrome P450 [CYP2C19] pharmacogenetic polymorphism)\n8\n, vonoprazan was chosen as part of the combination. Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker, which could provide fast and powerful acid inhibition, suggesting it might be possible to sustain a higher intragastric pH value. It was observed that a pH >4.0 status could be obtained at 4 h and to sustain for 24 h after the first administration of VPZ.\n13\n, \n23\n The effectiveness of VPZ and amoxicillin dual therapy used as first‐line treatment was pretty good with a eradication rates varied as 85%–90% in Japan, while there was little data on its effect on rescue treatment.\n3\n, \n13\n In our study, the dual therapy was composed of vonoprazan 20/40 mg per day (10/20 mg b.i.d) and amoxicillin 3000 mg per day (1000 mg t.i.d or 750 mg q.i.d). It was used as rescue therapy for patients who failed in their previous treatment, no matter how many times they had failed and what eradication regimens they had used before.\nIn this study, all patients had not used VA dual therapy, while some of them had used PPI + AMX dual therapy before (3/186). Altogether 186 subjects were enrolled, the eradication rate was 92.5% (172/186). Fourteen patients failed in VA dual therapy. We tried to identify factors that might be potential risk factors of eradication failure, such as gender, BMI, smoking, alcohol drinking, previous use of amoxicillin, or previous treatment failure times. It seemed that the previous use of amoxicillin and smoking might influence the eradication efficacy, but not statistically (Table 1). A recent meta‐analysis showed that although smoking increases the failure rate of H. pylori eradication treatment, however, when vonoprazan is used to treat the H. pylori infection, smoking has no effect on the eradication rate.\n24\n Although it inclined the more times the patients had failed, the less possibility they succeeded in VA dual therapy, previous failure times did not influence the efficacy of rescue treatment statistically (Figure 1).\nThe eradication rate of VA dual treatment according to different failure times previously. All patients had endured previous treatment failure. Most of them had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). Forty‐eight patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). The overall eradication rate of VA dual rescue therapy was 92.5% (172/186). The eradication rate was 92.9% (78 of 84) in patients who had failed one time, 96.3% (52 of 54) in patients who had failed two times, and 87.5% (42 of 48) in patients who had failed three or more times previously\nPrevalence of antimicrobial resistance of 25 cases who had been performed MIC tests. 5 cases had accepted the MIC test, most of H. pylori strains isolated from them were resistant to CLA (92%, 23 of 25), MTZ (96%, 24 of 25), LEV (88%, 22 of 25), and MOX (88%, 22 of 25). Three of 25 cases were resistant to AMX (12%) and none of them resistant to TET. Resistance to antibiotics was defined as: AMX resistant at MIC >0.125 mg/L; TET resistant at MIC >1 mg/L; CLA resistant at MIC >0.5 mg/L; MTZ resistant at MIC >8 mg/L; LEV resistant at MIC>1 mg/L; MOX resistant at MIC >1 mg/L. CLA: clarithromycin; MTZ: metronidazole; LEV: levofloxacin AMX: amoxicillin; TET: tetracycline; MOX: moxifloxacinMIC: Minimum Inhibitory Concentration\nEradication rates of patients who had or had not used amoxicillin in their previous treatment. In 186 patients, 83.3% of them (n = 155) had accepted previously treatments in which amoxicillin was included, while 16.7% of them (n = 31) did not get a regimen contained amoxicillin before. In all 186 patients, the overall eradication rate was 92.5%(172 of 186). In patients who had not used amoxicillin in their previous treatment, all of them got a successful eradication (100%, 31 of 31). While 91.0% (141 of 155) of patients who had used amoxicillin before eliminated the bacteria\nEradication rates of total 186 patients and patients who had accepted regimens with 10 mg bid or 20 mg b.i.d vonoprazan. In 186 patients, 23.1% of them (n = 43) had accepted 20 mg/d vonoprazan while 76.9% (n = 143) with 40 mg/d vonoprazan in their rescue treatment. In all 186 patients, the overall eradication rate was 92.5% (172 of 186). The eradication rates of different dose of vonoprazan were 95.3% (41 of 43) in 10 mg b.i.d and 91.6% (131 of 143) in 20 mg b.i.d, respectively\nIn 25 cases who got an MIC test during treatment, three cases were resistant to AMX with an MIC of .25 mg/L (resistance defined as a MIC >0.125 mg/L). Among them, one case failed in the VA dual treatment while two of them succeeded in eradication. All 14 who failed had used amoxicillin in their previous treatment, three of them got MIC test with results of .125 mg/L, .125 mg/L and .25 mg/L to AMX, with one of three was defined as resistant to AMX theoretically. Although we did not know the detail about it, it seemed that resistance to AMX was not absolute contraindication in VA dual therapy. There was an inclination that VA dual therapy might be more effective in patients who had not used amoxicillin before.\nTo our knowledge, this is the first real‐world study to reveal the efficacy of vonoprazan and amoxicillin dual regimen (VA dual regimen) as rescue treatment in China. In previous studies, dual therapy with VPZ was mostly used as first‐line treatment, and the eradication rates varied as 85% ~ 90% in Japan.\n13\n, \n25\n In clinical practices from Japan, VPZ was mostly used as component of triple therapy in first‐line (VPZ + amoxicillin + clarithromycin), second‐line (VPZ + amoxicillin + metronidazole), and third‐line (VPZ + amoxicillin + sitafloxacin) treatment of H. pylori infection. VPZ‐contained triple regimens had a relatively high eradication rate as 88.1%, 80.1%, and 75.8%, respectively.\n26\n Although vonoprazan appeared to restore the effectiveness of triple therapy, the improvement was almost entirely to improved effectiveness of amoxicillin dual therapy componentand resulted in the majority (>85% currently in Japan) of those receiving vonoprazan + amoxicillin plus a second antibiotic (e.g., clarithromycin, metronidazole, fluoroquinolone, or rifabutin) receiving no benefit from the second antibiotic.\n27\n It seemed that the only contribution of the second antibiotic is to increase global antimicrobial resistance.\n27\n, \n28\n, \n29\n\n\nFrom the data of our study, it was supposed that the VA dual therapy was effective as rescue treatment no matter what regimen had been given and which antibiotics had been used before. The VA dual therapy could be defined as a “simplified rescue therapy.”\nDuring the treatment course of the VA dual therapy, the adverse events happened were mild and did not cause quitting or failure of treatment. The administration mode was suitable since the compliance of patients was pretty good.\nThere were many limitations in our study. As a real‐world retrospective study, it's not a randomized controlled trial. The regimens were not consistent, whereas components of dual therapy were the same as vonoprazan + amoxicillin, the administration frequency or doses in VA dual therapy varied. The frequency of amoxicillin was given either t.i.d or q.i.d casually more than well‐designed, although the total dose was the same as 3000 mg per day. The total doses of vonoprazan also varied (20mg or 40 mg per day) with the body weight of patient. As to the MIC analysis, there was only 25 cases who got a successful H. pylori culture and antibacterial susceptibility test, depending on the patients' willingness. The MIC data were limited and not very representative. Based on the results of this retrospective study, a well‐designed random controlled prospective clinical trial could be anticipated being performed in the future.",
"The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before.",
"The authors have no competing interests."
] | [
null,
"materials-and-methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusions",
"COI-statement"
] | [
"amoxicillin",
"dual therapy",
"\nHelicobacter pylori infection",
"simplified rescue therapy",
"vonoprazan"
] | INTRODUCTION:
Helicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.
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The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.
Different from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.
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In recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.
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It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,
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which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.
MATERIALS AND METHODS:
Study design and participants A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.
Demographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy
Note: Data are n (%), or mean (SD, standard deviation).
Abbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.
*Compliance, taken >80% of tablets.
A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.
Demographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy
Note: Data are n (%), or mean (SD, standard deviation).
Abbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.
*Compliance, taken >80% of tablets.
Diagnosis of H. pylori infection and treatment regimen
H. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.
Vonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.
H. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.
Vonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.
Antibiotic susceptibility test Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.
Resistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.
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Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.
Resistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.
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Statistical analysis Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.
Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.
Study design and participants:
A real‐world, retrospective study was conducted in the Department of Gastroenterology in Peking University First Hospital, Beijing, China. Data were collected from May 2020 to June 2021. All patients who had accepted VA dual therapy as their rescue treatment were involved. The general data are shown in Table 1.The primary endpoint was the eradication rate, the secondary endpoint was the prevalence of adverse events, compliance, and related factors which might affect the cure efficacy of treatment.
Demographic and clinical data of all patients and patients who succeeded and failed in VA dual rescue therapy
Note: Data are n (%), or mean (SD, standard deviation).
Abbreviations: A, Amoxicillin; BMI, body mass index; V, Vonoprazan; VA, vonoprazan + amoxicillin dual therapy.
*Compliance, taken >80% of tablets.
Diagnosis of H. pylori infection and treatment regimen:
H. pylori infection was diagnosed as positive in 13C‐urease breath test (13C‐UBT)(75 mg 13C‐urea, Shenzhen Zhonghe Headway Bio‐Sci & Tech Co., Ltd.). As to the outcome of treatment, H. pylori status was determined by 13C‐UBT at least 6 weeks after completion of therapy.
Vonoprazan (VPZ, 20 mg/tablet, Takeda Pharmaceutical Co.) plus amoxicillin (250 mg/capsule, the United Laboratories International Holdings Limited) dual therapy (VA dual therapy) consisted of VPZ 10 mg twice daily (10 mg = half tablet with 20 mg/tablet, body weight ≤ 55 kg) or 20 mg twice daily (body weight > 55 kg) and amoxicillin 3000 mg per day (mostly 1000 mg t.i.d,750 mg q.i.d in few patients. The frequency of amoxicillin administration was given casually as t.i.d or q.i.d more than well‐designed). The treatment course was 14 days. VPZ was suggested to be taken half an hour before breakfast and dinner. Amoxicillin was suggested to be taken just after breakfast, lunch, and dinner and before sleep if q.i.d.
Antibiotic susceptibility test:
Some of the patients had got bacteria culture and antibiotic susceptibility test. Two biopsies were collected from the gastric antrum and corpus to culture H. pylori strains before treatment. When a positive culture was obtained, antibiotics’ susceptibility to amoxicillin (AMX), clarithromycin (CLA), metronidazole (MTZ), levofloxacin (LEV), moxifloxacin (MOX), and tetracycline (TET) was tested using Epsilometer test (E‐test) strips (BioMerienx, France) on Columbia blood agar plates containing 8% fresh defibrinated sheep blood. After 72 h of incubation under microaerobic atmosphere, the minimum inhibitory concentration (MIC) of each antibiotic was determined.
Resistance to AMX, CLA, MTZ, LEV, and TET was defined as MIC >0.125 mg/L, MIC >0.5 mg/L, MIC >8 mg/L, MIC >1 mg/L, and MIC >1 mg/L, respectively, according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for H. pylori (EUCAST, Breakpoint tables for interpretation of MICs and zone diameters, version 10.0, 2020, http://www.eucast.org/clinical_breakpoints/). Resistance to MOX was defined as MIC >1 mg/L according to literature reports.
9
,
10
Statistical analysis:
Data collected were analyzed using IBM SPSS Statistics SPSS 20.0 software (IBM Corp.,). Continuous variables were expressed as the mean ± standard deviation, and categorical variables were expressed as numbers and percentages. The significance of the p‐value was defined as less than .05 in the statistical analyses.
RESULTS:
Patients enrolled and baseline characteristics A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.
The demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.
A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.
The demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.
Eradication of H. pylori infection All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).
In 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).
According to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).
In 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4
).
All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).
In 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).
According to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).
In 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4
).
MIC to antibiotics of isolated H. pylori strains Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).
According to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.
Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).
According to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.
Compliance and adverse events Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.
Totally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.
Adverse events happened
Note: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.
Abbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).
Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.
Totally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.
Adverse events happened
Note: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.
Abbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).
Patients enrolled and baseline characteristics:
A total of 186 patients who accepted VA dual therapy as rescue treatment were enrolled, including 67 males and 119 females. All patients had failed in their previous treatment at least one time (average 2.1 times, range 1 ~ 7 times). Among them, most had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). 48 patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). Most of their previous treatment regimens were bismuth‐based quadruple regimens. The antibiotics mostly used were amoxicillin, clarithromycin, metronidazole, levofloxacin, or moxifloxacin, tetracycline and furazolidone were also used in some cases.
The demographic and clinical data of the patients are shown in Table 1. Thirty three of them had family history of gastric cancer. All patients were not allergic to penicillin, as they had taken amoxicillin before without side effects or proven to be safe with a negative penicillin allergy test. Although 134 patients (134/186, 72.0%) had one or more combined diseases, most of them (120/186, 64.5%) had no combined medicine during treatment. The most often combined diseases were hypertension, hyperlipidemia, and diabetes mellitus.
Eradication of H. pylori infection:
All the186 cases enrolled had completed the treatment. A total of 172 patients (172/186, 92.5%, 95% CI 87.4% to 95.7%) got successful eradication.Fourteen patients (14/186, 7.5%) failed in their VA dual therapy, all failed had used amoxicillin in their previous H. pylori treatment. According to demographic and clinical data of the patients, there were no significant risk factors of eradication failure including gender, BMI, smoking, or alcohol drinking status, family history of gastric cancer, endoscopy diagnosis, and previous treatment times (Table 1).
In 186 patients, 83.3% of them (n = 155) had used amoxicillin in their previous treatment, while 16.7% of them (n = 31) did not used it. All the patients who had not used amoxicillin before got a successful eradication(100%, 31/31), while only 91.0% (141/155) of the patients who had used amoxicillin before eliminated the bacteria (Figure 3). There was no statistical difference in eradication rate between the two groups (p = 0.08 by Chi‐square test and p = 0.132 by Fisher's analysis).
According to different treatment times endured before, there was little difference in eradication rate. In 186 cases involved, most of them had accepted treatment for one time (n = 85, 45.7%) or two times (n = 52, 27.9%) previously. Despite different treatment times before, the overall eradication rate was 92.5% (95% CI 87.4%–95.7%, 172/186). The eradication rate was 92.9% (95% CI 84.5%–97.1%, 78/84) in patients who had failed one time, 96.3% (95% CI 86.2%–99.4%, 52/54) in patients who had failed two times and 87.5%(95% CI 74.1%–94.9%, 42/48) in patients who had failed three or more times previously. There was no statistical difference in eradication rate in different treatment time groups (p = 0.403, two‐tailed significant tests).
In 186 patients, 23.1% of them (n = 43) had accepted vonoprazan 20 mg per day (10 mg b.i.d) with body weight ≤ 55 kg while 76.9% (n = 143) had accepted vonoprazan 40 mg per day (20 mg b.i.d) with body weight > 55 kg in their rescue treatment. The eradication rates of different dose of vonoprazan groups were 95.3% (95% CI 83.0%–99.2%, 41/43) in 10 mg b.i.d group and 91.6% (95% CI 85.5%–95.4%, 131/143) in 20 mg b.i.d group, respectively. There was no statistically difference between them (Figure 4
).
MIC to antibiotics of isolated H. pylori strains:
Twenty‐five cases had got the MIC test, most of H. pylori strains isolated from them were resistant to CLA, MTZ, LEV, and MOX. Resistance to AMX and TET was rare (Figure 2).
According to MIC of AMX in 25 cases, 88.0% (22 of 25) of them were susceptible to amoxicillin, while 44.0% (11 of 25) of them were super‐susceptible (MIC≤0.023 mg/L), 44.0% (11 of 25) of them were susceptible (0.023 mg/L < MIC≤0.125 mg/L), 12.0% (3 of 25) of them were resistant to amoxicillin with an MIC more than 0.125 mg/L. In three patients whose H. pylori strains were resistant to AMX, one failed and two succeeded in VA dual therapy.
Compliance and adverse events:
Of all 186 patients, 185 of them (99.5%) had good compliance (taken >80% of all tablets) (Table 1). All patient who got adverse events during their treatment had completed the whole course. The patient who quitted the treatment had failed one time in his previous treatment. The reason of quitting was that he had forgotten to take the drugs on time, not for the adverse events. He did not get the MIC test. Later he got a successful eradication in his third treatment with vonoprazan + bismuth + tetracycline +furazolidone quadruple therapy.
Totally 14 patients (7.5%, 95% CI 4.3%–12.6%) endured the adverse events (Table 2). The most happened adverse events were diarrhea (three of 14) and nausea (three of 14). All adverse events were mild and did not influence the completion of therapy. In all patients who suffered adverse events during treatment, only one patient (one of 14) with diarrhea failed in her eradication. Most adverse events were mild and reversible. All adverse events were spontaneously cured without intervention except one patient who had a successful treatment suffered mild skin rash occured 2 days after the end of the treatment and recovered after anti‐allergy treatment.
Adverse events happened
Note: Adverse events happened in 14 patients (7.5%), which were mild and did not affect the continuation of therapy.
Abbreviations: BMI, body mass index (kg/m2); Doses of VPZ, total doses of vonoprazan (mg).
DISCUSSION:
In our study, VA dual regimen was designed as rescue treatment used in patients who failed one or more times before, no matter what regimen they had used, including those who had used PPI + amoxicillin dual therapy. The overall eradication rate was 92.5% (95% CI 87.4%–95.7%) with minimal side effects (7.5%, 95% CI 4.3%–12.6%).
Causes of treatment failure of anti‐H. pylori therapy include antibiotics resistance, poor compliance of patients, low gastric pH, and high bacterial load.
11
,
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The prevalence of multidrug‐resistant H. pylori strains is increasing, especially in cases with multiple eradication failure, which makes rescue treatment difficult.
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However, since the resistance rate to amoxicillin is low even after the failure of eradication, amoxicillin can be a candidate of antimicrobial agent for the rescue therapy.
8
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15
,
16
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Beyond the traditional quadruple therapy, dual therapy, which was composed of PPI + amoxicillin, was testified to be an effective regimen used in treatment of H. pylori infection in recent years.
5
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16
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After the first report in 1989, the efficacy of AMX‐contained dual therapy was unstable and being abandoned for many years.
19
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While in recent 10 years, there were more and more studies showing that it could be pretty effective.
21
It was believed that there were two critical variables/factors that affected the efficacy of the treatment.
21
One was to achieve and maintain a relatively high intragastric pH value, in which the antibactericidal effect of amoxicillin would be stable to get a better bioavailability in gastric cavity. The second was the concentration of amoxicillin in stomach.
21
Amoxicillin is a time‐dependent antibiotic, which is rapidly absorbed into plasma and then to be excreted in 6 ~ 8 h after administration. Comparing with 1000 mg twice daily, a dosage of 500 ~ 750 mg per 6 h might be more likely to maintain a higher plasma concentration.
6
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22
According to the choice of acid inhibitors, since the intragastric pH value might vary according to the potency of different PPIs and ethnic difference in PPI metabolism (cytochrome P450 [CYP2C19] pharmacogenetic polymorphism)
8
, vonoprazan was chosen as part of the combination. Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker, which could provide fast and powerful acid inhibition, suggesting it might be possible to sustain a higher intragastric pH value. It was observed that a pH >4.0 status could be obtained at 4 h and to sustain for 24 h after the first administration of VPZ.
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The effectiveness of VPZ and amoxicillin dual therapy used as first‐line treatment was pretty good with a eradication rates varied as 85%–90% in Japan, while there was little data on its effect on rescue treatment.
3
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In our study, the dual therapy was composed of vonoprazan 20/40 mg per day (10/20 mg b.i.d) and amoxicillin 3000 mg per day (1000 mg t.i.d or 750 mg q.i.d). It was used as rescue therapy for patients who failed in their previous treatment, no matter how many times they had failed and what eradication regimens they had used before.
In this study, all patients had not used VA dual therapy, while some of them had used PPI + AMX dual therapy before (3/186). Altogether 186 subjects were enrolled, the eradication rate was 92.5% (172/186). Fourteen patients failed in VA dual therapy. We tried to identify factors that might be potential risk factors of eradication failure, such as gender, BMI, smoking, alcohol drinking, previous use of amoxicillin, or previous treatment failure times. It seemed that the previous use of amoxicillin and smoking might influence the eradication efficacy, but not statistically (Table 1). A recent meta‐analysis showed that although smoking increases the failure rate of H. pylori eradication treatment, however, when vonoprazan is used to treat the H. pylori infection, smoking has no effect on the eradication rate.
24
Although it inclined the more times the patients had failed, the less possibility they succeeded in VA dual therapy, previous failure times did not influence the efficacy of rescue treatment statistically (Figure 1).
The eradication rate of VA dual treatment according to different failure times previously. All patients had endured previous treatment failure. Most of them had accepted treatment for one time (n = 84, 45.7%) or two times (n = 54, 27.9%). Forty‐eight patients (26.3%) failed for three or more times: 26 cases failed three times (14.0%), 13 cases failed four times (7.0%), 7 cases failed five times (3.8%), 1 case failed six times (0.5%), 2 cases failed seven times (1.0%). The overall eradication rate of VA dual rescue therapy was 92.5% (172/186). The eradication rate was 92.9% (78 of 84) in patients who had failed one time, 96.3% (52 of 54) in patients who had failed two times, and 87.5% (42 of 48) in patients who had failed three or more times previously
Prevalence of antimicrobial resistance of 25 cases who had been performed MIC tests. 5 cases had accepted the MIC test, most of H. pylori strains isolated from them were resistant to CLA (92%, 23 of 25), MTZ (96%, 24 of 25), LEV (88%, 22 of 25), and MOX (88%, 22 of 25). Three of 25 cases were resistant to AMX (12%) and none of them resistant to TET. Resistance to antibiotics was defined as: AMX resistant at MIC >0.125 mg/L; TET resistant at MIC >1 mg/L; CLA resistant at MIC >0.5 mg/L; MTZ resistant at MIC >8 mg/L; LEV resistant at MIC>1 mg/L; MOX resistant at MIC >1 mg/L. CLA: clarithromycin; MTZ: metronidazole; LEV: levofloxacin AMX: amoxicillin; TET: tetracycline; MOX: moxifloxacinMIC: Minimum Inhibitory Concentration
Eradication rates of patients who had or had not used amoxicillin in their previous treatment. In 186 patients, 83.3% of them (n = 155) had accepted previously treatments in which amoxicillin was included, while 16.7% of them (n = 31) did not get a regimen contained amoxicillin before. In all 186 patients, the overall eradication rate was 92.5%(172 of 186). In patients who had not used amoxicillin in their previous treatment, all of them got a successful eradication (100%, 31 of 31). While 91.0% (141 of 155) of patients who had used amoxicillin before eliminated the bacteria
Eradication rates of total 186 patients and patients who had accepted regimens with 10 mg bid or 20 mg b.i.d vonoprazan. In 186 patients, 23.1% of them (n = 43) had accepted 20 mg/d vonoprazan while 76.9% (n = 143) with 40 mg/d vonoprazan in their rescue treatment. In all 186 patients, the overall eradication rate was 92.5% (172 of 186). The eradication rates of different dose of vonoprazan were 95.3% (41 of 43) in 10 mg b.i.d and 91.6% (131 of 143) in 20 mg b.i.d, respectively
In 25 cases who got an MIC test during treatment, three cases were resistant to AMX with an MIC of .25 mg/L (resistance defined as a MIC >0.125 mg/L). Among them, one case failed in the VA dual treatment while two of them succeeded in eradication. All 14 who failed had used amoxicillin in their previous treatment, three of them got MIC test with results of .125 mg/L, .125 mg/L and .25 mg/L to AMX, with one of three was defined as resistant to AMX theoretically. Although we did not know the detail about it, it seemed that resistance to AMX was not absolute contraindication in VA dual therapy. There was an inclination that VA dual therapy might be more effective in patients who had not used amoxicillin before.
To our knowledge, this is the first real‐world study to reveal the efficacy of vonoprazan and amoxicillin dual regimen (VA dual regimen) as rescue treatment in China. In previous studies, dual therapy with VPZ was mostly used as first‐line treatment, and the eradication rates varied as 85% ~ 90% in Japan.
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In clinical practices from Japan, VPZ was mostly used as component of triple therapy in first‐line (VPZ + amoxicillin + clarithromycin), second‐line (VPZ + amoxicillin + metronidazole), and third‐line (VPZ + amoxicillin + sitafloxacin) treatment of H. pylori infection. VPZ‐contained triple regimens had a relatively high eradication rate as 88.1%, 80.1%, and 75.8%, respectively.
26
Although vonoprazan appeared to restore the effectiveness of triple therapy, the improvement was almost entirely to improved effectiveness of amoxicillin dual therapy componentand resulted in the majority (>85% currently in Japan) of those receiving vonoprazan + amoxicillin plus a second antibiotic (e.g., clarithromycin, metronidazole, fluoroquinolone, or rifabutin) receiving no benefit from the second antibiotic.
27
It seemed that the only contribution of the second antibiotic is to increase global antimicrobial resistance.
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From the data of our study, it was supposed that the VA dual therapy was effective as rescue treatment no matter what regimen had been given and which antibiotics had been used before. The VA dual therapy could be defined as a “simplified rescue therapy.”
During the treatment course of the VA dual therapy, the adverse events happened were mild and did not cause quitting or failure of treatment. The administration mode was suitable since the compliance of patients was pretty good.
There were many limitations in our study. As a real‐world retrospective study, it's not a randomized controlled trial. The regimens were not consistent, whereas components of dual therapy were the same as vonoprazan + amoxicillin, the administration frequency or doses in VA dual therapy varied. The frequency of amoxicillin was given either t.i.d or q.i.d casually more than well‐designed, although the total dose was the same as 3000 mg per day. The total doses of vonoprazan also varied (20mg or 40 mg per day) with the body weight of patient. As to the MIC analysis, there was only 25 cases who got a successful H. pylori culture and antibacterial susceptibility test, depending on the patients' willingness. The MIC data were limited and not very representative. Based on the results of this retrospective study, a well‐designed random controlled prospective clinical trial could be anticipated being performed in the future.
CONCLUSION:
The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before.
CONFLICT OF INTEREST:
The authors have no competing interests.
| Background:
The currently recommended quadruple regimens as rescue therapy on Helicobacter pylori infection were not as effective as being supposed, especially in those who had failed two or more times. Dual regimen composed of vonoprazan (a potassium-competitive acid blocker) and amoxicillin might be an option since it's effective in eradication therapy as first-line treatment.
Methods:
From May 2020 to June 2021, the clinical data of patients who had failed in Helicobacter pylori infection treatment were collected in GI department of Peking University First Hospital, Beijing, China. Patients were given vonoprazan 20 mg or 40 mg per day and amoxicillin 3000 mg per day (VA dual therapy) for 14 days as rescue treatment. Helicobacter pylori status was evaluated by 13 C-urease breath test 6 weeks after treatment. All adverse effects during treatment were recorded.
Results:
A total of 186 patients were enrolled, including 67 males and 119 females. All of them had failed for 1 ~ 7 times in their previous treatment. Successful eradication was achieved in 172 patients (92.5%, 172/186). The adverse effects (referring to skin rash, abdominal pain, diarrhea, and headache), mainly mild and did not cause quit of treatment, occurred in 14 patients (7.5%, 14/186) and all symptoms relieved spontaneously.
Conclusions:
Dual regimen composed of vonoprazan and amoxicillin for 14 days was effective and safe as rescue therapy in Helicobacter pylori infection treatment. It could be chosen as a "simplified rescue therapy" with relatively high eradication rate no matter how many times the patients had failed and what regimens they had used previously. | INTRODUCTION:
Helicobacter pylori (H. pylori) infection is a major risk factor for development of gastritis, peptic ulcer, and gastric cancer. Successful eradication is an effective strategy to decrease the risk of gastric cancer.
1
,
2
The efficacy of bismuth‐based quadruple therapy as first‐line therapy has been clearly established, which is now been estimated as the mostly widely used regimen in China. As a country in which antibiotic resistance of H. pylori is pretty high especially in clarithromycin, metronidazole, and fluoroquinolone, it's relatively difficult to choose treatment regimen when it comes to the patients who had failed in their previous therapies. The recent clinical guidelines did not provide accordant advices for rescue therapy according to different populations with different antimicrobial resistance status and different regimens used before. Individual treatment with antibiotics‐sensitivity test would be ideal but not easy to be widely used in clinical practice.
Different from the other antibiotics, there is consistent reports that the primary and secondary resistance rates of H. pylori to amoxicillin maintained at a low level.
3
,
4
In recent years, proton‐pump inhibitor (PPI) plus amoxicillin dual therapy has gained increasing attention worldwide because of its effectiveness with a cure rate of 95.3% in first‐line treatment and 89.3% in second‐line treatment.
5
,
6
,
7
It is currently believed that the outcome of the dual therapy is pH‐dependent. Routine dose of PPIs has been proven to be unable to reliably maintain the intragastric pH value at a suitable level required by amoxicillin,
8
which might be the main reason dual therapy failed in mid‐ to late‐1990s. Vonoprazan (VPZ), a novel potassium‐competitive acid blocker, which became available in 2015, has significantly higher acid suppression effect by inhibiting the H + ‐K+ exchange directly to gain a predominant pH elevation for 24 h. Our study aimed to clarify the effectiveness and safety of VPZ plus amoxicillin dual regimen as simplified rescue treatment on the eradication of H. pylori infection no matter what regimens the patients had accepted in their previous treatment.
CONCLUSION:
The utility of vonoprazan to replace traditional proton‐pump inhibitor (PPI) as part of components in H. pylori treatment, especially in cases with multiple antibiotics resistance, was effective and safe. The VA dual therapy (vonoprazan 20/40 mg per day plus amoxicillin 3000 mg per day) would be effective and safe on treatment of H. pylori infection no matter how many times the patients had failed or which antibiotics they had used before. The safety of the regimen and compliance of patients were pretty good. To increase the eradication efficacy, we recommend 14 days VA dual regimen as a “simplified rescue therapy” on treatment of H. pylori infection, especially in those who had not used amoxicillin before. | Background:
The currently recommended quadruple regimens as rescue therapy on Helicobacter pylori infection were not as effective as being supposed, especially in those who had failed two or more times. Dual regimen composed of vonoprazan (a potassium-competitive acid blocker) and amoxicillin might be an option since it's effective in eradication therapy as first-line treatment.
Methods:
From May 2020 to June 2021, the clinical data of patients who had failed in Helicobacter pylori infection treatment were collected in GI department of Peking University First Hospital, Beijing, China. Patients were given vonoprazan 20 mg or 40 mg per day and amoxicillin 3000 mg per day (VA dual therapy) for 14 days as rescue treatment. Helicobacter pylori status was evaluated by 13 C-urease breath test 6 weeks after treatment. All adverse effects during treatment were recorded.
Results:
A total of 186 patients were enrolled, including 67 males and 119 females. All of them had failed for 1 ~ 7 times in their previous treatment. Successful eradication was achieved in 172 patients (92.5%, 172/186). The adverse effects (referring to skin rash, abdominal pain, diarrhea, and headache), mainly mild and did not cause quit of treatment, occurred in 14 patients (7.5%, 14/186) and all symptoms relieved spontaneously.
Conclusions:
Dual regimen composed of vonoprazan and amoxicillin for 14 days was effective and safe as rescue therapy in Helicobacter pylori infection treatment. It could be chosen as a "simplified rescue therapy" with relatively high eradication rate no matter how many times the patients had failed and what regimens they had used previously. | 8,712 | 319 | [
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Helicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY] | null | [CONTENT] amoxicillin | dual therapy |
Helicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY] | [CONTENT] amoxicillin | dual therapy |
Helicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY] | [CONTENT] amoxicillin | dual therapy |
Helicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY] | [CONTENT] amoxicillin | dual therapy |
Helicobacter pylori infection | simplified rescue therapy | vonoprazan [SUMMARY] | [CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY] | null | [CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY] | [CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY] | [CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY] | [CONTENT] Amoxicillin | Anti-Bacterial Agents | Clarithromycin | Drug Therapy, Combination | Female | Helicobacter Infections | Helicobacter pylori | Humans | Male | Potassium | Proton Pump Inhibitors | Pyrroles | Retrospective Studies | Sulfonamides | Treatment Outcome | Urease [SUMMARY] | [CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY] | null | [CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY] | [CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY] | [CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY] | [CONTENT] antibiotic resistance pylori | pylori therapy include | regimen treatment pylori | outcome treatment pylori | previous pylori treatment [SUMMARY] | [CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY] | null | [CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY] | [CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY] | [CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY] | [CONTENT] treatment | mg | patients | amoxicillin | therapy | failed | eradication | times | mic | dual [SUMMARY] | [CONTENT] different | ph | line | treatment | therapy | pylori | regimen | widely | plus amoxicillin dual | level [SUMMARY] | null | [CONTENT] times | treatment | patients | 95 | events | adverse | adverse events | failed | 186 | eradication [SUMMARY] | [CONTENT] effective safe | treatment pylori infection | safe | especially | effective | pylori | regimen | treatment pylori | treatment | day [SUMMARY] | [CONTENT] mg | treatment | patients | times | mic | amoxicillin | therapy | failed | eradication | dual [SUMMARY] | [CONTENT] mg | treatment | patients | times | mic | amoxicillin | therapy | failed | eradication | dual [SUMMARY] | [CONTENT] two ||| first [SUMMARY] | null | [CONTENT] 186 | 67 | 119 ||| 1 | 7 ||| 172 | 92.5% | 172/186 ||| 14 | 7.5% | 14/186 [SUMMARY] | [CONTENT] 14 days ||| [SUMMARY] | [CONTENT] two ||| first ||| May 2020 to June 2021 | GI | Peking University First Hospital | Beijing | China ||| 20 mg | 40 mg | 14 days ||| 13 | 6 weeks ||| ||| 186 | 67 | 119 ||| 1 | 7 ||| 172 | 92.5% | 172/186 ||| 14 | 7.5% | 14/186 ||| 14 days ||| [SUMMARY] | [CONTENT] two ||| first ||| May 2020 to June 2021 | GI | Peking University First Hospital | Beijing | China ||| 20 mg | 40 mg | 14 days ||| 13 | 6 weeks ||| ||| 186 | 67 | 119 ||| 1 | 7 ||| 172 | 92.5% | 172/186 ||| 14 | 7.5% | 14/186 ||| 14 days ||| [SUMMARY] |
|||||
Nationwide survey to evaluate the decision-making process in euthanasia requests in Belgium: do specifically trained 2nd physicians improve quality of consultation? | 25030375 | Following the 2002 enactment of the Belgian law on euthanasia, which requires the consultation of an independent second physician before proceeding with euthanasia, the Life End Information Forum (LEIF) was founded which provides specifically trained physicians who can act as mandatory consultants in euthanasia requests. This study assesses quality of consultations in Flanders and Brussels and compares these between LEIF and non-LEIF consultants. | BACKGROUND | A questionnaire was sent in 2009 to a random sample of 3,006 physicians in Belgium from specialties likely involved in the care of dying patients. Several questions about the last euthanasia request of one of their patients were asked. As LEIF serves the Flemish speaking community (i.e. region of Flanders and the bilingual Brussels Capital Region) and no similar counterpart is present in Wallonia, analyses were limited to Flemish speaking physicians in Flanders and Brussels. | METHODS | Response was 34%. Of the 244 physicians who indicated having received a euthanasia request seventy percent consulted a second physician in their last request; in 30% this was with a LEIF physician. Compared to non-LEIF physicians, LEIF physicians were more often not a colleague (69% vs 42%) and not a co-attending physician (89% vs 66%). They tended to more often discuss the request with the attending physician (100% vs 95%) and with the family (76% vs 69%), and also more frequently helped the attending physician with performing euthanasia (44% vs 24%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%) and examined the patient file (94% vs 97%). | RESULTS | In cases of explicit euthanasia requests in Belgium, the consultation procedure of another physician by the attending physician is not optimal and can be improved. Training and putting at disposal consultants through forums such as LEIF seems able to improve this situation. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Irrespective of whether euthanasia is a legal practice within a country, similar services may prove useful to also improve quality of consultations in various other difficult end-of-life decision-making situations. | CONCLUSION | [
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] | 4114442 | Background | Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002
[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission
[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)
[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting
[5].
While the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters
[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests
[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia
[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.
Previously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted
[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:
1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?
2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?
3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)? | Method | Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.
The researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders
[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.
To assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests
[17].
In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.
The researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders
[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.
To assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests
[17].
Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands
[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)
[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.
List of quality criteria as outlined by the Dutch consultation protocol
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands
[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)
[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.
List of quality criteria as outlined by the Dutch consultation protocol
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.
For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0. | Results | Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.
Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.
Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table
1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.
Number of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)
*1 to 17 missing cases. FL = Flanders, BXL = Brussels.
†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.
‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.
Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table
1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.
Number of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)
*1 to 17 missing cases. FL = Flanders, BXL = Brussels.
†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.
‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.
Quality of consultation Table
2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).
Extent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not
*1 to 16 missing cases.
†1 to 3 missing cases.
‡1 to 13 missing cases.
All the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table
3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.
Additional tasks performed by the second physician according to whether the second physician is a LEIF physician or not
*2 to 22 missing cases.
†1 to 4 missing cases.
‡1 to 16 missing cases.
Table
2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).
Extent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not
*1 to 16 missing cases.
†1 to 3 missing cases.
‡1 to 13 missing cases.
All the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table
3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.
Additional tasks performed by the second physician according to whether the second physician is a LEIF physician or not
*2 to 22 missing cases.
†1 to 4 missing cases.
‡1 to 16 missing cases.
Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table
4).
Advice of consultant according to whether the second physician is a LEIF physician or not
*3 missing cases.
†1 to 3 missing cases.
Overall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).
Sixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).
The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table
4).
Advice of consultant according to whether the second physician is a LEIF physician or not
*3 missing cases.
†1 to 3 missing cases.
Overall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).
Sixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table). | Conclusion | In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.
As we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making. | [
"Background",
"Study design",
"Questionnaire",
"\nList of quality criteria as outlined by the Dutch consultation protocol\n",
"Statistical analysis",
"Response rate and response bias",
"Physicians receiving euthanasia requests and consulting a second physician",
"Quality of consultation",
"Advice of the consultant and outcome of the request",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?",
"In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].",
"The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.",
"The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.",
"For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.",
"Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.",
"Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.",
"Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.",
"The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).",
"Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.",
"JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
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null,
null
] | [
"Background",
"Method",
"Study design",
"Questionnaire",
"\nList of quality criteria as outlined by the Dutch consultation protocol\n",
"Statistical analysis",
"Results",
"Response rate and response bias",
"Physicians receiving euthanasia requests and consulting a second physician",
"Quality of consultation",
"Advice of the consultant and outcome of the request",
"Discussion",
"Conclusion",
"Competing interests",
"Authors’ contributions",
"Pre-publication history"
] | [
"Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002\n[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission\n[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)\n[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting\n[5].\nWhile the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters\n[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests\n[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia\n[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.\nPreviously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted\n[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:\n1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?\n2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?\n3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?",
" Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\nIn March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].\n Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\n Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.\nFor all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.",
"In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.\nThe researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders\n[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.\nTo assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests\n[17].",
"The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands\n[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)\n[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.\n \nList of quality criteria as outlined by the Dutch consultation protocol\n The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.\nThe consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.",
"The consultant\n– was not a colleague of the attending physician\n– was not a co-attending physician\n– did not know the patient\n– talked to/examined the patient\n– considered the request*\n– talked about possible alternatives†\n– made a written report\n*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.\n†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.",
"For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.",
" Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\nOf the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.\n Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\nTwo hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.\n Quality of consultation Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\nTable \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.\n Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).\nThe consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).",
"Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.",
"Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table \n1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.\nNumber of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)\n*1 to 17 missing cases. FL = Flanders, BXL = Brussels.\n†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.\n‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.",
"Table \n2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).\nExtent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not\n*1 to 16 missing cases.\n†1 to 3 missing cases.\n‡1 to 13 missing cases.\nAll the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table \n3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.\nAdditional tasks performed by the second physician according to whether the second physician is a LEIF physician or not\n*2 to 22 missing cases.\n†1 to 4 missing cases.\n‡1 to 16 missing cases.",
"The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table \n4).\nAdvice of consultant according to whether the second physician is a LEIF physician or not\n*3 missing cases.\n†1 to 3 missing cases.\nOverall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).\nSixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).",
"This study is the first to examine the quality of consultation between attending physicians and consultants in euthanasia requests in Belgium. We found that in 70% of the euthanasia requests described the attending physician had consulted with a second physician. Of the (Dutch) criteria for good practice, independence of the consultant, either from the physician or the patients, is the one most often unmet. Life End Information Forum (LEIF) physicians, who had undergone training as consultants in euthanasia requests, were significantly more often independent from the attending physician and from the patient compared with those who had not received the LEIF training.\nAn important strength of this study is that we used a large sample of physicians from diverse specialties to ask questions about a very sensitive subject. To this end, we used a rigorous sampling and mailing procedure. The questionnaire was comprehensively tested. There are however also some limitations. First, the low response percentage makes it difficult to generalize the results to all physicians in Flanders and Brussels, although analyses of the non-response survey indicate that the sample of responders was comparable to the sample of non-responders regarding having received a euthanasia request. As the survey is retrospective, there may be recall bias, especially for requests from more than a year earlier. Furthermore, the information provided in this survey on the activities of the consultants stems only from the attending physician. An additional limitation is that, with the absence of an initiative of similar magnitude and organisation present in Wallonia or for French speaking Belgium, the analyses had to be limited to Flemish speaking physicians in Flanders and Brussels. The presence of an initiative for Flemish speaking physicians but absence of one for French speaking physicians may reflect the fact that Flanders shares the language of and is culturally closer to the Netherlands. In addition, a previous study also indicated more positive attitudes among Flemish physicians towards the due care criteria of the euthanasia law and a larger willingness to comply with these criteria\n[20]. As regional differences in the euthanasia practice likely also reflect cultural differences\n[20] a comparison of LEIF physicians versus non-LEIF physicians for the whole of Belgium (with all French speaking physicians thus being in the non-LEIF group) would have been contaminated by these differences. Hence, although it is likely that a service like LEIF would have similar positive effects in French speaking Belgium, our results cannot just be generalized to French speaking Belgium.\nWe found that 70% of the physicians who had received a euthanasia request had consulted with a mandatory second physician and for the cases where euthanasia was eventually performed this was 92%. A previous study in the Netherlands found consultation to take place in 87% of requests reported by GPs in the period 2000–2002 and in 97% of cases where euthanasia was performed\n[21]. Compared with the Netherlands, consultation in Flanders is thus rather low, especially when taking into account that the percentages in the Netherlands date from a period before 2002, when euthanasia was tolerated but not yet legalized in this country. That attending physicians do not consult a second physician in 30% of euthanasia requests can partly be attributed to the fact that in some cases they have already decided not to grant the request and consider they do not need a colleague to confirm their decision. It is open to discussion whether physicians need to consult with colleague physicians about every euthanasia request even if they feel it is not serious enough or feel reluctant to comply with it. The 8% of performed cases of euthanasia where no consultation took place are, however, problematic and do not comply with the legal due care criteria. Not consulting in these cases can possibly be attributed to a lack of knowledge of the procedure or to the reluctance of attending physicians to be scrutinized by a colleague\n[22].\nConsultants in Flanders and Brussels were found not always to have examined or talked to the patient and a written report was not made in over one third of consultations. As these are prescribed in the euthanasia law as tasks of the consultant, it is surprising that these are not always met. It may not be possible to talk to all patients, especially if they have lost competency in decision-making (a situation that can also qualify for euthanasia if there is a previous written request or euthanasia declaration); however, our question also asked about an examination of the patient and it could be expected that at least this would have been done in patients who lost competency. The number of cases where neither were done is, however, very low. The cases without a legally required written report is a more frequent problem and may be due to the lack of knowledge about this legal criterion and a lack of legal control mechanisms to ascertain that a written report is made. An additional potential problem is that independence in relation to the physician and patient seemed often not guaranteed. In one third of cases the consultant was a co-attending physician of the patient. While the law does not describe precisely what is meant by independence\n[1], being a co-attending physician seems incongruent with the intention of the law that the consultant is able to give independent advice without influence from the attending physician or the patient. The fact that attending physicians do sometimes not seek an independent consultant could indicate that they consider the consultation merely a formality.\nOur study indicated that involving consultants who followed a special training instead of those who did not could contribute to various aspects of the quality of consultation. The most important contribution seems to be in terms of the legally required independence: the LEIF physicians in our study were more often independent than non-LEIF physicians from the attending physician and the patient (i.e. they were more often unacquainted with them). The contact procedure through the central telephone number of LEIF, which is intended to assign a random LEIF physician (preferably from the region) to the attending physician, seems to create a better guarantee of independence as opposed to simply consulting a colleague from the same hospital or practice. However, this service appears to be called upon in particular by general practitioners, and much less often by specialists in hospitals who often call on their direct colleagues, which may be more practical and private but jeopardizes independence. Specialists therefore might especially benefit from a service such as LEIF in order to comply fully with this aspect of the law.\nAlthough differences were not statistically significant, LEIF physicians also tended to more often discuss the request with the attending physician and the family than non-LEIF physicians. While these are not strict due care requirements stated in the law or the Dutch consultation protocol\n[19] they can also be seen as aspects determining the quality of a consultation, contributing to a more careful and qualitative consultation practice.\nInvolving specifically trained consultants can thus benefit euthanasia consultations in a number of aspects. A number of found differences between LEIF and non-LEIF consultants are perhaps not necessarily indicative of differences in the quality of consultation but nevertheless suggest relevant differences in consultation practice that merit further discussion. While the fact that LEIF-physicians less frequently discussed euthanasia requests with other attending physicians (i.e. colleague-attending physicians) is a result of the fact that LEIF physicians are more often consulted by GPs rather than by hospital specialists, the most striking difference with non-LEIF consultations is that LEIF consultations significantly more often led to a positive advice on the euthanasia request (i.e. a judgement that the conditions for euthanasia have been met) and significantly more often involved help with performing euthanasia. It may be that these differences reflect a more positive attitude towards euthanasia in general in LEIF physicians. Not being fundamentally against euthanasia is a prerequisite to being admitted to the LEIF training programme\n[6]. It has to be acknowledged that underlying attitudinal aspects may result in different outcomes of a consultation, which is inherent to the fact that assessing the nature and validity of a euthanasia request does not merely involve checking off objective criteria but also involves a great deal of subjective and compassionate judgement. While some may find in this finding a proof for too high a compliance with euthanasia requests in LEIF physicians, others will argue that the higher reluctance of non-LEIF consultants to give a positive advice about a euthanasia request is due to their larger uncertainty about the due care criteria and the procedure since they lack the specific training and similar experience.\nSimilarly some will argue that helping with the performance of euthanasia is not and cannot be the responsibility of a consultant whereas others will see it as a pedagogic assistance for attending physicians. The function of role models in medical education, particularly in end-of-life care, and their influence on ethical decision-making has been described in other studies\n[23-27] and previous research indicated that LEIF consultants help with euthanasia for medico-technical reasons, because they consider it important that a more experienced colleague can show the attending physician how to perform a delicate act they are not prepared for through the medical curriculum\n[7].\nThe model of LEIF, as a training and consultation service, is strongly based and inspired on the model of SCEN (Support and Consultation for Euthanasia in the Netherlands) in the Netherlands\n[6], which was already developed prior to the euthanasia legalisation in 2002 in the Netherlands\n[28]. The fact that SCEN is more regulated, organised on a larger scale, more endorsed by government, and puts more focus on formal aspects of the consultation (e.g. the written consultation report)\n[6], but also the more specific stipulations in the Dutch euthanasia law about the activities that are required by a consultant in a euthanasia request may be explanations for the larger compliance with legal requirements in SCEN physicians and in the Netherlands in general\n[29].\nThe euthanasia law in Belgium has recently (and only after our study was conducted) been adapted to include competent minor patients\n[30]. Although the practice of euthanasia in minor patients will likely continue to be a very rare and marginal practice (a previous study including all deaths of minor persons in Flanders during one and a half year identified no case of euthanasia\n[31]), the specific difficulties and challenges a careful euthanasia practice in this population poses will require LEIF to pay specific attention to euthanasia requests in minor patients in its trainings.",
"In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.\nAs we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making.",
"Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.",
"JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6963/14/307/prepub\n"
] | [
null,
"methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
"discussion",
"conclusions",
null,
null,
null
] | [
"Euthanasia",
"Consultation",
"Referral practice",
"Terminal care"
] | Background:
Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002
[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission
[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)
[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting
[5].
While the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters
[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests
[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia
[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.
Previously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted
[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:
1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?
2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?
3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?
Method:
Study design In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.
The researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders
[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.
To assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests
[17].
In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.
The researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders
[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.
To assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests
[17].
Questionnaire The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands
[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)
[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.
List of quality criteria as outlined by the Dutch consultation protocol
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands
[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)
[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.
List of quality criteria as outlined by the Dutch consultation protocol
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
Statistical analysis For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.
For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.
Study design:
In March 2009 we sent a questionnaire to a random sample of 3,006 Belgian physicians by mail. The sampling frame was a weekly updated commercial register based on the register of the National Institute for Health and Disability Insurance (NIHDI). This commercial register was chosen because the prevailing privacy law made official registers from the NIHDI unavailable to researchers and because the quality and completeness of the register was judged as high based on a comparison with aggregate distributions from the NIHDI data. From this sampling frame we only selected registered medical practitioners who worked in Belgium, had graduated in their speciality at least 12 months before the sample was drawn, and were likely to be involved in the care of dying patients. The latter selection was done based on specialty. Those specialities for which little or no experience in the care for the dying could be expected were excluded. The following specialities were included: general practice, anaesthesiology, gynaecology, internal medicine, neurology, pulmonary medicine, gastroenterology, neuropsychiatry, psychiatry, cardiology, radiotherapy, and surgery. The random sample was stratified for province and speciality and represents a sampling fraction of 9.2%.
The researchers sent a questionnaire with a unique serial number to each physician in the sample. The physicians were instructed in a covering letter to send the questionnaire to an independent lawyer, guaranteeing complete anonymity while allowing for the sending of up to three reminders
[16]. The anonymity procedure and study protocol were approved by the Ethical Review Board of the University Hospital of the Vrije Universiteit Brussel.
To assess non-response bias, non-responders were sent a one-page form asking them for their reasons for not participating and requesting them to fill in two key questions from the original questionnaire, one about their attitude towards euthanasia, and another about their experience with euthanasia requests
[17].
Questionnaire:
The pre-structured, eight-page questionnaire with mainly closed-end questions was partly based on one previously used in the Netherlands
[18]. The questions were adapted to make them appropriate for the Belgian legal context and culture. Concerning their most recent euthanasia request, physicians were asked to answer questions on patient and request characteristics, consultation with a second physician, activities of the second physician and outcome of the request. Questions on quality criteria in accordance with the Belgian due care criteria for consultation were included (see List of quality criteria section)
[19]. In the questionnaire, euthanasia was defined as ‘the intentional ending of the patient’s life at his/her explicit request by the physician’; this definition corresponds to the legal definition of euthanasia in Belgium.
List of quality criteria as outlined by the Dutch consultation protocol
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
List of quality criteria as outlined by the Dutch consultation protocol
:
The consultant
– was not a colleague of the attending physician
– was not a co-attending physician
– did not know the patient
– talked to/examined the patient
– considered the request*
– talked about possible alternatives†
– made a written report
*This criterion was not questioned in the Belgian survey because it is too vague and not a task stipulated in de Belgian euthanasia law.
†This criterion was not questioned in the Belgian survey because it is not a requirement for the second physician in the Belgian law on euthanasia.
Statistical analysis:
For all analyses we selected only the responses from Dutch-speaking physicians from Flanders and Brussels, because LEIF offers its services and trainings in Dutch and provides its services in Brussels and Flanders. Fisher exact tests were performed to compare for LEIF and non-LEIF physicians’ independence and activities. P-values that were less than 0.05 were considered statistically significant. The analyses were performed using SPSS 19.0.
Results:
Response rate and response bias Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.
Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.
Physicians receiving euthanasia requests and consulting a second physician Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table
1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.
Number of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)
*1 to 17 missing cases. FL = Flanders, BXL = Brussels.
†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.
‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.
Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table
1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.
Number of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)
*1 to 17 missing cases. FL = Flanders, BXL = Brussels.
†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.
‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.
Quality of consultation Table
2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).
Extent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not
*1 to 16 missing cases.
†1 to 3 missing cases.
‡1 to 13 missing cases.
All the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table
3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.
Additional tasks performed by the second physician according to whether the second physician is a LEIF physician or not
*2 to 22 missing cases.
†1 to 4 missing cases.
‡1 to 16 missing cases.
Table
2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).
Extent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not
*1 to 16 missing cases.
†1 to 3 missing cases.
‡1 to 13 missing cases.
All the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table
3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.
Additional tasks performed by the second physician according to whether the second physician is a LEIF physician or not
*2 to 22 missing cases.
†1 to 4 missing cases.
‡1 to 16 missing cases.
Advice of the consultant and outcome of the request The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table
4).
Advice of consultant according to whether the second physician is a LEIF physician or not
*3 missing cases.
†1 to 3 missing cases.
Overall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).
Sixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).
The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table
4).
Advice of consultant according to whether the second physician is a LEIF physician or not
*3 missing cases.
†1 to 3 missing cases.
Overall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).
Sixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).
Response rate and response bias:
Of the 3,006 questionnaires sent, 222 physicians were unreachable, deceased or no longer in practice. From the non response survey another 57 were identified as no longer practising or not having received the questionnaire. As such, there were 2,726 eligible physicians from whom 914 questionnaires (34%) were returned. Small but significant differences between the responders of the survey and responders of the non-response survey were found for attitude toward euthanasia (90.4% vs 87.4% agreed on the statement concerning attitude toward euthanasia). No differences between these groups were found concerning having ever received a request.
Physicians receiving euthanasia requests and consulting a second physician:
Two hundred and forty-four Dutch-speaking physicians from Flanders and Brussels had received a euthanasia request since 2002 and described the most recent request they had received. Seventy per cent of these respondents had consulted with a second physician about this request (Table
1); for the cases where euthanasia was actually performed (N = 123) consultation had taken place in 91.9% (not in table). In 51 (30.0%) of the consultations, the consultant was a LEIF physician. General practitioners more often than specialists consulted with a LEIF physician. Physicians between 36 and 50 years old had significantly more often consulted a second physician than had their younger and older colleagues but this was less often a LEIF consultant.
Number of consultations with a second physician (LEIF and not LEIF) since the euthanasia law, according to physician’s characteristics (n = 244)
*1 to 17 missing cases. FL = Flanders, BXL = Brussels.
†Comparison with physicians who did not consult. Fisher exact test for statistically significant differences between categories vs all other categories within the variable. Significant differences in bold.
‡Fisher exact test for statistically significant differences between categories vs all other categories within the variable.
Quality of consultation:
Table
2 lists the extent to which the Dutch criteria for a good consultation were met by all physicians and by LEIF physicians. For all physicians, the consultant was not a direct colleague (i.e. same working environment) of the attending physician in 41.8% of cases and in two thirds of cases the consultant was not a co-attending physician of and did not know the patient. These criteria of independence in relation to both the attending physician and the patient were met significantly more often when the consultant was a LEIF physician as compared with a non-LEIF physician (respectively 68.6%, 88.7%, and 86.3% compared to 30.3%, 53.8%, and 49.2%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%), examined the patient file (94% vs 97%), or made a written report about their judgement regarding the euthanasia request (73% vs 67%).
Extent to which criteria for quality of consultation are met according to whether the second physician is a LEIF physician or not
*1 to 16 missing cases.
†1 to 3 missing cases.
‡1 to 13 missing cases.
All the LEIF physicians had discussed the request with the attending physician compared with 94.9% of the non-LEIF physicians (difference not statistically significant; Table
3). LEIF physicians tended to less often discuss the request with another attending physician or with a third physician than had non-LEIF physicians, although the difference was not statistically significant. LEIF physicians had helped the attending physician with performing the euthanasia more often than had non-LEIF physicians and tended to more often help with filling out the reporting form.
Additional tasks performed by the second physician according to whether the second physician is a LEIF physician or not
*2 to 22 missing cases.
†1 to 4 missing cases.
‡1 to 16 missing cases.
Advice of the consultant and outcome of the request:
The consultant gave a positive advice (i.e. concluded that the conditions for euthanasia were met) in 80.5% (N = 136) of all requests (Table
4).
Advice of consultant according to whether the second physician is a LEIF physician or not
*3 missing cases.
†1 to 3 missing cases.
Overall, LEIF physicians significantly more often gave a positive advice (i.e. concluded that the conditions for euthanasia were met) compared with non-LEIF physicians (90.2% vs 76.3%). When asked to what extent the advice of the consultant had influenced their final decision, 60% of respondents indicated that it had to some or to a great extent (56.8% LEIF and 61.4% non-LEIF).
Sixty-eight percent (N = 113) of the requests described in Flanders and Brussels resulted in euthanasia. Euthanasia was more often performed when a LEIF physician as opposed to a non-LEIF physician had been consulted but this difference was not significant (76.0% vs 64.1%, p = 0.151, not in table). Also, euthanasia was more often reported in case the consultant was a LEIF physician as compared to a non-LEIF physician but this difference was not significant either (86.5% vs 77.3%, p = 0.317, not in table).
Discussion:
This study is the first to examine the quality of consultation between attending physicians and consultants in euthanasia requests in Belgium. We found that in 70% of the euthanasia requests described the attending physician had consulted with a second physician. Of the (Dutch) criteria for good practice, independence of the consultant, either from the physician or the patients, is the one most often unmet. Life End Information Forum (LEIF) physicians, who had undergone training as consultants in euthanasia requests, were significantly more often independent from the attending physician and from the patient compared with those who had not received the LEIF training.
An important strength of this study is that we used a large sample of physicians from diverse specialties to ask questions about a very sensitive subject. To this end, we used a rigorous sampling and mailing procedure. The questionnaire was comprehensively tested. There are however also some limitations. First, the low response percentage makes it difficult to generalize the results to all physicians in Flanders and Brussels, although analyses of the non-response survey indicate that the sample of responders was comparable to the sample of non-responders regarding having received a euthanasia request. As the survey is retrospective, there may be recall bias, especially for requests from more than a year earlier. Furthermore, the information provided in this survey on the activities of the consultants stems only from the attending physician. An additional limitation is that, with the absence of an initiative of similar magnitude and organisation present in Wallonia or for French speaking Belgium, the analyses had to be limited to Flemish speaking physicians in Flanders and Brussels. The presence of an initiative for Flemish speaking physicians but absence of one for French speaking physicians may reflect the fact that Flanders shares the language of and is culturally closer to the Netherlands. In addition, a previous study also indicated more positive attitudes among Flemish physicians towards the due care criteria of the euthanasia law and a larger willingness to comply with these criteria
[20]. As regional differences in the euthanasia practice likely also reflect cultural differences
[20] a comparison of LEIF physicians versus non-LEIF physicians for the whole of Belgium (with all French speaking physicians thus being in the non-LEIF group) would have been contaminated by these differences. Hence, although it is likely that a service like LEIF would have similar positive effects in French speaking Belgium, our results cannot just be generalized to French speaking Belgium.
We found that 70% of the physicians who had received a euthanasia request had consulted with a mandatory second physician and for the cases where euthanasia was eventually performed this was 92%. A previous study in the Netherlands found consultation to take place in 87% of requests reported by GPs in the period 2000–2002 and in 97% of cases where euthanasia was performed
[21]. Compared with the Netherlands, consultation in Flanders is thus rather low, especially when taking into account that the percentages in the Netherlands date from a period before 2002, when euthanasia was tolerated but not yet legalized in this country. That attending physicians do not consult a second physician in 30% of euthanasia requests can partly be attributed to the fact that in some cases they have already decided not to grant the request and consider they do not need a colleague to confirm their decision. It is open to discussion whether physicians need to consult with colleague physicians about every euthanasia request even if they feel it is not serious enough or feel reluctant to comply with it. The 8% of performed cases of euthanasia where no consultation took place are, however, problematic and do not comply with the legal due care criteria. Not consulting in these cases can possibly be attributed to a lack of knowledge of the procedure or to the reluctance of attending physicians to be scrutinized by a colleague
[22].
Consultants in Flanders and Brussels were found not always to have examined or talked to the patient and a written report was not made in over one third of consultations. As these are prescribed in the euthanasia law as tasks of the consultant, it is surprising that these are not always met. It may not be possible to talk to all patients, especially if they have lost competency in decision-making (a situation that can also qualify for euthanasia if there is a previous written request or euthanasia declaration); however, our question also asked about an examination of the patient and it could be expected that at least this would have been done in patients who lost competency. The number of cases where neither were done is, however, very low. The cases without a legally required written report is a more frequent problem and may be due to the lack of knowledge about this legal criterion and a lack of legal control mechanisms to ascertain that a written report is made. An additional potential problem is that independence in relation to the physician and patient seemed often not guaranteed. In one third of cases the consultant was a co-attending physician of the patient. While the law does not describe precisely what is meant by independence
[1], being a co-attending physician seems incongruent with the intention of the law that the consultant is able to give independent advice without influence from the attending physician or the patient. The fact that attending physicians do sometimes not seek an independent consultant could indicate that they consider the consultation merely a formality.
Our study indicated that involving consultants who followed a special training instead of those who did not could contribute to various aspects of the quality of consultation. The most important contribution seems to be in terms of the legally required independence: the LEIF physicians in our study were more often independent than non-LEIF physicians from the attending physician and the patient (i.e. they were more often unacquainted with them). The contact procedure through the central telephone number of LEIF, which is intended to assign a random LEIF physician (preferably from the region) to the attending physician, seems to create a better guarantee of independence as opposed to simply consulting a colleague from the same hospital or practice. However, this service appears to be called upon in particular by general practitioners, and much less often by specialists in hospitals who often call on their direct colleagues, which may be more practical and private but jeopardizes independence. Specialists therefore might especially benefit from a service such as LEIF in order to comply fully with this aspect of the law.
Although differences were not statistically significant, LEIF physicians also tended to more often discuss the request with the attending physician and the family than non-LEIF physicians. While these are not strict due care requirements stated in the law or the Dutch consultation protocol
[19] they can also be seen as aspects determining the quality of a consultation, contributing to a more careful and qualitative consultation practice.
Involving specifically trained consultants can thus benefit euthanasia consultations in a number of aspects. A number of found differences between LEIF and non-LEIF consultants are perhaps not necessarily indicative of differences in the quality of consultation but nevertheless suggest relevant differences in consultation practice that merit further discussion. While the fact that LEIF-physicians less frequently discussed euthanasia requests with other attending physicians (i.e. colleague-attending physicians) is a result of the fact that LEIF physicians are more often consulted by GPs rather than by hospital specialists, the most striking difference with non-LEIF consultations is that LEIF consultations significantly more often led to a positive advice on the euthanasia request (i.e. a judgement that the conditions for euthanasia have been met) and significantly more often involved help with performing euthanasia. It may be that these differences reflect a more positive attitude towards euthanasia in general in LEIF physicians. Not being fundamentally against euthanasia is a prerequisite to being admitted to the LEIF training programme
[6]. It has to be acknowledged that underlying attitudinal aspects may result in different outcomes of a consultation, which is inherent to the fact that assessing the nature and validity of a euthanasia request does not merely involve checking off objective criteria but also involves a great deal of subjective and compassionate judgement. While some may find in this finding a proof for too high a compliance with euthanasia requests in LEIF physicians, others will argue that the higher reluctance of non-LEIF consultants to give a positive advice about a euthanasia request is due to their larger uncertainty about the due care criteria and the procedure since they lack the specific training and similar experience.
Similarly some will argue that helping with the performance of euthanasia is not and cannot be the responsibility of a consultant whereas others will see it as a pedagogic assistance for attending physicians. The function of role models in medical education, particularly in end-of-life care, and their influence on ethical decision-making has been described in other studies
[23-27] and previous research indicated that LEIF consultants help with euthanasia for medico-technical reasons, because they consider it important that a more experienced colleague can show the attending physician how to perform a delicate act they are not prepared for through the medical curriculum
[7].
The model of LEIF, as a training and consultation service, is strongly based and inspired on the model of SCEN (Support and Consultation for Euthanasia in the Netherlands) in the Netherlands
[6], which was already developed prior to the euthanasia legalisation in 2002 in the Netherlands
[28]. The fact that SCEN is more regulated, organised on a larger scale, more endorsed by government, and puts more focus on formal aspects of the consultation (e.g. the written consultation report)
[6], but also the more specific stipulations in the Dutch euthanasia law about the activities that are required by a consultant in a euthanasia request may be explanations for the larger compliance with legal requirements in SCEN physicians and in the Netherlands in general
[29].
The euthanasia law in Belgium has recently (and only after our study was conducted) been adapted to include competent minor patients
[30]. Although the practice of euthanasia in minor patients will likely continue to be a very rare and marginal practice (a previous study including all deaths of minor persons in Flanders during one and a half year identified no case of euthanasia
[31]), the specific difficulties and challenges a careful euthanasia practice in this population poses will require LEIF to pay specific attention to euthanasia requests in minor patients in its trainings.
Conclusion:
In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.
As we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making.
Competing interests:
Non-financial competing interests: Wim Distelmans is chair of the LEIF forum. He collaborated to the manuscript by providing all necessary information about the LEIF physicians and insights about possible explanations for the findings. However, he did not have any influence on the choice of data being presented and he committed a priori to not influence the interpretations the other authors chose to give to the findings.
Authors’ contributions:
JC, YVW, TS, JB, and LD designed the study. JC and LD obtained funding. JC, YVW, and TS were responsible for data acquisition and the data analyses. All authors contributed to the interpretation of data. JC and YVW drafted the manuscript and all authors critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6963/14/307/prepub
| Background:
Following the 2002 enactment of the Belgian law on euthanasia, which requires the consultation of an independent second physician before proceeding with euthanasia, the Life End Information Forum (LEIF) was founded which provides specifically trained physicians who can act as mandatory consultants in euthanasia requests. This study assesses quality of consultations in Flanders and Brussels and compares these between LEIF and non-LEIF consultants.
Methods:
A questionnaire was sent in 2009 to a random sample of 3,006 physicians in Belgium from specialties likely involved in the care of dying patients. Several questions about the last euthanasia request of one of their patients were asked. As LEIF serves the Flemish speaking community (i.e. region of Flanders and the bilingual Brussels Capital Region) and no similar counterpart is present in Wallonia, analyses were limited to Flemish speaking physicians in Flanders and Brussels.
Results:
Response was 34%. Of the 244 physicians who indicated having received a euthanasia request seventy percent consulted a second physician in their last request; in 30% this was with a LEIF physician. Compared to non-LEIF physicians, LEIF physicians were more often not a colleague (69% vs 42%) and not a co-attending physician (89% vs 66%). They tended to more often discuss the request with the attending physician (100% vs 95%) and with the family (76% vs 69%), and also more frequently helped the attending physician with performing euthanasia (44% vs 24%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%) and examined the patient file (94% vs 97%).
Conclusions:
In cases of explicit euthanasia requests in Belgium, the consultation procedure of another physician by the attending physician is not optimal and can be improved. Training and putting at disposal consultants through forums such as LEIF seems able to improve this situation. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Irrespective of whether euthanasia is a legal practice within a country, similar services may prove useful to also improve quality of consultations in various other difficult end-of-life decision-making situations. | Background:
Euthanasia, i.e. the intentional ending of life by a physician at a person’s explicit request, has been legal in Belgium under strict conditions since 2002
[1,2]. The person must be in a medically hopeless situation of persistent and unbearable physical or psychological suffering as a consequence of a serious and incurable medical condition, which cannot be alleviated otherwise. His or her request must be voluntary, well-considered and repeated. One of the procedural conditions to be followed before considering euthanasia is that the attending physician has to consult a second physician, the consultant, who must be independent from both the attending physician and the patient. This consultant has to read the medical file, examine the patient and ascertain that the patient’s suffering is unbearable and the request is voluntary, well-considered and repeated. He also has to write a report about his judgement. These are due care criteria to guarantee the safe practice of euthanasia by means of a control mechanism beforehand. If the attending physician and/or the consultant do not anticipate death of the person requesting euthanasia in the near future (e.g. in case of a quadriplegic person, or a person suffering from MS), a third independent physician who is a specialist in the disease or a psychiatrist, must be consulted and must perform the same tasks as the other consultant. After euthanasia has been performed, the attending physician must report it to the Federal Control and Evaluation Commission on Euthanasia using an available standard form, which serves as an a posteriori control mechanism. In 2009, for instance, a total of 822 euthanasia cases were officially reported to this commission
[3]. A large scale population-based representative study from 2007 estimated the total number of possible euthanasia cases to be almost twice the number of reported cases (although unreported cases were often not considered to be euthanasia by the physician)
[4]. Concerns have been raised about the actual adherence to the legal criteria of both the a priori consultation and a posteriori reporting
[5].
While the legislator regulated the consultation procedure in euthanasia requests it did not accommodate this procedure, for instance by providing adequate training for physicians to assess the due care criteria and other complex circumstances that would make a euthanasia legally acceptable. In Flanders, the Dutch speaking part of Belgium, a special service called Life End Information Forum (LEIF) was then established as a non-governmental initiative to provide information and training for health care professionals in end-of-life care matters such as euthanasia, since many did not have any experience in these matters
[6-9]. In 2003, the forum started organizing specific training for physicians to obtain the necessary skills and knowledge to act as independent consultants in euthanasia requests
[6]. The idea was that if a second physician was needed to appraise a request, that physician better had the necessary qualifications regarding both euthanasia and palliative care. As palliative care receives insufficient attention in the regular medical curriculum, and basic palliative care skills and knowledge were deemed necessary to evaluate options in a euthanasia request the training also paid attention to palliative care apart from euthanasia
[10-14]. On completing the training, these physicians become LEIF physicians. Attending physicians who receive a euthanasia request and who want to consult with an independent physician can call a central telephone number and a LEIF physician within their region is then assigned to them, or they can contact a LEIF physician directly. The Belgian euthanasia law does not specify, of course, that the consultant has to be a LEIF physician. LEIF is a Dutch speaking initiative and hence serves the Flemish speaking community, which lives in the regions of Flanders and the official bilingual Brussels Capital Region. No initiative of similar magnitude and organisation like LEIF is present in Wallonia.
Previously published data on how euthanasia requests are granted or not in Belgium have demonstrated that the advice of a second physician (the consultant) plays a key role in a euthanasia request being granted
[15]. In this study, we want to examine how often the consultant is a LEIF physician and whether consultation with a LEIF physician improves the quality of the consultation, by answering the following research questions:
1) How often is a LEIF physician consulted in euthanasia requests in Flanders and Brussels - the area covered by the LEIF physicians- and what are characteristics of the attending physician associated with consulting a LEIF physician instead of a non-LEIF physician?
2) To what extent are the legal requirements met in consultations with a second physician and is there a difference between those with LEIF and non-LEIF physicians?
3) Is there a difference in the consultations between LEIF and non-LEIF physicians in relation to additional non-mandatory consultation features (e.g. conversation with the family) and the outcome of the consultation (e.g. judgement about whether due care criteria were met and whether euthanasia was performed)?
Conclusion:
In cases of explicit euthanasia requests in Belgium, the consultation of an independent physician by the attending physician is not optimal and can be improved. Firstly, the proportion of consultations should be higher and secondly, there should be the required independence between the consultant and attending physician. The drafting of a written report about the judgement regarding the eligibility of the euthanasia request also needs to be more standardly performed by consultants. Although other legally prescribed consultation activities, such as a conversation or at least an examination of the patient, are almost always abided by, the norm should be 100%.
As we have demonstrated in this study, a service like the Life End Information Forum (LEIF) can contribute in some respects, by providing independent consultants but also by educating physicians on the consultation procedure in euthanasia requests. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Based on our findings a similar initiative to LEIF for French speaking physicians in Belgium seems warranted. While our findings have particular relevance to Belgium and other countries or regions having or considering legislation on euthanasia or assisted suicide, we believe that our findings have a wider relevance in pointing out how a consultation service and specific consultation training for various difficult end-of-life situations can contribute to end-of-life decision-making. | Background:
Following the 2002 enactment of the Belgian law on euthanasia, which requires the consultation of an independent second physician before proceeding with euthanasia, the Life End Information Forum (LEIF) was founded which provides specifically trained physicians who can act as mandatory consultants in euthanasia requests. This study assesses quality of consultations in Flanders and Brussels and compares these between LEIF and non-LEIF consultants.
Methods:
A questionnaire was sent in 2009 to a random sample of 3,006 physicians in Belgium from specialties likely involved in the care of dying patients. Several questions about the last euthanasia request of one of their patients were asked. As LEIF serves the Flemish speaking community (i.e. region of Flanders and the bilingual Brussels Capital Region) and no similar counterpart is present in Wallonia, analyses were limited to Flemish speaking physicians in Flanders and Brussels.
Results:
Response was 34%. Of the 244 physicians who indicated having received a euthanasia request seventy percent consulted a second physician in their last request; in 30% this was with a LEIF physician. Compared to non-LEIF physicians, LEIF physicians were more often not a colleague (69% vs 42%) and not a co-attending physician (89% vs 66%). They tended to more often discuss the request with the attending physician (100% vs 95%) and with the family (76% vs 69%), and also more frequently helped the attending physician with performing euthanasia (44% vs 24%). No significant differences were found in the extent to which they talked to the patient (96% vs 93%) and examined the patient file (94% vs 97%).
Conclusions:
In cases of explicit euthanasia requests in Belgium, the consultation procedure of another physician by the attending physician is not optimal and can be improved. Training and putting at disposal consultants through forums such as LEIF seems able to improve this situation. Adding stipulations in the law about the necessary competencies and tasks of consulting physicians may additionally incite improvement. Irrespective of whether euthanasia is a legal practice within a country, similar services may prove useful to also improve quality of consultations in various other difficult end-of-life decision-making situations. | 8,968 | 427 | [
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belgian | attending | request | non | attending physician | cases [SUMMARY] | [CONTENT] physician | leif | euthanasia | physicians | belgian | attending | request | non | attending physician | cases [SUMMARY] | [CONTENT] 2002 | Belgian | second | the Life End Information Forum | euthanasia ||| Flanders | Brussels [SUMMARY] | [CONTENT] 2009 | 3,006 | Belgium ||| one ||| Flemish | Flanders | Brussels Capital Region | Wallonia | Flemish | Flanders | Brussels [SUMMARY] | [CONTENT] Response | 34% ||| 244 | seventy percent | second | 30% ||| 69% | 42% | 89% | 66% ||| 100% | 95% | 76% | 69% | 44% | 24% ||| 96% | 93% | 94% | 97% [SUMMARY] | [CONTENT] Belgium ||| ||| ||| [SUMMARY] | [CONTENT] 2002 | Belgian | second | the Life End Information Forum | euthanasia ||| Flanders | Brussels ||| 2009 | 3,006 | Belgium ||| one ||| Flemish | Flanders | Brussels Capital Region | Wallonia | Flemish | Flanders | Brussels ||| Response | 34% ||| 244 | seventy percent | second | 30% ||| 69% | 42% | 89% | 66% ||| 100% | 95% | 76% | 69% | 44% | 24% ||| 96% | 93% | 94% | 97% ||| Belgium ||| ||| ||| [SUMMARY] | [CONTENT] 2002 | Belgian | second | the Life End Information Forum | euthanasia ||| Flanders | Brussels ||| 2009 | 3,006 | Belgium ||| one ||| Flemish | Flanders | Brussels Capital Region | Wallonia | Flemish | Flanders | Brussels ||| Response | 34% ||| 244 | seventy percent | second | 30% ||| 69% | 42% | 89% | 66% ||| 100% | 95% | 76% | 69% | 44% | 24% ||| 96% | 93% | 94% | 97% ||| Belgium ||| ||| ||| [SUMMARY] |
||||||
Knowledge, preventive behavior and risk perception regarding COVID-19: a self-reported study on college students. | 33623599 | There are a limited number of studies on the issues associated with the knowledge and self-practice preventive measures for COVID-19 among medical students. We aimed to determine the extent of knowledge, self-reported preventive behavior, and risk perception of the COVID-19 outbreak among college students in Libya. | INTRODUCTION | A cross-sectional study was conducted from April 20 to April 30, 2020. The participants were students of medical and non-medical subjects from Libyan educational institutes. Data on participants' characteristics, knowledge, preventive behavior, and risk perception were collected. | METHODS | Approximately 3669 participants completed the questionnaire, of which 2547 (69.4) were medical students and 1122 (30.6%) were non-medical students. The mean knowledge score on COVID-19 was 8.62 (SD: 1.26, range: 0-12), corresponding to 71.8% correct answers. A significant difference was observed between medical and non-medical students in terms of knowledge (p < 0.001). Overall, the knowledge score of the students differed significantly with respect to age, current year of study, and financial source (p < 0.05). The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. | RESULTS | Notably, college students were observed to have substantial knowledge, preventive behavior, and a positive attitude toward COVID-19. Government programs should aim to educate individuals from other sectors of the society to ensure the proper dissemination of knowledge on preventive safety measures, as this will help restrict and control the pandemic. | CONCLUSION | [
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] | 7875787 | Introduction | The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].
The development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.
Medical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya. | Methods | Study setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.
COVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.
Preventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.
Statistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.
Ethical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study. | Results | Out of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.
Characteristics of individuals in the study
Questionnaire on knowledge toward COVID-19
Differences in knowledge score according to study characteristics
The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.
Questionnaire on preventive behavior and attitude toward COVID-19
Table 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).
Differences in preventive score according to study characteristics | Conclusion | Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.
What is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
What this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.
Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups. | [
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"The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.",
"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups."
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] | [
"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].\nThe development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.\nMedical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.",
"Study setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.\nCOVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.\nPreventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.\nStatistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.\nEthical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study.",
"Out of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.\nCharacteristics of individuals in the study\nQuestionnaire on knowledge toward COVID-19\nDifferences in knowledge score according to study characteristics\nThe mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.\nQuestionnaire on preventive behavior and attitude toward COVID-19\nTable 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).\nDifferences in preventive score according to study characteristics",
"To the best of our knowledge, this is the first study conducted in Africa that discusses knowledge, self-preventive measures, and attitudes toward COVID-19 among college students, who constitute an integral part of an educated society. Overall, 71.8% of the study participants were aware of COVID-19, while more than 92.7% of both medical and non-medical students were taking preventive measures against COVID-19 to reduce the risk of infection transmission. Our study provides an overview of the knowledge of medical students, who will work as physicians in future, and are currently engaged in hospitals and at a high risk of infection transmission; the study compared their knowledge, preventive measures, and attitude toward COVID-19 to that of non-medical students to determine the difference. A majority of the participants (87.7% of non-medical students and 84.8% of medical students) opined that the COVID-19 pandemic could be controlled successfully, while a lower percentage of participants (74.6% non-medical students and 73.5% medical students) believed that the disease could be controlled in Libya. Contrastingly, the preventive behavior score among students was encouraging, as indicated by a high mean score of 7.4 ± 0.93 in non-medical students and 7.43 ± 0.96 of medical student out of 8, which corresponds to approximately 7.4/8 *100 = 92.5% of non-medical student and 7.43/8 *100 = 92.87% of medical students; this suggests that the students have a positive preventive behavior and attitude toward COVID-19. Students in their internship year had better knowledge and greater preventive behavior owing to the extent of time they spend at the medical department, where they work at a higher risk of exposure and encounter more cases. Therefore, their knowledge and attitude are attributed to their experience.\nApproximately 97% participants from both groups reported avoidance of crowds and shopping, which displays the high levels of caution among the individuals. In addition, we identified several factors that can be associated with the preventive behavior and knowledge of the participants toward COVID-19. Our findings are necessary to understand the current state of public health awareness and to determine the need for proper dissemination of knowledge and awareness among students who are medical practitioners of the future and may possibly come in contact with infected individuals during their rotation in the hospitals. As medical students are at a higher risk of contracting infection and disease transmission owing to the course of study in hospitals, their knowledge and awareness of preventive measures are crucial for reducing the risk of community spread of SARS-CoV-2. Despite the healthcare situation and civil war in Libya, college students achieved high scores in knowledge, preventive behavior, and attitude toward COVID-19 infection. There was no significant difference between the rates of correct answers to most questions asked to either medical or non-medical students, which displays the substantial overall knowledge among college students. Their level of knowledge, preventive behavior, and attitude owing to their active learning and sources of information on COVID-19 yielded positive results. A similar study conducted on a Chinese population reported an overall knowledge of 90% [18]. Another unpublished study conducted by Haque et al. on a Bangladeshi population reported an overall knowledge score of 54.87% toward COVID-19 [19]. In addition, another study conducted on the Nepalese population reported an overall knowledge score ranging from 60.0-98.7% and attitude scores ranging from 77.9-96.4%. The study also confirmed that participants with a medical degree had greater knowledge than non-medical participants [20]. Another study conducted on an Egyptian population comprising 559 participants reported the mean and standard deviation of knowledge score as 16.39±2.63, ranging from 7 to 22, which corresponds to approximately 74.5% overall knowledge among participants regarding COVID-19 [17]. Another study in Thailand reported 67.9% as the overall level of knowledge toward COVID-19 prevention and only 29.5% were reported to have substantial knowledge about the preventive measures for COVID-19, which is lower than that in our study findings [21]. Another study conducted among Iranian medical students showed that 79.6% of medical students had adequate knowledge about COVID-19 [14].\nOur study reported high scores of attitude and preventive behavior toward COVID-19, which corresponds to a lower potential risk rate for COVID-19. In addition, more than 88% of the study participants reported discussing the preventive measures with their families and friends, which can effectively increase awareness about the current situation. This indicates the importance of health education that could improve the prevention behavior toward COVID-19 in society. The health education approach is a common method adopted for reducing the risk of transmission of infectious diseases. The study had a large sample size and involved surveying students from approximately 15 colleges and universities during the COVID-19 outbreak in Libya. In addition, our sample was representative of those from major cities, and the study yielded a generalized result. However, our sample predominantly comprised female students, 2923 (79.7%) overall, between the two groups. In addition, we included only college students who are likely to have better knowledge and information about COVID-19. Therefore, this may have limited our estimation of the knowledge of people who do not have access to a college education or the general population. Additionally, the comparison between medical and non-medical students did not display significant differences for most questions despite the fact that medical students were more likely to have greater knowledge owing to the nature of their profession and academic pursuit; this could be attributed to the larger sample size of medical students, where 69.4% of the sample size comprised medical students and 30.6% were non-medical students. However, our study covered populations from major cities and universities and can be considered as a generalized representation of the Libyan population irrespective of residence state.",
"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.\nWhat is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nThe extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.\nWhat this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.\nOur study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.",
"The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;\nSeveral studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.",
"Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;\nThere was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.",
"The authors declare no competing interests."
] | [
"intro",
"methods",
"results",
"discussion",
"conclusion",
null,
null,
"COI-statement"
] | [
"SARS-CoV-2",
"knowledge",
"public health",
"COVID-19",
"behavior",
"pandemic",
"awareness"
] | Introduction:
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].
The development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.
Medical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.
Methods:
Study setting: a cross-sectional survey was conducted between April 20 and April 30, 2020 with data submitted by 5000 medical and non-medical students using a Google form shared through email, mobile messages, and in print with students from 15 universities and colleges in Libya. The survey was conducted anonymously, and the participants were unaware of the study outcomes and provided consent after the study designed was explained. Questions on demographic data, including age, gender, type of study, year of study, availability of a stable financial source, the effect of civil war on COVID-19, and on a friend/family member with COVID-19 were included. Missing data or incomplete answers were excluded from the study. Additionally, the study participants were medical and non-medical students from the following disciplines; dentistry, pharmacy, medical technology, nursing, and veterinary sciences.
COVID-19-related knowledge: the extent of COVID-19-related knowledge was assessed in the first section through 12 items based on an earlier study on COVID-19 in China [18]. The questions were related to the knowledge of COVID-19, which consisted of two questions on the signs and symptoms, one on treatment, one on disease progression, three on disease transmission, three on prevention, and two on isolation care. Each correct answer was awarded 1 point, while incorrect answers or unanswered questions were awarded 0. The total knowledge score was 12. Reliability was calculated based on a Cronbach´s alpha value of 0.87.
Preventive behavior and attitude: approximately eight items from a previous survey were used to assess the preventive behavior [14]. Two questions were related to avoidance of public gathering, one on reducing the use of public transport, one on limiting shopping, one on cough etiquette, two on cleaning and hand hygiene practices, and one on the discussion of preventive measures with family members and acquaintances. Choosing the option “yes” added one point, while 0 was added for a “no” in each behavior-related question. The maximum prevention score was 8, with a score >6 indicating higher preventive behavior and appropriate awareness with respect to COVID-19. According to the method followed in a previous study, reliability was calculated based on Cronbach´s alpha value of 0.72. Two questions were added on the attitude toward COVID-19 control ability.
Statistical analysis: descriptive statistical measures, including frequency, percentage, and mean score, were used to report the findings. The chi-square test was used to determine the association between the categories. The independent-samples t-test was used to determine the difference between the means of two independent groups. A one-way ANOVA was calculated on participants’ score of knowledge and preventive behavior. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Windows, Version 25.0; IBM Corp., Armonk, NY). P-value < 0.05 was considered statistically significant.
Ethical consideration: the ethical approval to conduct this study was obtained from the Bioethics Committee at the Biotechnology Research Center in Libya [Reference number: BEC-BTRC-141.4-8-2020]. All participants provided informed consent before participating in the study.
Results:
Out of the 5000 participants who received the questionnaire, approximately 3669 participants completed the questionnaire and provided completed data, with a response rate of 73.38% among the total study participants. Among those included, the mean age was 22.77 years (standard deviation (SD): 2.83, range: 17-30); out of 3669 included, 2923 (79.7%) were women, while 746 (20.3%) were male. The study participants included 2547 (69.4) medical students and 1122 (30.6%) non-medical students. The non-medical students were as follows: dentistry: 350 (8.3); pharmacy: 288 (7.8); medical technology: 271 (7.4); nursing: 250 (6.8); veterinary science: 8(0.2). The baseline characteristics are presented in Table 1. The mean score of knowledge on COVID-19 (out of 12) was 8.62 (SD: 1.26, range: 0-12), and the estimated overall knowledge was approximately 8.62/12 * 100, which corresponded to 71.8% correct answers. However, there was a significant difference between the answers provided by medical and non-medical students (p < 0.001). The mean ± SD score was 8.51 ± 1.15 for non-medical students and 8.67 ± 1.3 for medical students. In addition, for the answers to 9 of the 12 questions on knowledge, there was a significant difference between medical and non-medical students, where medical students provided correct answers at a higher rate. More than 94% of both categories of students were aware of the signs and symptoms of COVID-19. Additionally, there was a significant difference between the answers provided by medical and non-medical students to the question on disease progression; 95.1% of medical students were aware of the disease progression characteristics compared to 91.4% non-medical students. More than 97% of individuals from both groups were aware of the necessity of public transport avoidance to reduce the risk of transmission. Approximately 97% of medical students and 98% of non-medical students were aware of the 14-day isolation process. Table 2 presents an overview of the knowledge questions and answer rate differences between the two groups. Overall, the knowledge score for the students differed significantly based on age, the current year of study, and status of financial source (p < 0.05). Among non-medical students, the score was not significantly different across the baseline factors. Among medical students, the age, current year of study, financial source, and COVID-19 patient among friends and/or family members differed significantly between the groups. Table 3 presents an overview of the differences in knowledge between the groups with respect to the characteristics.
Characteristics of individuals in the study
Questionnaire on knowledge toward COVID-19
Differences in knowledge score according to study characteristics
The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students. Table 4 presents an overview of the questions on preventive measures and the answer rate differences between the two groups. However, there was a statistically significant difference between the two groups with respect to two questions. Approximately 97% individuals from both groups reported reducing the use of public transport and shopping activities. Approximately 96% of individuals from both groups reported avoiding coughing around other individuals and practicing cough etiquette. More than 98% of them reported avoidance of crowded places or places with mass gatherings. In addition, approximately 87% non-medical students and 84% medical students reported an increase in the use of cleaning and disinfecting items. In addition, more than 91% of all students reported washing their hands more frequently than usual. In addition, more than 88% reported engaging in discussions on COVID-19 prevention with their friends and family members.
Questionnaire on preventive behavior and attitude toward COVID-19
Table 5 outlines the difference in major characteristics according to the difference in the mean scores from the preventive measure questionnaire. There was a significant difference with respect to gender and age (p < 0.05) in the overall group. Among the non-medical students, the mean difference between the categories was statistically significant with respect to gender, financial source, and having a COVID-19-infected family member and/or friend. Among the medical students, there was a statistically significant difference with respect to age, gender, and year of study. Table 5 presents an overview of the differences in preventive behaviors based on the study characteristics. Approximately 84% of the non-medical students and 87.2% of the medical students reported canceling or postponing their meetings and outdoor activities as a preventive measure. The majority of participants agreed with the statement that COVID-19 could be controlled (87.7% of non-medical students and 84.8% of medical students). Approximately 74.6% of non-medical students and 73.5% of medical students agreed with the statement that the COVID-19 outbreak in Libya could be controlled. However, there was a significant association of the attitude score with both genders and with the financial status (P < 0.05). The categories of age, and having a friend/family member with COVID-19 had no significant association with the attitude score (p > 0.05).
Differences in preventive score according to study characteristics
Discussion:
To the best of our knowledge, this is the first study conducted in Africa that discusses knowledge, self-preventive measures, and attitudes toward COVID-19 among college students, who constitute an integral part of an educated society. Overall, 71.8% of the study participants were aware of COVID-19, while more than 92.7% of both medical and non-medical students were taking preventive measures against COVID-19 to reduce the risk of infection transmission. Our study provides an overview of the knowledge of medical students, who will work as physicians in future, and are currently engaged in hospitals and at a high risk of infection transmission; the study compared their knowledge, preventive measures, and attitude toward COVID-19 to that of non-medical students to determine the difference. A majority of the participants (87.7% of non-medical students and 84.8% of medical students) opined that the COVID-19 pandemic could be controlled successfully, while a lower percentage of participants (74.6% non-medical students and 73.5% medical students) believed that the disease could be controlled in Libya. Contrastingly, the preventive behavior score among students was encouraging, as indicated by a high mean score of 7.4 ± 0.93 in non-medical students and 7.43 ± 0.96 of medical student out of 8, which corresponds to approximately 7.4/8 *100 = 92.5% of non-medical student and 7.43/8 *100 = 92.87% of medical students; this suggests that the students have a positive preventive behavior and attitude toward COVID-19. Students in their internship year had better knowledge and greater preventive behavior owing to the extent of time they spend at the medical department, where they work at a higher risk of exposure and encounter more cases. Therefore, their knowledge and attitude are attributed to their experience.
Approximately 97% participants from both groups reported avoidance of crowds and shopping, which displays the high levels of caution among the individuals. In addition, we identified several factors that can be associated with the preventive behavior and knowledge of the participants toward COVID-19. Our findings are necessary to understand the current state of public health awareness and to determine the need for proper dissemination of knowledge and awareness among students who are medical practitioners of the future and may possibly come in contact with infected individuals during their rotation in the hospitals. As medical students are at a higher risk of contracting infection and disease transmission owing to the course of study in hospitals, their knowledge and awareness of preventive measures are crucial for reducing the risk of community spread of SARS-CoV-2. Despite the healthcare situation and civil war in Libya, college students achieved high scores in knowledge, preventive behavior, and attitude toward COVID-19 infection. There was no significant difference between the rates of correct answers to most questions asked to either medical or non-medical students, which displays the substantial overall knowledge among college students. Their level of knowledge, preventive behavior, and attitude owing to their active learning and sources of information on COVID-19 yielded positive results. A similar study conducted on a Chinese population reported an overall knowledge of 90% [18]. Another unpublished study conducted by Haque et al. on a Bangladeshi population reported an overall knowledge score of 54.87% toward COVID-19 [19]. In addition, another study conducted on the Nepalese population reported an overall knowledge score ranging from 60.0-98.7% and attitude scores ranging from 77.9-96.4%. The study also confirmed that participants with a medical degree had greater knowledge than non-medical participants [20]. Another study conducted on an Egyptian population comprising 559 participants reported the mean and standard deviation of knowledge score as 16.39±2.63, ranging from 7 to 22, which corresponds to approximately 74.5% overall knowledge among participants regarding COVID-19 [17]. Another study in Thailand reported 67.9% as the overall level of knowledge toward COVID-19 prevention and only 29.5% were reported to have substantial knowledge about the preventive measures for COVID-19, which is lower than that in our study findings [21]. Another study conducted among Iranian medical students showed that 79.6% of medical students had adequate knowledge about COVID-19 [14].
Our study reported high scores of attitude and preventive behavior toward COVID-19, which corresponds to a lower potential risk rate for COVID-19. In addition, more than 88% of the study participants reported discussing the preventive measures with their families and friends, which can effectively increase awareness about the current situation. This indicates the importance of health education that could improve the prevention behavior toward COVID-19 in society. The health education approach is a common method adopted for reducing the risk of transmission of infectious diseases. The study had a large sample size and involved surveying students from approximately 15 colleges and universities during the COVID-19 outbreak in Libya. In addition, our sample was representative of those from major cities, and the study yielded a generalized result. However, our sample predominantly comprised female students, 2923 (79.7%) overall, between the two groups. In addition, we included only college students who are likely to have better knowledge and information about COVID-19. Therefore, this may have limited our estimation of the knowledge of people who do not have access to a college education or the general population. Additionally, the comparison between medical and non-medical students did not display significant differences for most questions despite the fact that medical students were more likely to have greater knowledge owing to the nature of their profession and academic pursuit; this could be attributed to the larger sample size of medical students, where 69.4% of the sample size comprised medical students and 30.6% were non-medical students. However, our study covered populations from major cities and universities and can be considered as a generalized representation of the Libyan population irrespective of residence state.
Conclusion:
Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.
What is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
What this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.
Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.
What is known about this topic:
The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
What this study adds:
Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.
Competing interests:
The authors declare no competing interests.
| Background:
There are a limited number of studies on the issues associated with the knowledge and self-practice preventive measures for COVID-19 among medical students. We aimed to determine the extent of knowledge, self-reported preventive behavior, and risk perception of the COVID-19 outbreak among college students in Libya.
Methods:
A cross-sectional study was conducted from April 20 to April 30, 2020. The participants were students of medical and non-medical subjects from Libyan educational institutes. Data on participants' characteristics, knowledge, preventive behavior, and risk perception were collected.
Results:
Approximately 3669 participants completed the questionnaire, of which 2547 (69.4) were medical students and 1122 (30.6%) were non-medical students. The mean knowledge score on COVID-19 was 8.62 (SD: 1.26, range: 0-12), corresponding to 71.8% correct answers. A significant difference was observed between medical and non-medical students in terms of knowledge (p < 0.001). Overall, the knowledge score of the students differed significantly with respect to age, current year of study, and financial source (p < 0.05). The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students.
Conclusions:
Notably, college students were observed to have substantial knowledge, preventive behavior, and a positive attitude toward COVID-19. Government programs should aim to educate individuals from other sectors of the society to ensure the proper dissemination of knowledge on preventive safety measures, as this will help restrict and control the pandemic. | Introduction:
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in the Hubei Province of China as the causative agent of coronavirus disease (COVID-19), a form of severe viral pneumonia, in December 2019 [1,2]. This was followed by rapid transmission worldwide, and soon the disease was declared a pandemic by the World Health Organization (WHO) in February 2020 [3]. On April 22, 2020, the WHO announced that 2.5 million cases with more than 160,00 deaths had been recorded [4]. The probability of transmission of COVID-19 differs based on the form and duration of exposure. Therefore, in areas where population dissemination is prevalent, preventive measures are necessary to reduce the chances of unintended exposure in the COVID-19 risk population [5-7]. Such steps are necessary to determine the cases of infection and for the rapid assessment and monitoring of COVID-19 transmission sources, and these could slow the spread of infection. These steps require the understanding of and precautions taken by the public to prevent the spread of the infection, and include measures such as social distancing, avoiding public gathering, and avoiding direct contact with suspected carriers [8,9]. In addition, several preventive measures are suggested to reduce the risk of transmission and the subsequent risk of disease, such as hand washing, avoiding touching the face, covering mouth while coughing, self-quarantine in cases of contact with individuals suspected or confirmed as COVID-19-positive, and disinfecting surfaces that may harbor the pathogen [10,11].
The development of vaccines against SARS-CoV-2 faces several financial, scientific, and ethical challenges, and may require several months of research and testing [12,13]. Therefore, preventive measures and risk perceptions are required to limit community transmission until vaccine and drug development processes reach a stage at which the prevention and control COVID-19 community transmission can be realized. A limited number of researchers have addressed issues such as the knowledge and self-practice preventive measures for COVID-19 among medical students [14]. Several studies have focused on healthcare workers rather than on communities and individuals [15,16], while certain studies have focused on community knowledge, attitude, and practice toward prevention of COVID-19 infection [17,18]. Students of medicine are expected to have substantial knowledge and awareness compared to the public as they are at a higher risk of contracting the virus owing to the nature of their work. Therefore, the risk of transmission should not be underestimated, particularly since healthcare workers could be a potential source of disease transmission in the community.
Medical students are the healthcare professionals of the future. Therefore, their knowledge and self-prevention practices should be assessed to ensure the effectiveness of preventive measures on COVID-19 outbreaks and community transmission. Moreover, compliance with preventive measures is a necessity and depends on the knowledge, preventive behavior, and risk perception of COVID-19 among individuals. There is a compelling need to understand the importance of general awareness about COVID-19 to promote COVID-19 control in countries and facilitate preventive measures. Major concerns have been raised with respect to the effects of these measures on controlling the spread of disease. This paper aims to determine the knowledge, self-reported preventive behaviors, and risk perception of the COVID-19 outbreak among medical students in Libya.
Conclusion:
Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19. A majority of them also expressed their optimism regarding the control of COVID-19. In addition, there was a significant difference between the scores of the medical and non-medical student groups; however, the majority of correct answers did not differ significantly. Government programs should aim to educate individuals from other sectors of the society and the general population to ensure that preventive safety measures are adopted, as this will help in controlling the pandemic.
What is known about this topic The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
The extent of community knowledge, attitude, and preventive behavior in COVID-19 are major concerns;
Several studies have reported knowledge, attitude, and preventive behavior among individuals without specific emphasis on college students.
What this study adds Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups.
Our study revealed that college students have substantial knowledge, preventive behavior awareness, and a positive attitude toward COVID-19;
There was a significant difference between the knowledge, preventive behavior, and positive attitude scores between medical and non-medical student groups. | Background:
There are a limited number of studies on the issues associated with the knowledge and self-practice preventive measures for COVID-19 among medical students. We aimed to determine the extent of knowledge, self-reported preventive behavior, and risk perception of the COVID-19 outbreak among college students in Libya.
Methods:
A cross-sectional study was conducted from April 20 to April 30, 2020. The participants were students of medical and non-medical subjects from Libyan educational institutes. Data on participants' characteristics, knowledge, preventive behavior, and risk perception were collected.
Results:
Approximately 3669 participants completed the questionnaire, of which 2547 (69.4) were medical students and 1122 (30.6%) were non-medical students. The mean knowledge score on COVID-19 was 8.62 (SD: 1.26, range: 0-12), corresponding to 71.8% correct answers. A significant difference was observed between medical and non-medical students in terms of knowledge (p < 0.001). Overall, the knowledge score of the students differed significantly with respect to age, current year of study, and financial source (p < 0.05). The mean score of preventive behavioral measures toward COVID-19 (out of 8) was 7.42 (SD: 0.95, range: 0-8), and the overall preventive measure score was estimated to be approximately 7.42/8*100, which corresponds to 92.7% for both medical and non-medical students.
Conclusions:
Notably, college students were observed to have substantial knowledge, preventive behavior, and a positive attitude toward COVID-19. Government programs should aim to educate individuals from other sectors of the society to ensure the proper dissemination of knowledge on preventive safety measures, as this will help restrict and control the pandemic. | 3,748 | 335 | [
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] | [CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY] | [CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY] | [CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY] | [CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY] | [CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY] | [CONTENT] SARS-CoV-2 | knowledge | public health | COVID-19 | behavior | pandemic | awareness [SUMMARY] | [CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY] | [CONTENT] Adolescent | Adult | COVID-19 | Cross-Sectional Studies | Disease Outbreaks | Female | Health Knowledge, Attitudes, Practice | Humans | Libya | Male | Perception | Self Report | Students | Students, Medical | Surveys and Questionnaires | Universities | Young Adult [SUMMARY] | [CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY] | [CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY] | [CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY] | [CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY] | [CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY] | [CONTENT] coronavirus disease covid | awareness covid 19 | exposure covid 19 | prevention covid | covid 19 outbreaks [SUMMARY] | [CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY] | [CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY] | [CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY] | [CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY] | [CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY] | [CONTENT] medical | students | 19 | covid | covid 19 | knowledge | preventive | medical students | study | non [SUMMARY] | [CONTENT] covid 19 | covid | 19 | risk | transmission | measures | preventive measures | preventive | infection | self [SUMMARY] | [CONTENT] study | related | questions | score | covid 19 | 19 | covid | calculated | added | survey [SUMMARY] | [CONTENT] medical students | medical | students | non medical students | non medical | non | characteristics | score | approximately | table [SUMMARY] | [CONTENT] preventive | positive attitude | knowledge | attitude | preventive behavior | behavior | knowledge attitude preventive | knowledge attitude preventive behavior | college | college students [SUMMARY] | [CONTENT] medical | knowledge | preventive | students | 19 | covid 19 | covid | study | medical students | behavior [SUMMARY] | [CONTENT] medical | knowledge | preventive | students | 19 | covid 19 | covid | study | medical students | behavior [SUMMARY] | [CONTENT] COVID-19 ||| COVID-19 | Libya [SUMMARY] | [CONTENT] April 20 to April 30, 2020 ||| Libyan ||| [SUMMARY] | [CONTENT] Approximately 3669 | 2547 | 69.4 | 1122 | 30.6% ||| COVID-19 | 8.62 | 1.26 | 0-12 | 71.8% ||| ||| ||| COVID-19 | 8) | 7.42 | 0.95 | 0-8) | approximately 7.42/8*100 | 92.7% [SUMMARY] | [CONTENT] COVID-19 ||| [SUMMARY] | [CONTENT] COVID-19 ||| COVID-19 | Libya | April 20 to April 30, 2020 ||| Libyan ||| ||| Approximately 3669 | 2547 | 69.4 | 1122 | 30.6% ||| COVID-19 | 8.62 | 1.26 | 0-12 | 71.8% ||| ||| ||| COVID-19 | 8) | 7.42 | 0.95 | 0-8) | approximately 7.42/8*100 | 92.7% ||| COVID-19 ||| [SUMMARY] | [CONTENT] COVID-19 ||| COVID-19 | Libya | April 20 to April 30, 2020 ||| Libyan ||| ||| Approximately 3669 | 2547 | 69.4 | 1122 | 30.6% ||| COVID-19 | 8.62 | 1.26 | 0-12 | 71.8% ||| ||| ||| COVID-19 | 8) | 7.42 | 0.95 | 0-8) | approximately 7.42/8*100 | 92.7% ||| COVID-19 ||| [SUMMARY] |
||||||
Excessive proinflammatory cytokine and chemokine responses of human monocyte-derived macrophages to enterovirus 71 infection. | 22994237 | The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms. | BACKGROUND | Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR. | METHODS | Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs. | RESULTS | Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs. | CONCLUSIONS | [
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"Enterovirus A, Human",
"Gene Expression Profiling",
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"Interferon-Induced Helicase, IFIH1",
"Macrophages",
"Real-Time Polymerase Chain Reaction",
"Receptors, Immunologic",
"Toll-Like Receptors",
"Young Adult"
] | 3519709 | Background | Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].
Although the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.
Previous studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection. | Methods | Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.
Peripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.
The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.
Peripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.
Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.
Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.
Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.
MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.
Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.
Primer sequences and probes used in real-time PCR assay
Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.
Primer sequences and probes used in real-time PCR assay
Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).
Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).
Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.
Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant. | Results | Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.
A dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.
Viral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.
We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.
A dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.
Viral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.
Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.
Proinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.
Proinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).
Chemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).
Chemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.
A higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).
In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.
A higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01). | Conclusions | In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed. | [
"Background",
"Effective infection and viral replication of EV71 in human MDMs",
"Enhanced proinflammatory cytokine responses of human MDMs to EV71",
"Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71",
"Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs",
"Isolation and culture of monocyte-derived macrophages",
"Infection of MDMs with EV71",
"Immunofluorescence assay of viral VP1 protein in infected cells",
"Quantification of mRNA by real-time RT-PCR",
"Measurement of cytokines",
"Statistical analysis",
"Abbreviations",
"Competing interests",
"Authors' contributions",
"Pre-publication history"
] | [
"Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.",
"We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.",
"We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).",
"Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).",
"In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).",
"The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.",
"Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.",
"MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.",
"Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay",
"Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).",
"Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.",
"(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.",
"The authors declare that they have no competing interests.",
"Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\n"
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Results",
"Effective infection and viral replication of EV71 in human MDMs",
"Enhanced proinflammatory cytokine responses of human MDMs to EV71",
"Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71",
"Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs",
"Discussion",
"Conclusions",
"Methods",
"Isolation and culture of monocyte-derived macrophages",
"Infection of MDMs with EV71",
"Immunofluorescence assay of viral VP1 protein in infected cells",
"Quantification of mRNA by real-time RT-PCR",
"Measurement of cytokines",
"Statistical analysis",
"Abbreviations",
"Competing interests",
"Authors' contributions",
"Pre-publication history"
] | [
"Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].\nAlthough the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.\nPrevious studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.",
" Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\nWe first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.\n Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nWe subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\nChemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).\n Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).\nIn order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).",
"We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.\nA dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.\nViral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.",
"We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.\nProinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).",
"Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).\nChemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).",
"In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.\nA higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).",
"Macrophages, which are shown to support the infection of various viruses including HIV-1, influenza viruses, and poliovirus and so on, play a critical role in presentation of antigens, pathogen clearance, and induction of inflammation during the early phase of viral infection [21-23]. In this study, both viral gene and antigen of EV71 were detected. The increases of virus yields and the number of viral gene copies were observed in EV71-infected MDMs between the 6-h and 48-h POI. Excess cytokine and chemokine responses of MDMs were triggered by EV71. These findings suggested that macrophages may be not only the target cells but also the effectors during EV71 infection.\nIt is controversial whether TNF-α was involved in fatal EV71 infection. Significant or minor change of blood TNF-α in EV71 patient with both encephalitis and PE was reported by different clinical studies [24,25]. Significant stronger release of TNF-α from EV71-infected MDMs at 12-h POI than 24-h POI in the study, implying that TNF-α was induced by EV71 infection at early stage and maybe involved in its pathology. Consistent with clinical features of EV71 patients with encephalitis and PE, who are presented with higher levels of the proinflammatory cytokines in blood [9,24], IL-6, IL-1β and TNF-α were induced in EV71-infected MDMs. These proinflammatory cytokines are thought to be the potent pyrogens inducing fever, and the magnitude of febrile response correlates with the level of virus shedding in human and animals [26]. Notably, a transient increase of blood brain barrier (BBB) permeability and its injury were found at early stage of EV71 infection [27]. The pathology may be due to an augmentation of systemic and local TNF-α production, which exhibits detrimental effects by enhancing cell infiltration, cytopathic damage, or functioning as a paracellular pathway for the virus across the BBB [28]. Furthermore, the subsequent responses of acute phase proteins and chemokine activations mediated by IL-6, IL-1β and TNF-α could exacerbate virus induced inflammation and pathology [29]. Therefore, the rapid and strong proinflammatory response of MDMs to EV71 may partially explain the clinical severity.\nMacrophages or plasmacytoid DCs, specialized in secreting large amounts of type I IFN after virus infection, play an important role in viral infection. However, minor change of IFN-α in EV71-infected MDMs was detected in our study. It is likely that the 3C protein of EV71 virus inhibits type I interferon activation by viral nuclear acid or RIG-I signalling [30]. Although elevated level of IFN-γ in both plasma and cerebrospinal fluid was found in patients with PE [10], the IFN-γ from EV71-infected MDMs was undetectable here. The discrepancy may be a result of the main cellular source of IFN-γ in vivo from activated T and NK cells other than macrophages. A series of IFN-γ-responsive and inflammatory chemokines such as RANTES, IP-10 and IL-8 in MDMs were triggered by EV71 virus. Not only live- but also UV-inactivated EV71 can induce IL-8 releasing from MDMs, which suggested that viral proteins may also be involved in inducing of IL-8. IL-8 is a potent chemoattractant and activator of neutrophils, one of the major immune cells responsible for inflammation of CNS during meningitis or encephalitis [31]. Our in vitro findings support the clinical findings that a higher total WBC count, absolute neutrophil count and elevated IL-8 and IP-10 level in patients with BE or PE [11,25].\nTLRs and RLHs recognize distinct ligands and trigger host immune response in different virus infection [32]. The recognition of human rhinovirus, human parechovirus 1, rotavirus or coxsackie B virus by different host cells are mediated through elevated TLR2, 7, 8 and (or) Mda5 expression, which induce secretion of proinflammatory cytokines, chemokines, and interferons [16-19]. When TLR2, TLR7 and TLR8 were silenced, there was a considerable decrease in cytokine secretion in human airway epithelial cells with HRV-6 infection [18]. In our study, elevated TLR2, TLR7 and TLR8 expressions as well as increased proinflammatory cytokines and chemokines were observed in EV71-infected MDMs. Furthermore, enhanced IL-8 and TLR2 mRNA expression were also found in UV-inactivated virus treated-MDMs. It is likely that the interaction between TLR2 on cell surface and viral proteins rather than viral RNAs is necessary for the activation of MDMs. Significant up-regulations of mRNA for TLR7 and TLR8 were observed at different time points, and it suggested that there were differential kinetics between TLR7 and TLR8 involvements in EV71-infected MDMs. These results indicated the cytokine productions in EV71-infected MDMs may be partly through the activation of TLR2, TLR7 and (or) TLR8. Further investigations such as gene knockout experiments are needed to determine the exact roles of them.\nAs documented previously, younger children less than 5-year-old were the most susceptible groups to EV71 and usually presented with severe infection [3]. The underlying mechanisms for the severity remain unknown. And enhanced proinflammatory cytokines and chemokines were indeed found in children patients with encephalitis or PE [9,11,24]. Although there is differential expression level of chemokine receptors on adult and neonatal MDMs [33], one of possible factors for the age-related severity in avian influenza virus infection, a similar cytokines/chemokines profile was found in influenza virus-infected adult and neonatal MDMs and the levels of most of the cytokines/chemokines were comparable [23]. Moreover, the adults can also be infected with EV71 and the clinical severity in adult patients with acute encephalitis was similar to those of EV71 infection in children [34]. Therefore, the findings on adult MDMs infection model here may partially reflect natural in vivo infection.",
"In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed.",
" Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\nThe study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.\n Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\nEnterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.\n Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\nMDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.\n Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\nInfected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay\n Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\nConcentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).\n Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.\nStatistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.",
"The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.\nPeripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.",
"Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.",
"MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.",
"Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.\nPrimer sequences and probes used in real-time PCR assay",
"Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).",
"Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.",
"(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.",
"The authors declare that they have no competing interests.",
"Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.",
"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/12/224/prepub\n"
] | [
null,
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null,
null,
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"discussion",
"conclusions",
"methods",
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] | [
"Enterovirus 71",
"Macrophages",
"Proinflammatory cytokines",
"Chemokines",
"Toll-like receptors"
] | Background:
Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].
Although the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.
Previous studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.
Results:
Effective infection and viral replication of EV71 in human MDMs We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.
A dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.
Viral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.
We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.
A dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.
Viral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.
Enhanced proinflammatory cytokine responses of human MDMs to EV71 We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.
Proinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.
Proinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71 Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).
Chemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).
Chemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.
A higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).
In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.
A higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).
Effective infection and viral replication of EV71 in human MDMs:
We first determined the infectivity of MDMs to EV71 by immunofluorescence staining of viral VP1. As evidenced by the expression of viral VP1+ at 12-h POI. (data not shown), human MDMs could be infected by EV71 virus. Furthermore, the infection showed a dose-dependent for infectious dose, as evidenced by an increase of VP1 expression at 24-h POI. along with a MOI of 0.1, 0.5, 1 and 5. (Figure 1C-F). The VP1+ fluorescence signal was detected in the cytoplasm in EV71-infected MDMs at 24-h POI., but not in mock or UV-inactivated EV71-infected MDMs at MOI of 5(Figure 1). Then the viral gene copy was quantified. An increase of viral gene copy was detected between 6-h and 12-h POI. (range from 16,421 ± 5061 to 136,027 ± 54,473 copies/104 copies of beta-actin, p = 0.008, n = 8). The viral gene copies maintained at a high level at the following assessed time points, 24-h and 48-h POI, but showed no significant difference from that at 12-h POI. (p = 0.194, p = 0.273, n = 8) (Figure 2A). To identify whether MDMs produce infectious progeny particles or not, virus titers of culture supernatants were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula [20]. The results showed that MDMs were productively infected by EV71 with approximate 10-fold increase in virus titter by 48-h POI. (Figure 2B, n = 8). Our in vitro findings suggested that MDMs are susceptible to EV71 infection and maybe one of target cells in vivo during EV71 infection.
A dose-dependent infection of EV71 virus in human monocyte-derived macrophages (MDMs). Immunofluorescence staining of EV71 VP1 in mock- or EV71-infected MDMs was assayed at 24-h POI. A. mock, B. UV-inactivated EV71 infection, C. EV71 infection at a MOI of 0.1, D. EV71 infection at a MOI of 0.5, E. EV71 infection at a MOI of 1, F. EV71 infection at a MOI of 5.
Viral replication of EV71-infected human MDMs. Viral gene copies were quantified by real-time RT-PCR. Supernatants from infected cells were collected at designated time points post inoculation of EV71 at a MOI of 5 and the viral titter was measured by TCID50 analysis on Vero cells. A. the change of VP1 gene. The number of gene copy was normalized to 104copies of β-actin and expressed as mean ± standard error (SEM) from eight independent experiments, and each experiment was performed in triplicates. B. viral titters of EV71-infected MDMs cultures. The data were expressed as mean ± SEM from 8 independent experiments.
Enhanced proinflammatory cytokine responses of human MDMs to EV71:
We subsequently determine the functional response of human MDMs to live EV71 or UV-irradiated EV71. Significant higher level of TNF-α was rapidly induced by live EV71 infection other than mock or UV-irradiated EV71 at the 12-h POI. (72.34 ± 52.56 pg/ml vs. 6.68 ± 7.67 pg/ml, 31.74 ± 20.26 pg/ml, respectively, p = 0.001, p = 0.011, n = 8), and then gradually decreased over time (30.68 ± 21.50 pg/ml, at 24-h POI.). Both IL-1β and IL-6 from MDMs were triggered by live EV71, and maintained a higher level at 12-h (p = 0.018, p = 0.008, n = 8) and 24-h POI. (p = 0.007, p = 0.006, n = 8) as compared with that of mock (Figure 3). In addition, we found that EV71 infection failed to induce IFN-α releasing from human MDMs, and the IFN-γ was undetectable.
Proinflammatory cytokines IL-1β, IL-6 and TNF-α induced in EV71 infected MDMs. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5. Culture supernatants were harvested at 12-h and 24-h after infection to measure the indicated cytokines by CBA as described in Methods section. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Enhanced production of IL-8, RANTES and IP-10 from human MDMs infected with EV71:
Chemokines include a large superfamily and play multiple roles in shaping the innate and adaptive immune responses during viral infection. We measured the levels of the chemokines including CXCL-10/IP-10, CCL-2/MCP-1, CXCL-9/MIG, CXCL-8/IL-8, and CCL-5/RANTES in the supernatants at 12-h and 24-h POI. At early time points (12-h POI.), enhanced production of IL-8 and RANTES was detected in EV71-infected MDMs than in mock-infected MDMs (p = 0.003, p = 0.002, n = 8), and the concentrations of these chemokines increased along with time (Figure 4). At 24-h POI, much higher concentrations of IL-8, RANTES and IP-10 were found in EV71-infected MDMs than in those of mock( p = 0.013, p = 0.004, p = 0.001, n = 8). Moreover, compared with mock-infected MDMs, UV-irradiated EV71 can induce significantly high level of IL-8 production at 12-h and 24-h POI. than mock (p = 0.004, p = 0.002, n = 8).
Chemokines induced in MDMs infected with EV71 and (or) UV-inactivated EV71. MDMs were infected with or without EV71 (live or UV-inactivated) at MOI of 5 for 12-h and 24-h. The chemokines including IP-10, MCP-1, RANTES, IL-8, MIG concentration in the supernatants at 12-h and 24-h was measured by CBA. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates. (*, P < 0.05 and **, P < 0.01).
Up-regulation of TLR2, TLR7 and TLR8 mRNA expression in EV71 infected- MDMs:
In order to investigate the involvement of TLRs and RLHs in EV71-infected MDMs, we screened the TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1, Mda-5 mRNA expression by real-time quantitative RT-PCR. The results showed that the mRNA expression of TLR2 appeared to be significantly enhanced in EV71-infected human MDMs from 6-h to 24-h POI. Increased mRNA expressions of TLR2 were also observed in UV-inactivated virus-infected MDMs at 12-h to 24-h POI. as compared with mock (p = 0.014, p = 0.008, n = 8). Both TLR7 and TLR8 mRNA expressions were significantly enhanced in EV71-infected MDMs at different time points. In contrast to the findings that higher TLR7 mRNA level was induced at earlier time points (6-h and 12-h POI.), enhanced mRNA expression of TLR8 was observed at a relatively later stage, 24-h POI. (Figure 5). In addition, there was minor change in the mRNA expression of TLR3, TLR4, TLR6, TLR9, TLR10, RIG-1 and Mda-5 in the tested samples.
A higher mRNA expression of TLR2, TLR7 and TLR8 were induced in MDMs infection with EV71. The mRNA expression of TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-1 and MDA-5 were performed by SYBR green real-time RT-PCR. The relative change of mRNA expression was analyzed using △△CT method. To standardize results for variability in cDNA quantity, we expressed them with target gene/β-actin as 1 in mock at 6-h POI. Data are expressed as mean ± SEM from eight independent experiments performed in triplicates (*, P < 0.05 and **, P < 0.01).
Discussion:
Macrophages, which are shown to support the infection of various viruses including HIV-1, influenza viruses, and poliovirus and so on, play a critical role in presentation of antigens, pathogen clearance, and induction of inflammation during the early phase of viral infection [21-23]. In this study, both viral gene and antigen of EV71 were detected. The increases of virus yields and the number of viral gene copies were observed in EV71-infected MDMs between the 6-h and 48-h POI. Excess cytokine and chemokine responses of MDMs were triggered by EV71. These findings suggested that macrophages may be not only the target cells but also the effectors during EV71 infection.
It is controversial whether TNF-α was involved in fatal EV71 infection. Significant or minor change of blood TNF-α in EV71 patient with both encephalitis and PE was reported by different clinical studies [24,25]. Significant stronger release of TNF-α from EV71-infected MDMs at 12-h POI than 24-h POI in the study, implying that TNF-α was induced by EV71 infection at early stage and maybe involved in its pathology. Consistent with clinical features of EV71 patients with encephalitis and PE, who are presented with higher levels of the proinflammatory cytokines in blood [9,24], IL-6, IL-1β and TNF-α were induced in EV71-infected MDMs. These proinflammatory cytokines are thought to be the potent pyrogens inducing fever, and the magnitude of febrile response correlates with the level of virus shedding in human and animals [26]. Notably, a transient increase of blood brain barrier (BBB) permeability and its injury were found at early stage of EV71 infection [27]. The pathology may be due to an augmentation of systemic and local TNF-α production, which exhibits detrimental effects by enhancing cell infiltration, cytopathic damage, or functioning as a paracellular pathway for the virus across the BBB [28]. Furthermore, the subsequent responses of acute phase proteins and chemokine activations mediated by IL-6, IL-1β and TNF-α could exacerbate virus induced inflammation and pathology [29]. Therefore, the rapid and strong proinflammatory response of MDMs to EV71 may partially explain the clinical severity.
Macrophages or plasmacytoid DCs, specialized in secreting large amounts of type I IFN after virus infection, play an important role in viral infection. However, minor change of IFN-α in EV71-infected MDMs was detected in our study. It is likely that the 3C protein of EV71 virus inhibits type I interferon activation by viral nuclear acid or RIG-I signalling [30]. Although elevated level of IFN-γ in both plasma and cerebrospinal fluid was found in patients with PE [10], the IFN-γ from EV71-infected MDMs was undetectable here. The discrepancy may be a result of the main cellular source of IFN-γ in vivo from activated T and NK cells other than macrophages. A series of IFN-γ-responsive and inflammatory chemokines such as RANTES, IP-10 and IL-8 in MDMs were triggered by EV71 virus. Not only live- but also UV-inactivated EV71 can induce IL-8 releasing from MDMs, which suggested that viral proteins may also be involved in inducing of IL-8. IL-8 is a potent chemoattractant and activator of neutrophils, one of the major immune cells responsible for inflammation of CNS during meningitis or encephalitis [31]. Our in vitro findings support the clinical findings that a higher total WBC count, absolute neutrophil count and elevated IL-8 and IP-10 level in patients with BE or PE [11,25].
TLRs and RLHs recognize distinct ligands and trigger host immune response in different virus infection [32]. The recognition of human rhinovirus, human parechovirus 1, rotavirus or coxsackie B virus by different host cells are mediated through elevated TLR2, 7, 8 and (or) Mda5 expression, which induce secretion of proinflammatory cytokines, chemokines, and interferons [16-19]. When TLR2, TLR7 and TLR8 were silenced, there was a considerable decrease in cytokine secretion in human airway epithelial cells with HRV-6 infection [18]. In our study, elevated TLR2, TLR7 and TLR8 expressions as well as increased proinflammatory cytokines and chemokines were observed in EV71-infected MDMs. Furthermore, enhanced IL-8 and TLR2 mRNA expression were also found in UV-inactivated virus treated-MDMs. It is likely that the interaction between TLR2 on cell surface and viral proteins rather than viral RNAs is necessary for the activation of MDMs. Significant up-regulations of mRNA for TLR7 and TLR8 were observed at different time points, and it suggested that there were differential kinetics between TLR7 and TLR8 involvements in EV71-infected MDMs. These results indicated the cytokine productions in EV71-infected MDMs may be partly through the activation of TLR2, TLR7 and (or) TLR8. Further investigations such as gene knockout experiments are needed to determine the exact roles of them.
As documented previously, younger children less than 5-year-old were the most susceptible groups to EV71 and usually presented with severe infection [3]. The underlying mechanisms for the severity remain unknown. And enhanced proinflammatory cytokines and chemokines were indeed found in children patients with encephalitis or PE [9,11,24]. Although there is differential expression level of chemokine receptors on adult and neonatal MDMs [33], one of possible factors for the age-related severity in avian influenza virus infection, a similar cytokines/chemokines profile was found in influenza virus-infected adult and neonatal MDMs and the levels of most of the cytokines/chemokines were comparable [23]. Moreover, the adults can also be infected with EV71 and the clinical severity in adult patients with acute encephalitis was similar to those of EV71 infection in children [34]. Therefore, the findings on adult MDMs infection model here may partially reflect natural in vivo infection.
Conclusions:
In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed.
Methods:
Isolation and culture of monocyte-derived macrophages The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.
Peripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.
The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.
Peripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.
Infection of MDMs with EV71 Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.
Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.
Immunofluorescence assay of viral VP1 protein in infected cells MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.
MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.
Quantification of mRNA by real-time RT-PCR Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.
Primer sequences and probes used in real-time PCR assay
Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.
Primer sequences and probes used in real-time PCR assay
Measurement of cytokines Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).
Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).
Statistical analysis Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.
Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.
Isolation and culture of monocyte-derived macrophages:
The study was approved for human subject protection by the Ethics Committee of National Institute for Viral Disease Control and Prevention, China CDC. Following informed consent was written by participants.
Peripheral blood obtained from 8 healthy blood donors aged from 20 to 40 years old. Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll-Paque density gradient (Pharmacia Biotech, Uppsala, Sweden) to remove erythrocytes, platelets, and cell debris. Monocytes were isolated by plastic adherence, harvested, counted and seeded on tissue culture plates in RPMI 1640 (Invitrogen Life Technologies, Great Island, NY, USA) medium supplemented with 10% heat-inactivated autologous plasma at 106cells/ml. The purity of monocytes was determined by flow cytometry with anti-CD14 monoclonal antibody (Mab, PharMingen, San Diego, CA, USA) and was consistently above 90%. The monocytes were reseed with 2-5 × 105 cells per well onto a 24 well culture plate, and were allowed to differentiate into MDMs for 10–14 days in vitro.
Infection of MDMs with EV71:
Enterovirus 71 virus was propagated in RD cells (obtained from ATCC) in DMEM containing 2% fetal bovine serum (FBS, Invitrogen, Grand Island, NY, USA) and incubated at 35°C with 5%CO2. When 80% of the cells showed the typical enteroviral cytopathic effect (CPE), the infected cells were harvested after being frozen and thawed for three times. Cell debris was removed by centrifugation and filtration using a 0.22 μm membrane filter (Millipore, Billerica, CA). EV71 viruses were inactivated using ultraviolet radiation 3000 mj/cm2, 30 mins on ice (UV-inactivated EV71). Differentiated MDMs were infected by EV71 and UV-inactivated EV71 at MOI (multiplicity of infection) of 0.1, 0.5, 1 and 5. This is taken to be 0-h point of infection (POI.) for the experiments described below. Virus titters were performed by measuring the 50% tissue culture infective dose (TCID50) on Vero cells and calculated by using the Reed and Muench formula.
Immunofluorescence assay of viral VP1 protein in infected cells:
MDMs were fixed with methanol: acetone (1:1) for 5 min. The cell monolayer was incubated by anti-EV71 VP1 monoclonal antibody (MAB979, Millipore, Billerica, CA) at room temperature for 60 min, and followed by labelling FITC-conjugated goat anti-mouse IgG for 1-h. After being completely washed, the cells were observed under a fluorescence microscope.
Quantification of mRNA by real-time RT-PCR:
Infected MDMs cultured in macrophage serum-free medium (Invitrogen, Grand Island, NY, USA) were harvested at 2-h, 6-h, 12-h, 24-h and 48-h POI. Total RNAs were extracted using QIAGEN RNeasy mini kit(QIAGEN, Hilden, Germany). Reverse transcription was performed on DNase-treated total RNA. The cDNA was synthesized from mRNA with oligo(dT)12–18 primer and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). Specific primers and probes used in the real-time PCR assay were listed in Table 1 and Q-PCR were performed by Rote-Gene 3000 Sequence Detection System (QIAGEN, Hilden, Germany). Viral gene copies were quantified on the basis of a TaqMan Probes fluorescence signal after PCR. We expressed viral gene variability as the number of target gene copies per 104copies of β-actin. The relative changes of other human genes were analyzed by SYBR green real-time PCR. Dissociation curve analysis was performed after each assay, to ensure specific target detection.
Primer sequences and probes used in real-time PCR assay
Measurement of cytokines:
Concentration of cytokines from culture supernatants were determined by Cytometric Bead Array(CBA). IL-1β, IL-6, TNF-α, IFN-α, IFN-γ, IP-10, IL-8, RANTES, MCP-1, MIG Flex Set reagents (BD Biosciences, San Diego, CA) were used to measure cytokines by flow cytometry according to the manufacturer’s protocol. The results are presented as the means of assays performed in duplicate wells. Data were analyzed by using FCAP Array 0.1 and BD Cytometric Bead Array 1.4 software assay. The theoretical limits of detection were listed as follows: IL-1β(2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IFN-α (1.5 pg/ml), IFN-γ(1.8 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), MIG (1.1 pg/ml).
Statistical analysis:
Statistical significance was determined by Two-Way ANOVA or the Mann–Whitney rank sum tests. All analyses were performed using the Statistical Package for Social Sciences (SPSS13.0) software (SPSS Inc, IL, USA). A probability P < 0.05 was considered statistically significant.
Abbreviations:
(EV71): Enter virus; (TLRs): 71Toll-like receptors; (RIG-I): Retinoic acid-inducible gene I; (RLHs): RIG-I-like helicases; (MDMs): Monocyte-derived macrophages; (MDA5): Melamoma differentiation associated gene 5; (HFMD): Hand-foot-and-mouth disease; (BE): Brain stem encephalitis; (PE): Pulmonary edema; (CNS): Central nervous system; (BBB): Blood brain barrier; (MOI): Multiplicity of infection; (CSF): Cerebrospinal fluid; (PBMCs): Peripheral blood mononuclear cells.
Competing interests:
The authors declare that they have no competing interests.
Authors' contributions:
Conceived and designed the experiments: ZD, YJ, XG and JZ. Performed the experiments: XG and JZ. Analyzed the data: XG and JZ. Contributed reagents/materials/analysis tools: WZ, NL, JL and LL. Wrote the paper: XG, JZ, WZ and ZD. All authors read and approved the final manuscript.
Pre-publication history:
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2334/12/224/prepub
| Background:
The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms.
Methods:
Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR.
Results:
Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs.
Conclusions:
Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs. | Background:
Enterovirus 71, a positive-stranded RNA virus, is highly infectious and can cause hand-foot-and-mouth disease (HFMD), herpangina, neurological diseases with potentially more serious complications such as encephalitis, aseptic meningitis, brain stem encephalitis (BE) in infants and young children. Many of patients died from fulminant pulmonary edema (PE) or hemorrhage, which was based on nervous system injury [1,2]. In recent years, its prevalence in Malaysia, Taiwan, Singapore, China, Korea and so on, and continuous spreading widely provoked global concern [3-7].
Although the pathogenesis of neurogenic PE caused by EV71 remains unclear, host factors especially host immune response rather than EV71 itself or its genotype may be one of important determinants for the disease severity [1,8]. Excessive proinflammatory cytokine and chemokine responses were thought to contribute to the severity of EV71 infection [9]. Current findings implied that the inflammatory cytokines or chemokines are probably synthesized by infiltrated mononuclear cells (macrophages or T cells) in tissues or neuron-surrounding cells, such as microglia (the resident macrophages in central nervous system (CNS)) or astrocytes in EV71 infection [9-11]. Since monocyte-derived macrophages and microglia are derived from the common precursors in the bone marrow, express similar surface markers and perform roughly similar functions [12], both of them may be involved in the immune response to EV71 infection.
Previous studies have identified that the immune cells such as human peripheral blood mononuclear cells, human T cell line (Jurkat), monocytic cells, human immature dendritic cells can be infected with EV71 [13-15]. Macrophages play an important role in the innate immunity system, however, the interaction between macrophages and EV71 remains unknown. Furthermore, Toll-like receptors (TLRs) and RIG-I-like helicases (RLHs) recognize a number of viruses resulting in the activation of an innate immunity response that induce secretion of proinflammatory cytokines and chemokines [16-19]. In this study, we focused on the proinflammatory cytokine and chemokine responses of MDMs to EV71 infection. And we also investigated whether TLRs and RLHs were involved in EV71-infected MDMs, and explored the possible mechanisms of inflammatory responses to EV71 infection.
Conclusions:
In summary, effective viral replication in EV71-infected MDMs and excessive inflammatory cytokine and chemokine responses of MDMs to the virus were demonstrated for the first time in our study. The results indicate that macrophages are an important target for EV71, and they can trigger pro-inflammatory response and chemokine response against viral infection. However, inordinate macrophages response may be detrimental to the infected host due to exacerbate virus inflammation and pathology. TLR2, TLR7 and TLR8 may participate in the induction of cytokines/chemokines in EV71-infected MDMs. These data suggested that MDMs may play an important role in the pathogenesis of EV71 infection in vivo though more evidence is needed. | Background:
The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms.
Methods:
Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR.
Results:
Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs.
Conclusions:
Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs. | 9,660 | 335 | [
434,
569,
318,
339,
360,
199,
194,
72,
213,
189,
54,
113,
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67,
16
] | 19 | [
"ev71",
"mdms",
"infected",
"infection",
"poi",
"il",
"24",
"viral",
"infected mdms",
"12"
] | [
"virus inflammation pathology",
"cytokines chemokines ev71",
"macrophages ev71",
"enterovirus 71 positive",
"exacerbate virus inflammation"
] | [CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY] | [CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY] | [CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY] | [CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY] | [CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY] | [CONTENT] Enterovirus 71 | Macrophages | Proinflammatory cytokines | Chemokines | Toll-like receptors [SUMMARY] | [CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY] | [CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY] | [CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY] | [CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY] | [CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY] | [CONTENT] Adult | Cells, Cultured | Cytokines | DEAD Box Protein 58 | DEAD-box RNA Helicases | Enterovirus A, Human | Gene Expression Profiling | Humans | Interferon-Induced Helicase, IFIH1 | Macrophages | Real-Time Polymerase Chain Reaction | Receptors, Immunologic | Toll-Like Receptors | Young Adult [SUMMARY] | [CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY] | [CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY] | [CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY] | [CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY] | [CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY] | [CONTENT] virus inflammation pathology | cytokines chemokines ev71 | macrophages ev71 | enterovirus 71 positive | exacerbate virus inflammation [SUMMARY] | [CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY] | [CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY] | [CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY] | [CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY] | [CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY] | [CONTENT] ev71 | mdms | infected | infection | poi | il | 24 | viral | infected mdms | 12 [SUMMARY] | [CONTENT] ev71 | cells | ev71 infection | cells human | macrophages | infection | immune | proinflammatory | system | responses [SUMMARY] | [CONTENT] ml | pg ml | pg | il | usa | cells | pcr | assay | island ny usa | island ny [SUMMARY] | [CONTENT] ev71 | mdms | poi | infected | 24 | mock | mrna | expression | 24 poi | infection [SUMMARY] | [CONTENT] response | ev71 | inflammatory | chemokine | important | mdms | infected | macrophages | ev71 infected mdms | viral [SUMMARY] | [CONTENT] ev71 | mdms | infected | il | infection | pg ml | pg | ml | poi | cells [SUMMARY] | [CONTENT] ev71 | mdms | infected | il | infection | pg ml | pg | ml | poi | cells [SUMMARY] | [CONTENT] 71 ||| [SUMMARY] | [CONTENT] ||| Cytometric Bead Array(CBA ||| 5 | RT-PCR [SUMMARY] | [CONTENT] ||| 6 | 48 | POI ||| IL-1 | IL-6 | TNF | IFN | EV71 ||| IP-10 | 12 | 24-h | POI ||| TLR8 | TLR3 | TLR4 [SUMMARY] | [CONTENT] ||| [SUMMARY] | [CONTENT] 71 ||| ||| ||| Cytometric Bead Array(CBA ||| 5 | RT-PCR ||| ||| 6 | 48 | POI ||| IL-1 | IL-6 | TNF | IFN | EV71 ||| IP-10 | 12 | 24-h | POI ||| TLR8 | TLR3 | TLR4 ||| ||| [SUMMARY] | [CONTENT] 71 ||| ||| ||| Cytometric Bead Array(CBA ||| 5 | RT-PCR ||| ||| 6 | 48 | POI ||| IL-1 | IL-6 | TNF | IFN | EV71 ||| IP-10 | 12 | 24-h | POI ||| TLR8 | TLR3 | TLR4 ||| ||| [SUMMARY] |
||||||
Association of vancomycin trough concentration on the treatment outcome of patients with bacteremia caused by Enterococcus species. | 34702193 | Pharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia. | BACKGROUND | A retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality. | METHODS | A total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia. | RESULTS | In this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia. | CONCLUSIONS | [
"Anti-Bacterial Agents",
"Bacteremia",
"Enterococcus",
"Humans",
"Methicillin-Resistant Staphylococcus aureus",
"Microbial Sensitivity Tests",
"Retrospective Studies",
"Staphylococcal Infections",
"Treatment Outcome",
"Vancomycin"
] | 8547083 | Background | Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].
Vancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].
To our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species. | null | null | Results | A total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).
Table 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality
Characteristics
Total (n = 37)Survivors (n = 29)Non-survivors (n = 8)
P value
Demographics
Age (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)
Baseline demographic and clinical features of patients with enterococcal bacteremia
BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis
Continuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)
The 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.
In univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).
Table 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval
Multivariate logistic regression analysis for factors associated with 28-day mortality
OR, odds ratio; CI, confidence interval
Fig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level
Kaplan-Meier survival analysis of patients according to vancomycin trough level | Conclusions | In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species. | [
"Background",
"Methods",
"Study population and design",
"Definitions",
"Microbiological data",
"Vancomycin dosing and pharmacodynamics data",
"Statistical analysis",
""
] | [
"Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.",
"Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).",
"A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.",
"Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.",
"The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].",
"To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.",
"The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).",
"\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia."
] | [
null,
null,
null,
null,
null,
null,
null,
null
] | [
"Background",
"Methods",
"Study population and design",
"Definitions",
"Microbiological data",
"Vancomycin dosing and pharmacodynamics data",
"Statistical analysis",
"Results",
"Discussion",
"Conclusions",
"Supplementary Information",
""
] | [
"Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].\nVancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].\nTo our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.",
"Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nA retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.\nDefinitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nBacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.\nMicrobiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nThe species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].\nVancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nTo obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.\nStatistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).\nThe relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).",
"A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.",
"Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.",
"The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].",
"To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.\nDemographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.",
"The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).",
"A total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).\n\nTable 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality\nCharacteristics\nTotal (n = 37)Survivors (n = 29)Non-survivors (n = 8)\nP value\n\nDemographics\nAge (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nBaseline demographic and clinical features of patients with enterococcal bacteremia\nBMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis\nContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)\nThe 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.\nIn univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).\n\nTable 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval\nMultivariate logistic regression analysis for factors associated with 28-day mortality\nOR, odds ratio; CI, confidence interval\n\nFig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level\nKaplan-Meier survival analysis of patients according to vancomycin trough level",
"In this study, we found that vancomycin trough concentration (≤ 15 µg/mL) was significantly associated with increased mortality in patients with Enterococcus bacteremia.\nMany studies have examined pharmacokinetic/pharmacodynamic (PK/PD) parameters to maintain the efficiency of vancomycin and to minimize its adverse effects, but most of these studies have examined MRSA infection. Of the PK/PD parameters, the trough level and AUC24/MIC have been reported to describe the effectiveness of vancomycin [15, 16]. The clinical success and renal toxicity of vancomycin treatment are exposure-dependent and characterized by the AUC24. The exact range targeted by clinicians is influenced by the AUC24 estimation and bacterial MIC [15, 17]. Using AUC-based vancomycin therapeutic drug monitoring (TDM) helps to individualize the estimation of the AUC24/MIC and minimize the risk of toxicity due to unnecessary overexposure.\nAlthough trough concentration is a known surrogate marker for AUC24/MIC, the method using only trough-based vancomycin TDM is controversial for several reasons. First, the vancomycin trough concentration poorly characterizes the AUC24, and adequate vancomycin AUC24 levels can be obtained at trough concentrations < 15 mg/L. Vancomycin-associated nephrotoxicity also increases when the vancomycin trough concentration is >15 mg/L. These factors provide evidence for an AUC-based approach to vancomycin TDM [18]. Thus, according to the Infectious Diseases Society of America guidelines, the trough level does not correlate well with the AUC, and trough-only monitoring with a target of 15–20 µg/mL is no longer recommended based on its efficacy in patients with MRSA infections [10]. However, because it may be difficult to determine the AUC24/MIC in a clinical setting, trough serum concentrations are still monitored clinically. Moreover, there is insufficient evidence to recommend whether trough level-only or AUC/MIC-guided vancomycin monitoring should be used for patients with non-MRSA infections.\nIn one study of PK/PD determinants of vancomycin efficacy in enterococcal bacteremia, a vancomycin AUC/MIC ≥ 389 achieved within 72 h was associated with reduced mortality. However, the study found that vancomycin trough concentrations differed significantly between survivors and non-survivors [19]. According to Nakamura et al. [20] neither the AUC24/MIC nor the trough concentration of vancomycin is significantly associated with mortality in patients with E. faecium bacteremia. The trough concentration was higher in non-survivors than in survivors, and AUC24/MIC did not differ significantly between non-survivors and survivors.\nIn our study, vancomycin trough levels of 15 µg/mL or lower were associated with mortality in patients with enterococcal bacteremia, but the AUC24/MIC was not. Zelenitsky et al. reported that the survival rate was higher when the vancomycin trough concentration was maintained above 15 mg/L than when low trough concentrations were maintained [21]. In addition, Kullar et al. reported that the vancomycin treatment period was significantly shorter in the group in which the vancomycin trough concentration was higher than the group to which the vancomycin trough concentration was applied at 5 to 20 mg/L, and the rate at which the bacteria were negatively converted was also found to be significantly higher in the group that maintained the higher trough concentration [22]. According to a meta-analysis comparing high and low vancomycin serum trough regimen groups with gram-positive bacterial infections, there was no significant difference between the two in all-cause mortality or risk of clinical failure; however, a subgroup analysis confirmed that treatment failure was reduced in high vancomycin trough regimen groups [23]. Therefore, in enterococcal bacteremia, the trough level may be a good parameter for monitoring the therapeutic effects of vancomycin.\nAll enterococcal BSIs treated with vancomycin were included in this study to examine the therapeutic effect of vancomycin on Enterococcus species. Therefore, strains susceptible to ampicillin were added in this study. In most cases, vancomycin was used for ampicillin-susceptible Enterococcus species when beta-lactam antibiotics could not be used due to its side effects, or was used without checking the results of antimicrobial susceptibility tests. Ampicillin is the preferred antibiotic used to treat ampicillin-sensitive enterococcal infections. However, according to several studies comparing the therapeutic effects of beta-lactam antibiotics and vancomycin on ampicillin-susceptible enterococcal BSI, there was no significant difference in mortality [24]. Therefore, when needed, or when beta-lactam antibiotics are difficult to use, vancomycin can be considered as a treatment option for ampicillin-susceptible strains.\nAn important concern when using vancomycin is the occurrence of acute kidney injury. A higher vancomycin trough concentration increases the risk of VIN [20, 25]. Although most vancomycin-induced nephrotoxic events are reversible, many studies support there is increased mortality from AKI of any cause, including vancomycin-induced nephropathy [26]. While 6 of our 37 patients developed VIN, it was not statistically related to trough concentration (p = 0.09) or 28-day mortality. (p= 0.446).\nIn this study, mortality was higher at younger ages. Aging process causes structural and functional changes in multiple organ systems, especially the kidneys, and is known to affect PK/PD of drugs by causing changes in body composition, drug absorption, distribution, and clearance [27]. These changes may have affected the patient’s prognosis or treatment outcome.\nThis study has several limitations that are inherent to its retrospective design. As with any observational study, there remains a possibility that unmeasured confounders influenced our findings. According to several studies, it is well known that sepsis or septic shock affects drug absorption, distribution and other pharmacological processes due to pathophysiological changes such as decreased perfusion to body organs, interstitial edema or increased capillary permeability [28]. Therefore, the PK/PD alterations may not have been reflected depending on the patients’ disease severity. In addition, in the early stage of sepsis, extravascular volume expansion with fluid loading and capillary leak causes a change in volume distribution, which can cause hyperfiltration in the kidney and lead to an alteration of drug concentration. Thereby, it may have affected the prognosis of non-survivors with low trough levels. For these reasons, we cannot exclude the possibility that residual bias affected our findings. Another limitation of this study was that only patients with bacteremia were enrolled, and patients with non-bacteremic sepsis were not analyzed. In addition, the single-center nature of a referral tertiary hospital leads to selection bias in severe cases admitted to our hospital. A multicenter study with a larger sample size is needed.",
"In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species.",
" \nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\n\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.",
"\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.\nAdditional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia."
] | [
null,
null,
null,
null,
null,
null,
null,
"results",
"discussion",
"conclusion",
"supplementary-material",
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] | [
"Vancomycin",
"\nEnterococcus\n",
"Trough level",
"AUC/MIC"
] | Background:
Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].
Vancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].
To our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.
Methods:
Study population and design A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.
A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.
Definitions Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.
Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.
Microbiological data The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].
The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].
Vancomycin dosing and pharmacodynamics data To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.
Demographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.
To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.
Demographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.
Statistical analysis The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).
The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).
Study population and design:
A retrospective, single-center, cohort study was conducted at Severance Hospital, a 2400-bed, tertiary-care hospital in Seoul, South Korea. The electronic medical records of patients > 18 years of age with enterococcal bacteremia treated with vancomycin between January 1, 2007 and December 31, 2017 were reviewed. The age range of patients enrolled in this study was from 26 to 91 years. The exclusion criteria were as follows: presence of polymicrobial bacteremia, isolates resistant to vancomycin, missing trough vancomycin concentration, and vancomycin MIC data. Data collected from the patients’ medical records included demographic characteristics, comorbidities, source of bacteremia, antimicrobial treatment data, duration of bacteremia, relapse of bacteremia, and mortality. The primary outcome was 28-day all-cause mortality. The study was approved by the Institutional Review Board (IRB) of the Yonsei University Health System Clinical Trial Center (4-2020-0161). All methods were carried out in accordance with relevant guidelines and regulations under Ethics approval. Since the study was retrospective and the study subjects were anonymized, the IRB waived the requirement for written informed consent from the patients.
Definitions:
Bacteremia was defined as at least one positive blood culture with an identifiable source and clinical manifestations consistent with bacteremia, or at least two separate blood cultures positive for Enterococcus species. Persistent blood stream infection was defined as the case when the bacteria were continuously identified in two or more follow-up cultures after the first positive Enterococcus blood culture [13]. For patients with more than one episode of Enterococcus bacteremia, only the first episode during the study period was included. The duration of bacteremia was defined as the number of days from the first positive blood culture to the date of the first negative Enterococcus blood culture. Relapse occurred if the cultures became negative for more than 2 days and then became positive within 90 days. Vancomycin-induced nephropathy (VIN) was defined as an increase in the serum creatinine level of ≥ 0.5 mg/dL, or a 50% increase from baseline in consecutive daily readings, or a decrease in the calculated creatinine level of 50% from baseline on 2 consecutive days in the absence of an alternative explanation [6]. The inappropriate empirical antibiotics use means that the empirical antibiotics used before identification of the bacteria is not suitable for the drug susceptibility test result. Community-acquired bacteremia is defined as bacteremia occurred within 48 h of hospitalization. And hospital-acquired bacteremia is a bacteremia occurred 48 h after admission.
Microbiological data:
The species in the clinical isolates were identified using the VITEK®2 system with GNI cards (bioMérieux, Marcy-l’Étoile, France) until 2014 or Microflex MALDI-TOF mass spectrometry with Biotyper software 3.1 (Bruker Daltonics, Leipzig, Germany) after clinical adoption in 2014. Antimicrobial susceptibility tests were performed using the disk diffusion method or a VITEK-2 N131 card (bioMérieux). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [14].
Vancomycin dosing and pharmacodynamics data:
To obtain trough concentrations, blood samples were collected immediately before the next vancomycin dose. Samples for peak concentrations were collected 1 h after intravenous vancomycin infusion was completed. Based on the hospital guidelines, all venous blood samples used to determine steady-state serum concentrations were obtained after administering at least four doses of vancomycin. The serum vancomycin concentrations were analyzed with a chemiluminescence microparticle immunoassay using an Architect automated immunochemistry analyzer (Abbott Labs, Chicago, IL, USA). All pharmacokinetic calculations and modeling were performed using the MW/Pharm software package (ver. 3.82; Mediware, Zuidhorn, the Netherlands). And the trough level used to categorize in this study was measured through the first blood samples after at least four consecutive doses of vancomycin.
Demographic parameters, including weight, height, ethnicity, and sex, were imported from the hospital’s electronic records. Renal function was estimated using the most recent serum creatinine level. Estimated curves were fitted using the default settings of the posterior Bayesian estimation after the simulation. The area under the curve (AUC) of the estimated vancomycin concentration was calculated in a chronological manner. Patients on renal dialysis were screened for additional creatinine concentrations.
Statistical analysis:
The relationships between the vancomycin trough concentration or the AUC24/MIC and mortality in patients with E. faecium or E. faecalis bacteremia were analyzed using both continuous and categorical variables. The Kolmogorov-Smirnov test and Shapiro-Wilk test were conducted to verify the normality of the continuous variables. The independent t-test and the Mann–Whitney test were used to compare the continuous variables of the two groups. The chi-square test or Fisher’s exact test were used to compare categorical variables. Potentially significant variables identified in the univariate analysis were included in a multivariate logistic regression analysis to identify the risk factors associated with 28-day mortality. The Kaplan–Meier survival curve was used to compare mortality according to vancomycin trough concentrations. All statistical analyses were performed using SPSS Statistics ver. 25.0 (IBM Corp., Armonk, NY, USA).
Results:
A total of 209 patients had confirmed enterococcal bacteremia. Of these, 73 patients had polymicrobial bacteremia, 62 had vancomycin-resistant enterococci, and 37 did not perform therapeutic drug monitoring (TDM). Finally, 37 patients (22 men, 15 women; mean age, 60.4 years) were enrolled: 26 cases of E. faecium and 11 of E. faecalis. (Additional file 1: Table S1). The most common comorbidities were solid cancer (56.8%), followed by hypertension (40.5%), and diabetes mellitus (35.1%). The most common source of infection was biliary infection (40.5%), followed by peritonitis (21.6%).
Table 1Baseline demographic and clinical features of patients with enterococcal bacteremia28-day all mortality
Characteristics
Total (n = 37)Survivors (n = 29)Non-survivors (n = 8)
P value
Demographics
Age (year, mean±SD)60.5 ± 13.262.8 ± 2.352.3 ± 4.40.044BMI (mean±SD)21.4 ± 3.121.2 ± 0.622.4 ± 1.10.323Male (%)22 (59.5)18(62.1)4(50.0)0.412Community AB (%)9 (24.3)7 (24.1)2 (25.0)0.643Hospital AB (%)28 (75.7)22 (75.9)6 (75.0)0.643Comorbidities (%)Solid cancer21 (56.8)17 (58.6)4 (50.0)0.483HTN15 (40.5)11 (37.9)4 (50.0)0.412DM13 (35.1)11 (37.9)2 (25.0)0.408Chronic liver disease11 (29.7)8 (27.6)3 (37.5)0.444Organ transplantation9 (24.3)7 (24.1)2 (25.0)0.643Chronic renal disease9 (24.3)6 (20.7)3 (37.5)0.292Cerebrovascular disease7 (18.9)6 (20.7)1 (12.5)0.521Cardiovascular disease6 (16.2)5 (17.2)1 (12.5)0.613Hematologic malignancies4 (10.8)3 (10.3)1 (12.5)0.640CHF3 (8.1)2 (6.9)1 (12.5)0.530Hemiplegia2 (5.4)2 (6.9)0 (0.0)0.610PAOD1 (2.7)0 (0.0)1 (12.5)0.216ILD1 (2.7)1 (3.4)0 (0.0)0.784Antibiotic use in 30 day (%)27 (73.0)22 (75.9)5 (62.5)0.367Steroid use in 30 day (%)16 (43.2)12 (41.4)4 (50.0)0.483Anticancer drug use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Immunosuppressant use in 30 day (%)9 (24.3)7 (24.1)2 (25.0)0.643Vancomycin induced AKI (%)6 (16.2)4 (13.8)2 (25.0)0.591Septic shock (%)8 (21.6)4 (13.8)4 (50.0)0.049Source of bacteremia (%)Biliary15 (40.5)12 (41.4)3 (37.5)0.588Peritonitis8 (21.6)7 (24.1)1 (12.5)0.435UTI1 (2.7)0 (0.0)1 (12.5)0.216Skin1 (2.7)1 (3.4)0 (0.0)0.784CRBSI1 (2.7)1 (3.4)0 (0.0)0.784Foreign device1 (2.7)1 (3.4)0 (0.0)0.784Others2 (5.4)1 (3.4)1 (12.5)0.390Primary8 (21.6)6 (20.7)2 (25.0)0.565ICU stay (%)14 (37.8)10 (34.5)4 (50.0)0.343SOFA score (mean±SD)7.0 ± 4.86.2 ± 0.89.9 ± 1.90.056Persistent BSI (%)18 (48.6)15 (51.7)3 (37.5)0.379Recurrence of same BSI (%)5 (13.5)5 (17.2)0 (0.0)0.272Initial empirical inappropriate antibiotics (%)16 (43.2)13 (44.8)3 (37.5)0.517Combination therapy (%)8 (21.6)6 (20.7)2 (25.0)0.565PK/PD parameter (%)Trough level ≤15 µg/mL19 (51.4)12 (41.4)7 (87.5)Trough level>15 µg/mL18 (48.6)17 (58.6)1 (12.5)0.042AUC24/MIC≤3897 (18.9)5 (17.2)2 (25.0)AUC24/MIC>38930 (81.1)24 (82.8)6 (75.0)0.479BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditisContinuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)
Baseline demographic and clinical features of patients with enterococcal bacteremia
BMI, body mass index; Community AB, Community acquired bacteremia; Hospital AB, Hospital acquired bacteremia; HTN, hypertension; DM, diabetes mellitus; CHF, congestive heart failure; PAOD peripheral arterial occlusive disease; ILD, interstitial lung disease; AKI. Acute kidney disease, UTI, urinary tract infection; CRBSI, catheter-related blood stream infection; BSI, blood stream infection, AUC, area under curve; MIC minimum inhibitory concentration; Other cases of bacteremia include infective endocarditis
Continuous variables are shown as the mean ± standard deviation (SD) and categorical variables, as numbers (percentage)
The 28-day mortality rate was 21.6% (8/37). No significant differences were found in the baseline characteristics, except for age (Table 1). Vancomycin-induced nephropathy occurred in 6 of the 37 patients. In the MIC distributions for vancomycin, there were 3 cases of MIC 2 mg/L, 18 cases of MIC 1 mg/L, and 16 cases of MIC < 0.5 mg/L. The median value of AUC24 in this study was 474.21, the minimum value was 181.17, and the maximum was 1111.41.
In univariate analyses, vancomycin trough concentration (≤ 15 µg/mL) (p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality. The proportion of patients achieving AUC24/MIC ≤ 389 did not differ between the two groups (p = 0.479). In a multivariate analysis, vancomycin trough concentration (≤15 µg/mL) (p = 0.041; odds ratio (OR), 119.013; 95% confidence interval (CI), [1.207–11732.690]), and younger age (p = 0.031; OR, 1.212; 95% CI, [1.018–1.442]) were associated with mortality in patients with enterococcal bacteremia (Table 2). Moreover, in the Kaplan–Meier analysis, the group with vancomycin trough levels of 15 µg/mL or lower had a higher 28-day mortality (log rank = 0.027) (Fig. 1).
Table 2Multivariate logistic regression analysis for factors associated with 28-day mortalityVariablesOR95 % CIp valueYounger age1.2121.018~1.4420.031Vancomycin trough level≤15 µg/mL119.0131.207~11732.6900.041Septic shock18.3690.997~338.5740.050OR, odds ratio; CI, confidence interval
Multivariate logistic regression analysis for factors associated with 28-day mortality
OR, odds ratio; CI, confidence interval
Fig. 1Kaplan-Meier survival analysis of patients according to vancomycin trough level
Kaplan-Meier survival analysis of patients according to vancomycin trough level
Discussion:
In this study, we found that vancomycin trough concentration (≤ 15 µg/mL) was significantly associated with increased mortality in patients with Enterococcus bacteremia.
Many studies have examined pharmacokinetic/pharmacodynamic (PK/PD) parameters to maintain the efficiency of vancomycin and to minimize its adverse effects, but most of these studies have examined MRSA infection. Of the PK/PD parameters, the trough level and AUC24/MIC have been reported to describe the effectiveness of vancomycin [15, 16]. The clinical success and renal toxicity of vancomycin treatment are exposure-dependent and characterized by the AUC24. The exact range targeted by clinicians is influenced by the AUC24 estimation and bacterial MIC [15, 17]. Using AUC-based vancomycin therapeutic drug monitoring (TDM) helps to individualize the estimation of the AUC24/MIC and minimize the risk of toxicity due to unnecessary overexposure.
Although trough concentration is a known surrogate marker for AUC24/MIC, the method using only trough-based vancomycin TDM is controversial for several reasons. First, the vancomycin trough concentration poorly characterizes the AUC24, and adequate vancomycin AUC24 levels can be obtained at trough concentrations < 15 mg/L. Vancomycin-associated nephrotoxicity also increases when the vancomycin trough concentration is >15 mg/L. These factors provide evidence for an AUC-based approach to vancomycin TDM [18]. Thus, according to the Infectious Diseases Society of America guidelines, the trough level does not correlate well with the AUC, and trough-only monitoring with a target of 15–20 µg/mL is no longer recommended based on its efficacy in patients with MRSA infections [10]. However, because it may be difficult to determine the AUC24/MIC in a clinical setting, trough serum concentrations are still monitored clinically. Moreover, there is insufficient evidence to recommend whether trough level-only or AUC/MIC-guided vancomycin monitoring should be used for patients with non-MRSA infections.
In one study of PK/PD determinants of vancomycin efficacy in enterococcal bacteremia, a vancomycin AUC/MIC ≥ 389 achieved within 72 h was associated with reduced mortality. However, the study found that vancomycin trough concentrations differed significantly between survivors and non-survivors [19]. According to Nakamura et al. [20] neither the AUC24/MIC nor the trough concentration of vancomycin is significantly associated with mortality in patients with E. faecium bacteremia. The trough concentration was higher in non-survivors than in survivors, and AUC24/MIC did not differ significantly between non-survivors and survivors.
In our study, vancomycin trough levels of 15 µg/mL or lower were associated with mortality in patients with enterococcal bacteremia, but the AUC24/MIC was not. Zelenitsky et al. reported that the survival rate was higher when the vancomycin trough concentration was maintained above 15 mg/L than when low trough concentrations were maintained [21]. In addition, Kullar et al. reported that the vancomycin treatment period was significantly shorter in the group in which the vancomycin trough concentration was higher than the group to which the vancomycin trough concentration was applied at 5 to 20 mg/L, and the rate at which the bacteria were negatively converted was also found to be significantly higher in the group that maintained the higher trough concentration [22]. According to a meta-analysis comparing high and low vancomycin serum trough regimen groups with gram-positive bacterial infections, there was no significant difference between the two in all-cause mortality or risk of clinical failure; however, a subgroup analysis confirmed that treatment failure was reduced in high vancomycin trough regimen groups [23]. Therefore, in enterococcal bacteremia, the trough level may be a good parameter for monitoring the therapeutic effects of vancomycin.
All enterococcal BSIs treated with vancomycin were included in this study to examine the therapeutic effect of vancomycin on Enterococcus species. Therefore, strains susceptible to ampicillin were added in this study. In most cases, vancomycin was used for ampicillin-susceptible Enterococcus species when beta-lactam antibiotics could not be used due to its side effects, or was used without checking the results of antimicrobial susceptibility tests. Ampicillin is the preferred antibiotic used to treat ampicillin-sensitive enterococcal infections. However, according to several studies comparing the therapeutic effects of beta-lactam antibiotics and vancomycin on ampicillin-susceptible enterococcal BSI, there was no significant difference in mortality [24]. Therefore, when needed, or when beta-lactam antibiotics are difficult to use, vancomycin can be considered as a treatment option for ampicillin-susceptible strains.
An important concern when using vancomycin is the occurrence of acute kidney injury. A higher vancomycin trough concentration increases the risk of VIN [20, 25]. Although most vancomycin-induced nephrotoxic events are reversible, many studies support there is increased mortality from AKI of any cause, including vancomycin-induced nephropathy [26]. While 6 of our 37 patients developed VIN, it was not statistically related to trough concentration (p = 0.09) or 28-day mortality. (p= 0.446).
In this study, mortality was higher at younger ages. Aging process causes structural and functional changes in multiple organ systems, especially the kidneys, and is known to affect PK/PD of drugs by causing changes in body composition, drug absorption, distribution, and clearance [27]. These changes may have affected the patient’s prognosis or treatment outcome.
This study has several limitations that are inherent to its retrospective design. As with any observational study, there remains a possibility that unmeasured confounders influenced our findings. According to several studies, it is well known that sepsis or septic shock affects drug absorption, distribution and other pharmacological processes due to pathophysiological changes such as decreased perfusion to body organs, interstitial edema or increased capillary permeability [28]. Therefore, the PK/PD alterations may not have been reflected depending on the patients’ disease severity. In addition, in the early stage of sepsis, extravascular volume expansion with fluid loading and capillary leak causes a change in volume distribution, which can cause hyperfiltration in the kidney and lead to an alteration of drug concentration. Thereby, it may have affected the prognosis of non-survivors with low trough levels. For these reasons, we cannot exclude the possibility that residual bias affected our findings. Another limitation of this study was that only patients with bacteremia were enrolled, and patients with non-bacteremic sepsis were not analyzed. In addition, the single-center nature of a referral tertiary hospital leads to selection bias in severe cases admitted to our hospital. A multicenter study with a larger sample size is needed.
Conclusions:
In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species.
Supplementary Information:
Additional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.
Additional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.
Additional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.
Additional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.
:
Additional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.
Additional file 1: Table S1. Baseline characteristics and clinical features according to Enterococcus species in patients with enterococcal bacteremia.
| Background:
Pharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia.
Methods:
A retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality.
Results:
A total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia.
Conclusions:
In this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia. | Background:
Enterococcus species are ubiquitous in natural environments, including plants, soil, and water, and are part of the normal gastrointestinal flora of humans and other animals. Their broad distribution allows them to survive and persist in various environments [1]. Because of their ability to colonize medical devices and their high viability in nosocomial environments, Enterococcus species are a major cause of hospital-acquired infections [2]. Enterococci are important nosocomial pathogens worldwide, accounting for 14% of all hospital-acquired infections in the United States between 2011 and 2014 [3]. Enterococcus species cause infections of the bloodstream, urinary tract, and surgical sites, inter alia. These infections can lead to infective endocarditis, urinary tract infections, bacteremia, peritonitis, and prosthetic joint infections. In many centers, the proportion of ampicillin-resistant Enterococcus faecium exceeds 70%. Enterococcus faecalis resistant to ampicillin is rare, but resistance rate is increasing in nosocomial infections and the rate of ampicillin-resistant E. faecalis is reported to be 1.8%; hence, the use of vancomycin is increasing [3, 4].
Vancomycin is the commonly used glycopeptide to treat aminopenicillin-resistant gram-positive bacteria [5]. Because it has adverse effects such as nephrotoxicity, studies have examined the pharmacokinetics of vancomycin to minimize its toxicity and maximize its therapeutic effect [6]. The guidelines for vancomycin dosing strategies to assist clinicians and pharmacists have been revised. By attempting to implement therapeutic monitoring using current knowledge of vancomycin pharmacodynamics and in view of potential problems with efficacy, resistance, and toxicity, these guidelines are intended to improve patient outcomes [7, 8]. Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections [8, 9]. The pharmacodynamic parameter that best predicts vancomycin efficacy in invasive MRSA infections is the ratio of the 24 h area under the concentration-time curve to minimum inhibitory concentration (AUC24/MIC) [10]. Serum vancomycin trough concentrations have been used as a surrogate marker for identifying an AUC24/MIC of ≥ 400 [11], although several recent studies found that the trough concentration poorly reflects the AUC24/MIC target and that a high trough concentration increases the risk of nephrotoxicity [8, 12].
To our knowledge, few studies have examined the target vancomycin concentration in the treatment of non-MRSA gram-positive infections, especially those caused by Enterococcus species. Therefore, we investigated whether vancomycin concentration targets such as the trough level and AUC/MIC are associated with the treatment outcome in patients with bacteremia caused by Enterococcus species.
Conclusions:
In conclusion, a vancomycin trough level of 15 µg/mL or lower was significantly associated with 28-day mortality in patients with enterococcal bacteremia. However, larger prospective studies are needed to examine the association of vancomycin PK/PD parameters for treating other gram-positive pathogens, especially Enterococcus species. | Background:
Pharmacokinetic-pharmacodynamic (PK/PD) targets of vancomycin therapy have been recognized for methicillin-resistant Staphylococcus aureus infections but not for other gram-positive bacterial infections. Therefore, we investigated whether vancomycin concentration targets such as the trough level and ratio of the area under the curve to minimum inhibitory concentration (AUC/MIC) are associated with the treatment outcome in enterococcal bacteremia.
Methods:
A retrospective cohort analysis enrolled patients with bacteremia caused by vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis who were treated with vancomycin from January 2007 to December 2017 at a tertiary hospital located in Seoul, South Korea. Patients without vancomycin concentrations were excluded from the study. The primary outcome was 28-day all-cause mortality.
Results:
A total of 37 patients were enrolled-26 with E. faecium infection and 11 with E. faecalis infection. The 28-day all-cause mortality rate was 21.6 %. In univariate analysis, vancomycin trough level (≤ 15 µg/mL; p = 0.042), age (p = 0.044), and septic shock (p = 0.049) were associated with 28-day mortality but not AUC24/MIC (> 389; p = 0.479). In multivariate analysis, vancomycin trough concentration (≤ 15 µg/mL; p = 0.041) and younger age (p = 0.031) were associated with 28-day mortality in patients with enterococcal bacteremia.
Conclusions:
In this study, a vancomycin trough level of 15 µg/mL or lower was associated with 28-day mortality in enterococcal bacteremia. However, relatively large prospective studies are needed to examine the efficacy of vancomycin PK/PD parameters in patients with enterococcal bacteremia. | 6,069 | 328 | [
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|||||
Risk factors for prolonged virus shedding of respiratory tract and fecal in adults with severe acute respiratory syndrome coronavirus-2 infection. | 34390043 | The dynamic alteration and comparative study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding pattern during treatment are limited. This study explores the potential risk factors influencing prolonged viral shedding in COVID-19. | BACKGROUND | A total of 126 COVID-19 patients were enrolled in this retrospective longitudinal study. A multivariate logistic regression analysis was carried out to estimate the potential risk factors. | METHODS | 38.1% (48/126) cases presented prolonged respiratory tract viral shedding, and 30 (23.8%) cases presented prolonged rectal swab viral shedding. Obesity (OR, 3.31; 95% CI, 1.08-10.09), positive rectal swab (OR, 3.43; 95% CI, 1.53-7.7), treatment by lopinavir/ritonavir with chloroquine phosphate (OR, 2.5; 95% CI, 1.04-6.03), the interval from onset to antiviral treatment more than 7 days (OR, 2.26; 95% CI, 1.04-4.93), lower CD4+ T cell (OR, 0.92; 95% CI, 0.86-0.99) and higher NK cells (OR, 1.11; 95% CI, 1.02-1.20) were significantly associated with prolonged respiratory tract viral shedding. CD3-CD56+ NK cells (OR, 0.87; 95% CI, 0.76-0.99) were related with prolonged fecal shedding. | RESULTS | Obesity, delayed antiviral treatment, and positive SARS-CoV-2 for stool were independent risk factors for prolonged SARS-CoV-2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with chloroquine phosphate. CD4+ T cell and NK cells were significantly associated with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance. | CONCLUSIONS | [
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] | 8418473 | INTRODUCTION | Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.
Thus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration. | null | null | RESULTS | Demographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).
Characteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.
Abbreviations: BMI, body mass index; CT, computed tomography.
Epidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.
A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).
Characteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.
Abbreviations: BMI, body mass index; CT, computed tomography.
Epidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.
Laboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).
Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).
Treatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).
Treatment and outcomes of COVID‐19 patients
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.
Abbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.
We identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).
Dynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Dynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).
Treatment and outcomes of COVID‐19 patients
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.
Abbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.
We identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).
Dynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Dynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Risk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.
Multivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19
Univariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.
Abbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.
The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.
Multivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19
Univariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.
Abbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.
Risk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).
Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095). | null | null | [
"INTRODUCTION",
"Study design and participants",
"Data collection",
"Cutoff of respiratory and fecal viral shedding",
"Statistical analysis",
"Demographic and clinical manifestations",
"Laboratory findings and immunological indicators",
"Treatment and outcome",
"Risk factors with prolonged respiratory tract viral shedding",
"Risk factors with prolonged fecal viral shedding",
"AUTHOR CONTRIBUTIONS"
] | [
"Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.",
"This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.",
"Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.",
"The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.",
"Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.",
"A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.",
"Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).",
"All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset",
"The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.",
"Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).",
"Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content."
] | [
null,
null,
null,
null,
null,
null,
null,
null,
null,
null,
null
] | [
"INTRODUCTION",
"MATERIALS AND METHODS",
"Study design and participants",
"Data collection",
"Cutoff of respiratory and fecal viral shedding",
"Statistical analysis",
"RESULTS",
"Demographic and clinical manifestations",
"Laboratory findings and immunological indicators",
"Treatment and outcome",
"Risk factors with prolonged respiratory tract viral shedding",
"Risk factors with prolonged fecal viral shedding",
"DISCUSSION",
"CONFLICT OF INTEREST",
"AUTHOR CONTRIBUTIONS",
"Supporting information"
] | [
"Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.\nThus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.",
"Study design and participants This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\nThis longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.\nData collection Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\nDemographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.\nCutoff of respiratory and fecal viral shedding The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\nThe duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.\nStatistical analysis Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.\nContinuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.",
"This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.\nThis study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.\nThe privacy rights of human subjects are always observed.",
"Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7\n\nCases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.\nAll confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.",
"The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.",
"Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.",
"Demographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nA total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.\nLaboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nBaseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).\nTreatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nAll patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nRisk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nThe univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.\nRisk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).\nPatients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).",
"A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).\nCharacteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.\nAbbreviations: BMI, body mass index; CT, computed tomography.\nEpidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.",
"Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).",
"All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).\nTreatment and outcomes of COVID‐19 patients\nData are presented as the median and inter‐quartile range (IQR) and n (%).\np values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.\nAbbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.\nWe identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).\nDynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset\nDynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset",
"The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.\nMultivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19\nUnivariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.\nAbbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.",
"Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).",
"The viral shedding window of COVID‐19 remains largely uncharacterized, which poses challenges to reappraising discontinuation of quarantine. In our study, the dynamic detection of SARS‐CoV‐2 RNA on the respiratory tract and rectal swabs among 126 infected cases was conducted. We found that 38.1% (48/126) of confirmed cases presented prolonged respiratory tract viral shedding, and 23.8% (30/126) of cases presented prolonged fecal viral shedding. Obesity, positive detection of rectal swab species for viral RNA, delayed antiviral treatment, elevated NK, and decreased CD4+ T cell cells were significantly related to prolonged respiratory viral shedding, and patients treated by LPV/r combined with arbidol had remarkably shortened duration of respiratory viral shedding compared with those treated by LPV/r combined with CQ. We also identified that decreased CD3−CD56+ NK cells on admission were related to shorten fecal shedding.\nIn this study, the median duration from onset of symptoms to antiviral therapy was 3 days (IQR, 1–7.3 days), and the interval from onset to antiviral treatment more than 7 days was an independent risk factor for prolonged viral shedding. Our results were in line with the current report that timely detection and supportive care for symptomatic patients with COVID‐19 contributed to the clearance of viral RNA.15 Previous data indicated the complex interaction between obesity and increased pneumonia risk, and the underlying mechanism might involve immune system dysregulation.16, 17 A retrospective cohort study in French reported that the proportion of COVID‐19 patients who required invasive mechanical ventilation increased with BMI.18 Additionally, a previous study on influenza A H1N1 found obesity was associated with prolonged hospitalization and the need for IMV.19 In our cohort, the obesity rate was 13.5%, and obesity was an independent predisposition factor for prolonged respiratory tract viral RNA shedding after adjustment for age and gender. Obesity‐related low‐grade inflammation appeared to be associated with the prognosis of virus infection, and further study is to be warranted to elucidate etiological mechanisms.\nNo specific antiviral drugs have been approved to be safe and effective for the treatment of COVID‐19 so far. Some drugs with antiviral properties and immunomodulatory effects were officially recommended for COVID‐19, including LPV/r, arbidol, CQ, etc.12, 20 LPV/r and arbidol were previously recommended for patients with SARS and MERS‐CoV.21, 22 Some studies implied that the use of LPV/r and arbidol was associated with an apparent favorable clinical response to COVID‐19,23, 24 but controversy existed in different studies. For example, Cheng and his/her colleagues reported LPV/r had no advantage in shortening the duration of SARS‐CoV‐2 shedding in five mild patients in Taiwan.25 The US Center for Disease Control clarified that CQ inhibited viral replication by interfering with SARS‐CoV‐2 binding to the ACE2 receptor.26 CQ has been used to treat COVID‐19 with no benefits having been observed in a multinational registry analysis.27 Our findings demonstrated that the use of LPV/r in combination with arbidol was linked with a shorter hospital stay and duration of viral shedding compared with the use of LPV/r in combination with CQ.\nT lymphocyte plays a vital role in SARS‐CoV‐2 elimination by stimulating antigen‐(Ag) specific T lymphocytes.28 Emerging data indicated that lymphocytopenia was one of the important predictor factors influencing exacerbation and mortality of patients with COVID‐19.29, 30, 31 Likewise, our data identified that decreased CD4+ T cell was a significant risk factor for prolonged viral shedding of the respiratory tract. NK cells were one of the predominant lymphocyte subsets and accounted for the third of intrahepatic lymphocytes.32 Viral infection‐induced NK cells present activated and mature phenotypes with stronger cytotoxic capabilities in a murine model.33 However, our study showed the increased NK cell was a risk factor for prolonged viral shedding of the respiratory tract but a protective factor for fecal viral shedding. Our study also showed that 60 (47.6%) of 126 COVID‐19 patients detected positive fecal viral results, and 30 (23.8%) presented prolonged fecal viral shedding, and the positive SARS‐CoV‐2 for stool was related to prolonged duration of respiratory viral shedding. However, GI symptoms just occurred in 11(8.7%) of patients in our study. Likewise, a meta‐analysis reported that approximately 7.4% of patients with COVID‐19 experienced diarrhea, and about 30% to 50% of patients were detected for positive SARS‐CoV‐2 RNA.34 Chen et al.15 found diarrhea was independently associated with prolonged SARS‐CoV‐2 RNA shedding in the respiratory tract among 267 confirmed cases. It has been proven that ACE2 is abundantly expressed in the GI tract.35, 36 Positive detection of SARS‐CoV‐2 in digestive tract specimens suggested that pathogenicity of SARS‐CoV‐2 might be transmitted by the fecal‐oral route, not limited by droplet transmission.37 In our study, the viral shedding patterns of the respiratory and digestive tract were not consistent. Similarly, Wang et al.38 reported that the median duration of positive SARS‐CoV‐2 in fecal samples was 25 days, significantly longer than that in respiratory tract samples by approximately 9 days and that patients with SARS‐CoV‐2 RNA in fecal presented higher proportions of complete absorption and no lesion progression of chest CT results.\nThere were some limitations in this study. First, most patients enrolled in this study had mild clinical symptoms, so our findings may not apply to severe and critical patients. Second, immune indicators including T lymphocyte subsets and cytokines were not conducted in all patients, so the role of immunomodulatory effects in eliminating SARS‐CoV‐2 might be underestimated. Third, our study was a single‐center study and limited by a relatively small sample size. Fourth, the underlying reason for the unparallel detection results between the respiratory tract and fecal samples needs to be clarified in the future.\nIn conclusion, obesity, delayed antiviral treatment, and positive SARS‐CoV‐2 for stool were independent risk factors for prolonged SARS‐CoV‐2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with CQ. Decreased CD4+ T cell and elevated CD3−CD56+ NK cells were associated with prolonged respiratory tract RNA shedding, while decreased CD3−CD56+ NK cells might be linked with shortened fecal RNA shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.",
"None.",
"Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.",
"Fig S1\nClick here for additional data file.\nTab S1\nClick here for additional data file.\nTab S2\nClick here for additional data file.\nTab S3\nClick here for additional data file.\nTab S4\nClick here for additional data file."
] | [
null,
"materials-and-methods",
null,
null,
null,
null,
"results",
null,
null,
null,
null,
null,
"discussion",
"COI-statement",
null,
"supplementary-material"
] | [
"COVID‐19",
"longitudinal study",
"prolonged viral shedding",
"risk factor",
"SARS‐CoV‐2"
] | INTRODUCTION:
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) results in the coronavirus disease 2019 (COVID‐19) pandemic.1 The clinical and epidemiological characteristics of COVID‐19 and the structure of SARS‐CoV‐2 have been elucidated gradually.2, 3, 4, 5 Although people are generally susceptible to SARS‐CoV‐2, approximately 80% of COVID‐19 are mild.6 However, many cases were reported persistent positive for respiratory SARS‐CoV‐2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved.7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS‐CoV‐2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample.8, 9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding.10, 11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID‐19 are still elusive.
Thus, we performed a longitudinal retrospective study of 126 hospitalized laboratory‐confirmed COVID‐19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.
MATERIALS AND METHODS:
Study design and participants This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.
This study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.
The privacy rights of human subjects are always observed.
This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.
This study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.
The privacy rights of human subjects are always observed.
Data collection Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7
Cases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.
All confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.
Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7
Cases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.
All confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.
Cutoff of respiratory and fecal viral shedding The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.
The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.
Statistical analysis Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.
Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.
Study design and participants:
This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS‐CoV‐2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China.12 The final follow‐up was up to June 15, 2020.
This study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ‐NBEY‐KY‐2020–008–01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.
The privacy rights of human subjects are always observed.
Data collection:
Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated.7
Cases were confirmed by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result.2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT‐PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.
All confirmed cases were offered treatment, and the antiviral drugs mainly included LPV/r (400 mg/100 mg, orally, bid), arbidol (200 mg, orally, TID), chloroquine phosphate (CQ, 500 mg, orally, bid), and alpha‐interferon (5 million U, inhalation, QD). All data were double‐checked by two physicians.
Cutoff of respiratory and fecal viral shedding:
The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time‐dependent respiratory tract viral testing among 802 initial‐testing‐positive patients revealed that 50% of patients turned negative in 28 days.13 60 patients with both positive viral RNA test for respiratory tract and rectal swab specimens simultaneously showed that the duration of viral shedding was 28.5 days (IQR, 21.25–38) for respiratory tract and 27.5 days (IQR, 18.3–39) for rectal swabs. Thus, the cutoff of the prolonged respiratory tract or fecal viral shedding was determined as the duration of SARS‐CoV‐2 RNA positive for more than 28 days in this study.
Statistical analysis:
Continuous variables were presented median and inter‐quartile range (IQR), and the differences between groups were evaluated with the Mann‐Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi‐square tests or Fisher's exact test. Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding. All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two‐sided p < 0.05 was defined as statistical significance.
RESULTS:
Demographic and clinical manifestations A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).
Characteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.
Abbreviations: BMI, body mass index; CT, computed tomography.
Epidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.
A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).
Characteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.
Abbreviations: BMI, body mass index; CT, computed tomography.
Epidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.
Laboratory findings and immunological indicators Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).
Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).
Treatment and outcome All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).
Treatment and outcomes of COVID‐19 patients
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.
Abbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.
We identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).
Dynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Dynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).
Treatment and outcomes of COVID‐19 patients
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.
Abbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.
We identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).
Dynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Dynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Risk factors with prolonged respiratory tract viral shedding The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.
Multivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19
Univariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.
Abbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.
The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.
Multivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19
Univariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.
Abbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.
Risk factors with prolonged fecal viral shedding Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).
Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).
Demographic and clinical manifestations:
A total of 126 hospitalized patients with COVID‐19 were included, and 38.1% (48/126) of patients presented the duration of respiratory tract viral shedding >28 days (Table 1). Of these patients, 82 (65.1%) were women and the median age was 50 (IQR, 35.8–58.3), and 29 (23.0%) had one or more comorbidities. The median body mass index (BMI) was 23.5 kg/m2 (IQR, 20.9–26.0), and cases with a duration of respiratory tract viral shedding >28 days showed a significantly higher percentage of obesity (BMI >28 kg/m2) than the <28 days cases (20.8% vs 9%).
Characteristics of 126 Hospitalized Patients with SARS‐CoV‐2 infection
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from Mann‐Whitney U test, chi‐square test, and Fisher's exact test.
Abbreviations: BMI, body mass index; CT, computed tomography.
Epidemiological data showed that there were 101(80.2%) patients who had closely contacted with confirmed COVID‐19 patients, and most cases were temple gathering and familial cluster.14 The most common symptoms at the onset of illness were fever (84 [66.7%]), cough (62 [42.9%]), and fatigue (25 [19.8%]). 15 (11.9%), 106 (84.1%), and 5 (4.0%) patients were categorized into the group of mild, moderate, and severe types, respectively. Additionally, 60 (47.6%) cases were presented with positive viral RNA test for rectal swab, and 30 (23.8%) cases presented the duration of fecal samples viral shedding for more than 28 days (Table 1). For chest CT scans, more than half of the patients (67, 53.2%) showed bilateral pneumonia on admission.
Laboratory findings and immunological indicators:
Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non‐prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discovered in other laboratory findings between the two groups. The median Cq values on admission were 32.3 (IQR 28.3–36.3) for ORF1ab and 31.7 (IQR 28.1–35.4) for N‐gene, and there was no significant difference in baseline Cq values between the respiratory shedding prolonged and non‐prolonged groups (p ˃ 0.05) (Table S1).
Treatment and outcome:
All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1–7.3) from symptom onset (Table 2). Just 15.1% (19/126) of patients received systemic corticosteroid treatment. Based on the therapeutic regimen, 37(29.4%) patients were treated by LPV/r with arbidol and 72 (57.1%) by LPV/r with CQ. The median duration of respiratory tract SARS‐CoV‐2 shedding and the hospital stay in the non‐prolonged group (<28 days) were 19 days (IQR, 14.5–22.5) and 14 days (IQR, 12–18), respectively, which were significantly shorter than those in the prolonged group (>28 days) of 37.5 days (IQR, 32–46.8) and 21 days (IQR, 16.5–26.8) (p < 0.05) (Table 2).
Treatment and outcomes of COVID‐19 patients
Data are presented as the median and inter‐quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from the Mann‐Whitney U test and chi‐square test.
Abbreviations: CQ, chloroquine phosphate; LPV/r, lopinavir/ritonavir.
We identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient 35 and patient 36, had the duration of viral shedding of SARS‐CoV‐2 more than 90 days. Patient 35 was initially positive of respiratory virus and converted to negative at day 14 after onset, but later, reverted to positive from day 32 to day 48 and positive at day 75, day 84, and day 90 (Figure 1). The detection for rectal swab samples showed a conversion from an initial SARS‐CoV‐2 negative result to positive and back to negative in 12 days (Figure 2). Patient 36 repeatedly tested positive via rectal swab specimens for 68 days since onset, then test negative at day 72, suspected positive at day 78, positive again at day 84, suspected positive at day 90, and negative at day 96and 98 (Figure 2). However, the respiratory viral shedding time in patient 36 was just 12 days (Figure 1).
Dynamic changes in SARS‐CoV‐2 RNA for respiratory tract samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Dynamic changes in SARS‐CoV‐2 RNA for stool samples since illness onset. SARS‐CoV‐2 negative results are represented by blue dots, invalid results by yellow dots, and positive results are in red dots, plotted on a time scale from the date of the first test since illness onset
Risk factors with prolonged respiratory tract viral shedding:
The univariable analysis and multivariable logistic regression analysis were presented in Table 3 and Table S2. The multivariable logistic regression analysis suggested that obesity (BMI >28) (OR, 3.31; 95% CI, 1.08–10.09), positive detection of rectal swab species for SARS‐CoV‐2 RNA (OR, 3.43; 95% CI, 1.53–7.7), treatment by LPV/r with CQ (OR, 2.5; 95% CI, 1.04–6.03), the prolonged interval time of more than 7 days from illness onset to antiviral treatment (OR, 2.26; 95% CI, 1.04–4.93), CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99), and NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged viral shedding of respiratory tract even after the adjustment for age and gender.
Multivariable logistic regression analysis of factors associated with prolonged shedding of SARS‐CoV‐2 RNA among patients with COVID‐19
Univariate and adjusted multivariate logistic regression analysis were carried out to estimate the potential risk factors associated with prolonged duration of SARS‐CoV‐2 RNA shedding, and the age and sex were adjusted as covariates in adjusted model.
Abbreviations: BMI, body mass index; LPV/r, lopinavir/ritonavir.
Risk factors with prolonged fecal viral shedding:
Patients with positive detection of rectal swab species for SARS‐CoV‐2 accounted for almost 47.6% (60/126), and the white blood cell count (OR, 1.281; 95% CI, 1.030–1.593), hs‐CRP (OR, 0.975; 95% CI, 0.952–0.999), CD3+ CD8+ T cells (OR, 0.9; 95% CI, 0.812–0.997;), and CD4/CD8 ratio (OR, 2.016; 95% CI, 1.037–3.920) on admission were significantly associated with positive viral shedding in rectal swab species after adjustment for age and gender (Figure S1 and Table S3). Additionally, CD3−CD56+ NK cells (OR, 0.87; 95% CI: 0.76–0.99) on admission were linked to prolonged fecal shedding (Table 3 and Table S4). However, Spearman's correlation analysis showed no significant correlation between the duration of the respiratory tract and fecal viral shedding among 60 patients with both positive SARS‐CoV‐2 RNA in the respiratory tract and fecal specimens simultaneously (r = 0.218, p = 0.095).
DISCUSSION:
The viral shedding window of COVID‐19 remains largely uncharacterized, which poses challenges to reappraising discontinuation of quarantine. In our study, the dynamic detection of SARS‐CoV‐2 RNA on the respiratory tract and rectal swabs among 126 infected cases was conducted. We found that 38.1% (48/126) of confirmed cases presented prolonged respiratory tract viral shedding, and 23.8% (30/126) of cases presented prolonged fecal viral shedding. Obesity, positive detection of rectal swab species for viral RNA, delayed antiviral treatment, elevated NK, and decreased CD4+ T cell cells were significantly related to prolonged respiratory viral shedding, and patients treated by LPV/r combined with arbidol had remarkably shortened duration of respiratory viral shedding compared with those treated by LPV/r combined with CQ. We also identified that decreased CD3−CD56+ NK cells on admission were related to shorten fecal shedding.
In this study, the median duration from onset of symptoms to antiviral therapy was 3 days (IQR, 1–7.3 days), and the interval from onset to antiviral treatment more than 7 days was an independent risk factor for prolonged viral shedding. Our results were in line with the current report that timely detection and supportive care for symptomatic patients with COVID‐19 contributed to the clearance of viral RNA.15 Previous data indicated the complex interaction between obesity and increased pneumonia risk, and the underlying mechanism might involve immune system dysregulation.16, 17 A retrospective cohort study in French reported that the proportion of COVID‐19 patients who required invasive mechanical ventilation increased with BMI.18 Additionally, a previous study on influenza A H1N1 found obesity was associated with prolonged hospitalization and the need for IMV.19 In our cohort, the obesity rate was 13.5%, and obesity was an independent predisposition factor for prolonged respiratory tract viral RNA shedding after adjustment for age and gender. Obesity‐related low‐grade inflammation appeared to be associated with the prognosis of virus infection, and further study is to be warranted to elucidate etiological mechanisms.
No specific antiviral drugs have been approved to be safe and effective for the treatment of COVID‐19 so far. Some drugs with antiviral properties and immunomodulatory effects were officially recommended for COVID‐19, including LPV/r, arbidol, CQ, etc.12, 20 LPV/r and arbidol were previously recommended for patients with SARS and MERS‐CoV.21, 22 Some studies implied that the use of LPV/r and arbidol was associated with an apparent favorable clinical response to COVID‐19,23, 24 but controversy existed in different studies. For example, Cheng and his/her colleagues reported LPV/r had no advantage in shortening the duration of SARS‐CoV‐2 shedding in five mild patients in Taiwan.25 The US Center for Disease Control clarified that CQ inhibited viral replication by interfering with SARS‐CoV‐2 binding to the ACE2 receptor.26 CQ has been used to treat COVID‐19 with no benefits having been observed in a multinational registry analysis.27 Our findings demonstrated that the use of LPV/r in combination with arbidol was linked with a shorter hospital stay and duration of viral shedding compared with the use of LPV/r in combination with CQ.
T lymphocyte plays a vital role in SARS‐CoV‐2 elimination by stimulating antigen‐(Ag) specific T lymphocytes.28 Emerging data indicated that lymphocytopenia was one of the important predictor factors influencing exacerbation and mortality of patients with COVID‐19.29, 30, 31 Likewise, our data identified that decreased CD4+ T cell was a significant risk factor for prolonged viral shedding of the respiratory tract. NK cells were one of the predominant lymphocyte subsets and accounted for the third of intrahepatic lymphocytes.32 Viral infection‐induced NK cells present activated and mature phenotypes with stronger cytotoxic capabilities in a murine model.33 However, our study showed the increased NK cell was a risk factor for prolonged viral shedding of the respiratory tract but a protective factor for fecal viral shedding. Our study also showed that 60 (47.6%) of 126 COVID‐19 patients detected positive fecal viral results, and 30 (23.8%) presented prolonged fecal viral shedding, and the positive SARS‐CoV‐2 for stool was related to prolonged duration of respiratory viral shedding. However, GI symptoms just occurred in 11(8.7%) of patients in our study. Likewise, a meta‐analysis reported that approximately 7.4% of patients with COVID‐19 experienced diarrhea, and about 30% to 50% of patients were detected for positive SARS‐CoV‐2 RNA.34 Chen et al.15 found diarrhea was independently associated with prolonged SARS‐CoV‐2 RNA shedding in the respiratory tract among 267 confirmed cases. It has been proven that ACE2 is abundantly expressed in the GI tract.35, 36 Positive detection of SARS‐CoV‐2 in digestive tract specimens suggested that pathogenicity of SARS‐CoV‐2 might be transmitted by the fecal‐oral route, not limited by droplet transmission.37 In our study, the viral shedding patterns of the respiratory and digestive tract were not consistent. Similarly, Wang et al.38 reported that the median duration of positive SARS‐CoV‐2 in fecal samples was 25 days, significantly longer than that in respiratory tract samples by approximately 9 days and that patients with SARS‐CoV‐2 RNA in fecal presented higher proportions of complete absorption and no lesion progression of chest CT results.
There were some limitations in this study. First, most patients enrolled in this study had mild clinical symptoms, so our findings may not apply to severe and critical patients. Second, immune indicators including T lymphocyte subsets and cytokines were not conducted in all patients, so the role of immunomodulatory effects in eliminating SARS‐CoV‐2 might be underestimated. Third, our study was a single‐center study and limited by a relatively small sample size. Fourth, the underlying reason for the unparallel detection results between the respiratory tract and fecal samples needs to be clarified in the future.
In conclusion, obesity, delayed antiviral treatment, and positive SARS‐CoV‐2 for stool were independent risk factors for prolonged SARS‐CoV‐2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with CQ. Decreased CD4+ T cell and elevated CD3−CD56+ NK cells were associated with prolonged respiratory tract RNA shedding, while decreased CD3−CD56+ NK cells might be linked with shortened fecal RNA shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.
CONFLICT OF INTEREST:
None.
AUTHOR CONTRIBUTIONS:
Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.
Supporting information:
Fig S1
Click here for additional data file.
Tab S1
Click here for additional data file.
Tab S2
Click here for additional data file.
Tab S3
Click here for additional data file.
Tab S4
Click here for additional data file.
| Background:
The dynamic alteration and comparative study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding pattern during treatment are limited. This study explores the potential risk factors influencing prolonged viral shedding in COVID-19.
Methods:
A total of 126 COVID-19 patients were enrolled in this retrospective longitudinal study. A multivariate logistic regression analysis was carried out to estimate the potential risk factors.
Results:
38.1% (48/126) cases presented prolonged respiratory tract viral shedding, and 30 (23.8%) cases presented prolonged rectal swab viral shedding. Obesity (OR, 3.31; 95% CI, 1.08-10.09), positive rectal swab (OR, 3.43; 95% CI, 1.53-7.7), treatment by lopinavir/ritonavir with chloroquine phosphate (OR, 2.5; 95% CI, 1.04-6.03), the interval from onset to antiviral treatment more than 7 days (OR, 2.26; 95% CI, 1.04-4.93), lower CD4+ T cell (OR, 0.92; 95% CI, 0.86-0.99) and higher NK cells (OR, 1.11; 95% CI, 1.02-1.20) were significantly associated with prolonged respiratory tract viral shedding. CD3-CD56+ NK cells (OR, 0.87; 95% CI, 0.76-0.99) were related with prolonged fecal shedding.
Conclusions:
Obesity, delayed antiviral treatment, and positive SARS-CoV-2 for stool were independent risk factors for prolonged SARS-CoV-2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with chloroquine phosphate. CD4+ T cell and NK cells were significantly associated with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance. | null | null | 7,980 | 356 | [
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|||
Characterizing Help-Seeking Searches for Substance Use Treatment From Google Trends and Assessing Their Use for Infoveillance: Longitudinal Descriptive and Validation Statistical Analysis. | 36454620 | There is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUDs) seeking help within a given geographical area. This presents a challenge to policy makers in the effective deployment of resources for the treatment of SUDs. Internet search queries related to help seeking for SUDs using Google Trends may represent a low-cost, real-time, and data-driven infoveillance tool to address this shortfall in information. | BACKGROUND | We used negative binomial regression models to examine temporal trends in the annual SUD help-seeking internet search queries from Google Trends by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol from 2010 to 2020. To validate the value of these data for surveillance purposes, we then used negative binomial regression models to investigate the relationship between SUD help-seeking searches and state-level outcomes across the continuum of care (including lack of care). We started by looking at associations with self-reported treatment need using data from the National Survey on Drug Use and Health, a national survey of the US general population. Next, we explored associations with treatment admission rates from the Treatment Episode Data Set, a national data system on SUD treatment facilities. Finally, we studied associations with state-level rates of people experiencing and dying from an opioid overdose, using data from the Agency for Healthcare Research and Quality and the CDC WONDER database. | METHODS | Statistically significant differences in help-seeking searches were observed over time between 2010 and 2020 (based on P<.05 for the corresponding Wald tests). We were able to identify outlier states for each drug over time (eg, West Virginia for both opioids and methamphetamine), indicating significantly higher help-seeking behaviors compared to national trends. Results from our validation analyses across different outcomes showed positive, statistically significant associations for the models relating to treatment need for alcohol use, treatment admissions for opioid and methamphetamine use, emergency department visits related to opioid use, and opioid overdose mortality data (based on regression coefficients having P≤.05). | RESULTS | This study demonstrates the clear potential for using internet search queries from Google Trends as an infoveillance tool to predict the demand for substance use treatment spatially and temporally, especially for opioid use disorders. | CONCLUSIONS | [
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] | 9756118 | Introduction | Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].
Indirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.
A promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].
While the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.
We sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.
We tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts. | Methods | Extraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.
We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.
Statistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.
Descriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.
aQuery fractions (QFs) refer to queries per every 1 million total Google searches.
bMeth: methamphetamine.
cVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.
Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.
Descriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.
aQuery fractions (QFs) refer to queries per every 1 million total Google searches.
bMeth: methamphetamine.
cVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.
Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.
First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.
Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.
We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.
Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.
All analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].
We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.
All analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].
Ethical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).
Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX). | Results | Descriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].
Average help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.
Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].
Average help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.
Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).
Table 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).
Gini coefficient estimates from query fractions (QF) variables across substances and years.
Box and whisker plot of help-seeking searches for opioid use.
The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).
Table 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).
Gini coefficient estimates from query fractions (QF) variables across substances and years.
Box and whisker plot of help-seeking searches for opioid use.
Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”
Predictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.
Estimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.
The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”
Predictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.
Estimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.
Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.
In the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).
Estimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.
aQF: query fraction.
Predictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.
The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.
In the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).
Estimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.
aQF: query fraction.
Predictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.
Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).
Estimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.
aQF: query fraction.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).
Estimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.
aQF: query fraction. | Conclusions | This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs. | [
"Extraction of Google Search Query Data",
"Statistical Analysis of Google Search Query Data",
"Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1)",
"Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2)",
"Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)",
"Ethical Considerations",
"Descriptive Analysis of Google Search Query Data for SUD Help Seeking",
"Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking",
"Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1)",
"Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2)",
"Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)",
"Principal Findings",
"Limitations",
"Conclusions"
] | [
"We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.",
"Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.",
"First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.",
"We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.",
"We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].",
"Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).",
"Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.",
"The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.",
"The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.",
"The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.",
"The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.",
"To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].",
"Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].",
"This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs."
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] | [
"Introduction",
"Methods",
"Extraction of Google Search Query Data",
"Statistical Analysis of Google Search Query Data",
"Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1)",
"Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2)",
"Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)",
"Ethical Considerations",
"Results",
"Descriptive Analysis of Google Search Query Data for SUD Help Seeking",
"Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking",
"Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1)",
"Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2)",
"Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3)",
"Discussion",
"Principal Findings",
"Limitations",
"Conclusions"
] | [
"Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].\nIndirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.\nA promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].\nWhile the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.\nWe sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.\nWe tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.",
"Extraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nWe obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.\nStatistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nNegative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.\nValidation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nFirst, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.\nValidation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nWe investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.\nValidation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nWe investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].\nEthical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).\nEthical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).",
"We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.",
"Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.\nDescriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.\naQuery fractions (QFs) refer to queries per every 1 million total Google searches.\nbMeth: methamphetamine.\ncVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.",
"First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.",
"We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.",
"We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.\nAll analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].",
"Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).",
"Descriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nFigure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.\nStatistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nThe negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nThe analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.\nValidation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nThe analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.\nValidation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.",
"Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].\nAverage help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.",
"The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).\nTable 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).\nGini coefficient estimates from query fractions (QF) variables across substances and years.\nBox and whisker plot of help-seeking searches for opioid use.",
"The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”\nPredictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.\nEstimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.",
"The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.\nIn the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).\nEstimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.\naQF: query fraction.\nPredictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.",
"The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.\nThe analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).\nEstimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.\naQF: query fraction.",
"Principal Findings To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\nTo our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].\nLimitations Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\nOur study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].\nConclusions This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.\nThis study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.",
"To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.\nImportantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.\nFurther analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].",
"Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.\nWhile our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.\nImportantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.\nWhile the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].",
"This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs."
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null,
null,
null,
"discussion",
null,
null,
null
] | [
"internet",
"search",
"help-seeking",
"substance use treatment",
"surveillance",
"infoveillance",
"google trends"
] | Introduction:
Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].
Indirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.
A promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].
While the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.
We sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.
We tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.
Methods:
Extraction of Google Search Query Data We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.
We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.
Statistical Analysis of Google Search Query Data Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.
Descriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.
aQuery fractions (QFs) refer to queries per every 1 million total Google searches.
bMeth: methamphetamine.
cVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.
Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.
Descriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.
aQuery fractions (QFs) refer to queries per every 1 million total Google searches.
bMeth: methamphetamine.
cVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.
Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1) First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.
First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.
Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2) We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.
We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.
Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.
All analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].
We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.
All analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].
Ethical Considerations Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).
Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).
Extraction of Google Search Query Data:
We obtained Google queries in November 2020 originating from the United States that included the terms “quit,” “stop,” “rehab(s),” “rehabilitation,” “treatment(s),” “help,” or “detox” in combination with (A) alcohol (“alcohol,” “alcoholic,” or “alcoholism”), (B) cannabis (“cannabis” or “marijuana”), (C) cocaine (“cocaine”), (D) methamphetamine (“methamphetamine” or “meth”), or (E) opioids (“opioid(s),” “heroin,” “fentanyl,” “oxycontin,” “oxycodone,” “codeine,” “hydrocodone,” “morphine”) from January 1, 2010, to November 1, 2020. For example, “Where can I get help for alcoholism” would be included in the alcohol help-seeking search category. These searches were specified without quotation marks, and the data were obtained by selecting the “search terms” option, as opposed to the “topics” option. The search terms for our drugs of interest corresponded to the standard dictionary term for each (eg, methamphetamine), alongside other commonly used terms if relevant (eg, meth) based on the authors’ expertise in SUD and others’ contributions in this field [12,13,16]. For opioids, we also included names of most frequently used street drugs (ie, heroin, fentanyl) and prescription drugs with their brand name if very commonly used (eg, oxycodone and OxyContin). For alcohol, we also included “alcoholic” and alcoholism,” as these are part of the mainstream English lexicon used to describe alcohol use disorders. Despite the extensive range of slang terms used to describe drugs [25], these were not included, given that slang is ever evolving, its linguist survival is often short-lived, and it is typically context specific and limited in use within specific social settings [26]. Given our focus on treatment seeking (ie, a formal context), our broad geographical scale (ie, all US states), and our extended time scale (10 years), we opted to limit our search to the most standard terms to allow for consistency over time and space. The search query data were obtained for each calendar year between 2010 and 2020 from Google Trends using the Google Application Programming Interface (API) Client library in Python [27]. Trends in Google queries were measured in query fractions (QFs), which estimate the number of searches that mention substance-specific keywords, in combination with the help-seeking keywords, in the time frame and geography divided by the total number of searches in the same time frame and geography and expressed as a rate per 1 million searches. This approach facilitates comparability by adjusting for changes in Google usage over time, as well as differences across states and substance types.
Statistical Analysis of Google Search Query Data:
Negative binomial regression models were fitted to the QF data to make inferences regarding the significance of temporal changes in help-seeking queries. Negative binomial regression is commonly used to analyze count and rate data exhibiting over-dispersion (ie, variance greater than the mean) [28]. The QF data in this study were found to be overdispersed, as shown in Table 1; therefore, the negative binomial model was chosen to analyze these data. The model specifications included a main fixed effect for year (ie, 2010 through 2020). Random effects were included for intercept terms to account for differences between states at the beginning of the study and for correlations between data points collected in the same states over different years. Moreover, autocorrelated error terms were specified to account for correlations in the data between successive time points. A Wald test was performed to confirm whether the variable “year” was statistically significant for each of the models [29]. We calculated Gini coefficients [30] to quantify the dispersion of help-seeking queries across states for each substance and each year.
Descriptive statistics from 2010 to 2020 for annual search query fractions (QFs) by substance typea.
aQuery fractions (QFs) refer to queries per every 1 million total Google searches.
bMeth: methamphetamine.
cVariable estimated by combining QF statistics for opioid, methamphetamine, and cocaine use treatment seeking.
Validation of Google Search Queries as Indicators of Unmet Treatment Needs for Substance Use (Hypothesis 1):
First, an analysis exploring the number of people needing but not receiving treatment at a specialty facility for SUD in the past year was conducted using data from the National Survey on Drug Use and Health (NSDUH) for the years 2016 to 2019. The NSDUH is an annual state-level representative survey of the civilian, noninstitutionalized population aged 12 or older and is publicly accessible from the website of the Substance Abuse and Mental Health Services Administration (SAMHSA) [31]. To produce state-level estimates for variables collected in this survey (rounded to the nearest thousand), the Research Triangle Institute conducted an analysis of the sample data for each year using survey-weighted hierarchical Bayes methods [32]. The NSDUH separately enquires about needing but not receiving treatment at a specialty facility for alcohol and illicit drug use in the past year. Therefore, a negative binomial model was utilized to regress NSDUH estimates specific to alcohol use on the variables of alcohol QF and year, and a second analysis was conducted exploring illicit drug use, also using a negative binomial regression model. The main fixed effects included in the second model were a composite QF statistic, estimated by combining QF statistics for opioid, methamphetamine, and cocaine use unmet treatment need and the year corresponding to the data points. For both sets of analyses involving NSDUH data, random effects were specified for intercept terms and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33], which reflect the number of times the event could have potentially occurred. Additionally, interactions between the main fixed effects were assessed to infer if and how the association between alcohol QF and treatment need as well as the association between the composite illicit drug QF and treatment need varied across the years.
Validation of Google Search Queries as Indicators of Treatment Seeking for Substance Use Disorders (Hypothesis 2):
We investigated whether there was a positive association between treatment-seeking searches and the receipt of treatments for SUD. For the latter, data were obtained from the Treatment Episode Data Set: Admissions (TEDS-A) data sets (years 2012 to 2018) on the number of admissions to substance use facilities by primary substance for which treatment was sought [34]. Observations from these data were only selected for admissions involving individual referrals for treatment (ie, excluding mandated treatment visits). Negative binomial regression models were fitted to the admissions data with separate analyses for the different types of substance use (alcohol, cannabis, opioids, cocaine, and methamphetamine). In each model, the year and corresponding help-seeking QF variable (ie, substance-specific) were included as main fixed effects, along with intercepts for the states as random effects and the natural logarithm of states’ population estimates from the US Census Bureau as offset terms [33]. Additionally, interactions between the main fixed effects variables (ie, help-seeking QF and year) were assessed to infer if and how the association between treatment-seeking searches and admissions varied across the years.
Validation Of Google Search Queries as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3):
We investigated whether help-seeking searches were positively associated with nonfatal and fatal opioid overdose. Accordingly, data on the rate of ED encounters associated with opioid use per 100,000 people (mostly corresponding to nonfatal overdoses) were obtained from the Agency for Healthcare Quality and Research (AHRQ) [35], and data on the number of opioid-related overdose deaths were obtained from the CDC WONDER database (using the criteria set out in previous research [36]). For these analyses, we regressed state-specific opioid hospitalization rates and mortality count data, respectively, on the variables opioid QF and the year corresponding to the data points, using a negative binomial specification. Once again, random intercepts were included to account for correlations between repeated observations within states. The analysis of fatal opioid overdose data included an offset term corresponding to the log of the state-level population. This approach was not taken for the AHRQ data, as these data were obtained in the form of rates, rather than count data. The interaction terms between the main fixed effects (ie, opioid QF and year) were also assessed.
All analyses were conducted using R software (version 4.1.0.), and the negative binomial models were fitted using the glmmTMB package [28].
Ethical Considerations:
Ethical review was not required because the study relied on public, aggregated, and deidentified data. Given that this study relied on the use of secondary deidentified data (numbers were aggregated to the state level), the Institutional Review Board of the University of California San Diego determined that an ethics review was not required (Project #200332XX).
Results:
Descriptive Analysis of Google Search Query Data for SUD Help Seeking Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].
Average help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.
Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].
Average help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.
Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).
Table 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).
Gini coefficient estimates from query fractions (QF) variables across substances and years.
Box and whisker plot of help-seeking searches for opioid use.
The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).
Table 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).
Gini coefficient estimates from query fractions (QF) variables across substances and years.
Box and whisker plot of help-seeking searches for opioid use.
Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1) The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”
Predictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.
Estimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.
The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”
Predictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.
Estimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.
Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2) The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.
In the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).
Estimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.
aQF: query fraction.
Predictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.
The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.
In the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).
Estimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.
aQF: query fraction.
Predictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.
Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3) The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).
Estimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.
aQF: query fraction.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).
Estimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.
aQF: query fraction.
Descriptive Analysis of Google Search Query Data for SUD Help Seeking:
Figure 1 shows that QF values were highest on average in 2010 for all substances, except in the case of alcohol, where it was the second highest year for searching, and the highest levels were observed in 2020. Help-seeking searches were lowest in 2012 in the case of opioids, in 2013 in the case of alcohol and cannabis, and 2014 in the case of cocaine and methamphetamine. Figure 1 also shows the varying levels of completeness in the search query data across the different types of SUD. Missing data points can occur in cases involving very low search volumes [37].
Average help-seeking trends for substance use. Gray lines represent state-specific trends while black dots represent the mean estimates for states* with data across all time points. *Number of states (plus the District of Columbia) with nonmissing query data by substance: Alcohol=51, Opioid=41, Cannabis=44, Methamphetamine=32, Cocaine=25. Number of data points by substance: Alcohol=561, Opioid=461, Cannabis=484, Methamphetamine=382, Cocaine=285.
Statistical Analysis of Time and Geographic Trends in Google Search Query Data for SUD Help Seeking:
The negative binomial regression analyses of QF data (results shown in Table S1 in Multimedia Appendix 1) showed that the variable “year” was statistically significant for all substance types based on the resulting Wald tests (P<.001). This indicates that there were important variations in help-seeking searches between the various years. Pairwise comparisons tests for significant differences in the help-seeking search counts over consecutive years were performed by applying Bonferroni corrections to the outputs of the negative binomial regression analyses (results shown in Table S2 in Multimedia Appendix 1). All substances showed significant decreases from 2010 to 2011 (alcohol: 10%, cannabis: 21%, cocaine: 25%, methamphetamine: 43%, opioids: 26%). Aside from this, significant differences across consecutive years were found for alcohol (13% increase from 2015 to 2016 and 21% increase from 2019 to 2020) and methamphetamine (23% increase from 2019 to 2020).
Table 1 provides descriptive statistics for each of the QF variables to facilitate the interpretation of all subsequent regression analyses where these were employed as independent variables. Inequalities in help-seeking searches between states, as measured by Gini coefficients, were highest for methamphetamine across all years (Figure 2). Inequalities were lowest for alcohol for all years except those between 2016 and 2018, when cocaine was the lowest. A consistent trend observed across all substances was that inequalities were highest in 2010 and then reduced over time before sharply increasing again in 2020. These results can also be further understood by looking at the box and whisker plots, which show the spread of data points across states by substance type and year (see Figure 3 for opioid use and Figure S1 in Multimedia Appendix 1 for other substances).
Gini coefficient estimates from query fractions (QF) variables across substances and years.
Box and whisker plot of help-seeking searches for opioid use.
Validation of Google Search Queries for SUD Help Seeking as Indicators of Unmet Treatment Need for SUD (Hypothesis 1):
The analysis of NSDUH data showed a statistically significant (P=.004) association between QF and the number of people needing but not receiving treatment for alcohol use at a specialty facility (rate ratio changes are shown in Table 2, and regression outputs can be found in Tables S3 and S4 in Multimedia Appendix 1). The estimates were not significantly different from 0 in the case of illicit drug use (P=.26). The coefficient estimates for alcohol use and illicit drug use confirmed our expectation of a positive association between the QF variables and the number of people needing but not receiving treatment for their substance use. After adjusting for variations across years, both the analyses for alcohol use and illicit drug use showed that a 1-unit increase (ie, 1 additional search per million searches) in the composite QF variable approximately corresponded to a 1% increase in the expected rate of people needing but not receiving treatment for alcohol use and illicit drug use, respectively. Neither analysis showed statistically significant interactions between QF variables and the variable “year.”
Predictions were made for the model analyzing the number of individuals needing but not receiving treatment for alcohol use on account of the significant QF finding. No evaluation of predictive performance was conducted for the illicit drug use model because its association with the QF variable was not statistically significant. Comparisons between the predicted and observed rates of people needing but not receiving treatment for alcohol use are presented in Figure S2 in Multimedia Appendix 1. The predictive performance was also quantified by calculating root mean squared errors (RMSE), comparing observed and predicted rates. The mean RMSE was 697, which is low when compared to the mean rate of 5284 per 100,000 people needing but not receiving treatment for alcohol use. The resulting scatter index of 13%, which is calculated by dividing the mean RMSE by the mean rate and then multiplied by 100, suggests a reasonable predictive performance based on previously used benchmarks [38]. Predictive performance was also examined over time and across states/territories (Tables S5-S6 in Multimedia Appendix 1). It was shown to be best in 2019 for the states of Idaho, Virginia, Michigan, New York, and Kansas and worse in 2018 for the District of Columbia, Colorado, Oregon, Montana, Vermont, compared to other years and states, respectively.
Estimates of the rate ratio change in number of people needing but not receiving treatment (NSDUH) associated with a one unit increase in the query fractions (QFs) variable.
Validation of Google Search Queries for SUD Help Seeking as Indicators of Treatment Seeking for SUD (Hypothesis 2):
The analysis of TEDS-A data showed that the association between help-seeking searches and the receipt of treatments for SUD varied by substance type (rate ratios are shown in Table 3, and regression outputs can be found in Tables S7 to S11 in Multimedia Appendix 1). Statistically significant and positive associations between these variables were found for methamphetamine use (P<.001). Interactions between the methamphetamine QF and the year variable were nonsignificant and thus ruled out (P=.88 for Type III Wald test). The model outputs showed that a 1-unit increase in the methamphetamine QF variable approximately corresponded to a 26% increase in the expected rate of treatment episodes. Statistically significant and positive associations were also found for opioids (P<.001). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (P=.26 for Type III Wald test). The outputs from the model showed that a 1-unit increase in the opioid QF variable approximately corresponded to a 12% increase in the expected rate of treatment episodes.
In the case of cannabis, the association was also positive and slightly above a 5% statistical significance criterion (P=.07). Although the data did not allow strong inferences to be drawn from the analysis of cannabis data, the outputs from the model showed that a 1-unit increase in the cannabis QF variable approximately corresponded to a 3% increase in the expected rate of treatment episodes. Findings for the analyses of alcohol and cocaine use showed both nonsignificant association between treatment-seeking searches and the receipt of treatments (P=.92 for alcohol use and P=.22 for cocaine use). Neither model exhibited significant interactions between the QF and the year variable (P=.23 for alcohol use and P=.88 for cocaine use for Type III Wald test for the models of treatment).
Estimates of the rate ratio change in number of individual treatment referrals associated with a 1 unit increase in the query fractions (QFs) variable.
aQF: query fraction.
Predictions were made for the models analyzing admissions to treatment for methamphetamine and opioid use. Comparisons between the predicted and observed rates of admission to treatment are presented in Figures S3-S4 in Multimedia Appendix 1. The predictive performance of the models for opioid and methamphetamine use was also quantified by calculating RMSE. The mean RMSE for methamphetamine was 11.7, which indicates a poor predictive performance, given that the mean admission rate was 15.2 per 100,000 people. The predictive performance was also shown to be weak for opioids based on comparisons between the mean RMSE (77.9) and the mean admission rate (102.4 per 100,000 people). Predictive performance was also examined over time and across states (Tables S12 and S13 in Multimedia Appendix 1). For both substances, predictive performance was found to be best in 2011 compared to other years for both substances, and in the states of Indiana, Texas, New York, and Michigan for methamphetamine use and in Illinois, Ohio, Missouri, Utah for opioid use. It was the worst in 2018 for both substances, compared to other years, and generally worse among states with higher admission rates.
Validation Of Google Search Queries for SUD Help Seeking as Predictors of Health Harms Related to Unmet Treatment Need (Hypothesis 3):
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid-related emergency department visits using AHRQ data showed a positive and statistically significant association (P<.001, see Tables S14-S15 in Multimedia Appendix 1). However, statistically significant interactions between the opioid QF and the variable year were identified, indicating that the relationship was not stable over time (Type III Wald test P=.005). An evaluation of the simple main effects of the opioid QF by year showed a decreasing trend over time (Table S14 in Multimedia Appendix 1). In 2011, a 1-unit increase in the opioid QF variables was associated with a 6% increase in the expected rate of opioid-related emergency department visits, but by 2018, there was a nonsignificant association between these variables. No evaluation of predictive performance was conducted for this model, as the association with the QF variable was found to vary over time.
The analysis investigating the relationship between treatment-seeking searches for opioid use and opioid overdose mortality counts using CDC WONDER data showed a positive and statistically significant association (P<.001, see Table 4 and Table S16 in Multimedia Appendix 1). Interactions between the opioid QF and the year variable were nonsignificant and thus ruled out (Type III Wald test P=.11). The outputs from the model showed that a 1-unit increase in the opioid QF variable corresponded to a 11% increase in the expected overdose mortality count (Table 4). The predictive performance for the model was determined by estimating the RMSE. The relative difference between the mean RMSE (4.3) and the mean admission rate per 100,000 people (12.2) indicated a better predictive performance, on average, when compared to the models predicting treatment admission rates. Figure S5 in Multimedia Appendix 1 illustrates the differences between predicted and observed mortality rates across states. Predictive performance was best in 2013 compared to other years (Table S17 in Multimedia Appendix 1) and in the states of New York, Florida, Virginia, and Wisconsin compared to other states (Table S18 in Multimedia Appendix 1). It was worst in 2017 and in West Virginia, Ohio, Idaho, and Maryland, where opioid overdose mortality was very high (with the exception of Idaho).
Estimates of the rate ratio change in number of overdose deaths associated with a 1-unit increase in the opioid query fractions (QFs) variable.
aQF: query fraction.
Discussion:
Principal Findings To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.
Importantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.
Further analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].
To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.
Importantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.
Further analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].
Limitations Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.
While our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.
Importantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.
While the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].
Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.
While our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.
Importantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.
While the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].
Conclusions This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.
This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.
Principal Findings:
To our knowledge, this is the first study to retrospectively describe spatial and temporal changes in substance use searches in the United States and rigorously investigate their association with outcomes along the continuum of care (and absence of care) for SUD. In the future, monitoring of Google search queries with validated metrics may allow the prospective identification of variations by substance and state indicating specific SUD treatment information and linkage needs in the population, providing useful near real-time insights to public health organizations developing and delivering campaigns for SUD treatment. Key stakeholders (local health departments, harm reduction organizations, etc) could then better allocate resources to target SUD treatment needs (eg, a digital intervention in real time) for each substance in specific states. For instance, between 2010 and 2020, West Virginia (methamphetamine and opioids), New Mexico (methamphetamine), Delaware (opioids), and Connecticut (cocaine) were repeatedly found to exhibit high levels of demand for information on SUD treatments that were potentially unmet.
Importantly, the positive and significant associations we identified between help-seeking searches for opioid and methamphetamine use and admissions to substance use treatment facilities suggests that, at least for these 2 substances, internet search data represents a valuable resource to assess treatment seeking. Interpreting the magnitude of these associations should be considered in the context of the baseline rate of treatment admissions and the overall population size for a given state. For instance, the implications of a 1-unit increase in the rate of help-seeking searches for methamphetamine use in California differs vastly from that in Virginia. The average rate of treatment admissions per 100,000 across all years was 37.43 for California and 1.12 for Virginia. Given that a 1-unit increase in the rate of help-seeking searches is associated with a 26% increase in treatment admissions, this corresponds to 9.73 additional admissions per 100,000 for the average rate in California and 0.29 additional admissions per 100,000 for the average rate in Virginia. In absolute terms, this equates to over 3800 additional admissions for California and only 5 additional admissions for Virginia.
Further analyses showed significant associations between help-seeking searches for opioid use and data on health harms related to unmet treatment need. These findings have implications for both surveillance and treatment, as they demonstrate the clear potential of search query monitoring to fill existing gaps and indicate that the internet likely represents a strategic platform to link people in need of treatment to services. This is especially important given that there are well-documented challenges in estimating the prevalence and incidence of SUD [5,8]. As such, leveraging internet search platforms could make health agencies more responsive to both information and treatment referral needs. This potential can be realized by developing a surveillance platform for real-time monitoring and linkage to services that can allow users to rapidly evaluate fluctuating patterns in SUD help seeking and implement strategic outreach. To realize this potential, search data need to be measured in terms of QFs to ensure that data points are comparable over time and across states. This approach was achieved in this study by extracting search data using the Google API Client library. It is important to highlight that this is not achieved when data are extracted directly through the Google Trends website but rather when data are normalized according to the selected time frame and geographical region [37].
Limitations:
Our study is not without limitations. Several states were missing search query data for SUD help-seeking behavior because Google Trends will only report search queries if they are above a minimum threshold. There was variation in the predictive performance of our models over time and across states. In particular, performance was lowest in states where rates of treatment admissions or overdose mortality rates were very high, which is expected when using RMSE as the performance indicator since it penalizes large errors. Using search data may be subject to selection bias, as not all people access the internet equally. Although some queries may reflect general curiosity rather than help seeking, it is well known that internet search trends mirror many health-related behaviors [39], and in the specific case of SUD, that of family members, partners, and friends trying to help their loved one [39]. Another potential confounding factor is the fact that the Google search algorithm is nonstatic. Search patterns change over time due to the thousands of decisions being made by Google’s programmers as the company strives to test and improve its search algorithm [40]. This could lead to temporal changes in the likelihood of individuals successfully finding treatment following an online search. As such, this phenomenon could distort the association between search trends and treatment admissions.
While our approach may overcome many of the ongoing limitations in substance use surveillance (ie, a lack of timely, substance specific, and publicly available data), the finest granularity of aggregate Google search data is limited to designated marketing areas [41], so it does not necessarily align with the jurisdictional level of public health departments. The approach taken in this paper also assumes that search queries are made using standard terms for SUD in the context of treatment seeking, which disregards instances where people might use slang terms. It is also possible that the predictive value of specific terms varies between states and over time. However, given the nonpunitive nature of online help seeking for SUD (as compared with that of purchasing or selling drugs), we expect this to be limited. It is important to recognize the potential limitations of using data on the number of treatment-seeking visits from TEDS-A. Given that these data are collected from facilities receiving public funding, the findings from analyses using this data potentially misrepresent associations for states with greater reliance on private funding or nonspecialty settings such as office-based outpatient treatment. Other potential confounding factors include geographical and temporal variations in the number of help-seeking queries in other languages, the proportion of queries coming from surrogate seekers [42], and the use of alternative search engines. In particular, including searches using Spanish terms would have a heterogeneous impact across states, and the relationship between searches and health outcomes might be different depending on the policies and interventions in place to facilitate healthcare access among non-English speakers and those who are undocumented [43,44]. This warrants a separate study focusing on Spanish language terms and SUD-related health outcomes among Hispanic individuals.
Importantly, the strength and significance of associations between searches and outcomes along the continuum of care varied depending on the substance and outcome, as well as between states and through time. This is expected, given that there have been heterogeneities in drug policies over time and across States. Between 2010 and 2020, cannabis was made legal in 19 states for medical use, in 8 states for recreational use, and in 3 states for medical use first and then later for recreational use [45]. Given that the impacts of legalization on the social acceptability of treatment-seeking behaviors are still poorly understood [46], it is difficult to surmise whether changes in the legal status of cannabis across states and over the duration of the study may have had a distorting impact on the results. Another key policy area is the state adoption of naloxone access laws (NAL), which increased rapidly from 2013 onward [47]. By 2020, all 50 states and the District of Columbia had some form of NAL in place, although the laws varied significantly across states [48]. Despite the proven clinical benefits of naloxone for the reversal of opioid overdoses, its population-level impact depends on the effectiveness of distribution programs alongside multiple contextual factors [49,50]. For this reason, the impact NALs may have had on our results is unclear.
While the goal of this study was to validate the use of help-seeking queries as a surveillance tool across states, our findings call for further investigation within states to contextualize and interpret the results. The inclusion of additional covariates could potentially help to improve the predictive performance of the models developed in this paper and elucidate factors that determine variations in outcomes across years. A key challenge in this regard was the limited sample size, in that there was insufficient statistical power to include additional predictors. One potential remedy to this problem would be to obtain data with more granularity in terms of the time intervals between observations (eg, monthly data) or the geographical level under investigation (eg, county-level data) to increase the number of observations. Finally, while this study retrospectively analyzes SUD help-seeking internet search data to validate their value for surveillance and linkage to treatment, real-time analysis would be the most useful for informing public health agencies, as indicated by some examples investigating mental health–related outcomes during the COVID-19 pandemic [51,52].
Conclusions:
This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs.
| Background:
There is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUDs) seeking help within a given geographical area. This presents a challenge to policy makers in the effective deployment of resources for the treatment of SUDs. Internet search queries related to help seeking for SUDs using Google Trends may represent a low-cost, real-time, and data-driven infoveillance tool to address this shortfall in information.
Methods:
We used negative binomial regression models to examine temporal trends in the annual SUD help-seeking internet search queries from Google Trends by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol from 2010 to 2020. To validate the value of these data for surveillance purposes, we then used negative binomial regression models to investigate the relationship between SUD help-seeking searches and state-level outcomes across the continuum of care (including lack of care). We started by looking at associations with self-reported treatment need using data from the National Survey on Drug Use and Health, a national survey of the US general population. Next, we explored associations with treatment admission rates from the Treatment Episode Data Set, a national data system on SUD treatment facilities. Finally, we studied associations with state-level rates of people experiencing and dying from an opioid overdose, using data from the Agency for Healthcare Research and Quality and the CDC WONDER database.
Results:
Statistically significant differences in help-seeking searches were observed over time between 2010 and 2020 (based on P<.05 for the corresponding Wald tests). We were able to identify outlier states for each drug over time (eg, West Virginia for both opioids and methamphetamine), indicating significantly higher help-seeking behaviors compared to national trends. Results from our validation analyses across different outcomes showed positive, statistically significant associations for the models relating to treatment need for alcohol use, treatment admissions for opioid and methamphetamine use, emergency department visits related to opioid use, and opioid overdose mortality data (based on regression coefficients having P≤.05).
Conclusions:
This study demonstrates the clear potential for using internet search queries from Google Trends as an infoveillance tool to predict the demand for substance use treatment spatially and temporally, especially for opioid use disorders. | Introduction:
Understanding help-seeking behavior for substance use treatment is critical for the effective deployment of resources. This presents a challenge to researchers and policy makers because there is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUD) within a given geographical area [1]. A standard approach involves asking a sample of the general population questions about their substance use, either through surveys or in-depth interviews [2]. Unfortunately, these sources are subject to well-known limitations, such as low participation rates, lag time between data collection and published results, and data availability [3]. Additionally, survey scale-up is not always feasible given both costs and concerns about participant burden [4].
Indirect estimation approaches have been used, including capture-recapture [5], multiplier [6], and data triangulation methods [7], but these methods are also subject to limitations, either in the form of impractical data requirements or the potential for bias [7,8]. Finally, efforts have been made to collect data on drug-related harms, such as overdose statistics, although time-lags in their dissemination have meant that these initiatives have struggled to keep pace with the rapidly evolving opioid epidemic in the United States [9]. In view of this methodological backdrop, there has been limited scope to develop real-time surveillance mechanisms to guide policy responses.
A promising development has emerged in the use of internet search queries related to substance use [10]. One of the main benefits of this approach to substance use surveillance is that the data are publicly accessible and can be easily obtained in real time [11]. There is a growing body of research exploring the use of internet search data for the surveillance of substance use trends. A study in 2018 found strong and significant correlations between Google search data for novel psychoactive drugs and annual drug use prevalence, collected in a nationally representative US sample [10]. Two studies explored the relationship between drug-related internet search queries and opioid-related emergency department (ED) visits in the United States, and both demonstrated the predictive potential of internet search data [12,13]. Three further studies found strong associations between drug-related internet search queries and opioid-related overdose deaths at the national, state, and county levels [14-16]. Elsewhere, studies have demonstrated the potential use of opioid-related data from social media platforms, including Twitter and Reddit, to inform surveillance efforts [17-19].
While the previous literature has focused on the use of internet data as a proxy for real-time data on opioid-related health harms, this study provides new insights into the use of internet search data to explore SUD help seeking for a broad range of substances, including cocaine, methamphetamine, opioids, cannabis, and alcohol, and validate these against observed SUD indicators. These substances were chosen because they are the 5 most common types of substance that people are admitted to treatment for in the United States [20]. By validating surveillance of SUD help seeking as a methodological tool, it is our hope that key stakeholders, including local health departments, harm reduction organizations, and researchers, will be better able to proactively respond to need [21]. We first described help-seeking searches for cocaine, methamphetamine, opioids, cannabis, and alcohol at national and state level from 2010 to 2020 in the United States and characterized heterogeneity in these outcomes between states.
We sought to determine the feasibility of using search query data as a low-cost and real-time indicator of unmet treatment need, demand for treatment, and a predictor of the health harms related to unmet treatment needs. The exploratory nature of this study warrants a continuum of hypotheses to account for different outcomes that might be expected to occur depending on the relative demand versus capacity for treatment. If there is sufficient treatment capacity, one would expect to see a strong, positive association between help-seeking searches and treatment admissions. However, given the limited capacity for SUD treatment in the United States, it was important to consider additional hypotheses; if there is excess demand for treatment, we would expect to see a weaker association between help-seeking searches and treatment admissions but a stronger association with unmet treatment need and drug-related health harms. In addition, it is also key to acknowledge that treatment seeking for SUD is a complex process that involves moving, often nonlinearly, through different stages of behavior change [22]. Therefore, considering several outcomes also allows us to reflect the different situations that individuals with SUD, or those around them and trying to help, might be experiencing.
We tested 3 hypotheses, the first of which posits that treatment-seeking searches are positively associated with unmet treatment needs, as searching for help indicates that the person is struggling with their substance use and is considering treatment as an option but has not yet received help (ie, contemplation). Next, we tested the hypothesis that treatment-seeking searches are positively associated with treatment admissions, as searching for help is an indicator that the person is actively seeking to engage (ie, preparation/action) [22,23]. Finally, we tested the hypothesis that treatment-seeking searches are positively associated with nonfatal and fatal overdose, as expressing a treatment need often occurs in the latter stages of SUD, when symptoms are more severe, leading to an increased risk of overdose and death (ie, contemplation, preparation, or relapse) [24]. We identified relevant variables across different state-level data sources to validate the models for each of these outcomes and determine whether internet searches for substance use help seeking can be used to enhance SUD treatment need surveillance and treatment linkage efforts.
Conclusions:
This study examined temporal and spatial trends in the annual fractions of substance use help-seeking internet search queries by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol. Our investigations showed positive, statistically significant associations for the models relating to treatment need (but not receiving treatment) for alcohol use, treatment admissions for opioid and methamphetamine use, and overdose mortality data. In the wake of current substance use trends, it is critical that public health professionals learn from and respond to the millions of individuals searching for help online. The field should invest in and prioritize automated surveillance, including extensions of our approach, to understand evolving public health needs. | Background:
There is no recognized gold standard method for estimating the number of individuals with substance use disorders (SUDs) seeking help within a given geographical area. This presents a challenge to policy makers in the effective deployment of resources for the treatment of SUDs. Internet search queries related to help seeking for SUDs using Google Trends may represent a low-cost, real-time, and data-driven infoveillance tool to address this shortfall in information.
Methods:
We used negative binomial regression models to examine temporal trends in the annual SUD help-seeking internet search queries from Google Trends by US state for cocaine, methamphetamine, opioids, cannabis, and alcohol from 2010 to 2020. To validate the value of these data for surveillance purposes, we then used negative binomial regression models to investigate the relationship between SUD help-seeking searches and state-level outcomes across the continuum of care (including lack of care). We started by looking at associations with self-reported treatment need using data from the National Survey on Drug Use and Health, a national survey of the US general population. Next, we explored associations with treatment admission rates from the Treatment Episode Data Set, a national data system on SUD treatment facilities. Finally, we studied associations with state-level rates of people experiencing and dying from an opioid overdose, using data from the Agency for Healthcare Research and Quality and the CDC WONDER database.
Results:
Statistically significant differences in help-seeking searches were observed over time between 2010 and 2020 (based on P<.05 for the corresponding Wald tests). We were able to identify outlier states for each drug over time (eg, West Virginia for both opioids and methamphetamine), indicating significantly higher help-seeking behaviors compared to national trends. Results from our validation analyses across different outcomes showed positive, statistically significant associations for the models relating to treatment need for alcohol use, treatment admissions for opioid and methamphetamine use, emergency department visits related to opioid use, and opioid overdose mortality data (based on regression coefficients having P≤.05).
Conclusions:
This study demonstrates the clear potential for using internet search queries from Google Trends as an infoveillance tool to predict the demand for substance use treatment spatially and temporally, especially for opioid use disorders. | 18,201 | 433 | [
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||||||
Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action. | 35508030 | Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. | BACKGROUND | Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. | METHODS | The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). | FINDINGS | Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models. | MAIN CONCLUSIONS | [
"Animals",
"Antiprotozoal Agents",
"Flow Cytometry",
"Humans",
"Ketoconazole",
"Leishmania",
"Leishmaniasis",
"Mice",
"Mice, Inbred BALB C",
"Mitochondria"
] | 9060495 | null | null | null | null | RESULTS |
Ketoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.
Fig. 1:
Leishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).
Ketoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.
Fig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).
Ketoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.
Autophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.
Fig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).
Fig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).
Ketoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).
Fig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).
Ketoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.
Fig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.
Ketoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.
Fig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.
| null | null | [] | [] | [] | [
"MATERIALS AND METHODS",
"RESULTS",
"DISCUSSION"
] | [
"\nChemicals - Ketoconazole, dimethylsulfoxide (DMSO), penicillin, streptomycin, triton x-100, rhodamine 123, monodansylcadaverine (MDC), RPMI 1640 medium from Sigma Chemical Co. (USA), annexin V FITC apoptosis detection kit from BD Pharmingen, MitoTrackerR RedCMXRos from Invitrogen (USA), foetal bovine serum (FBS) from Cultilab (Brazil), and Schneider’s insect medium from LGC Biotecnologia (Brazil) were used in this study. Ketoconazole was dissolved in a 1 M stock of DMSO and stored at -20ºC. During assays new dilutions were carried out to ensure that the DMSO concentration in culture medium did not exceed 0.1%.\n\nAnimals - Male BALB/c mice (six-eight weeks old) had access to water and standard chow ad libitum. They were kept in a room with controlled temperature (25ºC) and luminosity (12 h light/dark cycles). The experimental procedures were analysed and approved by the Animal Ethics Committee of the Federal University of Uberlândia (CEUA/UFU) under the number 36/2013.\n\nCells culture - Promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8 strain) were cultured in Schneider’s insect medium pH 7.4 containing 10% heat-inactivated FBS, 1% penicillin (100 UI x mL−1), and streptomycin (100 µg x mL−1). Parasites were kept in a BOD chamber at 23 ± 0.5ºC.\nMurine macrophage cell line RAW264.7 (Rio de Janeiro Bank Cell) was cultured in RPMI 1640 medium pH 7.4 supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) in 75-cm2 flasks. All cell cultures were done at 37ºC in humidified air with 5% CO2.\nFree amastigotes were obtained from footpad of BALB/c mice previously infected with promastigote forms (1 × 107 cells/footpad) for five to six weeks.\n29\n These parasites were cultured in complete Schneider’s insect medium, at 32 ± 0.5ºC pH 5, ensuring that these parasites remained as axenic amastigote forms.\n30\n\n\n\nAntiproliferative and viability assays - The effect of ketoconazole on cellular proliferation was evaluated. Promastigotes (5 × 106 cells x mL−1) were cultured in 25 cm2 cell culture flasks containing medium with different drug concentrations (300 to 0.001 μM). After 24, 48, 72, 96 and 120 h of incubation with drug, the parasite concentrations were determined by blind counting using a Neubauer chamber. Three independent experiments were performed, each one in triplicate. The EC50 values with 95% confidence limits were calculated by GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, USA).\nViability assay was carried out on promastigote forms using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent. Parasites (5 × 106 cells x mL−1) were incubated in the absence (viability control) or presence of ketoconazole (200 to 0.097 µM) for 24, 48, and 72 h into 96 wells plate. The internal controls of the experiment were: culture medium only, DMSO only, parasites cultured in the presence of Triton X-100 (death control) and parasites cultured in the presence of culture medium (viability control) (data not shown to make the figure clean). Three independent experiments were performed, all in triplicate. The effective concentration of drug able to cause 50% of citotoxicity (EC50) was graphically determined by non-linear regression log in each independent experiment using the Graphpad Prism 5 software (United States); for this, all treatments and the viability control had their absorbances deducted from the absorbance value of the culture medium. Finally, the absorbance of the viability control deducted from the absorbance of the culture medium was used as reference parameter (100% of viability) and the percentages of treatments were calculated from then on.\nMoreover, another viability assay was performed: Trypan Blue exclusion test. Parasites (5 x 106 cells x mL−1) were cultured in medium for 48 h. Subsequently, the cells were treated with 10 µM ketoconazole (2xEC50 of 72 h growth curve) and the number of promastigotes was determined by blind counting using a Neubauer chamber for three consecutive days (72 h) and Trypan blue stain. Three independent experiments were performed, all in triplicate.\n\nMorphological and ultrastructural analysis - For evaluating ultrastructural changes, transmission electron (TEM) and scanning electron (SEM) microscopies were performed. For TEM, promastigotes (5 x 106 cells x mL−1, log phase) were incubated with or without (control) ketoconazole 10 μM for 72 h. After being treated, the parasites were fixed at 4ºC with 2.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS) pH 7.2 for 24 h, washed in PBS, post-fixed in PBS containing 0.8% potassium ferrocyanide and 1% osmium tetroxide (OsO4) for 1 h and washed once again. Subsequently, the parasites were dehydrated in a gradual series of acetone and set in resin. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate and analysed using a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany). For SEM, promastigotes treated or not with ketoconazole 10 μM for 72 h were dehydrated in ethanol, critical point-dried in CO2, mounted in stubs, sputtered with a thin gold layer, and analysed using a scanning electron microscope (Zeiss EVO MA10).\n\nEvaluation of mitochondrial damage - The fluorescent stain Rhodamine 123 was employed to evaluate the mitochondrial transmembrane potential (∆Ψ\nm\n ) of promastigotes. Initially, parasites (5 x 106 cells x mL−1) were treated with 10 µM ketoconazole or not (control) for 24, 48, and 72 h. Aliquots were incubated with Rhodamine 123 (15 µg x mL−1) for 15 min at room temperature and protected from light (dark). Subsequently, the parasites were washed twice with PBS. The cell population analysis was carried out in a flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. Three independent experiments were performed.\nFurthermore, promastigotes (5 x 106 cells x mL−1) treated or not (control) with ketoconazole (10 µM, 72 h) were incubated with MitoTrackerR RedCMXRos (300 nM) for 30 min in the dark. Subsequently, parasites were washed with PBS, fixed in paraformaldehyde 1% in cacodylate buffer, and washed twice with PBS. The cell population analysis was performed with a confocal fluorescence microscopy (Zeiss LSM510 Meta).\n\nDetection of autophagic vacuoles - The monodansylcadaverine (MDC) labeling was carried out to evaluate the autophagic vacuoles formation. Promastigotes (5 x 106 cells x mL−1, log phase) were cultured in absence (control) or presence of ketoconazole 10 μM for 24, 48, and 72 h. Subsequently, 100 µM MDC was added to parasites and incubated for 2 h in the dark. Afterwards, the parasites were washed with PBS, fixed in 1% paraformaldehyde in cacodylate buffer, washed twice with PBS, mounted on microscope slides, and analysed by fluorescence microscopy (excitation wavelength 358 nm and emission wavelength 463 nm). Two independent experiments were performed and the fluorescence intensity was quantified by the software Image J version 1.48.\n\nCell cycle analysis - Initially, the interference of ketoconazole on the cell cycle was evaluated by counting the number of nucleous, kinetoplast and flagella by parasite. Promastigotes (5 x 106 cells x mL-1) treated or not (control) with ketoconazole (10 uM, 72 h) labelled with DAPI (1:500, for 1 h) were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta). For a more detailed study, parasites (5 x 106 cells x mL−1, log phase) were incubated for 72 h with or without (control) ketoconazole (10 μM, 72 h) in medium containing 10% FBS. After incubation, parasites were washed and resuspended in ice-cold 70% ethanol in PBS and fixed for 18 h at 4ºC. Subsequently, the parasites were incubated with PBS containing 10 µg x mL−1 propidium iodide and 100 µg x mL−1 RNAse A for 45 min at 37ºC, protected from light. The cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. For each phase of the cell cycle, a respective percentage was obtained using the FlowJov10.0.7 software.\n\nCell death analysis - Promastigotes (5 x 106 cells x mL−1) were incubated for 72 h with or without (negative control) ketoconazole (10 μM, 72 h); while parasites incubated with formaldehyde 4% for 15 min were considered positive control. Subsequently, a binding buffer containing annexin V-FITC and 2 μg x mL−1 propidium iodide (BD Bioscience, Brazil) was added to the parasites and incubated for 15 min at 25ºC in the dark, according to the manufacturer’s instructions. Then, the parasites were washed and the cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA). Thirty thousand events were obtained from the region corresponding to the parasites. The percentages of apoptotic cells were determined using the FlowJo v10.0.7 software.\nFor Propidium Iodide (PI) and Annexin V (AV) labeling, promastigotes (5 x 106 cells x mL−1) treated with ketoconazole (10 μM, 72 h), medium (negative control) or formaldehyde 4% (positive control) were incubated in presence of PI (2 µg x mL-1), AV (1 µg x mL-1) or both for 15 min at 25ºC, protected from the light. Subsequently, parasites were fixed with 1% paraformaldehyde in a cacodylate buffer, washed and placed on glass coverslips. Finally, the parasites were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta).\n\nIntracellular amastigote assay - Murine macrophage cell line RAW264.7 (4 × 105/well) cultured in RPMI 1640 medium, supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) was placed in 24-well plates containing 13-mm diameter glass coverslips and infected with L. (L.) amazonensis free amastigotes isolated just before their use (two free amastigotes: 1 macrophage). After 1 h 30, plates were washed with PBS to remove non-internalised amastigotes and RPMI medium alone (control) or containing drugs (200 to 12,5 µM for ketoconazole, double serial dilutions) was added. Experiments were conducted at 37ºC in a 5% CO2 humidified incubator for 48 h. Cells on coverslips were fixed and stained with Giemsa stain modified solution for evaluation of the infectivity index; 100 cells per coverslip were blind counted and the infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage. This assay was carried out in quadruplicate and two independent experiments were performed.\nIt is worth highlighting the cytotoxic effect of ketoconazole on murine macrophages cell line RAW264.7 was, previously, performed by MTT test at the same drug concentrations used to assess the viability of promastigotes (section 2.4), but within 48 h. Since the macrophages were infected with amastigotes isolated from animal paw just before use and it was not necessary time to promastigote-amastigote differentiation, the infectivity assay was conducted at 48 h, time that allowed better visualisation and counting of the intramacrophage amastigotes.\n\nStatistics - Statistical analysis was conducted using GraphPad Prism 5. Each set of results was firstly checked for normal distribution using KolmogorovSmirnov, D’Agostinho and Pearson, and ShapiroWalk tests. Normally distributed data were analysed through one-way-ANOVA followed by Tukey’s post-test or Student test T. Statistically significant differences were assumed when at least p < 0.05.",
"\nKetoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.\n\nFig. 1:\nLeishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).\n\n\nKetoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.\n\nFig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).\n\n\nKetoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.\nAutophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.\n\nFig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).\n\n\nFig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).\n\n\nKetoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).\n\nFig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).\n\n\nKetoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.\n\nFig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.\n\n\nKetoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.\n\nFig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.\n",
"Considering the importance of ergosterol for the biology and pathogenesis of the Leishmania parasite, herein, we certified the antiproliferative/viability effects of ketoconazole against L. (L.) amazonensis both promastigotes\n31\n and intracellular amastigotes\n32\n in another parasite strain: IFLA/BR/67/PH8. Furthermore, we show in more detail the biological effects of the inhibitor, aiming at understanding the drug mechanism of action.\nRegarding ultrastructure, our results revealed rounded up shape, mitochondrial swelling, augmented accumulation of lipid droplets and acidocalcisomes, presence of multivesicular bodies and frequent truncated and/or double flagella. These data are similar to those previously obtained for L. amazonensis promastigotes treated with ketoconazole\n31\n\n,\n\n33\n\n,\n\n34\n and suggest mitochondrial damage.\nWhen parasites (Trypanosoma cruzi, Leishmania spp, Toxoplasma gondii) are treated with inhibitors of important enzymes of the ergosterol biosynthesis pathway, the mitochondrion is the organelle primarily affected.\n35\n In our study, the ketoconazole-treated parasites presented hyperpolarisation of the mitochondrial membrane potential, time-dependent up to 72 h. Mitochondrial membrane potential variations could be the result of diverse events: inhibition of electron transport chain (decrease); blockage of ATP synthase (increase); stimulation of uncoupling proteins (decrease); or permeabilisation of the inner membrane (decrease).\n36\n Macedo et al.\n37\n demonstrated that L. (L.) amazonensis promastigotes treated with itraconazole and posaconazole for 48 h presented collapse of the mitochondrial membrane potential associated with intense mitochondrial swelling, disorganisation and rupture of mitochondrial membranes. Another study showed that T. cruzi treated with ketoconazole (EC50 = 32 µM for 72 h) presented a gradual increase in Rho 123 time-dependent fluorescence and a confocal microscopy of parasites confirmed an intense proliferation of the inner mitochondrial membrane. This organelle became highly branched and compact and elevated levels of Rho 123 were accumulated within the cells.\n38\n In many cases, a transient hyperpolarisation occurs before mitochondrial depolarisation, as if it were an attempt of the cells to avoid death. This effect can be seen in great part of the heat-shocked Leishmania promastigotes.\n39\n\n\nThe mitochondrial membrane is hyperpolarised does not mean that the organelle is functioning normally. Our results showed that MitoTrackerR labeling, a probe which passively diffuses across the plasma membrane and accumulates in active mitochondria, revealed an outstanding intensity decrease in ketoconazole-treated promastigotes, indicating loss of mitochondrial activity in these promastigotes. This result was also confirmed by the MTT viability assay, which measures the activity of mitochondrial enzymes, and by the proliferation curve using Trypan blue, which takes into account only viable cells. Mitochondrion is essential for generating the necessary energy for the survival and proliferation of eukaryotic cells.\n40\n\n) In this way, malfunctioning mitochondria could impair the ATP synthesis and inorganic phosphate would accumulate in acidocalcisomes, which explains the increase in the amount of this organelle 72 h after ketoconazole treatment. Acidocalcisomes are organelles of acidic nature and high electron density important due to their storage of polyphosphates, calcium, magnesium, and other elements.\n41\n The augmented number of this organelle was already reported for ketoconazole and L. amazonensis (MHOM/Josefa/75/Br strain).\n42\n\n\nThe treatment of L. amazonensis with ketoconazole stressed the cells, especially early after the treatment (24 and 48 h), leading to the appearance of autophagic vacuoles. Itraconazole and posaconazole induced appearance of autophagosome-like structures in L. (L.) amazonensis\n\n37\n and Rodrigues et al.\n43\n described the effect of autophagy on L. amazonensis (MHOM/BR/75/Josefa strain) treated with 22,26-azasterol. Thus, the confirmed presence of autophagic vacuoles might represent an adaptive response of the parasite to treatment (stress condition). Initially, parasites would resort to autophagic vacuoles in an attempt to remodel/remove abnormal cellular constituents, degrading damaged structures.\n44\n Over time, directing energy to the parasite single mitochondrion may be more important than repairing cell damage. This would explain the fact that at 72 h there was less labeling for autophagic vacuoles and higher mitochondrial hyperpolarisation occurred. Autophagy is involved in turnover and recycling by removal of damaged cellular components, regulating homeostasis during crucial processes such as cell growth and differentiation and plays fundamental role in mitochondrial functionality.\n44\n\n,\n\n45\n\n\nResults of the growth curve and frequent ultrastructural alterations in flagellum (truncated and/or double flagella), despite the intact kinetoplast, suggest that ketoconazole interferes with the L. amazonensis cell cycle. Therefore, the cell cycle of the parasites was evaluated both by nucleus, kinetoplast, and flagellum (n-k-f) counts and by flow cytometry. Different from some data in the literature involving ketoconazole acting on other cell types\n46\n\n,\n\n47\n and azole compounds on L. amazonensis,\n37\n our results of the n-k-f score did not reveal differences between the control and ketoconazole-treated parasites. In addition, the cell cycle assay showed that the treatment was not able to alter the cycle phases (Sub G1, G1, S, and G2/M) in relation to control parasites suggesting that ketoconazole does not interfere with parasite replication under the tested conditions. When L. (L.) amazonensis was treated with itraconazole and posaconazole, some cells presented more than two flagella and significant alterations in kinetoplast were observed by ultrastructural analysis; however, the possible alteration in the cell cycle was not evaluated.\n37\n\n\nKetoconazole has been related to apoptosis in several normal and tumoral cells.\n48\n\n,\n\n49\n\n,\n\n50\n\n,\n\n51\n Haegler et al.\n52\n showed that ketoconazole and posaconazole presented hepatocellular toxicity, impairing the ∆Ψm, the function of enzyme complexes of electron transport chain, accumulating mitochondrial superoxide and inducing apoptosis. Possibly, mitochondrial dysfunction could be associated with hepatotoxicity caused by those azole compounds. Our data revealed that treatment of the parasites with ketoconazole, under the conditions previously described, showed a trend towards apoptosis, although there was a slight positive result for PI. Furthermore, few autophagic vacuoles were observed after treatment. Other researchers reported that treatment of T. cruzi with ketoconazole (EC50/72 h) resulted in neither necrosis nor apoptosis. However, high concentrations of the drug (EC100/24h) resulted in fast cell death due to necrosis.\n38\n\n\nSBIs effects on extracellular L. amazonensis parasites (promastigotes) appear to be more gradual than that observed with the intracellular form (amastigote);\n31\n thus, SBIs effects profile allows a more detailed study of the events in the promastigote form. In view of the effects provoked by ketoconazole on the L. (L.) amazonensis promastigote, the interference of drug on infectivity capacity of amastigotes, the clinically relevant form of the parasite, was evaluated and evidenced a susceptibility to treatment. Taken together, the infectivity results added to the data of mitochondrial damage, increase in the acidocalcisome and autophagic vacuoles amounts may be evidence that the susceptibility observed in vitro is related to mitochondrial dysfunction provoked by ketoconazole. However, future studies should be carried out on amastigotes to confirm this hypothesis.\nTo our knowledge, this study demonstrates the effects of ketoconazole on mitochondrion functioning, autophagic compartments, cell cycle and death of L. (L.) amazonensis promastigotes for the first time. We suggest that some damages are occurring in the parasite, mainly in the mitochondrion. In an attempt to remodel and/or destroy eventual damaged structures, parasites hyperpolarise mitochondrion and resort to autophagic vacuoles. However, this survival strategy adopted by the cell to direct efforts to maintain the cell energy is not sufficient because of the decrease in cell and mitochondria viabilities and increase in acidocalcisomes. Although these damages do not reflect directly in the parasite cell cycle, they lead the parasites to death, making them susceptible to in vitro treatment (Fig. 8).\n\nFig. 8:proposed mode action of ketoconazole on Leishmania (Leishmania) amazonensis. Parasites resort to autophagic vacuoles and mitochondrion hyperpolarisation in attempt to survive to damages mainly in the mitochondrion. The efforts to maintain the cell energy are not sufficient since the decrease of cell and mitochondria viability and increase of acidocalcisomes. These damages do not interfere with the parasite cell cycle, but they lead the parasites to death, making them susceptible to in vitro treatment conditions.\n"
] | [
"materials|methods",
"results",
"discussion"
] | [
"cutaneous leishmaniasis",
"ergosterol",
"mitochondrial damage",
"sterol biosynthesis inhibitor"
] | MATERIALS AND METHODS:
Chemicals - Ketoconazole, dimethylsulfoxide (DMSO), penicillin, streptomycin, triton x-100, rhodamine 123, monodansylcadaverine (MDC), RPMI 1640 medium from Sigma Chemical Co. (USA), annexin V FITC apoptosis detection kit from BD Pharmingen, MitoTrackerR RedCMXRos from Invitrogen (USA), foetal bovine serum (FBS) from Cultilab (Brazil), and Schneider’s insect medium from LGC Biotecnologia (Brazil) were used in this study. Ketoconazole was dissolved in a 1 M stock of DMSO and stored at -20ºC. During assays new dilutions were carried out to ensure that the DMSO concentration in culture medium did not exceed 0.1%.
Animals - Male BALB/c mice (six-eight weeks old) had access to water and standard chow ad libitum. They were kept in a room with controlled temperature (25ºC) and luminosity (12 h light/dark cycles). The experimental procedures were analysed and approved by the Animal Ethics Committee of the Federal University of Uberlândia (CEUA/UFU) under the number 36/2013.
Cells culture - Promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8 strain) were cultured in Schneider’s insect medium pH 7.4 containing 10% heat-inactivated FBS, 1% penicillin (100 UI x mL−1), and streptomycin (100 µg x mL−1). Parasites were kept in a BOD chamber at 23 ± 0.5ºC.
Murine macrophage cell line RAW264.7 (Rio de Janeiro Bank Cell) was cultured in RPMI 1640 medium pH 7.4 supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) in 75-cm2 flasks. All cell cultures were done at 37ºC in humidified air with 5% CO2.
Free amastigotes were obtained from footpad of BALB/c mice previously infected with promastigote forms (1 × 107 cells/footpad) for five to six weeks.
29
These parasites were cultured in complete Schneider’s insect medium, at 32 ± 0.5ºC pH 5, ensuring that these parasites remained as axenic amastigote forms.
30
Antiproliferative and viability assays - The effect of ketoconazole on cellular proliferation was evaluated. Promastigotes (5 × 106 cells x mL−1) were cultured in 25 cm2 cell culture flasks containing medium with different drug concentrations (300 to 0.001 μM). After 24, 48, 72, 96 and 120 h of incubation with drug, the parasite concentrations were determined by blind counting using a Neubauer chamber. Three independent experiments were performed, each one in triplicate. The EC50 values with 95% confidence limits were calculated by GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, USA).
Viability assay was carried out on promastigote forms using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent. Parasites (5 × 106 cells x mL−1) were incubated in the absence (viability control) or presence of ketoconazole (200 to 0.097 µM) for 24, 48, and 72 h into 96 wells plate. The internal controls of the experiment were: culture medium only, DMSO only, parasites cultured in the presence of Triton X-100 (death control) and parasites cultured in the presence of culture medium (viability control) (data not shown to make the figure clean). Three independent experiments were performed, all in triplicate. The effective concentration of drug able to cause 50% of citotoxicity (EC50) was graphically determined by non-linear regression log in each independent experiment using the Graphpad Prism 5 software (United States); for this, all treatments and the viability control had their absorbances deducted from the absorbance value of the culture medium. Finally, the absorbance of the viability control deducted from the absorbance of the culture medium was used as reference parameter (100% of viability) and the percentages of treatments were calculated from then on.
Moreover, another viability assay was performed: Trypan Blue exclusion test. Parasites (5 x 106 cells x mL−1) were cultured in medium for 48 h. Subsequently, the cells were treated with 10 µM ketoconazole (2xEC50 of 72 h growth curve) and the number of promastigotes was determined by blind counting using a Neubauer chamber for three consecutive days (72 h) and Trypan blue stain. Three independent experiments were performed, all in triplicate.
Morphological and ultrastructural analysis - For evaluating ultrastructural changes, transmission electron (TEM) and scanning electron (SEM) microscopies were performed. For TEM, promastigotes (5 x 106 cells x mL−1, log phase) were incubated with or without (control) ketoconazole 10 μM for 72 h. After being treated, the parasites were fixed at 4ºC with 2.5% glutaraldehyde in 0.1 M phosphate buffered saline (PBS) pH 7.2 for 24 h, washed in PBS, post-fixed in PBS containing 0.8% potassium ferrocyanide and 1% osmium tetroxide (OsO4) for 1 h and washed once again. Subsequently, the parasites were dehydrated in a gradual series of acetone and set in resin. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate and analysed using a Zeiss EM 109 transmission electron microscope (Zeiss, Oberkochen, Germany). For SEM, promastigotes treated or not with ketoconazole 10 μM for 72 h were dehydrated in ethanol, critical point-dried in CO2, mounted in stubs, sputtered with a thin gold layer, and analysed using a scanning electron microscope (Zeiss EVO MA10).
Evaluation of mitochondrial damage - The fluorescent stain Rhodamine 123 was employed to evaluate the mitochondrial transmembrane potential (∆Ψ
m
) of promastigotes. Initially, parasites (5 x 106 cells x mL−1) were treated with 10 µM ketoconazole or not (control) for 24, 48, and 72 h. Aliquots were incubated with Rhodamine 123 (15 µg x mL−1) for 15 min at room temperature and protected from light (dark). Subsequently, the parasites were washed twice with PBS. The cell population analysis was carried out in a flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. Three independent experiments were performed.
Furthermore, promastigotes (5 x 106 cells x mL−1) treated or not (control) with ketoconazole (10 µM, 72 h) were incubated with MitoTrackerR RedCMXRos (300 nM) for 30 min in the dark. Subsequently, parasites were washed with PBS, fixed in paraformaldehyde 1% in cacodylate buffer, and washed twice with PBS. The cell population analysis was performed with a confocal fluorescence microscopy (Zeiss LSM510 Meta).
Detection of autophagic vacuoles - The monodansylcadaverine (MDC) labeling was carried out to evaluate the autophagic vacuoles formation. Promastigotes (5 x 106 cells x mL−1, log phase) were cultured in absence (control) or presence of ketoconazole 10 μM for 24, 48, and 72 h. Subsequently, 100 µM MDC was added to parasites and incubated for 2 h in the dark. Afterwards, the parasites were washed with PBS, fixed in 1% paraformaldehyde in cacodylate buffer, washed twice with PBS, mounted on microscope slides, and analysed by fluorescence microscopy (excitation wavelength 358 nm and emission wavelength 463 nm). Two independent experiments were performed and the fluorescence intensity was quantified by the software Image J version 1.48.
Cell cycle analysis - Initially, the interference of ketoconazole on the cell cycle was evaluated by counting the number of nucleous, kinetoplast and flagella by parasite. Promastigotes (5 x 106 cells x mL-1) treated or not (control) with ketoconazole (10 uM, 72 h) labelled with DAPI (1:500, for 1 h) were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta). For a more detailed study, parasites (5 x 106 cells x mL−1, log phase) were incubated for 72 h with or without (control) ketoconazole (10 μM, 72 h) in medium containing 10% FBS. After incubation, parasites were washed and resuspended in ice-cold 70% ethanol in PBS and fixed for 18 h at 4ºC. Subsequently, the parasites were incubated with PBS containing 10 µg x mL−1 propidium iodide and 100 µg x mL−1 RNAse A for 45 min at 37ºC, protected from light. The cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA) and 10,000 events were obtained from the region corresponding to the parasites. For each phase of the cell cycle, a respective percentage was obtained using the FlowJov10.0.7 software.
Cell death analysis - Promastigotes (5 x 106 cells x mL−1) were incubated for 72 h with or without (negative control) ketoconazole (10 μM, 72 h); while parasites incubated with formaldehyde 4% for 15 min were considered positive control. Subsequently, a binding buffer containing annexin V-FITC and 2 μg x mL−1 propidium iodide (BD Bioscience, Brazil) was added to the parasites and incubated for 15 min at 25ºC in the dark, according to the manufacturer’s instructions. Then, the parasites were washed and the cell population was analysed by flow cytometer (BD Accuri C6 - Biosciences, CA, USA). Thirty thousand events were obtained from the region corresponding to the parasites. The percentages of apoptotic cells were determined using the FlowJo v10.0.7 software.
For Propidium Iodide (PI) and Annexin V (AV) labeling, promastigotes (5 x 106 cells x mL−1) treated with ketoconazole (10 μM, 72 h), medium (negative control) or formaldehyde 4% (positive control) were incubated in presence of PI (2 µg x mL-1), AV (1 µg x mL-1) or both for 15 min at 25ºC, protected from the light. Subsequently, parasites were fixed with 1% paraformaldehyde in a cacodylate buffer, washed and placed on glass coverslips. Finally, the parasites were analysed using a confocal fluorescence microscope (Zeiss LSM510 Meta).
Intracellular amastigote assay - Murine macrophage cell line RAW264.7 (4 × 105/well) cultured in RPMI 1640 medium, supplemented with 5% FBS, penicillin (100 UI x mL−1), streptomycin (100 μg x mL−1) was placed in 24-well plates containing 13-mm diameter glass coverslips and infected with L. (L.) amazonensis free amastigotes isolated just before their use (two free amastigotes: 1 macrophage). After 1 h 30, plates were washed with PBS to remove non-internalised amastigotes and RPMI medium alone (control) or containing drugs (200 to 12,5 µM for ketoconazole, double serial dilutions) was added. Experiments were conducted at 37ºC in a 5% CO2 humidified incubator for 48 h. Cells on coverslips were fixed and stained with Giemsa stain modified solution for evaluation of the infectivity index; 100 cells per coverslip were blind counted and the infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage. This assay was carried out in quadruplicate and two independent experiments were performed.
It is worth highlighting the cytotoxic effect of ketoconazole on murine macrophages cell line RAW264.7 was, previously, performed by MTT test at the same drug concentrations used to assess the viability of promastigotes (section 2.4), but within 48 h. Since the macrophages were infected with amastigotes isolated from animal paw just before use and it was not necessary time to promastigote-amastigote differentiation, the infectivity assay was conducted at 48 h, time that allowed better visualisation and counting of the intramacrophage amastigotes.
Statistics - Statistical analysis was conducted using GraphPad Prism 5. Each set of results was firstly checked for normal distribution using KolmogorovSmirnov, D’Agostinho and Pearson, and ShapiroWalk tests. Normally distributed data were analysed through one-way-ANOVA followed by Tukey’s post-test or Student test T. Statistically significant differences were assumed when at least p < 0.05.
RESULTS:
Ketoconazole inhibits growth parasite and interferes with parasite viability - For evaluation of the antiproliferative effect of ketoconazole, promastigotes were incubated with different concentrations of the drug and the growth curve was followed daily up to 120 h. The drug inhibited the growth of the promastigotes in a concentration-dependent way, most notably after 72 h of treatment, with inhibition rates around 70% for 10 µM and 85% for 100-300 µM from 72 h onwards. (Fig. 1D: EC50 after 72 h = 5.0 µM). Once evidenced the antiproliferative effect of ketoconazole, a viability assay by MTT was carried out. Thus, promastigotes were incubated with several concentrations of ketoconazole for different times (24, 48 and 72 h) in order to assess whether the interference with viability could be preceding the effect on growth observed from 72 h onwards. Ketoconazole caused a concentration- and time-dependent inhibition on the L. (L.) amazonensis promastigote viability after 24, 48 and 72 h of incubation presenting EC50 values of 15.97, 11.75, and 11.71 μM, respectively (Fig. 1A-C). Despite the interference in parasite viability, at the drug concentration capable of inhibit 50% of the proliferation of parasites after 72 h of treatment (ketoconazole 5 µM), the parasites were viable. To confirm this result, another viability assay was performed: the Trypan Blue exclusion test. In the assay, the growth curve was followed up to 72 h and a single and higher ketoconazole concentration was used: 10 µM. The concentration used is close to the EC50 value of viability (72 h) and twice the EC50 of proliferation (72 h), which would make it possible to better observe the drug effect. For the other tests, this concentration was used. When parasites cultured for 48 h in absence of drug were exposed to ketoconazole (10 µM), a reduced number of viable parasites was observed, which remained constant over time, while the control parasites exhibited exponential growth (Fig. 1E). In other words, the ketoconazole interfered with parasite viability, but it is not killing the parasites over the time.
Fig. 1:
Leishmania (Leishmania) amazonensis promastigotes viability and proliferation. (A-C) Concentration-effect curves of ketoconazole on parasite viability by MTT after 24 h (A), 48 h (B), and 72 h (C) of treatment; (D) the concentration-response curves of ketoconazole on parasite proliferation after 24, 48, 72, 96 and 120 h of treatment. The smaller graph displays the respective EC50 value for 72 h. The cell density was obtained by direct counting in a Neubauer chamber of fixed cells. E. Assay on viable parasite proliferation in presence of 10 µM of ketoconazole. The arrow indicates the cultivation time in which the drug was added to the medium. The viable cell density was obtained by direct counting in a Neubauer chamber using Trypan blue stain. Data are represented as mean ± scanning electron microscopy (SEM).
Ketoconazole causes outstanding morphological and ultrastructural alterations - In order to confirm the alterations previously observed and identify possible new damages, the ultrastructural analysis of the parasite was carried out. Promastigotes were incubated in absence (control) or presence of 10 µM of ketoconazole for 72 h. The control parasites exhibited normal cells with typical elongated and thin bodies, lengthened and single flagellum and mitochondrion (Fig. 2A, I). Ketoconazole-treated cells appeared rounded, swollen and with altered cell membrane morphology (Fig. 2 J-K) compared to non-treated ones, as revealed by scanning electron microscopy. Transmission electron microscopy revealed several morphological changes for treated-cells, such as significant mitochondrial swelling (Fig. 2B, L), vesicles associated to Golgi complex (Fig. 2C), augmented accumulation of acidocalcisomes (Fig. 2C, E) and of lipid droplets (Fig. 2D), intense activity of endo/exocytosis at flagellar pocket (Fig. 2F), flagellar alterations (stumpy and detached membrane) (Fig. 2G), and double flagella at flagellar pocket (Fig. 2H, J, K). However, normal kinetoplast (Fig. 2E) was observed.
Fig. 2:ultrastructural and morphological alterations observed in Leishmania (Leishmania) amazonensis promastigotes treated with ketoconazole. Transmission electron microscopy (TEM): (A) control parasite; (B-H) ketoconazole-treated parasites (10 µM); alterations are indicated by arrows: (B) mitochondrial swelling and intact cell membrane (black arrow); (C) vesicles associated to Golgi complex (white arrow) and acidocalcisomes (black arrows); (D) lipid droplets (black arrows); (E) acidocalcisomes (black arrows) and normal kinetoplast (white arrow); (F) intense activity of endo/exocitose at flagellar pocket (black arrow); (G) flagellar alterations (stumpy, detached membrane) (black arrow); (H) double flagella at flagellar pocket (black arrows). Scanning electron microscopy (SEM): (I) control parasite; (J-K) ketoconazole-treated parasites (10 µM) - Double flagella (black arrow). m = mitochondrion. (L) graph shows the quantification of mitochondrion area using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.0001).
Ketoconazole interferes with mitochondrial activity and induces autophagic vacuoles formation - In view of both the drug interference with the parasites viability and the significant mitochondrial swelling, the mitochondrial activities by Rhodamine 123 (Rho 123) and MitoTracker stains were evaluated. Furthermore, the autophagic vacuoles formation was assessed by MDC (monodansylcadaverine) labeling. Ketoconazole elevated mitochondrial activity, as suggested by the increased mitochondrial transmembrane potential (∆Ψm) measured by Rho123 dye. A time-dependent increase in fluorescence was induced by the drug until 72 h of treatment (Fig. 3A-C). The fluorescence intensities of Rho 123 at each time (24, 48 and 72 h) are showed in the graph (Fig. 3D). Moreover, an intense labeling with MitoTrackerR for control parasites and an outstanding decrease in intensity for ketoconazole-treated promastigotes for 72 h (Fig. 3E-K) could be observed, which indicates loss of mitochondrial activity in these parasites.
Autophagic vacuoles were intensely labeled at 24 and 48 h after drug incubation. However, such marking was shown to be reduced after 72 h (Fig. 4). There was statistically significant difference between ketoconazole-treated samples (24, 48, and 72h) compared to control non-treated, with p < 0.001.
Fig. 3:mitochondrial damage induced by ketoconazole on Leishmania (Leishmania) amazonensis. (A-C) flow cytometry histograms displaying changes in mitochondrial transmembrane potential (∆Ψm) measured by Rhodamine 123 (Rho123) in different times of drug exposure (ketoconazole, 10 µM): 24 h (A), 48 h (B), and 72 h (C); (E-J) images obtained by confocal microscopy showing parasites cultured in absence (control, panels E, F and G) or presence of ketoconazole (panels H, I and J) for 72 h and submitted to staining with MitotrackerR. Bar: 10 µm. (D and K) the graphs show the quantification of fluorescence emitted by Rhodamine 123 (D) or MitotrackerR. (K) using the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.05).
Fig. 4:ketoconazole-induced autophagic vacuoles on Leishmania (Leishmania) amazonensis promastigotes. Parasites were incubated with 10 µM ketoconazole for different times (24, 48, and 72 h) and then submitted to MDC staining. The fluorescence intensities were determined by the software ImageJ version 1.48. The graph shows the quantification of fluorescence. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was determined using ANOVA (p < 0.001).
Ketoconazole does not alter the parasite cell cycle - Due to growth inhibition and presence of double flagella and other flagellar alterations observed by electron microscopy analysis, the interference of the drug with the parasite cell cycle was investigated (treated parasites incubated with 10 µM of ketoconazole for 72 h). The interference of the drug with the cell cycle of L. (L.) amazonensis promastigotes, which was determined by counting of nucleus, kinetoplast and flagellum, did not reveal differences (data not shown) between treated and control parasites. The cell cycle analysis carried out by flow cytometry showed that the treatment did not cause alterations in a specific phase of cell cycle. In other words, the treatment does not interfere with the parasite cycle under the conditions tested (Fig. 5).
Fig. 5:cell cycle assay. (A-D) images obtained by confocal microscopy to nucleus, kinetoplast and flagellum counting showing parasites cultured in absence (control, panels A-B) or presence of ketoconazole (10 µM, 72 h) (panels C-D) and submitted to staining with DAPI. Bar: 10 µm. (E-F) typical DNA content frequency histograms representing Leishmania (Leishmania) amazonensis promastigotes incubated with: (E) medium, control or (F) ketoconazole (10 µM; 72 h). The cells were stained with propidium iodide (PI) and fluorescence of the PI-stained cells was measured. Cell cycle analysis provides the estimate of percentage of cells in Sub-G1, G1, S and G2/M phases of the cycle. G. The graph shows the quantification of fluorescence by the software ImageJ version 1.48. Data are expressed as the mean ± standard deviation (SD) and statistically significant difference compared to control was not observed using ANOVA (p < 0.05).
Ketoconazole tends to induce cell death by apoptosis - Given that ketoconazole causes several effects on L. (L.) amazonensis promastigotes (viability interference, mitochondrial swelling, increase in the amount of acidocalcisomes, induction of autophagic vacuoles), but does not alter the cell cycle, it was evaluated the type of cellular death that was occurring. The dot plot analysis of the assays (PI/Annexin V labeling and flow cytometry; treated parasites incubated with 10 µM of ketoconazole for 72 h; Fig. 6) showed that the distribution of viable, necrotic, and apoptotic cells after treatment with ketoconazole for 72 h was similar to that observed for untreated parasites (negative control), tending to cause apoptosis (4.94% versus 1.1%, statistically significant difference with p < 0.001 for ketoconazole and control, respectively), under the conditions tested.
Fig. 6:cell death assay. Images obtained by confocal microscopy showing parasites cultured in presence of medium (negative control), formaldehyde 4% (positive control) or ketoconazole (10 μM, 72 h) and submitted to staining with propidium iodide (PI) and/or Annexin V. Bar: 20 µm. Respective representative dot plots for Annexin V-FITC (A V) and/ or PI staining in Leishmania (Leishmania) amazonensis promastigotes. Lower left quadrant, viable cells (negative for both A V and PI); lower right quadrant, initial apoptotic cells (positive for A V and negative for PI); upper right quadrant, late apoptotic cells (positive for both A V and PI) and upper left quadrant, necrotic cells (positive for PI and negative for A V). The graph indicates the percentage of positive cells for PI, A V and both (A V/PI) for ketoconazole-treated parasites in relation to negative control. *: statistically significant difference compared to negative control with p < 0.001.
Ketoconazole is not toxic for host cells and interferes with infective capacity of amastigotes - In view of the effects induced by ketoconazole on the promastigote parasites, intracellular amastigotes, the clinically relevant form of the parasite, were treated with different concentrations of ketoconazole for 48 h in attempt to evaluate eventual interference of drug on infective capacity. For this purpose, a viability assay was previously carried out for the host cell (murine macrophage cell line RAW264.7). Concentration-dependent inhibition on macrophages viability was observed, with EC50 of 162.18 µM at 48 h. At infectivity condition, the drug interfered with infective capacity of amastigotes. After 48 h of treatment, similar drug concentrations: 11.75 µM (EC50 for promastigote - 48 h; Fig. 1B) and 12.5 µM (lowest tested concentration in the infectivity assay; Fig. 7A) inhibited 50% and 17% of promastigotes and intracellular amastigotes (infectivity), respectively. It is important to note that these concentrations were not toxic to the host cell, as the EC50 for macrophages was about 13-14 times higher.
Fig. 7:viability of murine macrophages cell line RAW264.7 and infective capacity of amastigotes (infectivity). Concentration-effect curve of ketoconazole on macrophages by MTT after 48 h of treatment (A). Infected macrophages were treated with ketoconazole (200-12.5 µM) for 48 h. The infectivity index was determined by multiplying the percentage of macrophages that had phagocytosed at least one parasite by the parasite average per infected macrophage (100 cells were examined) and are shown in the graph (B). Data are expressed as mean ± scanning electron microscopy (SEM). Statistically significant difference compared to negative control with p < 0.05.
DISCUSSION:
Considering the importance of ergosterol for the biology and pathogenesis of the Leishmania parasite, herein, we certified the antiproliferative/viability effects of ketoconazole against L. (L.) amazonensis both promastigotes
31
and intracellular amastigotes
32
in another parasite strain: IFLA/BR/67/PH8. Furthermore, we show in more detail the biological effects of the inhibitor, aiming at understanding the drug mechanism of action.
Regarding ultrastructure, our results revealed rounded up shape, mitochondrial swelling, augmented accumulation of lipid droplets and acidocalcisomes, presence of multivesicular bodies and frequent truncated and/or double flagella. These data are similar to those previously obtained for L. amazonensis promastigotes treated with ketoconazole
31
,
33
,
34
and suggest mitochondrial damage.
When parasites (Trypanosoma cruzi, Leishmania spp, Toxoplasma gondii) are treated with inhibitors of important enzymes of the ergosterol biosynthesis pathway, the mitochondrion is the organelle primarily affected.
35
In our study, the ketoconazole-treated parasites presented hyperpolarisation of the mitochondrial membrane potential, time-dependent up to 72 h. Mitochondrial membrane potential variations could be the result of diverse events: inhibition of electron transport chain (decrease); blockage of ATP synthase (increase); stimulation of uncoupling proteins (decrease); or permeabilisation of the inner membrane (decrease).
36
Macedo et al.
37
demonstrated that L. (L.) amazonensis promastigotes treated with itraconazole and posaconazole for 48 h presented collapse of the mitochondrial membrane potential associated with intense mitochondrial swelling, disorganisation and rupture of mitochondrial membranes. Another study showed that T. cruzi treated with ketoconazole (EC50 = 32 µM for 72 h) presented a gradual increase in Rho 123 time-dependent fluorescence and a confocal microscopy of parasites confirmed an intense proliferation of the inner mitochondrial membrane. This organelle became highly branched and compact and elevated levels of Rho 123 were accumulated within the cells.
38
In many cases, a transient hyperpolarisation occurs before mitochondrial depolarisation, as if it were an attempt of the cells to avoid death. This effect can be seen in great part of the heat-shocked Leishmania promastigotes.
39
The mitochondrial membrane is hyperpolarised does not mean that the organelle is functioning normally. Our results showed that MitoTrackerR labeling, a probe which passively diffuses across the plasma membrane and accumulates in active mitochondria, revealed an outstanding intensity decrease in ketoconazole-treated promastigotes, indicating loss of mitochondrial activity in these promastigotes. This result was also confirmed by the MTT viability assay, which measures the activity of mitochondrial enzymes, and by the proliferation curve using Trypan blue, which takes into account only viable cells. Mitochondrion is essential for generating the necessary energy for the survival and proliferation of eukaryotic cells.
40
) In this way, malfunctioning mitochondria could impair the ATP synthesis and inorganic phosphate would accumulate in acidocalcisomes, which explains the increase in the amount of this organelle 72 h after ketoconazole treatment. Acidocalcisomes are organelles of acidic nature and high electron density important due to their storage of polyphosphates, calcium, magnesium, and other elements.
41
The augmented number of this organelle was already reported for ketoconazole and L. amazonensis (MHOM/Josefa/75/Br strain).
42
The treatment of L. amazonensis with ketoconazole stressed the cells, especially early after the treatment (24 and 48 h), leading to the appearance of autophagic vacuoles. Itraconazole and posaconazole induced appearance of autophagosome-like structures in L. (L.) amazonensis
37
and Rodrigues et al.
43
described the effect of autophagy on L. amazonensis (MHOM/BR/75/Josefa strain) treated with 22,26-azasterol. Thus, the confirmed presence of autophagic vacuoles might represent an adaptive response of the parasite to treatment (stress condition). Initially, parasites would resort to autophagic vacuoles in an attempt to remodel/remove abnormal cellular constituents, degrading damaged structures.
44
Over time, directing energy to the parasite single mitochondrion may be more important than repairing cell damage. This would explain the fact that at 72 h there was less labeling for autophagic vacuoles and higher mitochondrial hyperpolarisation occurred. Autophagy is involved in turnover and recycling by removal of damaged cellular components, regulating homeostasis during crucial processes such as cell growth and differentiation and plays fundamental role in mitochondrial functionality.
44
,
45
Results of the growth curve and frequent ultrastructural alterations in flagellum (truncated and/or double flagella), despite the intact kinetoplast, suggest that ketoconazole interferes with the L. amazonensis cell cycle. Therefore, the cell cycle of the parasites was evaluated both by nucleus, kinetoplast, and flagellum (n-k-f) counts and by flow cytometry. Different from some data in the literature involving ketoconazole acting on other cell types
46
,
47
and azole compounds on L. amazonensis,
37
our results of the n-k-f score did not reveal differences between the control and ketoconazole-treated parasites. In addition, the cell cycle assay showed that the treatment was not able to alter the cycle phases (Sub G1, G1, S, and G2/M) in relation to control parasites suggesting that ketoconazole does not interfere with parasite replication under the tested conditions. When L. (L.) amazonensis was treated with itraconazole and posaconazole, some cells presented more than two flagella and significant alterations in kinetoplast were observed by ultrastructural analysis; however, the possible alteration in the cell cycle was not evaluated.
37
Ketoconazole has been related to apoptosis in several normal and tumoral cells.
48
,
49
,
50
,
51
Haegler et al.
52
showed that ketoconazole and posaconazole presented hepatocellular toxicity, impairing the ∆Ψm, the function of enzyme complexes of electron transport chain, accumulating mitochondrial superoxide and inducing apoptosis. Possibly, mitochondrial dysfunction could be associated with hepatotoxicity caused by those azole compounds. Our data revealed that treatment of the parasites with ketoconazole, under the conditions previously described, showed a trend towards apoptosis, although there was a slight positive result for PI. Furthermore, few autophagic vacuoles were observed after treatment. Other researchers reported that treatment of T. cruzi with ketoconazole (EC50/72 h) resulted in neither necrosis nor apoptosis. However, high concentrations of the drug (EC100/24h) resulted in fast cell death due to necrosis.
38
SBIs effects on extracellular L. amazonensis parasites (promastigotes) appear to be more gradual than that observed with the intracellular form (amastigote);
31
thus, SBIs effects profile allows a more detailed study of the events in the promastigote form. In view of the effects provoked by ketoconazole on the L. (L.) amazonensis promastigote, the interference of drug on infectivity capacity of amastigotes, the clinically relevant form of the parasite, was evaluated and evidenced a susceptibility to treatment. Taken together, the infectivity results added to the data of mitochondrial damage, increase in the acidocalcisome and autophagic vacuoles amounts may be evidence that the susceptibility observed in vitro is related to mitochondrial dysfunction provoked by ketoconazole. However, future studies should be carried out on amastigotes to confirm this hypothesis.
To our knowledge, this study demonstrates the effects of ketoconazole on mitochondrion functioning, autophagic compartments, cell cycle and death of L. (L.) amazonensis promastigotes for the first time. We suggest that some damages are occurring in the parasite, mainly in the mitochondrion. In an attempt to remodel and/or destroy eventual damaged structures, parasites hyperpolarise mitochondrion and resort to autophagic vacuoles. However, this survival strategy adopted by the cell to direct efforts to maintain the cell energy is not sufficient because of the decrease in cell and mitochondria viabilities and increase in acidocalcisomes. Although these damages do not reflect directly in the parasite cell cycle, they lead the parasites to death, making them susceptible to in vitro treatment (Fig. 8).
Fig. 8:proposed mode action of ketoconazole on Leishmania (Leishmania) amazonensis. Parasites resort to autophagic vacuoles and mitochondrion hyperpolarisation in attempt to survive to damages mainly in the mitochondrion. The efforts to maintain the cell energy are not sufficient since the decrease of cell and mitochondria viability and increase of acidocalcisomes. These damages do not interfere with the parasite cell cycle, but they lead the parasites to death, making them susceptible to in vitro treatment conditions.
| Background:
Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase.
Methods:
Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment.
Results:
The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain).
Conclusions:
Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models. | null | null | 6,408 | 208 | [] | 3 | [
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"promastigotes",
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"treated",
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] | [
"parasite certified antiproliferative",
"cell death assay",
"apoptosis given ketoconazole",
"incubated concentrations ketoconazole",
"dimethylsulfoxide dmso penicillin"
] | null | null | null | null | null | null | [CONTENT] cutaneous leishmaniasis | ergosterol | mitochondrial damage | sterol biosynthesis inhibitor [SUMMARY] | null | [CONTENT] cutaneous leishmaniasis | ergosterol | mitochondrial damage | sterol biosynthesis inhibitor [SUMMARY] | null | null | null | [CONTENT] Animals | Antiprotozoal Agents | Flow Cytometry | Humans | Ketoconazole | Leishmania | Leishmaniasis | Mice | Mice, Inbred BALB C | Mitochondria [SUMMARY] | null | [CONTENT] Animals | Antiprotozoal Agents | Flow Cytometry | Humans | Ketoconazole | Leishmania | Leishmaniasis | Mice | Mice, Inbred BALB C | Mitochondria [SUMMARY] | null | null | null | [CONTENT] parasite certified antiproliferative | cell death assay | apoptosis given ketoconazole | incubated concentrations ketoconazole | dimethylsulfoxide dmso penicillin [SUMMARY] | null | [CONTENT] parasite certified antiproliferative | cell death assay | apoptosis given ketoconazole | incubated concentrations ketoconazole | dimethylsulfoxide dmso penicillin [SUMMARY] | null | null | null | [CONTENT] ketoconazole | parasites | cell | 72 | control | cells | promastigotes | 48 | treated | mitochondrial [SUMMARY] | null | [CONTENT] ketoconazole | parasites | cell | 72 | control | cells | promastigotes | 48 | treated | mitochondrial [SUMMARY] | null | null | null | [CONTENT] ketoconazole | fig | 72 | µm | control | parasites | 10 | 48 | 10 µm | cell [SUMMARY] | null | [CONTENT] ketoconazole | parasites | cell | 72 | cells | control | ml | mitochondrial | 10 | fig [SUMMARY] | null | null | null | [CONTENT] first [SUMMARY] | null | [CONTENT] ||| ||| 14α | 14-demethylase ||| ||| ||| first ||| ||| [SUMMARY] | null |
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